Aberrant β-Catenin expression is related to invasive tumor growth in urothelial carcinoma of the urinary bladder

Aberrant β-Catenin expression is related to invasive tumor growth in urothelial carcinoma of the urinary bladder | 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 Aberrant β-Catenin expression is related to invasive tumor growth in urothelial carcinoma of the urinary bladder Florian Lutz, Resha Sharifi, Fiete Gehrisch, Natalia Gorbokon, and 32 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8639026/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 β-Catenin is a dual function protein with roles in cell cohesion and as a critical intracellular signal transducer in the Wnt signaling pathway. Cytoplasmic and nuclear translocation of the β-Catenin protein results in an increased transcription of cancer promoting genes. To study the prevalence and the potential role of aberrant β-Catenin staining patterns, more than 2,700 bladder tumors were analyzed by immunohistochemistry (IHC) in a tissue microarray format. The cohort included 636 patients with radical cystectomy for muscle-invasive disease (pT2-4) for which follow-up data were available. In normal urothelium, β-Catenin staining was always strong and largely limited to the cell membranes. A comparable staining pattern was also seen in the overwhelming majority of non-invasive pTa tumors, especially in case of low-grade neoplasms. Aberrant β-Catenin staining patterns were observed in 20.3% of tumors and included unequivocal (2.8%) and equivocal nuclear/cytoplasmic staining (10.6%), complete loss of β-Catenin staining (1.5%), and reduced β-Catenin staining (1+, 5.4%). β-Catenin staining was heterogeneous in 20.8%. All aberrant β-Catenin staining patterns were associated with invasive tumor growth (p = < 0.0001–0.0006), but unrelated to survival of patients with pT2-4 cancers. A complete loss of membranous β-Catenin staining was linked to UICC stage (p = 0.0106) and within pT2-4 tumors, to high pN (p = 0.0014) and pT p = 0.0100). Associations also occurred between heterogeneity and nodal metastasis (p = 0.0207), equivocal nuclear/cytoplasmic staining and high tumor grade (p = 0.0465), and low expression level and advanced pT (p = < 0.0001). It is concluded that clear-cut alterations of β-Catenin expression such as a nuclear and cytoplasmic translocation and a complete expression loss occur rarely in urothelial carcinomas of the urinary bladder. Patients with nuclear expression of β-Catenin in their tumors might benefit from specific therapies once Wnt pathway inhibitors should become safe and efficient. Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Urothelial carcinoma of the urinary bladder belongs to the most common malignant tumor types worldwide 1 . About 80% of patients present with non-invasive (pTa) or minimally invasive (pT1) cancers which can be removed by transurethral resection. In muscle-invasive carcinomas, radical cystectomy (RC) is the surgical standard of care although distant metastasis develops in up to 50% of these patients 2 . Most of these patients eventually die from their disease despite progress in systemic treatment approaches 3 . Significant shortcomings for appropriate individual treatment of bladder cancer patients include a poor predictability of the individual patient prognosis and a shortage of adequate systemic treatments for patients suffering from metastasis 4 , 5 . β-Catenin (CTNNB1) alterations are of potential interest as a prognostic feature and an option for therapeutic targeting 6 . β-Catenin is a dual function protein with relevant roles in both cell-cell adhesion and gene transcription 7 . At the cell membrane, β-Catenin associates with cadherin class cell-adhesion proteins to regulate cell adhesion 8 . In the cell, β-Catenin acts as a critical signal transducer in the Wingless-Type MMTV Integration Site Family (Wnt) signaling pathway 9 . In the absence of Wnt signaling, β-Catenin is restricted to the plasma membrane because cytoplasmic β-Catenin is degraded subsequent to ubiquitination 7 . β-Catenin ubiquitination is induced by glycogen synthase kinase 3 alpha (GSK3α) and beta (GSK3β) which are dependent on the Axin and adenomatous polyposis coli (APC) proteins as a scaffold 7 , 9 – 11 . Jointly these proteins form the “β-Catenin destruction complex”. In case of active Wnt signaling, β-Catenin accumulates in the cytoplasm and subsequently translocates to the nucleus where it acts as a transcriptional co-activator and binds to multiple transcription factors including members of the TCF/LEF family 6 , 12 . Aberrant nuclear β-Catenin translocation and subsequent activation of β-Catenin regulated genes can be caused by loss of function mutations of genes encoding proteins of the β-Catenin destruction complex 13 or by gain of function mutations of CTNNB1 14 . Affected cancers can be recognized by immunohistochemistry (IHC) due to their aberrant nuclear/cytoplasmic β-Catenin staining 15 – 19 . Earlier IHC studies on 4-739 urothelial tumors have found conflicting data and described a loss of β-Catenin expression in 0.0–66.0% of cases 20 – 39 , a nuclear or cytoplasmic staining in 0.0-37.5% of cases 20 , 24 , 26 , 28 , 32 , 38 – 44 , and an aberrant staining pattern in 15.0-92.9% of cases 22 , 27 , 33 , 34 , 45 , 46 (Table 1). Some of these studies have described associations between reduced 21 , 23 , 25 , 43 or aberrant 22 , 24 , 26 , 27 , 33 , 45 , 46 β-Catenin expression with unfavorable tumor features or patient prognosis, others could not confirm these observations 29 – 31 , 47 (Table 1). To learn more on the prevalence and the potential diagnostic and prognostic role of reduced or aberrant β-Catenin expression, a previously collected cohort of more than 2,700 urothelial bladder carcinomas was analyzed by IHC in a tissue microarray (TMA) format, and the results were compared with histopathological parameters of cancer aggressiveness and patient outcome data. Materials and Methods Tissue Microarrays (TMAs). Our set of TMAs contained one sample each from 2,710 urothelial tumors of the bladder archived at the Institute of Pathology, University Hospital Hamburg, Germany, Institute of Pathology, Charité Berlin, Germany, Department of Pathology, Academic Hospital Fuerth, Germany, or Department of Pathology, Helios Hospital Bad Saarow, Germany, and/or treated at Department of Urology, University Hospital Hamburg, Germany, Department of Urology, Charité Berlin, Germany, Department of Urology, Helios Hospital Bad Saarow, Germany, Department of Urology, Albertinen Hospital, Hamburg, Germany, and Department of Urology and Urological Oncology, Pomeranian Medical University, Szczecin, Poland. Patients at each center were treated according to the guidelines at the time. In brief, patients with pTa/pT1 disease underwent a transurethral resection of the bladder tumor with or without postoperative or adjuvant instillation therapy, while most patients with pT2-4 disease were treated by RC. Available histopathological data including grade (G), tumor stage (pT) and lymph node status (pN) are shown in Table 2. The grading of pTa tumors included both a classification according to WHO 2004 48 and Mostofi 1973 49 which were valid at the time of the respective diagnoses. Clinical follow up data (overall survival; OS) were available from 636 evaluable patients with pT2-4 carcinomas treated by cystectomy. The tissues were fixed in 4% buffered formalin and then embedded in paraffin. The TMA manufacturing process has been described 50 , 51 . In brief, one tissue spot (diameter: 0.6 mm) was transmitted from a cancer containing donor block into an empty recipient paraffin block. According to local regulations (HmbKHG, § 12) the responsible IRB (Ethics Committee of the Hamburg Medical Association, reference number: WF-049/09) approved the use of archived remnants of diagnostic tissues for manufacturing of TMAs and their analysis for research purposes as well as patient data analysis and waived the need for consent to participate in this study. All work has been carried out in compliance with the Helsinki Declaration. Immunohistochemistry (IHC). Freshly prepared TMA sections were immunostained on one day in one experiment. Slides were deparaffinized with xylol, rehydrated through a graded alcohol series and exposed to heat-induced antigen retrieval for 5 minutes in an autoclave at 121°C in pH 7.8 DakoTarget Retrieval Solution™ (Agilent, CA, USA; #S2367). Endogenous peroxidase activity was blocked with Dako Peroxidase Blocking Solution™ (Agilent, CA, USA; #52023) for 10 minutes. Primary antibody specific for β-Catenin (rabbit recombinant