Enhanced detection of bladder cancer using combined circulating tumor cells, urine-derived epithelial cells, and molecular biomarkers

Enhanced detection of bladder cancer using combined circulating tumor cells, urine-derived epithelial cells, and molecular biomarkers | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Enhanced detection of bladder cancer using combined circulating tumor cells, urine-derived epithelial cells, and molecular biomarkers Zhijie Jiang, Cheng Yuan, Honggang Yuan, Chengchen Qin, Yanqing Li, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8819995/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract Purpose The sensitivity of bladder cancer detection using a single biomarker from single sample type is limited. This study aimed to investigate whether a combined approach utilizing multiple biomarkers from different clinical samples could improve detection sensitivity. Methods A total of 85 patients with bladder cancer and 30 healthy individuals were enrolled in this study. Urine and blood samples were collected for the isolation of urine-derived epithelial cells(UDECs)and circulating tumor cells (CTCs). These cells were then analyzed via PD-L1 assay and fluorescence in situ hybridization (FISH) targeting chromosomes 7 and 8. In parallel, matched urine samples from patients underwent conventional urine exfoliation cytology testing (UEC). All data were analyzed in conjunction with pathological information using specialized statistical software. Results Analysis of CTCs demonstrated a significantly higher bladder cancer detection rate (78.6%) compared to UEC (36.7%). The combination of UDEC-FISH and CTC analysis utilizing urine and blood samples achieved a higher detection rate (94.1%) than the combination of UDEC-FISH with UEC performed on the same urine sample (79.8%). Furthermore, combined analysis of three markers of CTC, UEC, and UDEC-FISH (96.5%) or CTC, UEC, and UDEC-PD-L1 (90.6%) yielded significantly higher detection rates than any single biomarker analysis alone. Conclusion Integrating multiple biomarkers from distinct sample types significantly enhances the detection sensitivity for bladder cancer. Chromosome FISH (fluorescence in situ hybridization) analysis circulating tumor cells programmed cell death ligand 1 (PD-L1) urine-derived epithelial cells (UDECs) urine-derived tumor cells (UDTCs) urinary exfoliative cytology (UEC) Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Bladder cancer ranks as the ninth most common cancer globally, with approximately 6.13 million new cases and 2.3 million deaths reported annually worldwide (Bray et al. 2024). It is also one of the most common tumors of the urinary system, with the majority of patients diagnosed with non-muscle invasive bladder cancer (NMIBC) (Hoffmann and Schulz 2021; Miyazaki et al. 2017). The conventional diagnostic methods for urothelial carcinoma include endoscopy, urinary exfoliative cytology, or a combination of both; however, their detection rates remain low with limited heterogeneity (Białek et al. 2022). These methods have limited sensitivity and rely heavily on experienced examiners and precise instruments, primarily detecting high-grade bladder cancer or preinvasive lesions (Fiorentino et al. 2023). Malignant cells in bladder lesions typically exhibit weak cohesion, allowing single tumor cells to detach from the primary lesion and shed into the urine. Moreover, urine contains relatively few other cell types, theoretically facilitating the screening of tumor cells in urine samples (Zhang et al. 2021). Circulating tumor cell (CTC) detection is also applied in screening cancers of the urinary system, including renal pelvic carcinoma, bladder cancer, and ureteral carcinoma (Wang and Ding 2023; Koguchi et al. 2022; Wang et al. 2023). CTCs are closely associated with tumor recurrence and metastasis, making them potential biomarkers for diagnosis and prognosis. The presence of CTCs in the peripheral blood of bladder cancer patients often indicates a significantly elevated risk of local recurrence or disease progression, particularly in NMIBC. Studies comparing preoperative and postoperative dynamic CTC monitoring in bladder cancer patients have demonstrated the clinical potential of CTCs in assessing treatment efficacy, correlating with tumor infiltration depth, and reflecting pathological grading (Alva et al. 2015; Cui and Cao 2023). Consequently, CTC detection has emerged as a valuable tool to enhance clinical cancer management. Despite these advances, there remains a need for more sensitive and precise diagnostic and therapeutic approaches for bladder cancer. Urine-derived tumor cells (UDTCs) have therefore garnered increasing research attention, with studies reporting higher diagnostic sensitivity for urinary system cancers, even in low-grade subgroups (Gao et al. 2023; Lee et al. 2007; Gutierrez et al. 2022; Wolfs et al. 2021; Wang et al. 2020). Currently, the diagnostic accuracy of UDTCs largely relies on fluorescence in situ hybridization (FISH), a DNA probe-based molecular technique approved by the U.S. Food and Drug Administration (FDA) (Riesz et al. 2007; Ke et al. 2014; Caraway et al. 2010; Junker et al. 1999; Jia et al. 2012). Multiple studies have shown that FISH detection exhibits higher sensitivity and specificity than urinary exfoliative cytology, enabling the diagnosis of early-stage NMIBC (Zheng et al. 2022; Lin et al. 2019; Liem et al. 2017; Maffezzini et al. 2010). This study, we aimed to evaluate whether combining the analysis of CTCs in blood with urine-derived tumor cells (UDTCs) in urine could improve the diagnostic sensitivity and accuracy for bladder cancer. Material and Methods patients A total of 85 bladder cancer patients were enrolled in this study. Additionally, 30 healthy volunteers were recruited as controls. All participants provided written informed consent, and the study was approved by the local ethics committee, and all experiments were performed in accordance with relevant named guidelines and regulations. Each patient provided two types of samples: 4 mL of peripheral blood and 30 mL of urine. Blood samples were collected in EDTA anticoagulation tubes, while urine samples were collected in specialized storage tubes. Tumor tissues were collected during surgery and immediately placed in stationary liquid. All tumor tissues, blood, and urine samples were stored at 2–6℃ immediately after collection and then processed for subsequent laboratory analyses (Fig. 1 , supplementary data Table S1 ). Materials Instruments and kits for target cell enrichment and isolation, as well as PD-L1 expression analysis, were obtained from Advanced Gene Diagnostics (Hubei, China). The gene probe kit for chromosome 7 and 8 analyses was purchased from Diaglogic Biolabs (Fujian, China). The acridine orange staining reagent kit was obtained from Sigma (MO, USA). Methods Analysis of urinary exfoliative cytology Urinary exfoliative cytology was performed using a simplified acridine orange staining method. Briefly, 3 mL of acridine orange was added to 47 mL of PBS to prepare the staining buffer. Each urine sample was transferred to a 50 mL tube and centrifuged at 2000 rpm for 5 min at 4°C. The supernatant was discarded, and the sediment was resuspended in 30 mL PBS and centrifuged again under the same conditions. After discarding the supernatant, the remaining sediment was spread on a clean microscope slide and air-dried. The slides were then fixed in 90% ethanol for 10 min, washed with PBS, and stained in acridine orange staining buffer for 3 min. After washing with PBS, slides were immersed in glacial acetic acid for several minutes, followed by PBS washing, and then soaked in calcium chloride solution for 1 min with a final PBS wash. Slides were covered with coverslips and examined under a fluorescence microscope by a professional pathologist to identify tumor cells. CTCs analysis For CTC analysis, 4 mL of blood was centrifuged at 1500 rpm for 5 min at 4°C, and the supernatant was discarded. 