Potential Utility of ADNP in Circulating Tumor Cells as Biomarker for Prognostics in Non-muscle-invasive Bladder Cancer

Potential Utility of ADNP in Circulating Tumor Cells as Biomarker for Prognostics in Non-muscle-invasive Bladder Cancer | 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 Article Potential Utility of ADNP in Circulating Tumor Cells as Biomarker for Prognostics in Non-muscle-invasive Bladder Cancer Yuheng Wen, Zhihao Ming, Hong Li, Shuai Zhu, Jian Cao, Mingji Ye, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4009102/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Aug, 2024 Read the published version in Scientific Reports → Version 1 posted 13 You are reading this latest preprint version Abstract Objective This study aims to evaluate the prognostic utility of Activity-dependent neuroprotective protein (ADNP) expression in Circulating Tumor Cells (CTCs) inpatients with Non-muscle-invasive Bladder Cancer (NMIBC) undergoing Transurethral Resection of Bladder Tumor (TURBT). Methods A prospective cohort of 74 bladder cancer patients and 22 non-cancer controls were enrolled. The expression of ADNP mRNA was detected by immunomagnetic beads-droplet digital PCR. The ADNP mRNA expression was evaluated in patients with high-risk NMIBC and those with indeterminate invasion depth post 2nd TURBT. Primary cultured bladder cancer cells and PBMCs from healthy donors were immunofluorescence stained. Results Our findings suggest that baseline ADNP mRNA level in CTCs shows potential as a prognostic marker for NMIBC with a sensitivity of 83.33% and a specificity of 73.58%. In comparison to baseline, ADNP mRNA expression increased post 2nd TURBT in 5 patients, where 2 experienced recurrence. Meanwhile, among the 12 patients with decreased levels, only one patient relapsed. A considerable limitation of this study entails the small sample size. Conclusions The Immuno-magnetic beads-ddPCR technique provided a viable method for ADNP mRNA detection in CTCs from bladder cancer patients. The preoperative ADNP mRNA level in CTCs was identified as a prognostic indicator for NMIBC. Longitudinal monitoring of ADNP mRNA in CTCs of bladder cancer patients shows promise in evaluating treatment responses and predicting prognosis. Health sciences/Molecular medicine Health sciences/Urology Non-muscle invasive bladder cancer (NMIBC) ADNP ddPCR Longitudinal monitoring Prognosis Figures Figure 1 Figure 2 Figure 3 Figure 4 1. INTRODUCTION Bladder cancer (BC) is one of the most prevalent forms of cancer globally, with an estimated 570,000 new cases and 210,000 deaths reported in 2020 alone[ 1 ]. Dominantly, Non-muscle invasive bladder cancer (NMIBC), inclusive of carcinoma in situ (CIS), as well as stages Ta and T1, stands for approximately 75% of all new BC cases. Despite a high recurrence rate of 50–70% within five years of diagnosis, only 10–20% of these patients progress to the more severe Muscle-invasive bladder cancer (MIBC) [ 2 ]. Notably, MIBC presents an even more daunting prognosis, with the potential for metastases development in up to 50% of patients within two years of operative intervention, despite initial first-line treatment with cisplatin-based neoadjuvant chemotherapy followed by radical cystectomy and lymph node dissection[ 3 ]. This metastases development further coincides with a survival rate of 36% − 48% at the five-year mark post-surgery[ 4 ]. The principal causal factor for mortality in BC patients is metastatic bladder cancer [ 5 , 6 ]. The challenge with MBC is the difficulty in early detection of tumor cell spread, implying that timely and potential effective interventions are often unattainable[ 7 – 9 ]. Current clinical management strategies for BC heavily rely on data from the tumor obtained via Transurethral Resection of the Bladder Tumor (TURBT), which may not provide sufficient information to determine optimal individualized treatment approaches[ 3 , 10 , 11 ]. Radical bladder surgery, currently widely used for early bladder cancer management, offers optimal cure chances for invasive NMIBC yet remains an excessive procedure for a subgroup of patients[ 12 ]. Circulating Tumor Cells (CTCs), originating either from primary tumors or metastatic lesions, are perceived as metastatic seeds[ 13 – 16 ]. Current evidence from meta-analysis studies indicates CTCs as promising prognostic markers for aggressive tumor advancement and reduced survival rates for BC patients[ 17 ]. The Cell Search system, frequently utilized for CTCs detection through tumor surface markers, has limitations, as it may not capture all CTCs subtypes simultaneously due to CTC heterogeneity resulting from the Epithelial-mesenchymal transition. Furthermore, the potential of biomarker analysis within bladder cancer CTCs has not been thoroughly scrutinized[ 18 , 19 ]. The Activity-dependent neuroprotective protein (ADNP), initially characterized as a neuroprotective protein, has been associated with various neural developmental disorders and cancers[ 20 ]. The ADNP gene, located in chromosome 20q12–13.2—an area linked with aggressive tumor growth and commonly amplified in numerous neoplasms, including bladder cancer—expresses itself highly in malignant cells and is crucial to cell survival[ 21 , 22 ]. ADNP can trigger proliferation of BC cells via the AKT pathway, and ADNP mRNA and protein overexpression can induce cell migration and EMT, resulting in cisplatin resistance potentially associated with the TGF-β/Smad signaling pathway [ 23 , 24 ]. Consequently, in this study, we employed Immunomagnetic Beads (IMBs) for tumor cell enrichment within blood and utilized Droplet Digital PCR (ddPCR) for ADNP mRNA expression detection in CTCs. Our objective is to explore the ADNP mRNA expression level in peripheral blood CTCs in bladder cancer patients, its clinical importance, and whether it has any correlation with prognosis. 2.MATERIALS AND METHODS 2.1 Blood samples The study protocol was approved supervised by the Ethics Committee of Hunan Cancer Hospital, Changsha, China. The study was conducted in accordance with the STROBE guidelines in compliance with the national legislation(Good Clinical Practice (GCP)) and the Code of Ethical Principles for Medical Research Involving Human Subjects of the World Medical Association (Declaration of Helsinki). Peripheral blood samples were collected with the consent of patients in the Department of Urology, Hunan Cancer Hospital from April 2021 to December 2021. All patients were informed of the use and final treatment of blood samples before blood collection, and signed informed consent. The primary bladder cancer cells were obtained from Sun Yat-Sen University Cancer Centre (State Key Laboratory of Oncology in South China, Guangzhou, China). 2.2 Eligibility criteria Age 18–80 years. Patients with Eastern Cooperative Oncology Group (ECOG) score ≤ 2 points. No bladder cancer patient underwent antitumor therapy before diagnosis. All the treatments were performed in accordance with the Guidelines for the Diagnosis and Treatment of Urinary Surgery Diseases in China (2014 edition). The stage of bladder cancer was classified according to the International Union Against Cancer (UICC) TNM staging of bladder cancer (2017, 8th edition) and the grade was assessed according to the World Health Organization 2004 bladder cancer grading system. 