monoclonal, clone MSVA-578R, cat. #3609-578R, MS Validated Antibodies GmbH, Hamburg, Germany) was applied at 37°C for 60 minutes at a dilution of 1:2000. Bound antibody was then visualized using the EnVision Kit™ (Agilent, CA, USA; #K5007) according to the manufacturer’s directions. The sections were counterstained with haemalaun. For tumor tissues, the average staining intensity of unequivocally neoplastic cells was estimated as 0, 1+, 2+, 3+. Additional cytoplasmic and nuclear staining was recorded as either equivocal or unequivocal depending on the presence of clear-cut or equivocal nuclear staining in at least a subset of cells. In addition, staining heterogeneity was recorded if present. For the classification of a tumor as completely negative (0), unequivocal β-Catenin staining in tumor adjacent endothelial cells was required. Tumors with complete absence of β-Catenin staining in cancerous and endothelial cells were considered “non-informative”. Statistics . Statistical calculations were performed with JMP 16 software (SAS Institute Inc., NC, USA). Contingency tables and the chi²-test were performed to search for associations between β-Catenin expression loss, tumor phenotype, and other molecular parameters. Survival curves were calculated according to Kaplan-Meier. The Log-Rank test was applied to detect significant differences between groups. Results Technical issues. Of 2710 urothelial cancer samples, 1999 (73.8%) (Table 3) were interpretable for β-Catenin IHC. Reasons for non-informative cases included absence of β-Catenin staining in tumor and endothelial cells, lack of tissue spots, or absence of unequivocal cancer cells in the TMA spot. β-Catenin immunostaining. In normal urothelium, β-Catenin staining was always strong and largely limited to the cell membranes. Membranous β-Catenin staining was only absent at the luminal surface of umbrella cells, at least in most samples. A comparable staining pattern was also seen in the overwhelming majority of non-invasive pTa tumors, especially in case of low-grade or G2 neoplasms, and also in a large fraction of G3 and invasive cancers (Fig. 1 B/C). Patterns that deviated from these obviously normal findings occurred in 20.3% of our tumors, and included the following: unequivocal nuclear and cytoplasmic β-Catenin staining (2.8%; often accompanied by a reduced membranous positivity, indicated as mutated in Table 3, Fig. 1 D), a complete absence/loss of β-Catenin staining while staining was retained in tumor associated endothelial cells (1.5%; Fig. 1 E), reduced membranous β-Catenin staining (1+) (5.4%; Fig. 1 F), and equivocal cytoplasmic β-Catenin staining (sometimes accompanied by equivocal nuclear staining) (10.6%, Fig. 1 G/H). Irrespective of these findings, the staining was considered heterogeneous in 20.8% of our cancers (Table 3). Within the group of heterogeneous tumors some characteristic patterns were observed. These included a gradual decrease of β-Catenin staining intensity from the periphery to the center of tumor cell nests (Fig. 2 A), an increase of staining intensity from the periphery to the center of tumor cell nests (Fig. 2 B/C), a lack of β-Catenin staining at the peripheral cell membranes of otherwise strongly positive tumor cell nests (Fig. 2DE) and a marked decrease of staining from solid tumor areas towards areas with a more dispersed tumor growth (Fig. 2 F). The relationship between these findings and tumor phenotype are shown in Table 3. Aberrant β-Catenin staining including a complete loss (p = 0.0006), reduced staining (p < 0.0001), staining heterogeneity (p < 0.0001), as well as both unequivocal (p < 0.0001) and equivocal nuclear and cytoplasmic staining (p < 0.0001) was always associated with invasive tumor growth (p overall < 0.0001) while none of these parameters were significantly linked to patient survival in patients with pT2-4 cancers (Fig. 3 /4). Within pT2-4 tumors, a complete loss of membranous β-Catenin staining was linked to high pN (p = 0.0014) and pT (p = 0.0100). A complete loss of membranous staining was also linked to UICC stage (p = 0.0106). Statistically significant associations were also seen between heterogeneity and nodal metastasis (p = 0.0207), equivocal nuclear/cytoplasmic staining and high tumor grade (p = 0.0465), and low expression level and advanced pT (p = < 0.0001). Discussion Unequivocal nuclear and cytoplasmic staining of β-Catenin protein is typically associated with cancer-driving mutations in β-Catenin or other genes of the β-Catenin destruction complex 13 , 18 , 19 , 52 – 54 . That 3.7% of our muscle-invasive urothelial carcinomas of the urinary bladder had a clear-cut nuclear and cytoplasmic β-Catenin staining is only slightly below expectations. Data obtained by next-generation sequencing had found activating CTNNB1 mutations in < 2.0% 55–70 . Potentially inactivating mutations of the β-Catenin destruction complex (DC) (APC, GSK-3, CK1, PP2A, β-TrCP, Axin, and SCF) 71 that might lead to similar IHC findings were found in 2.8% while deep deletions of genes involved in DC proteins were reported in 5.1%, suggesting functionally relevant Wnt pathway mutations in up to 9% of invasive urothelial carcinomas. Given the typical IHC pattern of nuclear/cytoplasmic staining of β-Catenin can be focal 71 , it is possible that our approach resulted in a slight underestimate. It is of note that other investigators had earlier reported an average of 35.6% of cases with nuclear β-Catenin staining in 4 cohorts of 13–31 urothelial carcinomas in patients with pT2-4 carcinomas (Table 1, Supplementary table 1 ) 26,39,42,43 . The higher prevalence of aberrant β-Catenin staining in pT2–pT4 than in pTa carcinomas in our cohort, however, is in line with most 20 , 26 , 27 , 30 , 39 , 42 , 43 , 46 earlier studies (Table 1, Supplementary table 1 ). The large number of tumors analyzed in this study enabled us to assess the potential clinical significance of our findings despite their low prevalence. The absence of a clear-cut association between unequivocal nuclear β-Catenin expression and both patient prognosis and parameters of aggressive disease among pT2-4 carcinomas is in line with the notorious difficulty in finding molecular prognostic parameters for these cancers. Various other molecular features such as p53 alterations 72 , 73 , HER2 overexpression 74 , 75 , or c-Myc overexpression 76 , which are strongly linked to prognosis in other tumor entities have failed to demonstrate a prognostic impact in bladder cancer. That not even histologic grading has a confirmed prognostic role in pT2-4 urothelial cancers lead to the ongoing WHO recommendation not to grade these advanced-stage tumors since 2004 77 . It is of note that several prior studies described poor prognosis in patients with aberrant β-Catenin immunostaining in urothelial carcinomas although their definitions of aberrant staining varied widely, ranging from complete loss of staining to nuclear and/or cytoplasmic accumulation, and even altered membranous distribution 27 , 33 , 45 . Moreover, meta-analyses have suggested an unfavorable prognostic role of nuclear β-Catenin immunostaining in other cancer types such as colorectal adenocarcinoma 78 , gastric adenocarcinoma 79 , non-small cell lung cancer 80 , breast cancer 81 , ovarian carcinoma 82 and in head and neck carcinomas 83 . Most clinical potential of nuclear β-Catenin translocation as a feature of excessive Wnt pathway activation lies in the potential druggability of the intranuclear β-Catenin which exerts its tumor promoting role predominantly through interactions with TCF (T-cell factor)/LEF transcription factors 9 , 84 . Inhibitors can either block the nuclear import of β-Catenin 85 , interfere with its interaction with transcription factors 86 – 89 , or promote its degradation 90 – 94 . FOG-001, an inhibitor that blocks nuclear β-Catenin’s interaction with the TCF family of transcription factors is now being evaluated in a clinical phase I/II trial recruiting patients with advanced solid tumors carrying Wnt pathway activating mutation 95 . ICG-001 is another small molecule inhibitor that disrupts the interaction of CBP with nuclear β-Catenin, thereby antagonizing β-Catenin/TCF-mediated transcription 88 . Its clinical derivative PRI-724 in combination with gemcitabine demonstrated clinical activity in a phase 1b trial for patients with metastatic pancreatic cancer 96 . Tegavivint (BC2059), which selectively disrupts the β-Catenin–TBL1/TBLR1 interaction and leads to degradation of nuclear β-Catenin, achieved an objective response rate of 18% in adults with progressive, unresectable desmoid tumors 97 . The tankyrase inhibitor