4ml of cell preservation solution was added to blood, mixed gently, and centrifuged again at 1900 rpm for 10 min at 4°C. The supernatant was discarded, and cell preservation solution was added to 4 mL. Then, 96 µL of cell preservation solution containing 4 µL of EpCAM-antibody magnetic beads was added, mixed, and incubated at room temperature for 30 min. The sample was transferred to a well of a six-well plate and placed on a MagCapturer® Cell isolator to capture CTCs (followed manufacture instruction, Advanced Gene Diagnostics Ltd, Hubei, China). Captured cells were fixed for 10 min, stained with fluorescent antibodies (anti-CK, anti-CD45, anti-AF488, anti-AF568) for 1 hour, and DAPI for 5 min. After washing with PBS, the cells were resuspended in six-well plate with 1 mL PBS and scanned using an automatic fluorescence scanning system. Urine-derived epithelial cells (UDECs) analysis Urine samples (30 mL) were centrifuged once at 1900 rpm for 10 min at 4°C, and the supernatant was discarded. The remaining procedures were performed as described for CTC analysis. PD-L1 analysis on target cells and Chromosome 7 FISH assay PD-L1 expression and chromosome 7 (Chr7) and 8 (Chr8) FISH assays were performed using liquid-phase technology. Detailed procedures are described in Supplementary Figures S2–S4. Statistical analysis All data were analyzed using GraphPad Prism 10 and SPSS software. P-values < 0.05 were considered statistically significant (Fisher’s exact test). In statistical graphs and tables, “ns” indicates no statistical significance, “*” indicates P < 0.05, “**” indicates P < 0.01, and “***” indicates P < 0.001. Results CTCs analysis detected high rate of bladder cancer among the single biomarker analysis In a comparative analysis of matched samples and methods, significant differences were observed between healthy individuals and bladder cancer patients for CTCs, urinary exfoliative cytology, hematuria, average urine-derived epithelial cells (UDECs) number, PD-L1 expression, Chr7/8 abnormalities, average UDECs size, and average proportion of cell nucleus to cell volume. No significant differences were observed for UDECs positive rate or average nucleus size. The analysis of abnormalities in chromosomes 7/8, and CTCs showed significant high detection rates (82.9%, p < 0.0001 and 78.6%, p < 0.001, respectively), followed by PD-L1 (41.4%, p < 0.001) and UEC (36.7%, p < 0.001) when compared to controls (Table 1). Figures 2 and 3 show representative examples of PD-L1 positivity/negativity, normal and abnormal chromosomes 7/8 status, and CTC detection in both healthy individuals and bladder cancer patients. The PD-L1 and chromosomes 7/8 FISH assays were successfully performed in liquid phase, with simplified procedures and reduced processing time (Supplementary Fig. S1 -S3). CTC analysis outperformed urinary exfoliative cytology across pathological classes When comparing detection rates across pathological classes of the bladder cancer patients, CTC analysis was significantly superior to urinary exfoliative cytology in all the pathological classes bladder cancers (high-grade: p = 0.008; low-grade: p < 0.001; invasive: p = 0.007; non-invasive: p < 0.001; multiple intracavitary: p < 0.001, and single intracavitary: p < 0.001) (Table 2). CTC detection rates did not differ significantly between pathological subgroups (high-grade vs. low-grade: p = 0.541; invasive vs. non-invasive: p = 0.909; multiple vs. single intracavitary: p = 0.746). In contrast, urinary exfoliative cytology showed significant bias toward high-grade and invasive tumors (high-grade vs. low-grade: 46.5% vs. 25%, p < 0.05; invasive vs. non-invasive: 48.7% vs. 25%, p < 0.05) but not for multiple vs. single intracavitary tumors (41.2% vs. 28.6%, p = 0.266). Overall, the detection rate of CTCs was significantly higher than urinary exfoliative cytology (78.6% vs. 36.6%, p < 0.001). Comparison of CTCs and UDECs with Chr7/8 abnormality across pathological classes In a comparative analysis, the detection rates of CTCs and chromosomes 7/8 analysis were not significantly different overall or across pathological classes (Table 3) except for high-grade. Chromosomes 7/8 abnormalities did not exhibit significant pathological class bias, with higher rates in high-grade vs low-grade (90.0% vs 73.3%, p = 0.131), invasive vs non-invasive (91.7% vs 73.5%, p = 0.09), multiple intracavitary vs single intracavitary (85.1% vs 78.3%, p = 0.475). Chromosomes 7/8 FISH analysis was successfully performed using a liquid-phase method (Fig. 2 , Supplementary Fig. S3). Chromosomes 7/8 abnormality detection in CTCs and UDECs In the matched chromosome 7/8 FISH analysis, abnormalities were detected in all 12 bladder cancer patients’ CTC samples (positive rate 100%). Among the 70 UDECs samples, 82.9% exhibited these abnormalities (three or more copies); however, this difference in detection rate between CTCs and UDECs samples was not statistically significant (100% vs. 82.9%, p = 0.128). Also, no significant difference was observed between pathological classes for the chromosomes 7/8 analysis (p > 0.05) in both CTCs and UDECs samples (Table 4, Fig. 2 – 3 ). Combined analysis enhanced detection rate significantly for bladder cancer patients The combined biomarker analysis significantly improved detection rates compared with single marker (Table 5). The combination of CTC positivity (CTC+) or urinary exfoliative cytology positivity (UEC+), or chromosomal FISH-positive UDECs (UDEC-FISH+) yielded a detection rate of 96.47%. Similarly, the combination of a positive result in any one of the three assays-CTC, UEC, or UDECs-PD-L1 increased the detection rate to 90.6%. Both combined rates were significantly higher (p < 0.05*) than the rate from any single assay alone (Table 1, 5, Fig. 2 – 4 ). Detection rates were compared between single biomarker analysis (UEC+: urine epithelial cell positive; CTC+: circulating tumor cell positive) and combined biomarker analysis (UEC+/FISH+: UEC positive or chromosomal FISH positive; UEC+/FISH+/CTC+: UEC positive or chromosome FISH positive or CTC positive; UEC+/CTC+: UEC positive or CTC positive; UEC+/CTC+/PD-L1+: UEC positive or CTC positive or PD-L1 positive). The analysis of combined biomarkers demonstrated a significantly higher detection rate compared to that of single biomarker analysis. Discussion Tissue biopsy following cystoscopy remains the gold standard for bladder cancer diagnosis (Ahmadi et al. 2021). However, this invasive method can cause considerable discomfort and complications, such as hematuria, urinary tract infection, and urinary discomfort (Xu et al. 2021; Shkolyar et al. 2025). Non-invasive imaging techniques, including ultrasonography, computerized tomography (CT), magnetic resonance imaging (MRI), intravenous urography, along with cytological examination and tumor marker detection, are also employed for diagnosis (Ahmadi et al. 2021; DeGeorge et al. 2017). With advances in cellular and molecular diagnostics, non-invasive liquid biopsy approaches for urinary system tumors have gained increasing attention (Wu et al. 2023). Urinary exfoliative cytology is a convenient and non-invasive test, but it has limited sensitivity and heterogeneity, particularly for low-grade (G1) bladder carcinoma, and requires evaluation by skilled pathologists (Crocetto et al. 2022; Dey 2024; Muhammad et al. 2019; Filva-Ferreira et al. 2024; Liu et al. 2024). Previous studies have highlighted the importance of detecting tumor cells in urine for early diagnosis of bladder cancer (Gao et al. 2023; Liang et al. 2018). In our study, UDECs with chromosome 7/8 abnormalities demonstrated significantly higher cancer rates than urinary exfoliative cytology (82.9% vs. 36.7%, p < 0.001). The CTC assay reliably detected all six pathological classes of