2.3 The ADNP mRNA expression was detected by ddPCR. a)Immunomagnetic beads enriched peripheral blood CTCs: Peripheral blood mononuclear cells (PBMCs) were isolated by Lymphoprep density gradient centrifugation (STEMCELL，Cat 07851) with 6ml blood sample. Add EasySep Hu CD45 Depletion Buffer (STEMCELL，Cat 17898) antibody to remove white blood cells and get 100ul liquid containing CTCs. b)RNA extraction from CTCs: CTC samples were subjected to RNA extraction using the RNeasy Plus Micro Kit (Qiagen，Cat 74034). The collected RNA (20ul) was used for one-step probe RT-dPCR detection or immediately stored in the refrigerator at -80℃. c)ddPCR was used to detect ADNP mRNA expression in CTCs 1. The synthesis of primers was entrusted to Sangon Biotech Co. Ltd., (Shanghai, China) and the primer sequences of differential genes were shown in Table 1. 2. Each RNA sample was analysed in triplicate(5ul×3=15ul). All reagents are prepared and thoroughly mixed at room temperature according to the following table (Table 2) (110% of the required amount is recommended). 3. ddPCR was performed by the ddPCR platform system (TargetingOne Biotech. Co. Ltd. Beijing, China) which includes the Drop Maker M1 and the Chip Reader R1. A total of 15μl RNA was added to the 75μl mixture to make 90ul of the reaction solution, which was then added in aliquots to three reaction tubes. 30ul of the reaction solution was added to the aqueous phase reaction well of the microdroplet generation chip, 180ul of microdroplet generated oil was added to the oil phase well (TargetingOne，Cat 10002). Samples were prepared as nanoliter droplets in the Drop Maker M1 sample preparator. Finally, the microdroplets were stored in 8-row tube. 4. The 8-row tube containing microdroplets were placed into an ETC811 PCR apparatus(EASTWIN, Suzhou, China). The steps of reverse transcription and amplification were as follows: Stage1: 55 °C, 15 min, 1 cycle; Stage 2: 95°C, 10min, 1 cycle; Stage 3: 40 cycles at 94°C for 30s, 55-60°C for 1min; Stage 4: 12°C, 5min 1 cycle. 5. The amplified samples were put into the Chip Reader R1 analyzer to read the fluorescence signal values of ADNP-FAM and beta-actin-VIC. After Poisson statistical analysis, the FAM and VIC copy numbers were obtained. The ratio of ADNP copy number (FAM) to beta-actin copy number (VIC) was used as the relative expression level of ADNP mRNA, and the mean value of three parallel experiments was used as the relative expression level of ADNP mRNA in CTCs of individual peripheral blood. The coefficient of variation should not exceed 20%. 2.4 Immunofluorescence staining (IF) 1. Cells were released in 100ul PBS and coated specimen frame of microscope slide (Cytelligen, San Diego, CA, USA). The slide was placed in a constant temperature drying oven at 37 ℃for 20min, so that the cells precipitate adhered to the slide. 2. Then fixed with 400ul,4% PFA (Kingmorn; Cat# KA1440) for 12 min. Slide was washed twice in PBS for 5min each time. 3. Next, the slide was placed on a wet box and permeabilized with 5% Triton X-100 (Solarbio; Cat T8200) for 20 min, washed three times with PBS for 5 min each time. 4. incubated in a 3% BSA blocking solution (5%BSA was diluted to 3%BSA in PBS, Solarbio, Cat SW3015) for 30 min, then washed three times with PBS for 5 min each time. 5. followed by incubation with 150ul ADNP antibody(1:50,Bioss,Cat bs-0039R-FITC) for 60 min at room temperature, then washed three times with PBS for 5min each time. 6. stained with 10ul DAPI diluted for 3 min, and rinsed twice with PBS. Nuclei were stained with 10ul DAPI and immediately observed under a fluorescence microscope or preserved in a refrigerator at 4°C and shielded from light. 2.5 statistical analysis GraphPad Prism 8.0.2 statistical software was used. The Receiver operating characteristic curve (ROC) was used to analyze the subgroups and determine the optimal threshold value. Progression-free survival (PFS) was defined as the duration from the time of surgery to cancer recurrence/progression. Kaplan-Meier survival plots were generated based on the relative expression of ADNP before and after surgery. Gehan-Breslow-Wilcoxon test was used. *P < 0.05 and **P < 0.01 were considered statistically significant and significant differences, respectively. All p values are two-sided. 3. RESULTS 3.1 Characteristics of the Study Population The clinicopathological characteristics of patients are listed in Table 3. 3.2 Sensitivity and Specificity of ddPCR in Detecting ADNP mRNA Expression in CTCs Utilizing immunofluorescence staining, we determined the ADNP expression in peripheral blood mononuclear cells (PBMCs) derived from healthy donors and primary bladder cancer cells. The analysis unveiled that only a few cells in PBMCs from healthy donors demonstrated ADNP positivity (Figure 1A). In contrast, all the primary bladder cancer cells exhibited ADNP positivity post-immunofluorescence staining (Figure 1B). In the modeled CTC samples generated by adding 0-20 primary bladder cancer cells to 6ml of healthy donor peripheral blood, as was shown in Table 4, our assay efficiently detected the ADNP mRNA expression in the CTCs, even when the sample contained 10 or fewer primary bladder cancer cells. To supplement the specificity of our method, we elected beta-actin gene, which is ubiquitously expressed in normal cells, to be the internal reference gene. Simultaneously, we evaluated ADNP mRNA expression and expression of the beta-actin gene using fluorescence signals from FAM and VIC respectively. The results were documented in Figure 2. Utilizing ddPCR, we assessed the ADNP mRNA expression and beta-actin gene expression in the peripheral blood samples obtained from healthy subjects. We detected 12115 copies of the beta-actin gene while ADNP mRNA was only detected in 5 copies (Figure 2B). In contrast, following the addition of solely 10 CTCs to the same volume of healthy peripheral blood, we identified 25 ADNP mRNA copies using ddPCR, of which 12 showcased high expression of the beta-actin gene (Figure 2C). Furthermore, we implemented this method on a bladder cancer patient and the results, as depicted in Figure 2D, indicated a marked increase in ADNP mRNA expression compared to healthy individuals (Figure 2B), while the expression of beta-actin gene remained relatively the same. The aforementioned results highlighted the sensitivity and specificity of ddPCR in the detection of ADNP mRNA expression in CTCs, and also exemplified its potential relevance in the clinical diagnosis of bladder cancer. 3.3 The expression of ADNP mRNA in peripheral blood CTCs of patients with bladder cancer was detected. Expanding upon the method's efficacy in detecting ADNP mRNA expression in CTCs, we assessed the relative ADNP mRNA expression in two distinct cohorts: a non-cancer group encompassing healthy donors and patients with benign urinary lesions, and a cancer group. The non-cancer group (n=22) exhibited a mean of 0.000836±0.000453 ADNP mRNA expression (median=0.000857), while the cancer group (n=74) had a mean of 0.001558±0.000909 (median=0.001331). The statistical significance was striking, as indicated by p=0.0001 (Figure 3A). We subsequently found no notable variances in the relative ADNP mRNA expressions amongst the bladder cancer patient groups (Figure 3B). Similarly, the ADNP mRNA expression in NMIBC patients after the second TURBT did not remarkably diverge from that in healthy subjects (Figure 3C). Moreover, despite a 24% decrease of ADNP mRNA levels following Resection-TURBT (before TURBT: 0.001378±0.000772 vs. after R-TURBT: 0.001054±0.000581), the paired t-test indicated no statistical significance (p=0.11, Figure 3D). This could be due to the limited sample size. This assessment of our method suggests application into clinical practice, potentially as a sensitive biomarker for the diagnosis of bladder cancer. However, its discriminating ability across different BC groups and stages warrant further clarification. 