RK-582 which selectively inhibits β-Catenin-mediated transcription by stabilizing Axin within the destruction complex 90 , 98 is currently being evaluated in a first clinical phase I trial for patients with unresectable colorectal cancer (ClinicalTrials.gov ID NCT06853496). Numerous studies had described various aberrant β-Catenin expression patterns including overexpression, reduced or absent expression, and cytoplasmic and/or nuclear expression to be pathogenic in cancer 79 , 99 . To better understand the significance of these patterns, all of them were recorded and compared to histopathological features of malignancy and clinical outcome in our study. That reduced expression, loss of expression, heterogeneous expression, unequivocal and even equivocal nuclear/cytoplasmic staining were strongly linked to invasive tumor growth is in line with the well-known fundamental differences between non-invasive and invasive urothelial carcinomas 100 . Most molecular parameters that were previously analyzed in urothelial carcinomas have displayed distinct differences between pTa and pT2-4 cancers 63 , 101 – 103 . Among all evaluated immunostaining patterns, a complete loss of membranous β-catenin expression was most tightly linked to unfavorable tumor features and almost reached the level of statistical significance as a prognostic feature in pT2-4 carcinomas. β-Catenin expression loss apparently represents a disruptive alteration of β-Catenin function affecting cell cohesion 104 , 105 . The 1.5% (Table 3) of cases with a complete β-Catenin loss in our study are in the lower range of published studies which described a β-Catenin negativity in 0–66% (average 25.0%) in cohorts of 5-739 urothelial cancers (Table 1) 20 , 21 , 23 – 26 , 28 , 29 , 31 , 32 , 34 – 39 . Potential causes for these inconsistent data include differences in antibody properties, staining protocols, and thresholds for defining expression loss 106 . It is of note that our IHC assay had previously been validated according to the guidelines of the International Working Group of Antibody Validation 107 by comparing with an independent second antibody on 76 different normal tissue categories (Lutz et al, manuscript in preparation). That β-Catenin expression loss was most closely linked to invasive tumor growth and nodal metastasis fits well with data from experimental models suggesting a loss of cell cohesion 108 , invasive growth 109 , increased metastatic spread 109 , and epithelial mesenchymal transition 110 – 112 in tumor cells lacking membranous β-Catenin. It is of note, however, that – in contrast to our data – Salomon et al. have found evidence for a tumor suppressive role of a loss of membranous β-Catenin to a blockade of the tumor promoting Wnt pathway 113 . In summary, our study demonstrates that clear-cut alterations of β-Catenin expression such as a nuclear and cytoplasmic staining, and a complete expression loss occur rarely in urothelial carcinoma of the urinary bladder. While the 3.7% of patients with nuclear translocation of β-Catenin in their pT2-4 cancers might benefit from specific therapies once Wnt pathway inhibitors should become safe and efficient, a membranous expression loss may represent a distinct driver lesion for increased tumor aggressiveness. Abbreviations RC radical cystectomy CTNNB1 β-Catenin Wnt Wingless-Type MMTV Integration Site Family GSK3α glycogen synthase kinase 3 alpha GSK3β glycogen synthase kinase 3 beta APC adenomatous polyposis coli IHC immunohistochemistry TMA tissue microarray DC destruction complex TCF T-cell factor Declarations Ethics approval and consent to participate. According to local regulations (HmbKHG, §12) the responsible IRB (Ethics Committee of the Hamburg Medical Association / Ethik-Kommission der Ärztekammer Hamburg, reference number: WF-049/09) approved the use of archived remnants of diagnostic tissues for manufacturing of TMAs and their analysis for research purposes as well as patient data analysis and waived the need for consent to participate in this study. All work has been carried out in compliance with the Helsinki Declaration. Consent for publication. All authors have read and approved the final version of tis manuscript and agree to its submission for publication. Availability of data and materials. All data generated or analyzed during this study are included in this published article. Conflict of Interest. The rabbit recombinant monoclonal antibody, clone MSVA-578R was provided from MS Validated Antibodies GmbH (owned by a family member of GS). Funding. Not applicable. Authors Contribution Statement FL, ReS, FG, RoS, GS, BR: contributed to conception, design, data collection, data analysis and manuscript writing. Fl, ReS, FG, NG, SB, CK, HP, KF, SW, AnF, JJ, TJVS, IML, JCK, NH, FR, SS, SE, ML, AHM, HS, MF, MR, HZ, MS, KK, TE, NA, RS, GS, JW, TS, DH, MK, SM, BR: participated in pathology data analysis, data interpretation, and collection of samples. FL, RoS, GS, BR: study supervision. All authors agree to be accountable for the content of the work. Acknowledgements. We are grateful to Melanie Steurer, Laura Behm, Inge Brandt, Sünje Seekamp for excellent technical assistance. References Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, Bray F. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin. 2021;71:209–49. 10.3322/caac.21660 . Stein JP, Lieskovsky G, Cote R, Groshen S, Feng AC, Boyd S, Skinner E, Bochner B, Thangathurai D, Mikhail M, et al. 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Supplementary Files Table1.xlsx Table 1. β-Catenin immunostaining results and patterns from previous β-Catenin studies. Additional information, including subgroup analysis are listed in Supplementary table 1. Table2.xlsx Table 2. Patient cohort. Table3.xlsx Table 3. β-Catenin immunostaining and tumor phenotype. Supplementarytable1.xlsx Supplementary table 1. β-Catenin immunostaining results and patterns from previous β-Catenin studies including all subgroup analysis. Blue indicates the reference cohort, while cohorts for subgroup analysis are listed below. Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 12 Mar, 2026 Reviewers agreed at journal 08 Mar, 2026 Reviewers agreed at journal 02 Mar, 2026 Reviewers invited by journal 20 Feb, 2026 Editor assigned by journal 16 Feb, 2026 Editor invited by journal 27 Jan, 2026 Submission checks completed at journal 27 Jan, 2026 First submitted to journal 27 Jan, 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8639026","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":595474047,"identity":"763dd823-b097-4051-be0c-7da762004dbf","order_by":0,"name":"Florian Lutz","email":"","orcid":"","institution":"University Medical Center Hamburg-Eppendorf","correspondingAuthor":false,"prefix":"","firstName":"Florian","middleName":"","lastName":"Lutz","suffix":""},{"id":595474051,"identity":"40a3cf0b-ab16-432c-886a-1fc975a4236b","order_by":1,"name":"Resha Sharifi","email":"","orcid":"","institution":"University Medical Center Hamburg-Eppendorf","correspondingAuthor":false,"prefix":"","firstName":"Resha","middleName":"","lastName":"Sharifi","suffix":""},{"id":595474058,"identity":"7e160449-d03a-4d8f-8fe1-0eca1df89d27","order_by":2,"name":"Fiete Gehrisch","email":"","orcid":"","institution":"University Medical Center Hamburg-Eppendorf","correspondingAuthor":false,"prefix":"","firstName":"Fiete","middleName":"","lastName":"Gehrisch","suffix":""},{"id":595474059,"identity":"edd22403-26cd-4bfa-9791-53c2efbd0f2a","order_by":3,"name":"Natalia Gorbokon","email":"","orcid":"","institution":"University Medical Center Hamburg-Eppendorf","correspondingAuthor":false,"prefix":"","firstName":"Natalia","middleName":"","lastName":"Gorbokon","suffix":""},{"id":595474062,"identity":"cb83c6db-2946-4880-88fc-a57cb0f92e67","order_by":4,"name":"Seyma Büyücek","email":"","orcid":"","institution":"University Medical Center Hamburg-Eppendorf","correspondingAuthor":false,"prefix":"","firstName":"Seyma","middleName":"","lastName":"Büyücek","suffix":""},{"id":595474065,"identity":"524206be-5418-4bca-b911-42b95b90823d","order_by":5,"name":"Christian Kähler","email":"","orcid":"","institution":"University Medical Center Hamburg-Eppendorf","correspondingAuthor":false,"prefix":"","firstName":"Christian","middleName":"","lastName":"Kähler","suffix":""},{"id":595474077,"identity":"4d92893e-79f7-47a6-80e1-bb7df726eab5","order_by":6,"name":"Henning Plage","email":"","orcid":"","institution":"Charité - 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University Medicine Berlin","correspondingAuthor":false,"prefix":"","firstName":"Jonathan","middleName":"","lastName":"Jeutner","suffix":""},{"id":595474093,"identity":"90727565-1fa7-47e4-94f3-4cd44ef78b94","order_by":11,"name":"Till