bladder cancer (detection range: 75%–81.6%), whereas urinary exfoliative cytology showed notably lower sensitivity in low-grade vs high-grade tumors (25% vs. 46.5%, p < 0.05) and for non-invasive vs invasive tumors (25% vs. 48.7%, p < 0.05) (Tables 1–2, Fig. 4 ). Chromosome 7/8 abnormalities were frequently detected in both CTCs and patients’ UDECs, with no significant bias across different pathological classes (Table 3, 4). In contrast, the UEC detection rate differed significantly between high-grade and low-grade, as well as between invasive and non-invasive patients (p < 0.05) (Table 2). This indicates that the diagnostic sensitivity of UEC is more limited by tumor heterogeneity. Chromosome FISH serves as a primary and effective method for confirming the origin of CTCs and identifying shared chromosomal abnormalities in bladder cancer patients (Kim et al. 2019). While this chromosome-level analysis can reveal more aggressive tumor cell characteristics, it may fail to detect tumor cells with single-gene mutations. In contrast, CTCs analysis may capture a wider spectrum of tumor heterogeneity by detecting both single-gene variations and chromosome-level changes, providing a more comprehensive representation of cancerous cell properties. Therefore, a combined approach integrating CTC detection, chromosomal FISH, UEC, and PD-L1assays will have better sensitivity and heterogeneity for cancer detection suggests this will be advanced for monitoring patients with diverse clinical features (Tables 1, 5). Conclusion CTC and chromosome analyses alone demonstrated a significantly higher detection rate than urinary exfoliative cytology for bladder cancer. Combining CTCs, urinary exfoliative cytology, and UDECs with FISH or PD-L1 significantly improved detection compared with any single assay. Selecting an appropriate combination of these biomarkers can enhance bladder cancer diagnosis and facilitate clinical monitoring of patients with varying disease characteristics. Declarations Acknowledgments: We thank Dr. Mehmet Toner (Harvard University) for his valuable insights and discussions regarding the experimental aims of this study. We are also grateful to Dr. Kongming Wu (Huazhong University of Science and Technology) and Dr. Hongbing Zhang (Peking Union Medical College) for their participation in the discussion of the results. Additionally, we thank Dr. Stefanie Jeffrey (Stanford University School of Medicine) and Dr. Brian Deng (Medical Center, The Ohio State University) for their assistance in language editing. Funding: This research was partially supported by the National Natural Science Foundation of China (2023035 and 202446 to GD). Author contributions: Conceptualization: KY, ZJ, CY, HY, GD. Methodology: ZJ, CQ, YL, YH, CS, JD, FD, CL. Visualization: XL, JZ, GD. Funding acquisition: GD, JZ. Project administration: KY, CY, GD. Supervision: KY, GD. Writing – original draft: ZJ, QC, GD. Writing – review & editing: XL, XP. Competing interests: Authors declare that they have no competing interest. Ethical approval All participants provided written informed consent using a form specifically approved for this research by the Ethics/Institutional Review Board of Yichang Central People's Hospital. Availability of data and materials: All data associated with this study are presented in the paper or the Supplementary Materials. References Ahmadi H, Duddalwar V et al (2021) Diagnosis and Staging of Bladder Cancer. Hematol Oncol Clin of North Am 35(3):531–541. Alva A, Friedlander T et al (2015) Circulating tumor cells as potential biomarkers in bladder cancer. J Urol 194(3):790–798. Białek Ł, Bilski K et al (2022) Non-Invasive biomarkers in the diagnosis of upper urinary tract urothelial carcinoma-A systematic review. Cancers (Basel). 14(6):1520. Bray F, Laversanne M et al (2024) Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 74(3):229–263. Caraway NP, Khanna A et al (2010) Fluorescence in situ hybridization for detecting urothelial carcinoma: a clinicopathologic study. Cancer Cytopathol 118(5):259–268. Crocetto F, Barone B et al (2022) Liquid biopsy in bladder cancer: State of the art and future perspectives. Crit Rev Oncol Hematol 170:103577. Cui Y, Cao M (2023) Liquid Biopsy in Bladder Cancer. Methods Mol Biol 2695:111–120. DeGeorge KC, Holt HR et al (2017) Bladder Cancer: Diagnosis and Treatment. Am Fam Physician 96(8):507–514. Dey P (2004) Urinary markers of bladder carcinoma. Clin Chim Acta 340(1–2): 57–65. Fiorentino V, Pizzimenti C et al (2023) Bladder Epicheck Test: A novel tool to support urothelial carcinoma diagnosis in urine samples. Int J Mol Sci 24(15):12489. Gao F, Wang J et al (2023) Comprehensive optimization of urinary exfoliated tumor cells tests in bladder cancer with a promising microfluidic platform. Cancer Med 12(6):7283–7293. Gutierrez C, Pinson X et al (2022) Characterization of the peri-membrane fluorescence phenomenon allowing the detection of urothelial tumor cells in urine. Cancers (Basel) 14(9):2171. Hoffmann MJ, Schulz WA (2021) Alterations of Chromatin Regulators in the Pathogenesis of Urinary Bladder Urothelial Carcinoma. Cancers (Basel) 13(23):6040. Jia X-Y, Yu Q et al (2012) Targeting bladder tumor cells in voided urine of Chinese patients with FITC-CSNRDARRC peptide ligand. OncoTargets Ther 5:85–90. Junker K, Werner W et al (1999) Interphase cytogenetic diagnosis of bladder cancer on cells from urine and bladder washing. Int J Oncol 14(2):309–313. Ke Z, Lai Y et al (2014) Diagnosis of bladder cancer from the voided urine specimens using multi-target fluorescence in situ hybridization. Oncol Lett 7(2):325–330. Kim T-J, Moon HW et al (2019) Urovysion FISH Could Be Effective and Useful Method to Confirm the Identity of Cultured Circulating Tumor Cells from Bladder Cancer Patients. J of Cancer 10(14):3259–3266. Koguchi D, Matsumoto K et al (2022) Diagnostic potential of circulating tumor cells, urinary microRNA, and urinary cell-free DNA for bladder cancer: A review. Int J Mol Sci 23(16):9148. Lee S-M, Lee E-J et al (2007) Targeting bladder tumor cells in vivo and in the urine with a peptide identified by phage display. Mol Cancer Res 5(1):11–19. Liang L, Wang Y et al (2018) Microchips for detection of exfoliated tumor cells in urine for identification of bladder cancer. Anal Chim Acta 1044:93–101. Liem EIML, Baard J et al (2017) Fluorescence in situ hybridization as prognostic predictor of tumor recurrence during treatment with Bacillus Calmette-Guérin therapy for intermediate- and high-risk non-muscle-invasive bladder cancer. Med Oncol 34(10):172. Lin T, Jin H et al (2019) Surveillance of non-muscle invasive bladder cancer using fluorescence in situ hybridization: Protocol for a systematic review and meta-analysis. Medicine (Baltimore) 98(7):e14573. Liu T-J, Yang W-C et al (2024) Evaluating artificial intelligence–enhanced digital urine cytology for bladder cancer diagnosis. Cancer Cytopathol 132(11):686–695. Maffezzini M, Campodonico F et al (2010) Prognostic significance of fluorescent in situ hybridization in the follow-up of non-muscle-invasive bladder cancer. Anticancer Res 30(11):4761–4765. Miyazaki J, and Nishiyama H (2017) Epidemiology of urothelial carcinoma. Int J Urol 24(10):730–734. Muhammad AS, Mungadi IA et al (2019) Effectiveness of bladder tumor antigen quantitative test in the diagnosis of bladder carcinoma in a schistosome endemic area. Urol Ann 11(2):143–148. Riesz P, Lotz G et al (2007) Detection of bladder cancer from the urine using fluorescence in situ hybridization technique. Pathol Oncol Res 13(3):187–194. Shkolyar E, Zhou SR et al (2025) Optimizing cystoscopy and TURBT: enhanced imaging and artificial intelligence. Nat Rev Urol 22(1):46–54. Silva-Ferreira M, Carvalho JA et al (2024) Diagnostic Test Accuracy of Urinary DNA Methylation-based Biomarkers for the Detection of Primary and Recurrent Bladder Cancer: A Systematic Review and Meta-analysis. Eura