3.4 Survival analysis of ADNP mRNA expression in CTCs in NMIBC patients. We carried out an analysis of the ROC curve to assess the predictive efficacy of preoperative ADNP mRNA levels. The Area Under the Curve (AUC) was 0.7704 with a 95% confidence interval [CI] of 0.6047-0.9362 (Figure 4A). The most favorable cut-off value was deduced at 0.001517, exhibiting a sensitivity of 83.33% and a specificity of 73.58% (Figure 4B). It is worthy to note that all ADNP mRNA tests were completed within 3 days prior to the operation. Such an early detection timeline yielded a sensitivity of 83.33% and a specificity of 73.58% in forecasting tumor recurrence. During the follow-up, conventional cystoscopy and urine cytology were utilized to detect tumor recurrence. For the NMIBC patients who experienced recurrence, neither of the two customary methods was capable of promptly discovering the progress of tumor recurrence. As indicated in Figure 4C, the average time for cystoscopy to detect tumor recurrence amounted to 5.1 months, and it is even longer with urine cytology. The molecular diagnostic approach leveraging ADNP mRNA detection expedited prediction of tumor recurrence, underlining the modality's potential for early-stage recurrence diagnosis. Nonetheless, the predictive utility of postoperative ADNP mRNA levels in the portrayal of recurrence or progression was indeterminate among NMIBC patients who underwent secondary electrocautery, potentially due to the limited sample size (n=17, P=0.1015).Our cohort of 17 NMIBC patients reflected an increase in the baseline ADNP mRNA level in 5 patients following the secondary Transurethral Resection of Bladder Tumor (TURBT), with two of this cluster showing recurrence in the follow-up period. On the contrary, only one patient manifested recurrence among the twelve patients who saw a decrease in expression level (Fisher's exact test P=0.053). 4. Discussion Bladder cancer is a multifaceted disease, and developments in sequencing and gene expression studies have facilitated the discovery of a range of DNA, RNA, and protein biomarkers. Notably, multiple distinct molecular signatures and subtypes that provide an accurate prediction of disease progression and therapeutic response have been identified[ 25 ]. Historically, the detection of tumor cells in peripheral blood was achieved through the identification of specific gene mutations using immunomagnetic beads [ 26 ]. However, advancements in PCR technology have led to the introduction of Real-time quantitative Reverse Transcription PCR (qRT-PCR)[ 27 ]. Renowned for its pronounced sensitivity and specificity, ddPCR can be utilized to typify target nucleic acid and discern an actual copy number [ 28 ]. Our assay effectively detects ADNP expression in a minimum of ten BC cells, thereby establishing a valid CTC model. The present study identified that the ADNP protein was expressed in some PBMCs and ubiquitously in primary bladder cancer cells. Additionally, no correlation was found with white blood cell count or subtype. Several studies have utilized flow cytometry to analyze ADNP expression in various immune system cells in healthy donors, revealing prominent intracellular ADNP expression in monocytes, B cells, and T cells [ 29 ]. ADNP mRNA is found to be highly expressed in the CTCs of BC patients in comparison to healthy individuals. With a sensitivity of 48.6% and specificity of 90.9%, the relative expression of ADNP mRNA shows promise as a tumor marker for bladder cancer diagnosis. Transurethral resection, which removes all visible tumors, is the initial management step for non-muscle-invasive bladder cancer [ 30 ]. Our findings revealed that the post-operative relative expression of ADNP mRNA in NMIBC patients dropped relative to preoperative levels, indicating that TURBT can reduce tumor burden and the risk of metastasis. Preoperative ADNP mRNA expression levels could predict NMIBC recurrence or progression with a sensitivity of 83.33% and specificity of 73.58%. Our study supports clinical longitudinal monitoring in predicting patient prognosis and assessing treatment responses. Liquid biopsies present an advantage over tissue biopsies by allowing real-time tracking of treatment response and disease progression. 5. Conclusion We have shown that the novel ddPCR assay showed superior sensitivity in the detection of ADNP mRNA in CTCs. Immunomagnetic beads-ddPCR provides a reliable tool for the molecular characterization of CTCs in bladder cancer. There is a significant difference in the expression of ADNP mRNA in CTCs between healthy people and patients with bladder cancer. Although, few patients with NMIBC after second transurethral resection were tested, longitudinal monitoring of ADNP mRNA expression in CTCs has the potential to facilitate the management of NMIBC patients. Abbreviations ADNP, Activity-dependent neuroprotective protein; BC, Bladder cancer; CTCs, Circulating Tumor Cells; ddPCR, Droplet Digital PCR; ECOG, Eastern Cooperative Oncology Group; IF, Immunofluorescence staining; IMBs, Immunomagnetic Beads; MIBC, Muscle-invasive bladder cancer; NMIBC, Non-muscle invasive bladder cancer; PFS, Progression-free survival; qRT-PCR, quantitative Reverse Transcription PCR; TURBT, Transurethral Resection of the Bladder Tumor; UICC, Union Against Cancer Declarations Acknowledgements This study was approved by the Medical Ethical Committee of Hunan Cancer Hospital, with the ethical approval number KYJJ-2023-127. This work was supported by the National Cancer Center Clinical Research in “Climbing” Foundation of China [NCC201818A55], and the Natural Science Foundation Health Union Foundation of Hunan Province, China [2021JJ70030]. Author contribution XY conceived and designed the project. XY and YZ supervised the project. YHW performed microscopic analysis and conducted all dd-PCR analyses. ZHM analyzed data, including all statistical analyses, and prepared all figures and tables. HL, SZ, MJY and JC provided serum samples from patients with BC, as well as clinicopathological data. TG and XQS provide guidance for students. YHW and ZHM wrote the manuscript. All authors critically revised and approved the manuscript. Data Availability The datasets used and analysed during the current study available from the corresponding author on reasonable request. Conflict of interest The authors declare no conflict of interest. References Sung H, Ferlay J, Siegel RL, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA: a cancer journal for clinicians 2021; 71:209-249. Babjuk M, Burger M, Capoun O, et al. European Association of Urology Guidelines on Non-muscle-invasive Bladder Cancer (Ta, T1, and Carcinoma in Situ). 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Cite Share Download PDF Status: Published Journal Publication published 21 Aug, 2024 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 18 Jun, 2024 Reviews received at journal 17 Jun, 2024 Reviewers agreed at journal 06 Jun, 2024 Reviews received at journal 16 May, 2024 Reviewers agreed at journal 08 May, 2024 Reviewers agreed at journal 06 May, 2024 Reviewers agreed at journal 12 Apr, 2024 Reviewers agreed at journal 25 Mar, 2024 Reviewers invited by journal 17 Mar, 2024 Editor assigned by journal 17 Mar, 2024 Editor invited by journal 17 Mar, 2024 Submission checks completed at journal 17 Mar, 2024 First submitted to journal 03 Mar, 2024 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. 