JV Schmidt","email":"","orcid":"","institution":"Charité - University Medicine Berlin","correspondingAuthor":false,"prefix":"","firstName":"Till","middleName":"JV","lastName":"Schmidt","suffix":""},{"id":595474095,"identity":"c5e6019f-8a95-46e0-ac0c-332c032a0fc0","order_by":12,"name":"Julia C Kaulfuss","email":"","orcid":"","institution":"Charité - University Medicine Berlin","correspondingAuthor":false,"prefix":"","firstName":"Julia","middleName":"C","lastName":"Kaulfuss","suffix":""},{"id":595474097,"identity":"36e84368-0d44-4c66-b848-5175b21bc2c1","order_by":13,"name":"Isabel M Lichy","email":"","orcid":"","institution":"Charité - University Medicine Berlin","correspondingAuthor":false,"prefix":"","firstName":"Isabel","middleName":"M","lastName":"Lichy","suffix":""},{"id":595474100,"identity":"f147ce71-40e9-410d-ae27-72d728d5f9ab","order_by":14,"name":"Nicolas Hertzsprung","email":"","orcid":"","institution":"Charité - 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University Medicine Berlin","correspondingAuthor":false,"prefix":"","firstName":"Henrik","middleName":"","lastName":"Zecha","suffix":""},{"id":595474154,"identity":"3fb96560-7f8c-490b-a92a-b6555533ba8c","order_by":24,"name":"Marcin Slojewski","email":"","orcid":"","institution":"Pomeranian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Marcin","middleName":"","lastName":"Slojewski","suffix":""},{"id":595474156,"identity":"f04eb6e2-d9ab-44d6-98ab-f0dbe7997101","order_by":25,"name":"Krystian Kaczmarek","email":"","orcid":"","institution":"Pomeranian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Krystian","middleName":"","lastName":"Kaczmarek","suffix":""},{"id":595474158,"identity":"9e9df06c-8743-4842-9d5e-2ccbdcecbc5b","order_by":26,"name":"Thorsten Ecke","email":"","orcid":"","institution":"Helios Hospital Bad Saarow","correspondingAuthor":false,"prefix":"","firstName":"Thorsten","middleName":"","lastName":"Ecke","suffix":""},{"id":595474160,"identity":"f220eaba-b942-41fb-91c8-c01a39913790","order_by":27,"name":"Nico Adamini","email":"","orcid":"","institution":"Albertinen Hospital","correspondingAuthor":false,"prefix":"","firstName":"Nico","middleName":"","lastName":"Adamini","suffix":""},{"id":595474163,"identity":"f53bb592-859b-4cb0-baf6-cf45b53d183c","order_by":28,"name":"Ronald Simon","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAArUlEQVRIiWNgGAWjYJCCA0Asx8ADJHlI0WJMmhYQSGwgWot5A/vDQzcq7qRvOHP4AMObCiK0yBzgMTicc+ZZ7oazbQmMc84QoUUC6JjDuW2Hczec5zFg5m0jSgv7A5CWdIPz/B+Yef8RpYXBAKQlweBsDwMzbwMxWpjBfjlsOPPMMYODc44Ro4W9/fHnnIrD8nxnkh8+eFNDhBYGZiT2AWI0jIJRMApGwSggAgAAvfQ3M41YIOUAAAAASUVORK5CYII=","orcid":"","institution":"University Medical Center Hamburg-Eppendorf","correspondingAuthor":true,"prefix":"","firstName":"Ronald","middleName":"","lastName":"Simon","suffix":""},{"id":595474167,"identity":"55be09e1-0a50-4519-9849-6d0614a0bfa9","order_by":29,"name":"Guido Sauter","email":"","orcid":"","institution":"University Medical Center Hamburg-Eppendorf","correspondingAuthor":false,"prefix":"","firstName":"Guido","middleName":"","lastName":"Sauter","suffix":""},{"id":595474171,"identity":"5ad12b7e-2f39-48e9-a705-71dc58108222","order_by":30,"name":"Joachim Weischenfeldt","email":"","orcid":"","institution":"Charité - University Medicine Berlin","correspondingAuthor":false,"prefix":"","firstName":"Joachim","middleName":"","lastName":"Weischenfeldt","suffix":""},{"id":595474175,"identity":"3b1afca7-4dd3-48b1-8378-1e4160daec30","order_by":31,"name":"Thorsten Schlomm","email":"","orcid":"","institution":"Charité - University Medicine Berlin","correspondingAuthor":false,"prefix":"","firstName":"Thorsten","middleName":"","lastName":"Schlomm","suffix":""},{"id":595474184,"identity":"9e3b7b70-6889-48aa-b4ce-5ebe5b6fa9ae","order_by":32,"name":"David Horst","email":"","orcid":"","institution":"Charité - University Medicine Berlin","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"","lastName":"Horst","suffix":""},{"id":595474186,"identity":"76159025-e1e0-43b4-ba8a-677d6d702deb","order_by":33,"name":"Martina Kluth","email":"","orcid":"","institution":"University Medical Center Hamburg-Eppendorf","correspondingAuthor":false,"prefix":"","firstName":"Martina","middleName":"","lastName":"Kluth","suffix":""},{"id":595474194,"identity":"38207d58-02cd-41e4-a2bd-249ade4efd2c","order_by":34,"name":"Sarah Minner","email":"","orcid":"","institution":"University Medical Center Hamburg-Eppendorf","correspondingAuthor":false,"prefix":"","firstName":"Sarah","middleName":"","lastName":"Minner","suffix":""},{"id":595474196,"identity":"772db2a5-4f36-47ed-a860-7bcb374fcc89","order_by":35,"name":"Bernhard Ralla","email":"","orcid":"","institution":"Charité - University Medicine Berlin","correspondingAuthor":false,"prefix":"","firstName":"Bernhard","middleName":"","lastName":"Ralla","suffix":""}],"badges":[],"createdAt":"2026-01-19 11:40:45","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8639026/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8639026/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103439589,"identity":"3fd68436-9c24-42e2-9814-974f681e5b67","added_by":"auto","created_at":"2026-02-25 17:05:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":6010898,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eβ-Catenin immunostaining in normal and neoplastic bladder tissue.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStrong and membranous staining in normal urothelium (A), a non-invasive (pTa) tumor (B) and an invasive (pT2) tumor (C). Unequivocal nuclear and cytoplasmic staining (pT2) (D). Loss of tumoral staining with retained staining positivity in endothelial cells (E) and reduced membranous staining (F) (pT3). Equivocal strong cytoplasmic (\u0026lt;5%) (G) (pT2) and equivocal cytoplasmic/nuclear staining (H) (pT3).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8639026/v1/298ea0205d82d395867d0c93.png"},{"id":103508208,"identity":"1ff63674-6dcf-41ef-8803-26e8b4c74c31","added_by":"auto","created_at":"2026-02-26 13:47:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4261154,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHeterogeneous patterns of β-Catenin immunostaining in neoplastic bladder tissue.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe panels show a decrease of staining intensity from the periphery to the center of tumor cell nests (A) (pT3), an increase of staining intensity from the periphery to the center of tumor cell nests (B, C) (pT2, pT3), a lack of β-Catenin staining at the peripheral cell membranes of otherwise strongly positive tumor cell nests (D, E) (pT4, pT3), and a marked decrease of β-Catenin staining from solid tumor areas towards areas with a more dispersed tumor growth (F) (pT2).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8639026/v1/0788affb22f90bc7602ec24d.png"},{"id":103439582,"identity":"9a69cf30-42b6-4024-abc8-d33cd5dd8fcb","added_by":"auto","created_at":"2026-02-25 17:05:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":410311,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eβ-Catenin immunostaining patterns and patient prognosis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe panels show the impact of different β-Catenin immunostaining patterns on the overall survival (OS) of patients with pT2-4 urothelial bladder carcinoma. (A) Unequivocal nuclear/cytoplasmic, (B) loss of staining, (C) equivocal nuclear/cytoplasmic and (D) heterogeneous staining. † indicates that patients with unequivocal staining and expression loss were excluded from the analysis.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8639026/v1/326fa17f029574db04209474.png"},{"id":103439590,"identity":"138a908b-fbbe-4bf4-9f0d-4b9f12de8f60","added_by":"auto","created_at":"2026-02-25 17:05:08","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":43043,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eβ-Catenin immunostaining and patient prognosis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe graph shows the impact of β-Catenin immunostaining intensity on the overall survival (OS) of patients with pT2-4 urothelial bladder carcinoma.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8639026/v1/8dee46944603694c3a284ca0.png"},{"id":103511958,"identity":"cf8bcd3e-ee43-49d9-b889-7fd5093d13e6","added_by":"auto","created_at":"2026-02-26 14:11:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":15324459,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8639026/v1/09c96067-13ae-498a-8943-86ab9c6c3d47.pdf"},{"id":103439586,"identity":"56a54fb1-4776-47e6-aa86-d6bf2cc14c98","added_by":"auto","created_at":"2026-02-25 17:05:08","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14683,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable 1. β-Catenin immunostaining results and patterns from previous β-Catenin studies.