Urol Focus 10(6):992 − 934. Wang L, Ding D (2023) Correlation between mesenchymal circulating tumor cells and prognosis of urologic malignancies: a single-center retrospective analysis. Am J Transl Res 15(1):502–510. Wang Q, Li Z et al (2023) Clinical values of circulating tumor cells count in localized renal cell carcinoma. Transl Cancer Res 12(9):2351–2360. Wang X, Gu Y et al (2020) Unbiased enrichment of urine exfoliated cells on nanostructured substrates for sensitive detection of urothelial tumor cells. Cancer Med 9(1):290–301. Wolfs JRE, Hermans TJN et al (2021) Novel urinary biomarkers ADXBLADDER and bladder EpiCheck for diagnostics of bladder cancer: A review. Urol Oncol 39(3):161–170. Wu S, Li R et al (2023) Liquid biopsy in urothelial carcinoma: Detection techniques and clinical applications. Biomed Pharmacother 165:115027. Xu Y, Luo C et al (2021) Application of nanotechnology in the diagnosis and treatment of bladder cancer. J Nanobiotechnology 19(1):393. Zhang ML, VandenBussche CJ et al (2021) A review of urinary cytology in the setting of upper tract urothelial carcinoma. J Am Soc Cytopathol 10(1):29–35. Zheng W, Lin T et al (2022) An updated systematic review and meta-analysis. Diagnostics (Basel) 12(8):2005. Tables Table 1 to 5 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Tables.docx SupplementaryMaterialJCRCO.doc Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 06 Mar, 2026 Reviews received at journal 25 Feb, 2026 Reviewers agreed at journal 13 Feb, 2026 Reviewers agreed at journal 12 Feb, 2026 Reviewers invited by journal 12 Feb, 2026 Editor assigned by journal 12 Feb, 2026 Submission checks completed at journal 11 Feb, 2026 First submitted to journal 11 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Technology","correspondingAuthor":false,"prefix":"","firstName":"Xinghua","middleName":"","lastName":"Pan","suffix":""},{"id":592007034,"identity":"5302e5b5-23ba-4156-97ee-149f763fc55d","order_by":14,"name":"Kejun Yan","email":"","orcid":"","institution":"China Three Gorges University","correspondingAuthor":false,"prefix":"","firstName":"Kejun","middleName":"","lastName":"Yan","suffix":""},{"id":592007035,"identity":"a36f6d14-cfdb-45be-8444-a9459b3ded28","order_by":15,"name":"Glenn Deng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzElEQVRIiWNgGAWjYDACdhDBBmI0Nj74QJQWZpgWnsPNhjNI0yKR3ibNQYwOeWfmY48Lymzy5CMfNkgzMNjJ6TYQ0GJ4mC3deMa5tGLD24kNxgUMycZmBwhpaeYxk+ZtO5y4cXZiQ/IMhgOJ2whr4f8G1PI/cePMgw2HeYjRIs/MwwbUciBxvgRjYzNRWgyY2cykec4lJ27gSWxmnGFAhF/k25ufSfOU2SXObz/+/MeHCjs5gloMDqAwDAgoB9vSgM4YBaNgFIyCUYAOABXnQKOB+qFQAAAAAElFTkSuQmCC","orcid":"","institution":"Ningbo No. 2 hospital","correspondingAuthor":true,"prefix":"","firstName":"Glenn","middleName":"","lastName":"Deng","suffix":""}],"badges":[],"createdAt":"2026-02-08 08:08:48","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8819995/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8819995/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102893232,"identity":"1e8d8511-86b5-4ad3-9b79-f66b5345a04b","added_by":"auto","created_at":"2026-02-18 05:40:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":423937,"visible":true,"origin":"","legend":"\u003cp\u003eImages of view in surgery of single intracavitary or multiple intracavitary tumors in bladder cancer patients\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e Single intracavitary. \u003cstrong\u003eB\u003c/strong\u003e Multiple intracavitary.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8819995/v1/b1ea066db2afd1b7fafee23e.png"},{"id":102963549,"identity":"ce66fa7c-8833-4204-afc4-ac1376cca5c9","added_by":"auto","created_at":"2026-02-19 04:18:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":272458,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of PD-L1 expression and abnormality of chromosomes 7/8 FISH on urine-derived epithelial cells (UDECs)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e PD-L1-（negative） UDECs in healthy individuals. \u003cstrong\u003eB\u003c/strong\u003e Normal chromosome 7/8 (Chr7/8) and negative PD-L1 in UDECs of healthy individuals. \u003cstrong\u003eC\u003c/strong\u003e UDECs with abnormal chromosome 7/8 (Chr7/8) but no detectable PD-L1 expression. \u003cstrong\u003eD\u003c/strong\u003e UDECs with abnormal chromosomes 7/8 (Chr7/8) and positive PD-L1 expression.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8819995/v1/948b9cc533a046c879577c53.png"},{"id":102893234,"identity":"2815b961-8ea4-47a9-86aa-9683af6bff36","added_by":"auto","created_at":"2026-02-18 05:40:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":379883,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of chromosome FISH and PD-L1 in CTCs and UDECs\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA \u003c/strong\u003eNormal chromosome 7/8 in white blood cells. \u003cstrong\u003eB \u003c/strong\u003eCirculating tumor cells with abnormal chromosome 7/8 and positive PD-L1. \u003cstrong\u003eC \u003c/strong\u003eCirculating tumor cells with abnormal chromosome 7/8 and negative PD-L1. \u0026nbsp;\u003cstrong\u003eD\u003c/strong\u003e UDECs (urine-derived epithelial cells) with positive PD-L1. \u003cstrong\u003eE\u003c/strong\u003e UDECs (urine-derived epithelial cells) with negative PD-L1.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8819995/v1/8f89f588eb907cd78966c5d3.png"},{"id":102963607,"identity":"9d8c160c-32b2-46a8-97f5-b355b0820be1","added_by":"auto","created_at":"2026-02-19 04:19:21","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5713,"visible":true,"origin":"","legend":"\u003cp\u003eDetection Rate for Bladder Cancer Using Single and Combined Biomarker Analysis\u003c/p\u003e\n\u003cp\u003eDetection rates were compared between single biomarker analysis (UEC+: urine epithelial cell positive; CTC+: circulating tumor cell positive) and combined biomarker analysis (UEC+/FISH+: UEC positive or chromosomal FISH positive; UEC+/FISH+/CTC+: UEC positive or chromosome FISH positive or CTC positive; UEC+/CTC+: UEC positive or CTC positive; UEC+/CTC+/PD-L1+: UEC positive or CTC positive or PD-L1 positive). The analysis of combined biomarkers demonstrated a significantly higher detection rate compared to that of single biomarker analysis.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8819995/v1/65b41ce656591721724dcb26.png"},{"id":103049641,"identity":"5c2413c1-c792-476c-9332-97e7510ed204","added_by":"auto","created_at":"2026-02-20 07:44:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2110316,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8819995/v1/57b2c432-1621-46e8-8922-c531e3303e41.pdf"},{"id":102893230,"identity":"b7cfffd0-02bd-46fc-8f85-5ba4897ae7da","added_by":"auto","created_at":"2026-02-18 05:40:57","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":49452,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-8819995/v1/31b579a18833200a1e99230d.docx"},{"id":102893235,"identity":"036af53d-2c60-4114-86e3-8217302c330d","added_by":"auto","created_at":"2026-02-18 05:40:57","extension":"doc","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":286350,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterialJCRCO.doc","url":"https://assets-eu.researchsquare.com/files/rs-8819995/v1/12713f15ca616f5271d231c4.doc"}],"financialInterests":"No competing interests reported.","formattedTitle":"Enhanced detection of bladder cancer using combined circulating tumor cells, urine-derived epithelial cells, and molecular biomarkers","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBladder cancer ranks as the ninth most common cancer globally, with approximately 6.13\u0026nbsp;million new cases and 2.3\u0026nbsp;million deaths reported annually worldwide (Bray et al. 2024). It is also one of the most common tumors of the urinary system, with the majority of patients diagnosed with non-muscle invasive bladder cancer (NMIBC) (Hoffmann and Schulz 2021; Miyazaki et al. 2017). The conventional