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of Medicine, Central South University, Hunan Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Tian","middleName":"","lastName":"Gan","suffix":""},{"id":281002398,"identity":"7a472009-f25a-4e42-8c3a-5085949ae912","order_by":7,"name":"Xiangqun She","email":"","orcid":"","institution":"The Second People's Hospital of Huaihua","correspondingAuthor":false,"prefix":"","firstName":"Xiangqun","middleName":"","lastName":"She","suffix":""},{"id":281002400,"identity":"0094d6c1-af6c-40df-b8d1-3ea3e23538bb","order_by":8,"name":"Yong Zeng","email":"","orcid":"","institution":"Central South University, Hunan Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yong","middleName":"","lastName":"Zeng","suffix":""},{"id":281002401,"identity":"9b79b1af-3f0f-4149-bb22-d932a42dfdd4","order_by":9,"name":"Yu Xie","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAy0lEQVRIiWNgGAWjYDACZhDBY8PDz8DYQIoWmTQ5yQaitYCBzWFjgwPEKjY4znxMmieHOXHz+cNtD34w2MnpErJMspktTZrnDFvithuJ7YY9DMnGZoSs42fmMZPm7eEBamFsk+BhOJC4jZAWNmb+b9K8/yQSN/cfbJP8Q4wWoC1s0jw8BsYGDIlt0kTZAvSLseUcngQ5iRtALTIGRPjF4Pzhhzfe8Pzn4e8//kzyTYWdHEEtQMAigWQCYeUgwPyBOHWjYBSMglEwYgEASiw51jDsztkAAAAASUVORK5CYII=","orcid":"","institution":"The Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Hunan Cancer Hospital","correspondingAuthor":true,"prefix":"","firstName":"Yu","middleName":"","lastName":"Xie","suffix":""}],"badges":[],"createdAt":"2024-03-03 15:45:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4009102/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4009102/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-70379-6","type":"published","date":"2024-08-21T15:57:46+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":53016456,"identity":"5de7e5af-385d-42b6-b8bd-ad1602b37bb0","added_by":"auto","created_at":"2024-03-19 16:05:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":318653,"visible":true,"origin":"","legend":"\u003cp\u003eImmunofluorescence staining of ADNP (A) The ADNP of some cells in peripheral blood mononuclear cells of healthy people showed intracellular staining (green). (B) Immunofluorescence staining of ADNP on primary bladder cancer cells.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4009102/v1/753ad2339a9a6652791dd4bc.png"},{"id":53015598,"identity":"c960226a-2036-42b3-abdb-e128c6ac97c4","added_by":"auto","created_at":"2024-03-19 15:57:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":232477,"visible":true,"origin":"","legend":"\u003cp\u003eddPCR detection of ADNP mRNA expression 2D scatter plot. (A) In the blank control group, DEPC water was used instead of RNA template. The number of ADNP positive droplets ( ADNP-FAM ) was 0, the number of internal reference gene beta-actin positive droplets ( beta-actin-VIC ) was 1, and the black scatter represented the number of negative droplets. (B) In healthy human peripheral blood PBMCs, blue dots represented the number of ADNP-positive droplets ( ADNP-FAM ) was 5, and green dots represented the number of beta-actin-positive droplets ( beta-actin-VIC ) was 12115. (C) In the peripheral blood CTCs model, the number of ADNP-positive droplets ( ADNP-FAM ) in blue dots was 13, the number of both ADNP-FAM / beta-actin positive droplets ( ADNP-FAM / beta-actin-VIC ) in orange dots was 12, and the number of beta-actin-positive droplets ( beta-actin-VIC ) in green dots was 16637. (D) Results of ADNP mRNA expression in a patient with bladder cancer.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4009102/v1/7f260649ca4b2790c96ac082.png"},{"id":53015599,"identity":"1108f045-a5ed-452a-812d-30b89c1fa28e","added_by":"auto","created_at":"2024-03-19 15:57:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":232336,"visible":true,"origin":"","legend":"\u003cp\u003e(A) There was a significant difference in ADNP mRNA level between non-cancer group and bladder cancer group. (B) There was no difference in the expression of ADNP mRNA between bladder cancer subgroups. (C) There was no significant difference in the expression of ADNP mRNA in CTCs between healthy donors and NMIBC patients after secondary TURBT. (D) The expression of ADNP mRNA in CTCs of NMIBC patients after secondary TURBT decreased by 24 % compared with that before operation (baseline), but there was no statistical difference.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4009102/v1/191a4c6ce4142ae650c72b1b.png"},{"id":53015596,"identity":"96c8f4f6-5406-4f10-b7e8-b3f97459da9c","added_by":"auto","created_at":"2024-03-19 15:57:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":283869,"visible":true,"origin":"","legend":"\u003cp\u003eThe relative expression of ADNP mRNA in CTCs was used to analyze the survival of NMIBC patients. (A) ROC curve of ADNP mRNA expression level in preoperative CTCs for predicting the prognosis of recurrence or progression in NMIBC patients. (B) When the optimal threshold was 0.001517, the sensitivity was 83.33 % and the specificity was 73.58 %. (C) Depiction of the timeline for the risk of tumor recurrence for patients with NMIBC as per differing methods throughout the follow-up period.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4009102/v1/2cdcef02c25cb8473785a8eb.png"},{"id":63300366,"identity":"0ef3f59b-2aa1-4226-aabe-35bd75436126","added_by":"auto","created_at":"2024-08-26 16:13:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1559573,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4009102/v1/36667cc0-f8f7-43ce-a40a-403f6b788e20.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Potential Utility of ADNP in Circulating Tumor Cells as Biomarker for Prognostics in Non-muscle-invasive Bladder Cancer","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eBladder cancer (BC) is one of the most prevalent forms of cancer globally, with an estimated 570,000 new cases and 210,000 deaths reported in 2020 alone[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Dominantly, Non-muscle invasive bladder cancer (NMIBC), inclusive of carcinoma in situ (CIS), as well as stages Ta and T1, stands for approximately 75% of all new BC cases. Despite a high recurrence rate of 50\u0026ndash;70% within five years of diagnosis, only 10\u0026ndash;20% of these patients progress to the more severe Muscle-invasive bladder cancer (MIBC) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Notably, MIBC presents an even more daunting prognosis, with the potential for metastases development in up to 50% of patients within two years of operative intervention, despite initial first-line treatment with cisplatin-based neoadjuvant chemotherapy followed by radical cystectomy and lymph node dissection[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. This metastases development further coincides with a survival rate of 36% \u0026minus;\u0026thinsp;48% at the five-year mark post-surgery[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe principal causal factor for mortality in BC patients is metastatic bladder cancer [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The challenge with MBC is the difficulty in early detection of tumor cell spread, implying that timely and potential effective interventions are often unattainable[\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Current clinical management strategies for BC heavily rely on data from the tumor obtained via Transurethral Resection of the Bladder Tumor (TURBT), which may not provide sufficient information to determine optimal individualized treatment approaches[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRadical bladder surgery, currently widely used for early bladder cancer management, offers optimal cure chances for invasive NMIBC yet remains an excessive procedure for a subgroup of patients[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Circulating Tumor Cells (CTCs), originating either from primary tumors or metastatic lesions, are perceived as metastatic seeds[\u003cspan additionalcitationids=\"CR14 CR15\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Current evidence from meta-analysis studies indicates CTCs as