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdditional information, including subgroup analysis are listed in Supplementary table 1.\u003c/p\u003e","description":"","filename":"Table1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8639026/v1/00d2b79b7ae6d8e620bf2a4e.xlsx"},{"id":103439585,"identity":"cea3dd95-b57a-4caf-9e29-04018a6ece2c","added_by":"auto","created_at":"2026-02-25 17:05:08","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":10030,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable 2. Patient cohort.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Table2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8639026/v1/38b2b964203f97478847193a.xlsx"},{"id":103507898,"identity":"9bbb5526-28c4-4cb6-8d56-330ce3e19687","added_by":"auto","created_at":"2026-02-26 13:46:19","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":20067,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable 3. β-Catenin immunostaining and tumor phenotype.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Table3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8639026/v1/2ccdda723cc5f1a17407d8c6.xlsx"},{"id":103439587,"identity":"8e88a260-05d6-44ce-9d05-45b119357be0","added_by":"auto","created_at":"2026-02-25 17:05:08","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":20467,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary table 1. β-Catenin immunostaining results and patterns from previous β-Catenin studies including all subgroup analysis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBlue indicates the reference cohort, while cohorts for subgroup analysis are listed below.\u003c/p\u003e","description":"","filename":"Supplementarytable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8639026/v1/1b2c03ddf1396322a47c8ca0.xlsx"}],"financialInterests":"Competing interest reported. The rabbit recombinant monoclonal antibody, clone MSVA-578R was provided from MS Validated Antibodies GmbH (owned by a family member of GS).","formattedTitle":"Aberrant β-Catenin expression is related to invasive tumor growth in urothelial carcinoma of the urinary bladder","fulltext":[{"header":"Introduction","content":"\u003cp\u003eUrothelial carcinoma of the urinary bladder belongs to the most common malignant tumor types worldwide\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. About 80% of patients present with non-invasive (pTa) or minimally invasive (pT1) cancers which can be removed by transurethral resection. In muscle-invasive carcinomas, radical cystectomy (RC) is the surgical standard of care although distant metastasis develops in up to 50% of these patients\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Most of these patients eventually die from their disease despite progress in systemic treatment approaches\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Significant shortcomings for appropriate individual treatment of bladder cancer patients include a poor predictability of the individual patient prognosis and a shortage of adequate systemic treatments for patients suffering from metastasis\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eβ-Catenin (CTNNB1) alterations are of potential interest as a prognostic feature and an option for therapeutic targeting\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. β-Catenin is a dual function protein with relevant roles in both cell-cell adhesion and gene transcription\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. At the cell membrane, β-Catenin associates with cadherin class cell-adhesion proteins to regulate cell adhesion\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. In the cell, β-Catenin acts as a critical signal transducer in the Wingless-Type MMTV Integration Site Family (Wnt) signaling pathway\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. In the absence of Wnt signaling, β-Catenin is restricted to the plasma membrane because cytoplasmic β-Catenin is degraded subsequent to ubiquitination\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. β-Catenin ubiquitination is induced by glycogen synthase kinase 3 alpha (GSK3α) and beta (GSK3β) which are dependent on the Axin and adenomatous polyposis coli (APC) proteins as a scaffold\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Jointly these proteins form the \u0026ldquo;β-Catenin destruction complex\u0026rdquo;. In case of active Wnt signaling, β-Catenin accumulates in the cytoplasm and subsequently translocates to the nucleus where it acts as a transcriptional co-activator and binds to multiple transcription factors including members of the TCF/LEF family\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Aberrant nuclear β-Catenin translocation and subsequent activation of β-Catenin regulated genes can be caused by loss of function mutations of genes encoding proteins of the β-Catenin destruction complex\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e or by gain of function mutations of CTNNB1\u003csup\u003e14\u003c/sup\u003e. Affected cancers can be recognized by immunohistochemistry (IHC) due to their aberrant nuclear/cytoplasmic β-Catenin staining\u003csup\u003e\u003cspan additionalcitationids=\"CR16 CR17 CR18\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Earlier IHC studies on 4-739 urothelial tumors have found conflicting data and described a loss of β-Catenin expression in 0.0\u0026ndash;66.0% of cases\u003csup\u003e\u003cspan additionalcitationids=\"CR21 CR22 CR23 CR24 CR25 CR26 CR27 CR28 CR29 CR30 CR31 CR32 CR33 CR34 CR35 CR36 CR37 CR38\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, a nuclear or cytoplasmic staining in 0.0-37.5% of cases\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan additionalcitationids=\"CR39 CR40 CR41 CR42 CR43\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e, and an aberrant staining pattern in 15.0-92.9% of cases\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e (Table\u0026nbsp;1). Some of these studies have described associations between reduced\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e or aberrant\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e β-Catenin expression with unfavorable tumor features or patient prognosis, others could not confirm these observations\u003csup\u003e\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e (Table\u0026nbsp;1).\u003c/p\u003e \u003cp\u003eTo learn more on the prevalence and the potential diagnostic and prognostic role of reduced or aberrant β-Catenin expression, a previously collected cohort of more than 2,700 urothelial bladder carcinomas was analyzed by IHC in a tissue microarray (TMA) format, and the results were compared with histopathological parameters of cancer aggressiveness and patient outcome data.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cb\u003eTissue Microarrays (TMAs).\u003c/b\u003e Our set of TMAs contained one sample each from 2,710 urothelial tumors of the bladder archived at the Institute of Pathology, University Hospital Hamburg, Germany, Institute of Pathology, Charit\u0026eacute; Berlin, Germany, Department of Pathology, Academic Hospital Fuerth, Germany, or Department of Pathology, Helios Hospital Bad Saarow, Germany, and/or treated at Department of Urology, University Hospital Hamburg, Germany, Department of Urology, Charit\u0026eacute; Berlin, Germany, Department of Urology, Helios Hospital Bad Saarow, Germany, Department of Urology, Albertinen Hospital, Hamburg, Germany, and Department of Urology and Urological Oncology, Pomeranian Medical University, Szczecin, Poland. Patients at each center were treated according to the guidelines at the time. In brief, patients with pTa/pT1 disease underwent a transurethral resection of the bladder tumor with or without postoperative or adjuvant instillation therapy, while most patients with pT2-4 disease were treated by RC. Available histopathological data including grade (G), tumor stage (pT) and lymph node status (pN) are shown in Table\u0026nbsp;2. The grading of pTa tumors included both a classification according to WHO 2004\u003csup\u003e48\u003c/sup\u003e and Mostofi 1973\u003csup\u003e49\u003c/sup\u003e which were valid at the time of the respective diagnoses. Clinical follow up data (overall survival; OS) were available from 636 evaluable patients with pT2-4 carcinomas treated by cystectomy. The tissues were fixed in 4% buffered formalin and then embedded in paraffin. The TMA manufacturing process has been described\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. In brief, one tissue spot (diameter: 0.6 mm) was transmitted from a cancer containing donor block into an empty recipient paraffin block. According to local regulations (HmbKHG, \u0026sect;\u0026nbsp;12) the responsible IRB (Ethics Committee of the Hamburg Medical Association, reference number: WF-049/09) approved the use of archived remnants of diagnostic tissues for manufacturing of TMAs and their analysis for research purposes as well as patient data analysis and waived the need for consent to participate in this study. All work has been carried out in compliance with the Helsinki Declaration.