diagnostic methods for urothelial carcinoma include endoscopy, urinary exfoliative cytology, or a combination of both; however, their detection rates remain low with limited heterogeneity (Białek et al. 2022). These methods have limited sensitivity and rely heavily on experienced examiners and precise instruments, primarily detecting high-grade bladder cancer or preinvasive lesions (Fiorentino et al. 2023). Malignant cells in bladder lesions typically exhibit weak cohesion, allowing single tumor cells to detach from the primary lesion and shed into the urine. Moreover, urine contains relatively few other cell types, theoretically facilitating the screening of tumor cells in urine samples (Zhang et al. 2021). Circulating tumor cell (CTC) detection is also applied in screening cancers of the urinary system, including renal pelvic carcinoma, bladder cancer, and ureteral carcinoma (Wang and Ding 2023; Koguchi et al. 2022; Wang et al. 2023). CTCs are closely associated with tumor recurrence and metastasis, making them potential biomarkers for diagnosis and prognosis. The presence of CTCs in the peripheral blood of bladder cancer patients often indicates a significantly elevated risk of local recurrence or disease progression, particularly in NMIBC. Studies comparing preoperative and postoperative dynamic CTC monitoring in bladder cancer patients have demonstrated the clinical potential of CTCs in assessing treatment efficacy, correlating with tumor infiltration depth, and reflecting pathological grading (Alva et al. 2015; Cui and Cao 2023). Consequently, CTC detection has emerged as a valuable tool to enhance clinical cancer management. Despite these advances, there remains a need for more sensitive and precise diagnostic and therapeutic approaches for bladder cancer. Urine-derived tumor cells (UDTCs) have therefore garnered increasing research attention, with studies reporting higher diagnostic sensitivity for urinary system cancers, even in low-grade subgroups (Gao et al. 2023; Lee et al. 2007; Gutierrez et al. 2022; Wolfs et al. 2021; Wang et al. 2020). Currently, the diagnostic accuracy of UDTCs largely relies on fluorescence in situ hybridization (FISH), a DNA probe-based molecular technique approved by the U.S. Food and Drug Administration (FDA) (Riesz et al. 2007; Ke et al. 2014; Caraway et al. 2010; Junker et al. 1999; Jia et al. 2012). Multiple studies have shown that FISH detection exhibits higher sensitivity and specificity than urinary exfoliative cytology, enabling the diagnosis of early-stage NMIBC (Zheng et al. 2022; Lin et al. 2019; Liem et al. 2017; Maffezzini et al. 2010).\u003c/p\u003e \u003cp\u003eThis study, we aimed to evaluate whether combining the analysis of CTCs in blood with urine-derived tumor cells (UDTCs) in urine could improve the diagnostic sensitivity and accuracy for bladder cancer.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003epatients\u003c/h2\u003e \u003cp\u003eA total of 85 bladder cancer patients were enrolled in this study. Additionally, 30 healthy volunteers were recruited as controls. All participants provided written informed consent, and the study was approved by the local ethics committee, and all experiments were performed in accordance with relevant named guidelines and regulations. Each patient provided two types of samples: 4 mL of peripheral blood and 30 mL of urine. Blood samples were collected in EDTA anticoagulation tubes, while urine samples were collected in specialized storage tubes. Tumor tissues were collected during surgery and immediately placed in stationary liquid. All tumor tissues, blood, and urine samples were stored at 2\u0026ndash;6℃ immediately after collection and then processed for subsequent laboratory analyses (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, supplementary data Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMaterials\u003c/h3\u003e\n\u003cp\u003eInstruments and kits for target cell enrichment and isolation, as well as PD-L1 expression analysis, were obtained from Advanced Gene Diagnostics (Hubei, China). The gene probe kit for chromosome 7 and 8 analyses was purchased from Diaglogic Biolabs (Fujian, China). The acridine orange staining reagent kit was obtained from Sigma (MO, USA).\u003c/p\u003e\n\u003ch3\u003eMethods\u003c/h3\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of urinary exfoliative cytology\u003c/h2\u003e \u003cp\u003eUrinary exfoliative cytology was performed using a simplified acridine orange staining method. Briefly, 3 mL of acridine orange was added to 47 mL of PBS to prepare the staining buffer. Each urine sample was transferred to a 50 mL tube and centrifuged at 2000 rpm for 5 min at 4\u0026deg;C. The supernatant was discarded, and the sediment was resuspended in 30 mL PBS and centrifuged again under the same conditions. After discarding the supernatant, the remaining sediment was spread on a clean microscope slide and air-dried. The slides were then fixed in 90% ethanol for 10 min, washed with PBS, and stained in acridine orange staining buffer for 3 min. After washing with PBS, slides were immersed in glacial acetic acid for several minutes, followed by PBS washing, and then soaked in calcium chloride solution for 1 min with a final PBS wash. Slides were covered with coverslips and examined under a fluorescence microscope by a professional pathologist to identify tumor cells.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCTCs analysis\u003c/h3\u003e\n\u003cp\u003eFor CTC analysis, 4 mL of blood was centrifuged at 1500 rpm for 5 min at 4\u0026deg;C, and the supernatant was discarded. 4ml of cell preservation solution was added to blood, mixed gently, and centrifuged again at 1900 rpm for 10 min at 4\u0026deg;C. The supernatant was discarded, and cell preservation solution was added to 4 mL. Then, 96 \u0026micro;L of cell preservation solution containing 4 \u0026micro;L of EpCAM-antibody magnetic beads was added, mixed, and incubated at room temperature for 30 min. The sample was transferred to a well of a six-well plate and placed on a MagCapturer\u0026reg; Cell isolator to capture CTCs (followed manufacture instruction, Advanced Gene Diagnostics Ltd, Hubei, China). Captured cells were fixed for 10 min, stained with fluorescent antibodies (anti-CK, anti-CD45, anti-AF488, anti-AF568) for 1 hour, and DAPI for 5 min. After washing with PBS, the cells were resuspended in six-well plate with 1 mL PBS and scanned using an automatic fluorescence scanning system.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eUrine-derived epithelial cells (UDECs) analysis\u003c/h2\u003e \u003cp\u003eUrine samples (30 mL) were centrifuged once at 1900 rpm for 10 min at 4\u0026deg;C, and the supernatant was discarded. The remaining procedures were performed as described for CTC analysis.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePD-L1 analysis on target cells and Chromosome 7 FISH assay\u003c/h3\u003e\n\u003cp\u003ePD-L1 expression and chromosome 7 (Chr7) and 8 (Chr8) FISH assays were performed using liquid-phase technology. Detailed procedures are described in Supplementary Figures S2\u0026ndash;S4.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll data were analyzed using GraphPad Prism 10 and SPSS software. P-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant (Fisher\u0026rsquo;s exact test). In statistical graphs and tables, \u0026ldquo;ns\u0026rdquo; indicates no statistical significance, \u0026ldquo;*\u0026rdquo; indicates P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u0026ldquo;**\u0026rdquo; indicates P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and \u0026ldquo;***\u0026rdquo; indicates P\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eCTCs analysis detected high rate of bladder cancer among the single biomarker analysis\u003c/h2\u003e\n \u003cp\u003eIn a comparative analysis of matched samples and methods, significant differences were observed between healthy individuals and bladder cancer patients for CTCs, urinary exfoliative cytology, hematuria, average urine-derived epithelial cells (UDECs) number, PD-L1 expression, Chr7/8 abnormalities, average UDECs size, and average proportion of cell nucleus to cell volume. No significant differences were observed for UDECs positive rate or average nucleus size.