promising prognostic markers for aggressive tumor advancement and reduced survival rates for BC patients[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe Cell Search system, frequently utilized for CTCs detection through tumor surface markers, has limitations, as it may not capture all CTCs subtypes simultaneously due to CTC heterogeneity resulting from the Epithelial-mesenchymal transition. Furthermore, the potential of biomarker analysis within bladder cancer CTCs has not been thoroughly scrutinized[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe Activity-dependent neuroprotective protein (ADNP), initially characterized as a neuroprotective protein, has been associated with various neural developmental disorders and cancers[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The ADNP gene, located in chromosome 20q12\u0026ndash;13.2\u0026mdash;an area linked with aggressive tumor growth and commonly amplified in numerous neoplasms, including bladder cancer\u0026mdash;expresses itself highly in malignant cells and is crucial to cell survival[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. ADNP can trigger proliferation of BC cells via the AKT pathway, and ADNP mRNA and protein overexpression can induce cell migration and EMT, resulting in cisplatin resistance potentially associated with the TGF-β/Smad signaling pathway [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eConsequently, in this study, we employed Immunomagnetic Beads (IMBs) for tumor cell enrichment within blood and utilized Droplet Digital PCR (ddPCR) for ADNP mRNA expression detection in CTCs. Our objective is to explore the ADNP mRNA expression level in peripheral blood CTCs in bladder cancer patients, its clinical importance, and whether it has any correlation with prognosis.\u003c/p\u003e"},{"header":"2.MATERIALS AND METHODS","content":"\u003cp\u003e\u003cstrong\u003e2.1\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eBlood\u0026nbsp;samples\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study protocol was approved supervised by the Ethics Committee of Hunan Cancer Hospital, Changsha, China. The study was conducted in accordance with the STROBE guidelines in compliance with the national legislation(Good Clinical Practice (GCP)) and the Code of Ethical Principles for Medical Research Involving Human Subjects of the World Medical Association (Declaration of Helsinki). Peripheral blood samples were collected with the consent of patients in the Department of Urology, Hunan Cancer Hospital from April 2021 to December 2021. All patients were informed of the use and final treatment of blood samples before blood collection, and signed informed consent. The primary bladder cancer cells were obtained from Sun Yat-Sen University Cancer Centre (State Key Laboratory of Oncology in South China, Guangzhou, China).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Eligibility criteria\u003c/strong\u003e\u003c/p\u003e\n\u003col\u003e\n \u003cli\u003eAge 18\u0026ndash;80 years.\u003c/li\u003e\n \u003cli\u003ePatients with Eastern Cooperative Oncology Group (ECOG) score\u0026thinsp;\u0026le;\u0026thinsp;\u003cem\u003e2\u003c/em\u003e points.\u003c/li\u003e\n \u003cli\u003eNo bladder cancer patient underwent antitumor therapy before diagnosis. All the treatments were performed in accordance with the Guidelines for the Diagnosis and Treatment of Urinary Surgery Diseases in China (2014 edition).\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eThe stage of bladder cancer was classified according to the International Union Against Cancer (UICC) TNM staging of bladder cancer (2017, 8th edition) and the grade was assessed according to the World Health Organization 2004 bladder cancer grading system. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;The ADNP mRNA expression was detected by ddPCR.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea)Immunomagnetic beads enriched peripheral blood CTCs:\u0026nbsp;Peripheral blood mononuclear cells (PBMCs) were isolated by\u0026nbsp;Lymphoprep density gradient centrifugation\u0026nbsp;(STEMCELL，Cat 07851) with 6ml blood sample.\u0026nbsp;Add EasySep Hu CD45 Depletion Buffer (STEMCELL，Cat 17898) antibody to remove white blood cells and get 100ul liquid containing CTCs.\u003c/p\u003e\n\u003cp\u003eb)RNA extraction from CTCs: CTC samples were subjected to RNA extraction using the RNeasy Plus Micro Kit (Qiagen，Cat 74034).\u0026nbsp;The collected RNA (20ul) was used for one-step probe RT-dPCR detection or immediately stored in the refrigerator at -80℃.\u003c/p\u003e\n\u003cp\u003ec)ddPCR was used to detect ADNP mRNA expression in CTCs\u003c/p\u003e\n\u003cp\u003e1. The synthesis of primers was entrusted to Sangon Biotech Co. Ltd., (Shanghai, China) and the primer sequences of differential genes were shown in Table 1.\u003c/p\u003e\n\u003cp\u003e2.\u0026nbsp;Each RNA sample was analysed in triplicate(5ul\u0026times;3=15ul). All reagents are prepared and thoroughly mixed at room temperature according to the following table (Table 2) (110% of the required amount is recommended).\u003c/p\u003e\n\u003cp\u003e3. ddPCR was performed by the ddPCR platform system (TargetingOne Biotech. Co. Ltd. Beijing, China) which includes the Drop Maker M1 and the Chip Reader R1. A total of 15\u0026mu;l RNA was added to the 75\u0026mu;l mixture to make 90ul of the reaction solution, which was then added in aliquots to three reaction tubes. 30ul of the reaction solution was added to the aqueous phase reaction well of the microdroplet generation chip, 180ul of microdroplet generated oil was added to the oil phase well (TargetingOne，Cat 10002). Samples were prepared as nanoliter droplets in the Drop Maker M1 sample preparator. Finally, the microdroplets were stored in 8-row tube.\u003c/p\u003e\n\u003cp\u003e4. The 8-row tube\u0026nbsp;containing microdroplets were placed into an ETC811 PCR apparatus(EASTWIN, Suzhou, China). The steps of reverse transcription and amplification were as follows:\u0026nbsp;Stage1: 55 \u0026deg;C, 15 min, 1 cycle; Stage 2: 95\u0026deg;C, 10min, 1 cycle;\u0026nbsp;Stage 3: 40 cycles at 94\u0026deg;C for 30s, 55-60\u0026deg;C for 1min;\u0026nbsp;Stage 4: 12\u0026deg;C, 5min 1 cycle.\u003c/p\u003e\n\u003cp\u003e5. The amplified samples were put into the Chip Reader R1 analyzer to read the fluorescence signal values of ADNP-FAM and beta-actin-VIC. After Poisson statistical analysis, the FAM and VIC copy numbers were obtained. The ratio of ADNP copy number (FAM) to beta-actin copy number (VIC) was used as the relative expression level of ADNP mRNA, and the mean value of three parallel experiments was used as the relative expression level of ADNP mRNA in CTCs of individual peripheral blood. The coefficient of variation should not exceed 20%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 Immunofluorescence staining (IF)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e1.\u0026nbsp;Cells were released in 100ul PBS and\u0026nbsp;coated specimen frame of microscope slide\u0026nbsp;(Cytelligen, San Diego, CA, USA).\u0026nbsp;The slide was placed in a constant temperature drying oven at 37 ℃for 20min, so that the cells precipitate adhered to the slide.\u003c/p\u003e\n\u003cp\u003e2. Then fixed with 400ul,4% PFA (Kingmorn; Cat# KA1440) for 12 min. Slide was washed twice in PBS for 5min each time.\u003c/p\u003e\n\u003cp\u003e3. Next,\u0026nbsp;the slide was placed on a wet box\u0026nbsp;and permeabilized with 5% Triton X-100 (Solarbio; Cat T8200) for 20 min,\u0026nbsp;washed\u0026nbsp;three times with PBS for 5 min each time.\u003c/p\u003e\n\u003cp\u003e4. incubated in a 3%\u0026nbsp;BSA blocking solution (5%BSA was diluted to 3%BSA in PBS,\u0026nbsp;Solarbio, Cat SW3015) for 30 min, then washed three times with PBS for 5 min each time.