\u003c/p\u003e \u003cp\u003e \u003cb\u003eImmunohistochemistry (IHC).\u003c/b\u003e Freshly prepared TMA sections were immunostained on one day in one experiment. Slides were deparaffinized with xylol, rehydrated through a graded alcohol series and exposed to heat-induced antigen retrieval for 5 minutes in an autoclave at 121\u0026deg;C in pH 7.8 DakoTarget Retrieval Solution\u0026trade; (Agilent, CA, USA; #S2367). Endogenous peroxidase activity was blocked with Dako Peroxidase Blocking Solution\u0026trade; (Agilent, CA, USA; #52023) for 10 minutes. Primary antibody specific for β-Catenin (rabbit recombinant monoclonal, clone MSVA-578R, cat. #3609-578R, MS Validated Antibodies GmbH, Hamburg, Germany) was applied at 37\u0026deg;C for 60 minutes at a dilution of 1:2000. Bound antibody was then visualized using the EnVision Kit\u0026trade; (Agilent, CA, USA; #K5007) according to the manufacturer\u0026rsquo;s directions. The sections were counterstained with haemalaun. For tumor tissues, the average staining intensity of unequivocally neoplastic cells was estimated as 0, 1+, 2+, 3+. Additional cytoplasmic and nuclear staining was recorded as either equivocal or unequivocal depending on the presence of clear-cut or equivocal nuclear staining in at least a subset of cells. In addition, staining heterogeneity was recorded if present. For the classification of a tumor as completely negative (0), unequivocal β-Catenin staining in tumor adjacent endothelial cells was required. Tumors with complete absence of β-Catenin staining in cancerous and endothelial cells were considered \u0026ldquo;non-informative\u0026rdquo;.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistics\u003c/b\u003e. Statistical calculations were performed with JMP 16 software (SAS Institute Inc., NC, USA). Contingency tables and the chi\u0026sup2;-test were performed to search for associations between β-Catenin expression loss, tumor phenotype, and other molecular parameters. Survival curves were calculated according to Kaplan-Meier. The Log-Rank test was applied to detect significant differences between groups.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eTechnical issues.\u003c/b\u003e Of 2710 urothelial cancer samples, 1999 (73.8%) (Table\u0026nbsp;3) were interpretable for β-Catenin IHC. Reasons for non-informative cases included absence of β-Catenin staining in tumor and endothelial cells, lack of tissue spots, or absence of unequivocal cancer cells in the TMA spot.\u003c/p\u003e \u003cp\u003e \u003cb\u003eβ-Catenin immunostaining.\u003c/b\u003e In normal urothelium, β-Catenin staining was always strong and largely limited to the cell membranes. Membranous β-Catenin staining was only absent at the luminal surface of umbrella cells, at least in most samples. A comparable staining pattern was also seen in the overwhelming majority of non-invasive pTa tumors, especially in case of low-grade or G2 neoplasms, and also in a large fraction of G3 and invasive cancers (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB/C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePatterns that deviated from these obviously normal findings occurred in 20.3% of our tumors, and included the following: unequivocal nuclear and cytoplasmic β-Catenin staining (2.8%; often accompanied by a reduced membranous positivity, indicated as mutated in Table\u0026nbsp;3, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), a complete absence/loss of β-Catenin staining while staining was retained in tumor associated endothelial cells (1.5%; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE), reduced membranous β-Catenin staining (1+) (5.4%; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF), and equivocal cytoplasmic β-Catenin staining (sometimes accompanied by equivocal nuclear staining) (10.6%, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG/H). Irrespective of these findings, the staining was considered heterogeneous in 20.8% of our cancers (Table\u0026nbsp;3). Within the group of heterogeneous tumors some characteristic patterns were observed. These included a gradual decrease of β-Catenin staining intensity from the periphery to the center of tumor cell nests (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), an increase of staining intensity from the periphery to the center of tumor cell nests (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB/C), a lack of β-Catenin staining at the peripheral cell membranes of otherwise strongly positive tumor cell nests (Fig.\u0026nbsp;2DE) and a marked decrease of staining from solid tumor areas towards areas with a more dispersed tumor growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe relationship between these findings and tumor phenotype are shown in Table\u0026nbsp;3. Aberrant β-Catenin staining including a complete loss (p\u0026thinsp;=\u0026thinsp;0.0006), reduced staining (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), staining heterogeneity (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), as well as both unequivocal (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and equivocal nuclear and cytoplasmic staining (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) was always associated with invasive tumor growth (p overall\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) while none of these parameters were significantly linked to patient survival in patients with pT2-4 cancers (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e/4). Within pT2-4 tumors, a complete loss of membranous β-Catenin staining was linked to high pN (p\u0026thinsp;=\u0026thinsp;0.0014) and pT (p\u0026thinsp;=\u0026thinsp;0.0100). A complete loss of membranous staining was also linked to UICC stage (p\u0026thinsp;=\u0026thinsp;0.0106). Statistically significant associations were also seen between heterogeneity and nodal metastasis (p\u0026thinsp;=\u0026thinsp;0.0207), equivocal nuclear/cytoplasmic staining and high tumor grade (p\u0026thinsp;=\u0026thinsp;0.0465), and low expression level and advanced pT (p\u0026thinsp;=\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eUnequivocal nuclear and cytoplasmic staining of β-Catenin protein is typically associated with cancer-driving mutations in β-Catenin or other genes of the β-Catenin destruction complex\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan additionalcitationids=\"CR53\" citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. That 3.7% of our muscle-invasive urothelial carcinomas of the urinary bladder had a clear-cut nuclear and cytoplasmic β-Catenin staining is only slightly below expectations. Data obtained by next-generation sequencing had found activating CTNNB1 mutations in \u0026lt;\u0026thinsp;2.0%\u003csup\u003e55\u0026ndash;70\u003c/sup\u003e. Potentially inactivating mutations of the β-Catenin destruction complex (DC) (APC, GSK-3, CK1, PP2A, β-TrCP, Axin, and SCF)\u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e that might lead to similar IHC findings were found in 2.8% while deep deletions of genes involved in DC proteins were reported in 5.1%, suggesting functionally relevant Wnt pathway mutations in up to 9% of invasive urothelial carcinomas. Given the typical IHC pattern of nuclear/cytoplasmic staining of β-Catenin can be focal\u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e, it is possible that our approach resulted in a slight underestimate. It is of note that other investigators had earlier reported an average of 35.6% of cases with nuclear β-Catenin staining in 4 cohorts of 13\u0026ndash;31 urothelial carcinomas in patients with pT2-4 carcinomas (Table\u0026nbsp;1, Supplementary table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003e1\u003c/span\u003e)\u003csup\u003e26,39,42,43\u003c/sup\u003e. The higher prevalence of aberrant β-Catenin staining in pT2\u0026ndash;pT4 than in pTa carcinomas in our cohort, however, is in line with most \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e earlier studies (Table\u0026nbsp;1, Supplementary table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe large number of tumors analyzed in this study enabled us to assess the potential clinical significance of our findings despite their low prevalence. The absence of a clear-cut association between unequivocal nuclear β-Catenin expression and both patient prognosis and parameters of aggressive disease among pT2-4 carcinomas is in line with the notorious difficulty in finding molecular prognostic parameters for these cancers. Various other molecular features such as p53 alterations\u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e,\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e, HER2 overexpression\u003csup\u003e\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e,\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e, or c-Myc overexpression\u003csup\u003e\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e, which are strongly linked to prognosis in other tumor entities have failed to demonstrate a prognostic impact in bladder cancer. That not even histologic grading has a confirmed prognostic role in pT2-4 urothelial cancers lead to the ongoing WHO recommendation not to grade these advanced-stage tumors since 2004\u003csup\u003e77\u003c/sup\u003e. It is of note that several prior studies described poor prognosis in patients with aberrant β-Catenin immunostaining in urothelial carcinomas although their definitions of aberrant staining varied widely, ranging from complete loss of staining to nuclear and/or cytoplasmic accumulation, and even altered membranous distribution\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Moreover, meta-analyses have suggested an unfavorable prognostic role of nuclear β-Catenin immunostaining in other cancer types such as colorectal adenocarcinoma\u003csup\u003e\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e\u003c/sup\u003e, gastric adenocarcinoma\u003csup\u003e\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e, non-small cell lung cancer\u003csup\u003e\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e\u003c/sup\u003e, breast cancer\u003csup\u003e\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e\u003c/sup\u003e, ovarian carcinoma\u003csup\u003e\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e\u003c/sup\u003e and in head and neck carcinomas\u003csup\u003e\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMost clinical potential of nuclear β-Catenin translocation as a feature of excessive Wnt pathway activation lies in the potential druggability of the intranuclear β-Catenin which exerts its tumor promoting role predominantly through interactions with TCF (T-cell factor)/LEF transcription factors\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e\u003c/sup\u003e. Inhibitors can either block the nuclear import of β-Catenin\u003csup\u003e\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e\u003c/sup\u003e, interfere with its interaction with transcription factors\u003csup\u003e\u003cspan additionalcitationids=\"CR87 CR88\" citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e\u003c/sup\u003e, or promote its degradation\u003csup\u003e\u003cspan additionalcitationids=\"CR91 CR92 CR93\" citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e94\u003c/span\u003e\u003c/sup\u003e. FOG-001, an inhibitor that blocks nuclear β-Catenin\u0026rsquo;s interaction with the TCF family of transcription factors is now being evaluated in a clinical phase I/II trial recruiting patients with advanced solid tumors carrying Wnt pathway activating mutation\u003csup\u003e\u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e\u003c/sup\u003e. ICG-001 is another small molecule inhibitor that disrupts the interaction of CBP with nuclear β-Catenin, thereby antagonizing β-Catenin/TCF-mediated transcription\u003csup\u003e\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e\u003c/sup\u003e. Its clinical derivative PRI-724 in combination with gemcitabine demonstrated clinical activity in a phase 1b trial for patients with metastatic pancreatic cancer\u003csup\u003e\u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e96\u003c/span\u003e\u003c/sup\u003e. Tegavivint (BC2059), which selectively disrupts the β-Catenin\u0026ndash;TBL1/TBLR1 interaction and leads to degradation of nuclear β-Catenin, achieved an objective response rate of 18% in adults with progressive, unresectable desmoid tumors \u003csup\u003e\u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e97\u003c/span\u003e\u003c/sup\u003e. The tankyrase inhibitor RK-582 which selectively inhibits β-Catenin-mediated transcription by stabilizing Axin within the destruction complex\u003csup\u003e\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e,\u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e98\u003c/span\u003e\u003c/sup\u003e is currently being evaluated in a first clinical phase I trial for patients with unresectable colorectal cancer (ClinicalTrials.gov ID NCT06853496).\u003c/p\u003e \u003cp\u003eNumerous studies had described various aberrant β-Catenin expression patterns including overexpression, reduced or absent expression, and cytoplasmic and/or nuclear expression to be pathogenic in cancer\u003csup\u003e\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e,\u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e99\u003c/span\u003e\u003c/sup\u003e. To better understand the significance of these patterns, all of them were recorded and compared to histopathological features of malignancy and clinical outcome in our study. That reduced expression, loss of expression, heterogeneous expression, unequivocal and even equivocal nuclear/cytoplasmic staining were strongly linked to invasive tumor growth is in line with the well-known fundamental differences between non-invasive and invasive urothelial carcinomas\u003csup\u003e\u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e100\u003c/span\u003e\u003c/sup\u003e. Most molecular parameters that were previously analyzed in urothelial carcinomas have displayed distinct differences between pTa and pT2-4 cancers\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e,\u003cspan additionalcitationids=\"CR102\" citationid=\"CR101\" class=\"CitationRef\"\u003e101\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR103\" class=\"CitationRef\"\u003e103\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAmong all evaluated immunostaining patterns, a complete loss of membranous β-catenin expression was most tightly linked to unfavorable tumor features and almost reached the level of statistical significance as a prognostic feature in pT2-4 carcinomas. β-Catenin expression loss apparently represents a disruptive alteration of β-Catenin function affecting cell cohesion\u003csup\u003e\u003cspan citationid=\"CR104\" class=\"CitationRef\"\u003e104\u003c/span\u003e,\u003cspan citationid=\"CR105\" class=\"CitationRef\"\u003e105\u003c/span\u003e\u003c/sup\u003e. The 1.5% (Table\u0026nbsp;3) of cases with a complete β-Catenin loss in our study are in the lower range of published studies which described a β-Catenin negativity in 0\u0026ndash;66% (average 25.0%) in cohorts of 5-739 urothelial cancers (Table\u0026nbsp;1)\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan additionalcitationids=\"CR24 CR25\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan additionalcitationids=\"CR35 CR36 CR37 CR38\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Potential causes for these inconsistent data include differences in antibody properties, staining protocols, and thresholds for defining expression loss\u003csup\u003e\u003cspan citationid=\"CR106\" class=\"CitationRef\"\u003e106\u003c/span\u003e\u003c/sup\u003e. It is of note that our IHC assay had previously been validated according to the guidelines of the International Working Group of Antibody Validation\u003csup\u003e\u003cspan citationid=\"CR107\" class=\"CitationRef\"\u003e107\u003c/span\u003e\u003c/sup\u003e by comparing with an independent second antibody on 76 different normal tissue categories (Lutz et al, manuscript in preparation). That β-Catenin expression loss was most closely linked to invasive tumor growth and nodal metastasis fits well with data from experimental models suggesting a loss of cell cohesion\u003csup\u003e\u003cspan citationid=\"CR108\" class=\"CitationRef\"\u003e108\u003c/span\u003e\u003c/sup\u003e, invasive growth\u003csup\u003e\u003cspan citationid=\"CR109\" class=\"CitationRef\"\u003e109\u003c/span\u003e\u003c/sup\u003e, increased metastatic spread\u003csup\u003e\u003cspan citationid=\"CR109\" class=\"CitationRef\"\u003e109\u003c/span\u003e\u003c/sup\u003e, and epithelial mesenchymal transition\u003csup\u003e\u003cspan additionalcitationids=\"CR111\" citationid=\"CR110\" class=\"CitationRef\"\u003e110\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR112\" class=\"CitationRef\"\u003e112\u003c/span\u003e\u003c/sup\u003e in tumor cells lacking membranous β-Catenin. It is of note, however, that \u0026ndash; in contrast to our data \u0026ndash; Salomon et al. have found evidence for a tumor suppressive role of a loss of membranous β-Catenin to a blockade of the tumor promoting Wnt pathway\u003csup\u003e\u003cspan citationid=\"CR113\" class=\"CitationRef\"\u003e113\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn summary, our study demonstrates that clear-cut alterations of β-Catenin expression such as a nuclear and cytoplasmic staining, and a complete expression loss occur rarely in urothelial carcinoma of the urinary bladder. While the 3.7% of patients with nuclear translocation of β-Catenin in their pT2-4 cancers might benefit from specific therapies once Wnt pathway inhibitors should become safe and efficient, a membranous expression loss may represent a distinct driver lesion for increased tumor aggressiveness.