\u003c/p\u003e\n \u003cp\u003eThe analysis of abnormalities in chromosomes 7/8, and CTCs showed significant high detection rates (82.9%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 and 78.6%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, respectively), followed by PD-L1 (41.4%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and UEC (36.7%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) when compared to controls (Table 1). Figures \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e show representative examples of PD-L1 positivity/negativity, normal and abnormal chromosomes 7/8 status, and CTC detection in both healthy individuals and bladder cancer patients. The PD-L1 and chromosomes 7/8 FISH assays were successfully performed in liquid phase, with simplified procedures and reduced processing time (Supplementary Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e-S3).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eCTC analysis outperformed urinary exfoliative cytology across pathological classes\u003c/h2\u003e\n \u003cp\u003eWhen comparing detection rates across pathological classes of the bladder cancer patients, CTC analysis was significantly superior to urinary exfoliative cytology in all the pathological classes bladder cancers (high-grade: p\u0026thinsp;=\u0026thinsp;0.008; low-grade: p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; invasive: p\u0026thinsp;=\u0026thinsp;0.007; non-invasive: p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; multiple intracavitary: p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, and single intracavitary: p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Table\u0026nbsp;2).\u003c/p\u003e\n \u003cp\u003eCTC detection rates did not differ significantly between pathological subgroups (high-grade vs. low-grade: p\u0026thinsp;=\u0026thinsp;0.541; invasive vs. non-invasive: p\u0026thinsp;=\u0026thinsp;0.909; multiple vs. single intracavitary: p\u0026thinsp;=\u0026thinsp;0.746). In contrast, urinary exfoliative cytology showed significant bias toward high-grade and invasive tumors (high-grade vs. low-grade: 46.5% vs. 25%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; invasive vs. non-invasive: 48.7% vs. 25%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) but not for multiple vs. single intracavitary tumors (41.2% vs. 28.6%, p\u0026thinsp;=\u0026thinsp;0.266). Overall, the detection rate of CTCs was significantly higher than urinary exfoliative cytology (78.6% vs. 36.6%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eComparison of CTCs and UDECs with Chr7/8 abnormality across pathological classes\u003c/h2\u003e\n \u003cp\u003eIn a comparative analysis, the detection rates of CTCs and chromosomes 7/8 analysis were not significantly different overall or across pathological classes (Table 3) except for high-grade. Chromosomes 7/8 abnormalities did not exhibit significant pathological class bias, with higher rates in high-grade vs low-grade (90.0% vs 73.3%, p\u0026thinsp;=\u0026thinsp;0.131), invasive vs non-invasive (91.7% vs 73.5%, p\u0026thinsp;=\u0026thinsp;0.09), multiple intracavitary vs single intracavitary (85.1% vs 78.3%, p\u0026thinsp;=\u0026thinsp;0.475). Chromosomes 7/8 FISH analysis was successfully performed using a liquid-phase method (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, Supplementary Fig. S3).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eChromosomes 7/8 abnormality detection in CTCs and UDECs\u003c/h2\u003e\n \u003cp\u003eIn the matched chromosome 7/8 FISH analysis, abnormalities were detected in all 12 bladder cancer patients\u0026rsquo; CTC samples (positive rate 100%). Among the 70 UDECs samples, 82.9% exhibited these abnormalities (three or more copies); however, this difference in detection rate between CTCs and UDECs samples was not statistically significant (100% vs. 82.9%, p\u0026thinsp;=\u0026thinsp;0.128). Also, no significant difference was observed between pathological classes for the chromosomes 7/8 analysis (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) in both CTCs and UDECs samples (Table 4, Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003eCombined analysis enhanced detection rate significantly for bladder cancer patients\u003c/h2\u003e\n \u003cp\u003eThe combined biomarker analysis significantly improved detection rates compared with single marker (Table 5). The combination of CTC positivity (CTC+) or urinary exfoliative cytology positivity (UEC+), or chromosomal FISH-positive UDECs (UDEC-FISH+) yielded a detection rate of 96.47%. Similarly, the combination of a positive result in any one of the three assays-CTC, UEC, or UDECs-PD-L1 increased the detection rate to 90.6%. Both combined rates were significantly higher (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05*) than the rate from any single assay alone (Table 1, 5, Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eDetection rates were compared between single biomarker analysis (UEC+: urine epithelial cell positive; CTC+: circulating tumor cell positive) and combined biomarker analysis (UEC+/FISH+: UEC positive or chromosomal FISH positive; UEC+/FISH+/CTC+: UEC positive or chromosome FISH positive or CTC positive; UEC+/CTC+: UEC positive or CTC positive; UEC+/CTC+/PD-L1+: UEC positive or CTC positive or PD-L1 positive). The analysis of combined biomarkers demonstrated a significantly higher detection rate compared to that of single biomarker analysis.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eTissue biopsy following cystoscopy remains the gold standard for bladder cancer diagnosis (Ahmadi et al. 2021). However, this invasive method can cause considerable discomfort and complications, such as hematuria, urinary tract infection, and urinary discomfort (Xu et al. 2021; Shkolyar et al. 2025). Non-invasive imaging techniques, including ultrasonography, computerized tomography (CT), magnetic resonance imaging (MRI), intravenous urography, along with cytological examination and tumor marker detection, are also employed for diagnosis (Ahmadi et al. 2021; DeGeorge et al. 2017).\u003c/p\u003e \u003cp\u003eWith advances in cellular and molecular diagnostics, non-invasive liquid biopsy approaches for urinary system tumors have gained increasing attention (Wu et al. 2023). Urinary exfoliative cytology is a convenient and non-invasive test, but it has limited sensitivity and heterogeneity, particularly for low-grade (G1) bladder carcinoma, and requires evaluation by skilled pathologists (Crocetto et al. 2022; Dey 2024; Muhammad et al. 2019; Filva-Ferreira et al. 2024; Liu et al. 2024).\u003c/p\u003e \u003cp\u003ePrevious studies have highlighted the importance of detecting tumor cells in urine for early diagnosis of bladder cancer (Gao et al. 2023; Liang et al. 2018). In our study, UDECs with chromosome 7/8 abnormalities demonstrated significantly higher cancer rates than urinary exfoliative cytology (82.9% vs. 36.7%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The CTC assay reliably detected all six pathological classes of bladder cancer (detection range: 75%\u0026ndash;81.6%), whereas urinary exfoliative cytology showed notably lower sensitivity in low-grade vs high-grade tumors (25% vs. 46.5%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and for non-invasive vs invasive tumors (25% vs. 48.7%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Tables\u0026nbsp;1\u0026ndash;2, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eChromosome 7/8 abnormalities were frequently detected in both CTCs and patients\u0026rsquo; UDECs, with no significant bias across different pathological classes (Table\u0026nbsp;3, 4). In contrast, the UEC detection rate differed significantly between high-grade and low-grade, as well as between invasive and non-invasive patients (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Table\u0026nbsp;2). This indicates that the diagnostic sensitivity of UEC is more limited by tumor heterogeneity.