\u003c/p\u003e\n\u003cp\u003e5. followed by incubation with 150ul ADNP antibody(1:50,Bioss,Cat bs-0039R-FITC) for 60 min at room temperature, then washed three times with PBS for 5min each time.\u003c/p\u003e\n\u003cp\u003e6. stained with 10ul DAPI diluted for 3 min, and rinsed twice with PBS. Nuclei were stained with 10ul DAPI and immediately observed under a fluorescence microscope or preserved in a refrigerator at 4\u0026deg;C and shielded from light.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGraphPad Prism 8.0.2 statistical software was used. The Receiver operating characteristic curve (ROC) was used to analyze the subgroups and determine the optimal threshold value. Progression-free survival (PFS) was defined as the duration from the time of surgery to cancer recurrence/progression. Kaplan-Meier survival plots were generated based on the relative expression of ADNP before and after surgery. Gehan-Breslow-Wilcoxon test was used. *P \u0026lt; 0.05 and **P \u0026lt; 0.01 were considered statistically significant and significant differences, respectively. All p values are two-sided.\u003c/p\u003e"},{"header":"3. RESULTS","content":"\u003cp\u003e\u003cstrong\u003e3.1\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eCharacteristics of the Study Population\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe clinicopathological characteristics of patients are listed in Table 3. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eSensitivity and Specificity of ddPCR in Detecting ADNP mRNA Expression in CTCs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUtilizing immunofluorescence staining, we determined the ADNP expression in peripheral blood mononuclear cells (PBMCs) derived from healthy donors and primary bladder cancer cells. The analysis unveiled that only a few cells in PBMCs from healthy donors demonstrated ADNP positivity (Figure 1A). In contrast, all the primary bladder cancer cells exhibited ADNP positivity post-immunofluorescence staining (Figure 1B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the modeled CTC samples generated by adding 0-20 primary bladder cancer cells to 6ml of healthy donor peripheral blood, as was shown in Table 4, our assay efficiently detected the ADNP mRNA expression in the CTCs, even when the sample contained 10 or fewer primary bladder cancer cells. To supplement the specificity of our method, we elected beta-actin gene, which is ubiquitously expressed in normal cells, to be the internal reference gene. Simultaneously, we evaluated ADNP mRNA expression and expression of the beta-actin gene using fluorescence signals from FAM and VIC respectively. The results were documented in Figure 2. Utilizing ddPCR, we assessed the ADNP mRNA expression and beta-actin gene expression in the peripheral blood samples obtained from healthy subjects. We detected 12115 copies of the beta-actin gene while ADNP mRNA was only detected in 5 copies (Figure 2B). In contrast, following the addition of solely 10 CTCs to the same volume of healthy peripheral blood, we identified 25 ADNP mRNA copies using ddPCR, of which 12 showcased high expression of the beta-actin gene (Figure 2C).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFurthermore, we implemented this method on a bladder cancer patient and the results, as depicted in Figure 2D, indicated a marked increase in ADNP mRNA expression compared to healthy individuals (Figure 2B), while the expression of beta-actin gene remained relatively the same. The aforementioned results highlighted the sensitivity and specificity of ddPCR in the detection of ADNP mRNA expression in CTCs, and also exemplified its potential relevance in the clinical diagnosis of bladder cancer.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eThe expression of ADNP mRNA in peripheral blood CTCs of patients with bladder cancer was detected.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExpanding upon the method\u0026apos;s efficacy in detecting ADNP mRNA expression in CTCs, we assessed the relative ADNP mRNA expression in two distinct cohorts: a non-cancer group encompassing healthy donors and patients with benign urinary lesions, and a cancer group. The non-cancer group (n=22) exhibited a mean of 0.000836\u0026plusmn;0.000453 ADNP mRNA expression (median=0.000857), while the cancer group (n=74) had a mean of 0.001558\u0026plusmn;0.000909 (median=0.001331). The statistical significance was striking, as indicated by p=0.0001 (Figure 3A).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe subsequently found no notable variances in the relative ADNP mRNA expressions amongst the bladder cancer patient groups (Figure 3B). Similarly, the ADNP mRNA expression in NMIBC patients after the second TURBT did not remarkably diverge from that in healthy subjects (Figure 3C). Moreover, despite a 24% decrease of ADNP mRNA levels following Resection-TURBT (before TURBT: 0.001378\u0026plusmn;0.000772 vs. after R-TURBT: 0.001054\u0026plusmn;0.000581), the paired t-test indicated no statistical significance (p=0.11, Figure 3D). This could be due to the limited sample size.\u003c/p\u003e\n\u003cp\u003eThis assessment of our method suggests application into clinical practice, potentially as a sensitive biomarker for the diagnosis of bladder cancer. However, its discriminating ability across different BC groups and stages warrant further clarification.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 Survival analysis of ADNP mRNA expression in CTCs in NMIBC patients.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe carried out an analysis of the ROC curve to assess the predictive efficacy of preoperative ADNP mRNA levels. The Area Under the Curve (AUC) was 0.7704 with a 95% confidence interval [CI] of 0.6047-0.9362 (Figure 4A). The most favorable cut-off value was deduced at 0.001517, exhibiting a sensitivity of 83.33% and a specificity of 73.58% (Figure 4B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIt is worthy to note that all ADNP mRNA tests were completed within 3 days prior to the operation. Such an early detection timeline yielded a sensitivity of 83.33% and a specificity of 73.58% in forecasting tumor recurrence. During the follow-up, conventional cystoscopy and urine cytology were utilized to detect tumor recurrence. For the NMIBC patients who experienced recurrence, neither of the two customary methods was capable of promptly discovering the progress of tumor recurrence. As indicated in Figure 4C, the average time for cystoscopy to detect tumor recurrence amounted to 5.1 months, and it is even longer with urine cytology. The molecular diagnostic approach leveraging ADNP mRNA detection expedited prediction of tumor recurrence, underlining the modality\u0026apos;s potential for early-stage recurrence diagnosis.\u003c/p\u003e\n\u003cp\u003eNonetheless, the predictive utility of postoperative ADNP mRNA levels in the portrayal of recurrence or progression was indeterminate among NMIBC patients who underwent secondary electrocautery, potentially due to the limited sample size (n=17, P=0.1015).Our cohort of 17 NMIBC patients reflected an increase in the baseline ADNP mRNA level in 5 patients following the secondary Transurethral Resection of Bladder Tumor (TURBT), with two of this cluster showing recurrence in the follow-up period. On the contrary, only one patient manifested recurrence among the twelve patients who saw a decrease in expression level (Fisher\u0026apos;s exact test P=0.053).