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eRC radical cystectomy\u003c/p\u003e\n\u003cp\u003eCTNNB1 \u0026beta;-Catenin\u003c/p\u003e\n\u003cp\u003eWnt Wingless-Type MMTV Integration Site Family\u003c/p\u003e\n\u003cp\u003eGSK3\u0026alpha; glycogen synthase kinase 3 alpha\u003c/p\u003e\n\u003cp\u003eGSK3\u0026beta; glycogen synthase kinase 3 beta\u003c/p\u003e\n\u003cp\u003eAPC adenomatous polyposis coli\u003c/p\u003e\n\u003cp\u003eIHC immunohistochemistry\u003c/p\u003e\n\u003cp\u003eTMA tissue microarray\u003c/p\u003e\n\u003cp\u003eDC destruction complex\u003c/p\u003e\n\u003cp\u003eTCF T-cell factor\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate. \u003c/strong\u003eAccording to local regulations (HmbKHG, \u0026sect;12) the responsible IRB (Ethics Committee of the Hamburg Medical Association / Ethik-Kommission der \u0026Auml;rztekammer Hamburg, reference number: WF-049/09) approved the use of archived remnants of diagnostic tissues for manufacturing of TMAs and their analysis for research purposes as well as patient data analysis and waived the need for consent to participate in this study. All work has been carried out in compliance with the Helsinki Declaration.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eConsent for publication. \u003c/strong\u003eAll authors have read and approved the final version of tis manuscript and agree to its submission for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials. \u003c/strong\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eConflict of Interest. \u003c/strong\u003eThe rabbit recombinant monoclonal antibody, clone MSVA-578R was provided from MS Validated Antibodies GmbH (owned by a family member of GS).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eFunding. \u003c/strong\u003eNot applicable.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAuthors\u003c/strong\u003e \u003cstrong\u003eContribution Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFL, ReS, FG, RoS, GS, BR: contributed to conception, design, data collection, data analysis and manuscript writing.\u003c/p\u003e\n\u003cp\u003eFl, ReS, FG, NG, SB, CK, HP, KF, SW, AnF, JJ, TJVS, IML, JCK, NH, FR, SS, SE, ML, AHM, HS, MF, MR, HZ, MS, KK, TE, NA, RS, GS, JW, TS, DH, MK, SM, BR: participated in pathology data analysis, data interpretation, and collection of samples.\u003c/p\u003e\n\u003cp\u003eFL, RoS, GS, BR: study supervision.\u003c/p\u003e\n\u003cp\u003eAll authors agree to be accountable for the content of the work.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAcknowledgements. \u003c/strong\u003eWe are grateful to Melanie Steurer, Laura Behm, Inge Brandt, S\u0026uuml;nje Seekamp for excellent technical assistance.\u003c/p\u003e\n\n"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, Bray F. 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Oncotarget. 2015;6:11421\u0026ndash;33. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.18632/oncotarget.3222\u003c/span\u003e\u003cspan address=\"10.18632/oncotarget.3222\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables are available in the Supplementary Files section.\u003c/p\u003e\n"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-urology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"buro","sideBox":"Learn more about [BMC Urology](http://bmcurol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/buro/default.aspx","title":"BMC Urology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8639026/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8639026/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eβ-Catenin is a dual function protein with roles in cell cohesion and as a critical intracellular signal transducer in the Wnt signaling pathway. Cytoplasmic and nuclear translocation of the β-Catenin protein results in an increased transcription of cancer promoting genes. To study the prevalence and the potential role of aberrant β-Catenin staining patterns, more than 2,700 bladder tumors were analyzed by immunohistochemistry (IHC) in a tissue microarray format. The cohort included 636 patients with radical cystectomy for muscle-invasive disease (pT2-4) for which follow-up data were available. In normal urothelium, β-Catenin staining was always strong and largely limited to the cell membranes. A comparable staining pattern was also seen in the overwhelming majority of non-invasive pTa tumors, especially in case of low-grade neoplasms. Aberrant β-Catenin staining patterns were observed in 20.3% of tumors and included unequivocal (2.8%) and equivocal nuclear/cytoplasmic staining (10.6%), complete loss of β-Catenin staining (1.5%), and reduced β-Catenin staining (1+, 5.4%). β-Catenin staining was heterogeneous in 20.8%. All aberrant β-Catenin staining patterns were associated with invasive tumor growth (p\u0026thinsp;=\u0026thinsp;\u0026lt;\u0026thinsp;0.0001\u0026ndash;0.0006), but unrelated to survival of patients with pT2-4 cancers. A complete loss of membranous β-Catenin staining was linked to UICC stage (p\u0026thinsp;=\u0026thinsp;0.0106) and within pT2-4 tumors, to high pN (p\u0026thinsp;=\u0026thinsp;0.0014) and pT p\u0026thinsp;=\u0026thinsp;0.0100). Associations also occurred between heterogeneity and nodal metastasis (p\u0026thinsp;=\u0026thinsp;0.0207), equivocal nuclear/cytoplasmic staining and high tumor grade (p\u0026thinsp;=\u0026thinsp;0.0465), and low expression level and advanced pT (p\u0026thinsp;=\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). It is concluded that clear-cut alterations of β-Catenin expression such as a nuclear and cytoplasmic translocation and a complete expression loss occur rarely in urothelial carcinomas of the urinary bladder. Patients with nuclear expression of β-Catenin in their tumors might benefit from specific therapies once Wnt pathway inhibitors should become safe and efficient.\u003c/p\u003e","manuscriptTitle":"Aberrant β-Catenin expression is related to invasive tumor growth in urothelial carcinoma of the urinary bladder","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-25 17:05:03","doi":"10.21203/rs.3.rs-8639026/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-03-12T13:32:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"230056341173927468346711123001310457830","date":"2026-03-08T13:30:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"86559961556644481273154701326929300183","date":"2026-03-02T05:50:16+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-20T15:20:52+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-16T14:22:32+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-01-27T09:14:49+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-27T08:49:11+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Urology","date":"2026-01-27T08:36:05+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-urology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"buro","sideBox":"Learn more about [BMC Urology](http://bmcurol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/buro/default.aspx","title":"BMC Urology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7e16ee86-7d3f-47fb-825f-bb3c0050a76a","owner":[],"postedDate":"February 25th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-02-25T17:05:03+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-25 17:05:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8639026","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8639026","identity":"rs-8639026","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}