\u003c/p\u003e \u003cp\u003eChromosome FISH serves as a primary and effective method for confirming the origin of CTCs and identifying shared chromosomal abnormalities in bladder cancer patients (Kim et al. 2019). While this chromosome-level analysis can reveal more aggressive tumor cell characteristics, it may fail to detect tumor cells with single-gene mutations. In contrast, CTCs analysis may capture a wider spectrum of tumor heterogeneity by detecting both single-gene variations and chromosome-level changes, providing a more comprehensive representation of cancerous cell properties. Therefore, a combined approach integrating CTC detection, chromosomal FISH, UEC, and PD-L1assays will have better sensitivity and heterogeneity for cancer detection suggests this will be advanced for monitoring patients with diverse clinical features (Tables\u0026nbsp;1, 5).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eCTC and chromosome analyses alone demonstrated a significantly higher detection rate than urinary exfoliative cytology for bladder cancer. Combining CTCs, urinary exfoliative cytology, and UDECs with FISH or PD-L1 significantly improved detection compared with any single assay. Selecting an appropriate combination of these biomarkers can enhance bladder cancer diagnosis and facilitate clinical monitoring of patients with varying disease characteristics.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u0026nbsp;\u003c/strong\u003eWe thank Dr. Mehmet Toner (Harvard University) for his valuable insights and discussions regarding the experimental aims of this study. We are also grateful to Dr. Kongming Wu (Huazhong University of Science and Technology) and Dr. Hongbing Zhang (Peking Union Medical College) for their participation in the discussion of the results. Additionally, we thank Dr. Stefanie Jeffrey (Stanford University School of Medicine) and Dr. Brian Deng (Medical Center, The Ohio State University) for their assistance in language editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This research was partially supported by the National Natural Science Foundation of China (2023035 and 202446 to GD).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u003c/strong\u003e\u0026nbsp; Conceptualization: KY, ZJ, CY, HY, GD. Methodology: ZJ, CQ, YL, YH, CS, JD, FD, CL. Visualization: XL, JZ, GD. Funding acquisition: GD, JZ. Project administration: KY, CY, GD. Supervision: KY, GD. Writing \u0026ndash; original draft: ZJ, QC, GD. Writing \u0026ndash; review \u0026amp; editing: XL, XP.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003eAuthors declare that they have no competing interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e All participants provided written informed consent using a form specifically approved for this research by the Ethics/Institutional Review Board of Yichang Central People\u0026apos;s Hospital.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u003c/strong\u003e All data associated with this study are presented in the paper or the Supplementary Materials.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAhmadi H, Duddalwar V et al (2021) Diagnosis and Staging of Bladder Cancer. Hematol Oncol Clin of North Am 35(3):531\u0026ndash;541.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlva A, Friedlander T et al (2015) Circulating tumor cells as potential biomarkers in bladder cancer. J Urol 194(3):790\u0026ndash;798.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBiałek Ł, Bilski K et al (2022) Non-Invasive biomarkers in the diagnosis of upper urinary tract urothelial carcinoma-A systematic review. Cancers (Basel). 14(6):1520.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBray F, Laversanne M et al (2024) Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 74(3):229\u0026ndash;263.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCaraway NP, Khanna A et al (2010) Fluorescence in situ hybridization for detecting urothelial carcinoma: a clinicopathologic study. Cancer Cytopathol 118(5):259\u0026ndash;268.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCrocetto F, Barone B et al (2022) Liquid biopsy in bladder cancer: State of the art and future perspectives. Crit Rev Oncol Hematol 170:103577.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCui Y, Cao M (2023) Liquid Biopsy in Bladder Cancer. Methods Mol Biol 2695:111\u0026ndash;120.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeGeorge KC, Holt HR et al (2017) Bladder Cancer: Diagnosis and Treatment. Am Fam Physician 96(8):507\u0026ndash;514.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDey P (2004) Urinary markers of bladder carcinoma. Clin Chim Acta 340(1\u0026ndash;2): 57\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFiorentino V, Pizzimenti C et al (2023) Bladder Epicheck Test: A novel tool to support urothelial carcinoma diagnosis in urine samples. Int J Mol Sci 24(15):12489.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGao F, Wang J et al (2023) Comprehensive optimization of urinary exfoliated tumor cells tests in bladder cancer with a promising microfluidic platform. Cancer Med 12(6):7283\u0026ndash;7293.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGutierrez C, Pinson X et al (2022) Characterization of the peri-membrane fluorescence phenomenon allowing the detection of urothelial tumor cells in urine. Cancers (Basel) 14(9):2171.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHoffmann MJ, Schulz WA (2021) Alterations of Chromatin Regulators in the Pathogenesis of Urinary Bladder Urothelial Carcinoma. Cancers (Basel) 13(23):6040.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJia X-Y, Yu Q et al (2012) Targeting bladder tumor cells in voided urine of Chinese patients with FITC-CSNRDARRC peptide ligand. OncoTargets Ther 5:85\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJunker K, Werner W et al (1999) Interphase cytogenetic diagnosis of bladder cancer on cells from urine and bladder washing. Int J Oncol 14(2):309\u0026ndash;313.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKe Z, Lai Y et al (2014) Diagnosis of bladder cancer from the voided urine specimens using multi-target fluorescence in situ hybridization. Oncol Lett 7(2):325\u0026ndash;330.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim T-J, Moon HW et al (2019) Urovysion FISH Could Be Effective and Useful Method to Confirm the Identity of Cultured Circulating Tumor Cells from Bladder Cancer Patients. J of Cancer 10(14):3259\u0026ndash;3266.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKoguchi D, Matsumoto K et al (2022) Diagnostic potential of circulating tumor cells, urinary microRNA, and urinary cell-free DNA for bladder cancer: A review. Int J Mol Sci 23(16):9148.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee S-M, Lee E-J et al (2007) Targeting bladder tumor cells in vivo and in the urine with a peptide identified by phage display. Mol Cancer Res 5(1):11\u0026ndash;19.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiang L, Wang Y et al (2018) Microchips for detection of exfoliated tumor cells in urine for identification of bladder cancer. Anal Chim Acta 1044:93\u0026ndash;101.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiem EIML, Baard J et al (2017) Fluorescence in situ hybridization as prognostic predictor of tumor recurrence during treatment with Bacillus Calmette-Gu\u0026eacute;rin therapy for intermediate- and high-risk non-muscle-invasive bladder cancer. Med Oncol 34(10):172.