\u0026nbsp;\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eBladder cancer is a multifaceted disease, and developments in sequencing and gene expression studies have facilitated the discovery of a range of DNA, RNA, and protein biomarkers. Notably, multiple distinct molecular signatures and subtypes that provide an accurate prediction of disease progression and therapeutic response have been identified[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHistorically, the detection of tumor cells in peripheral blood was achieved through the identification of specific gene mutations using immunomagnetic beads [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. However, advancements in PCR technology have led to the introduction of Real-time quantitative Reverse Transcription PCR (qRT-PCR)[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Renowned for its pronounced sensitivity and specificity, ddPCR can be utilized to typify target nucleic acid and discern an actual copy number [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Our assay effectively detects ADNP expression in a minimum of ten BC cells, thereby establishing a valid CTC model.\u003c/p\u003e \u003cp\u003eThe present study identified that the ADNP protein was expressed in some PBMCs and ubiquitously in primary bladder cancer cells. Additionally, no correlation was found with white blood cell count or subtype. Several studies have utilized flow cytometry to analyze ADNP expression in various immune system cells in healthy donors, revealing prominent intracellular ADNP expression in monocytes, B cells, and T cells [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. ADNP mRNA is found to be highly expressed in the CTCs of BC patients in comparison to healthy individuals. With a sensitivity of 48.6% and specificity of 90.9%, the relative expression of ADNP mRNA shows promise as a tumor marker for bladder cancer diagnosis.\u003c/p\u003e \u003cp\u003eTransurethral resection, which removes all visible tumors, is the initial management step for non-muscle-invasive bladder cancer [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Our findings revealed that the post-operative relative expression of ADNP mRNA in NMIBC patients dropped relative to preoperative levels, indicating that TURBT can reduce tumor burden and the risk of metastasis. Preoperative ADNP mRNA expression levels could predict NMIBC recurrence or progression with a sensitivity of 83.33% and specificity of 73.58%.\u003c/p\u003e \u003cp\u003eOur study supports clinical longitudinal monitoring in predicting patient prognosis and assessing treatment responses. Liquid biopsies present an advantage over tissue biopsies by allowing real-time tracking of treatment response and disease progression.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eWe have shown that the novel ddPCR assay showed superior sensitivity in the detection of ADNP mRNA in CTCs. Immunomagnetic beads-ddPCR provides a reliable tool for the molecular characterization of CTCs in bladder cancer. There is a significant difference in the expression of ADNP mRNA in CTCs between healthy people and patients with bladder cancer. Although, few patients with NMIBC after second transurethral resection were tested, longitudinal monitoring of ADNP mRNA expression in CTCs has the potential to facilitate the management of NMIBC patients.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eADNP, Activity-dependent neuroprotective protein; BC, Bladder cancer; CTCs, Circulating Tumor Cells; ddPCR, Droplet Digital PCR; ECOG, Eastern Cooperative Oncology Group; IF, Immunofluorescence staining; IMBs, Immunomagnetic Beads; MIBC, Muscle-invasive bladder cancer; NMIBC, Non-muscle invasive bladder cancer; PFS, Progression-free survival; qRT-PCR, quantitative Reverse Transcription PCR; TURBT, Transurethral Resection of the Bladder Tumor; UICC, Union Against Cancer\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Medical Ethical Committee of Hunan Cancer Hospital, with the ethical approval number KYJJ-2023-127. This work was supported by the National Cancer Center Clinical Research in \u0026ldquo;Climbing\u0026rdquo; Foundation of China [NCC201818A55], and the Natural Science Foundation Health Union Foundation of Hunan Province, China [2021JJ70030].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXY conceived and designed the project. XY and YZ supervised the project. YHW performed microscopic analysis and conducted all dd-PCR analyses. ZHM analyzed data, including all statistical analyses, and prepared all figures and tables. HL, SZ, MJY and JC provided serum samples from patients with BC, as well as clinicopathological data. TG and XQS provide guidance for students. YHW and ZHM wrote the manuscript. All authors critically revised and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and analysed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSung H, Ferlay J, Siegel RL, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA: a cancer journal for clinicians 2021; 71:209-249.\u003c/li\u003e\n\u003cli\u003eBabjuk M, Burger M, Capoun O, et al. European Association of Urology Guidelines on Non-muscle-invasive Bladder Cancer (Ta, T1, and Carcinoma in Situ). European urology 2022; 81:75-94.\u003c/li\u003e\n\u003cli\u003eLenis AT, Lec PM, Chamie K, Mshs MD. Bladder Cancer: A Review. Jama 2020; 324:1980-1991.\u003c/li\u003e\n\u003cli\u003eSternberg CN, Apolo AB. Everything old is new again! Neoadjuvant chemotherapy in the treatment of muscle-invasive bladder cancer. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 2014; 32:1868-1870.\u003c/li\u003e\n\u003cli\u003ePantel K, Alix-Panabi\u0026egrave;res C, Riethdorf S. Cancer micrometastases. Nature reviews Clinical oncology 2009; 6:339-351.\u003c/li\u003e\n\u003cli\u003eJubber I, Ong S, Bukavina L, et al. Epidemiology of Bladder Cancer in 2023: A Systematic Review of Risk Factors. European urology 2023; 84:176-190.\u003c/li\u003e\n\u003cli\u003eChristensen E, Birkenkamp-Demtr\u0026ouml;der K, Sethi H, et al. Early Detection of Metastatic Relapse and Monitoring of Therapeutic Efficacy by Ultra-Deep Sequencing of Plasma Cell-Free DNA in Patients With Urothelial Bladder Carcinoma. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 2019; 37:1547-1557.\u003c/li\u003e\n\u003cli\u003eBirkenkamp-Demtr\u0026ouml;der K, Christensen E, Nordentoft I, et al. Monitoring Treatment Response and Metastatic Relapse in Advanced Bladder Cancer by Liquid Biopsy Analysis. European urology 2018; 73:535-540.\u003c/li\u003e\n\u003cli\u003eMetzenmacher M, V\u0026aacute;raljai R, Heged\u0026uuml;s B, et al. Plasma Next Generation Sequencing and Droplet Digital-qPCR-Based Quantification of Circulating Cell-Free RNA for Noninvasive Early Detection of Cancer. Cancers 2020; 12.\u003c/li\u003e\n\u003cli\u003ePatel VG, Oh WK, Galsky MD. Treatment of muscle-invasive and advanced bladder cancer in 2020. CA: a cancer journal for clinicians 2020; 70:404-423.\u003c/li\u003e\n\u003cli\u003eComp\u0026eacute;rat E, Amin MB, Cathomas R, et al. Current best practice for bladder cancer: a narrative review of diagnostics and treatments. Lancet (London, England) 2022; 400:1712-1721.\u003c/li\u003e\n\u003cli\u003eSylvester RJ. How well can you actually predict which non-muscle-invasive bladder cancer patients will progress? European urology 2011; 60:431-433; discussion 433-434.\u003c/li\u003e\n\u003cli\u003eKo JMY, Ng HY, Lam KO, et al. Liquid Biopsy Serial Monitoring of Treatment Responses and Relapse in Advanced Esophageal Squamous Cell Carcinoma. Cancers 2020; 12.\u003c/li\u003e\n\u003cli\u003eFu G, Cheng KS, Chen A, et al. Microfluidic Assaying of Circulating Tumor Cells and Its Application in Risk Stratification of Urothelial Bladder Cancer. Frontiers in oncology 2021; 11:701298.\u003c/li\u003e\n\u003cli\u003eMorelli MB, Amantini C, Rossi de Vermandois JA, et al. Correlation between High PD-L1 and EMT/Invasive Genes Expression and Reduced Recurrence-Free Survival in Blood-Circulating Tumor Cells from Patients with Non-Muscle-Invasive Bladder Cancer. Cancers 2021; 13.\u003c/li\u003e\n\u003cli\u003eAlix-Panabi\u0026egrave;res C, Pantel K. Liquid Biopsy: From Discovery to Clinical Application. Cancer discovery 2021; 11:858-873.\u003c/li\u003e\n\u003cli\u003eJiang H, Gu X, Zuo Z, Tian G, Liu J. Prognostic value of circulating tumor cells in patients with bladder cancer: A meta-analysis. PloS one 2021; 16:e0254433.\u003c/li\u003e\n\u003cli\u003eAzevedo R, Soares J, Peixoto A, et al. Circulating tumor cells in bladder cancer: Emerging technologies and clinical implications foreseeing precision oncology. Urologic oncology 2018; 36:221-236.\u003c/li\u003e\n\u003cli\u003eGorin MA, Verdone JE, van der Toom E, Bivalacqua TJ, Allaf ME, Pienta KJ. Circulating tumour cells as biomarkers of prostate, bladder, and kidney cancer. Nature reviews Urology 2017; 14:90-97.\u003c/li\u003e\n\u003cli\u003eGozes I, Yeheskel A, Pasmanik-Chor M. Activity-dependent neuroprotective protein (ADNP): a case study for highly conserved chordata-specific genes shaping the brain and mutated in cancer. Journal of Alzheimer\u0026apos;s disease : JAD 2015; 45:57-73.\u003c/li\u003e\n\u003cli\u003eGozes I. Activity-dependent neuroprotective protein: from gene to drug candidate. Pharmacology \u0026amp; therapeutics 2007; 114:146-154.\u003c/li\u003e\n\u003cli\u003eZamostiano R, Pinhasov A, Gelber E, et al. Cloning and characterization of the human activity-dependent neuroprotective protein. The Journal of biological chemistry 2001; 276:708-714.\u003c/li\u003e\n\u003cli\u003eZhu S, Xu Z, Zeng Y, et al. ADNP Upregulation Promotes Bladder Cancer Cell Proliferation via the AKT Pathway. Frontiers in oncology 2020; 10:491129.\u003c/li\u003e\n\u003cli\u003eXie Y, Zhu S, Zang J, et al. ADNP prompts the cisplatin-resistance of bladder cancer via TGF-\u0026beta;-mediated epithelial-mesenchymal transition (EMT) pathway. Journal of Cancer 2021; 12:5114-5124.\u003c/li\u003e\n\u003cli\u003eTran L, Xiao JF, Agarwal N, Duex JE, Theodorescu D. Advances in bladder cancer biology and therapy. Nature reviews Cancer 2021; 21:104-121.\u003c/li\u003e\n\u003cli\u003eHardingham JE, Kotasek D, Farmer B, et al. Immunobead-PCR: a technique for the detection of circulating tumor cells using immunomagnetic beads and the polymerase chain reaction. Cancer research 1993; 53:3455-3458.\u003c/li\u003e\n\u003cli\u003eGuo J, Xiao B, Zhang X, et al. Combined use of positive and negative immunomagnetic isolation followed by real-time RT-PCR for detection of the circulating tumor cells in patients with colorectal cancers. Journal of molecular medicine (Berlin, Germany) 2004; 82:768-774.\u003c/li\u003e\n\u003cli\u003eMa Y, Luk A, Young FP, et al. Droplet Digital PCR Based Androgen Receptor Variant 7 (AR-V7) Detection from Prostate Cancer Patient Blood Biopsies. International journal of molecular sciences 2016; 17.\u003c/li\u003e\n\u003cli\u003eBraitch M, Kawabe K, Nyirenda M, et al. Expression of activity-dependent neuroprotective protein in the immune system: possible functions and relevance to multiple sclerosis. Neuroimmunomodulation 2010; 17:120-125.\u003c/li\u003e\n\u003cli\u003eKamat AM, Hahn NM, Efstathiou JA, et al. Bladder cancer. Lancet (London, England) 2016; 388:2796-2810.\u003c/li\u003e\n\u003c/ol\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":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Non-muscle invasive bladder cancer (NMIBC), ADNP, ddPCR, Longitudinal monitoring, Prognosis","lastPublishedDoi":"10.21203/rs.3.rs-4009102/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4009102/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eThis study aims to evaluate the prognostic utility of Activity-dependent neuroprotective protein (ADNP) expression in Circulating Tumor Cells (CTCs) inpatients with Non-muscle-invasive Bladder Cancer (NMIBC) undergoing Transurethral Resection of Bladder Tumor (TURBT).\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eA prospective cohort of 74 bladder cancer patients and 22 non-cancer controls were enrolled. The expression of ADNP mRNA was detected by immunomagnetic beads-droplet digital PCR. The ADNP mRNA expression was evaluated in patients with high-risk NMIBC and those with indeterminate invasion depth post 2nd TURBT. Primary cultured bladder cancer cells and PBMCs from healthy donors were immunofluorescence stained.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eOur findings suggest that baseline ADNP mRNA level in CTCs shows potential as a prognostic marker for NMIBC with a sensitivity of 83.33% and a specificity of 73.58%. In comparison to baseline, ADNP mRNA expression increased post 2nd TURBT in 5 patients, where 2 experienced recurrence. Meanwhile, among the 12 patients with decreased levels, only one patient relapsed. A considerable limitation of this study entails the small sample size.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe Immuno-magnetic beads-ddPCR technique provided a viable method for ADNP mRNA detection in CTCs from bladder cancer patients. The preoperative ADNP mRNA level in CTCs was identified as a prognostic indicator for NMIBC. Longitudinal monitoring of ADNP mRNA in CTCs of bladder cancer patients shows promise in evaluating treatment responses and predicting prognosis.\u003c/p\u003e","manuscriptTitle":"Potential Utility of ADNP in Circulating Tumor Cells as Biomarker for Prognostics in Non-muscle-invasive Bladder Cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-19 15:57:37","doi":"10.21203/rs.3.rs-4009102/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-06-18T04:36:53+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-17T16:07:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"69312134880329399320350491559082652581","date":"2024-06-07T03:32:02+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-16T11:27:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"40cc6ad8-d865-4a2e-bb39-794c2ff0ad7c","date":"2024-05-08T11:54:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"2ec13de6-7880-4cab-b7e4-c70ce88d4c12","date":"2024-05-07T01:37:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"5ee25d2b-2394-418d-8ed9-33acd357d2cf","date":"2024-04-12T07:18:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"5f779137-acc9-4a12-941c-1cb936c99783","date":"2024-03-25T09:00:02+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-03-17T23:08:57+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-03-17T23:02:46+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-03-17T17:08:52+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-03-17T17:05:34+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-03-03T15:10:19+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8786d346-2af8-4cf2-bf5f-363bc805784d","owner":[],"postedDate":"March 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":29589865,"name":"Health sciences/Molecular medicine"},{"id":29589866,"name":"Health sciences/Urology"}],"tags":[],"updatedAt":"2024-08-26T16:04:31+00:00","versionOfRecord":{"articleIdentity":"rs-4009102","link":"https://doi.org/10.1038/s41598-024-70379-6","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-08-21 15:57:46","publishedOnDateReadable":"August 21st, 2024"},"versionCreatedAt":"2024-03-19 15:57:37","video":"","vorDoi":"10.1038/s41598-024-70379-6","vorDoiUrl":"https://doi.org/10.1038/s41598-024-70379-6","workflowStages":[]},"version":"v1","identity":"rs-4009102","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4009102","identity":"rs-4009102","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}