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin T, Jin H et al (2019) Surveillance of non-muscle invasive bladder cancer using fluorescence in situ hybridization: Protocol for a systematic review and meta-analysis. Medicine (Baltimore) 98(7):e14573.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu T-J, Yang W-C et al (2024) Evaluating artificial intelligence\u0026ndash;enhanced digital urine cytology for bladder cancer diagnosis. Cancer Cytopathol 132(11):686\u0026ndash;695.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaffezzini M, Campodonico F et al (2010) Prognostic significance of fluorescent in situ hybridization in the follow-up of non-muscle-invasive bladder cancer. Anticancer Res 30(11):4761\u0026ndash;4765.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiyazaki J, and Nishiyama H (2017) Epidemiology of urothelial carcinoma. Int J Urol 24(10):730\u0026ndash;734.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMuhammad AS, Mungadi IA et al (2019) Effectiveness of bladder tumor antigen quantitative test in the diagnosis of bladder carcinoma in a schistosome endemic area. Urol Ann 11(2):143\u0026ndash;148.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRiesz P, Lotz G et al (2007) Detection of bladder cancer from the urine using fluorescence in situ hybridization technique. Pathol Oncol Res 13(3):187\u0026ndash;194.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShkolyar E, Zhou SR et al (2025) Optimizing cystoscopy and TURBT: enhanced imaging and artificial intelligence. Nat Rev Urol 22(1):46\u0026ndash;54.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSilva-Ferreira M, Carvalho JA et al (2024) Diagnostic Test Accuracy of Urinary DNA Methylation-based Biomarkers for the Detection of Primary and Recurrent Bladder Cancer: A Systematic Review and Meta-analysis. Eura Urol Focus 10(6):992\u0026thinsp;\u0026minus;\u0026thinsp;934.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang L, Ding D (2023) Correlation between mesenchymal circulating tumor cells and prognosis of urologic malignancies: a single-center retrospective analysis. Am J Transl Res 15(1):502\u0026ndash;510.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Q, Li Z et al (2023) Clinical values of circulating tumor cells count in localized renal cell carcinoma. Transl Cancer Res 12(9):2351\u0026ndash;2360.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang X, Gu Y et al (2020) Unbiased enrichment of urine exfoliated cells on nanostructured substrates for sensitive detection of urothelial tumor cells. Cancer Med 9(1):290\u0026ndash;301.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWolfs JRE, Hermans TJN et al (2021) Novel urinary biomarkers ADXBLADDER and bladder EpiCheck for diagnostics of bladder cancer: A review. Urol Oncol 39(3):161\u0026ndash;170.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu S, Li R et al (2023) Liquid biopsy in urothelial carcinoma: Detection techniques and clinical applications. Biomed Pharmacother 165:115027.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu Y, Luo C et al (2021) Application of nanotechnology in the diagnosis and treatment of bladder cancer. J Nanobiotechnology 19(1):393.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang ML, VandenBussche CJ et al (2021) A review of urinary cytology in the setting of upper tract urothelial carcinoma. J Am Soc Cytopathol 10(1):29\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZheng W, Lin T et al (2022) An updated systematic review and meta-analysis. Diagnostics (Basel) 12(8):2005.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 to 5 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-cancer-research-and-clinical-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jocr","sideBox":"Learn more about [Journal of Cancer Research and Clinical Oncology](https://www.springer.com/journal/432)","snPcode":"432","submissionUrl":"https://submission.nature.com/new-submission/432/3","title":"Journal of Cancer Research and Clinical Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Chromosome FISH (fluorescence in situ hybridization) analysis, circulating tumor cells, programmed cell death ligand 1 (PD-L1), urine-derived epithelial cells (UDECs), urine-derived tumor cells (UDTCs), urinary exfoliative cytology (UEC)","lastPublishedDoi":"10.21203/rs.3.rs-8819995/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8819995/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eThe sensitivity of bladder cancer detection using a single biomarker from single sample type is limited. This study aimed to investigate whether a combined approach utilizing multiple biomarkers from different clinical samples could improve detection sensitivity.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eA total of 85 patients with bladder cancer and 30 healthy individuals were enrolled in this study. Urine and blood samples were collected for the isolation of urine-derived epithelial cells(UDECs)and circulating tumor cells (CTCs). These cells were then analyzed via PD-L1 assay and fluorescence in situ hybridization (FISH) targeting chromosomes 7 and 8. In parallel, matched urine samples from patients underwent conventional urine exfoliation cytology testing (UEC). All data were analyzed in conjunction with pathological information using specialized statistical software.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eAnalysis of CTCs demonstrated a significantly higher bladder cancer detection rate (78.6%) compared to UEC (36.7%). The combination of UDEC-FISH and CTC analysis utilizing urine and blood samples achieved a higher detection rate (94.1%) than the combination of UDEC-FISH with UEC performed on the same urine sample (79.8%). Furthermore, combined analysis of three markers of CTC, UEC, and UDEC-FISH (96.5%) or CTC, UEC, and UDEC-PD-L1 (90.6%) yielded significantly higher detection rates than any single biomarker analysis alone.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eIntegrating multiple biomarkers from distinct sample types significantly enhances the detection sensitivity for bladder cancer.\u003c/p\u003e","manuscriptTitle":"Enhanced detection of bladder cancer using combined circulating tumor cells, urine-derived epithelial cells, and molecular biomarkers","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-18 05:40:52","doi":"10.21203/rs.3.rs-8819995/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-06T07:34:04+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-25T21:58:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"284640855669489375271353984519777259800","date":"2026-02-14T03:41:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"310649684912064560821219531284455364510","date":"2026-02-12T10:52:31+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-12T10:19:19+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-12T06:43:46+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-11T14:57:35+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Cancer Research and Clinical Oncology","date":"2026-02-11T08:05:27+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-cancer-research-and-clinical-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jocr","sideBox":"Learn more about [Journal of Cancer Research and Clinical Oncology](https://www.springer.com/journal/432)","snPcode":"432","submissionUrl":"https://submission.nature.com/new-submission/432/3","title":"Journal of Cancer Research and Clinical Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"73c2978d-0951-4ee3-9541-de2a99775643","owner":[],"postedDate":"February 18th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-23T07:42:12+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-18 05:40:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8819995","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8819995","identity":"rs-8819995","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}