Mesenchymal Transition as a Potential Mechanism of Late Immune Evasion in Neuroblastoma: A Case of Late Relapse after Allogeneic Hematopoietic Stem Cell Transplantation

Mesenchymal Transition as a Potential Mechanism of Late Immune Evasion in Neuroblastoma: A Case of Late Relapse after Allogeneic Hematopoietic Stem Cell Transplantation | 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 Case Report Mesenchymal Transition as a Potential Mechanism of Late Immune Evasion in Neuroblastoma: A Case of Late Relapse after Allogeneic Hematopoietic Stem Cell Transplantation han hu, Liping Zhan, Wenxia Wang, Xiaomin Peng, Xilin Xiong, Honggui Xu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9280514/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract Background Neuroblastoma is the most common extracranial solid malignant tumor in children, and recurrence is not uncommon in high-risk cases, with immune evasion considered a key mechanism underlying relapse and drug resistance. Neural crest progenitor cells differentiate into adrenergic or mesenchymal phenotypes. MES-type neuroblastoma exhibits greater resistance to therapy and enhanced migratory capacity, leading to poorer prognosis. This report describes a child with high-risk neuroblastoma who experienced late relapse after allogeneic hematopoietic stem cell transplantation (allo-HSCT), with flow cytometry indicating mesenchymal transition in the recurrent tumor cells. By integrating clinical course and molecular characteristics, we explore the potential role of mesenchymal transition in mediating immune escape, aiming to provide new insights into the mechanisms of immune evasion in neuroblastoma and inform optimization of immunotherapeutic strategies. Case Presentation We present a patient with stage M neuroblastoma who achieved complete remission after surgery, chemotherapy, radiotherapy, and allogeneic hematopoietic stem cell transplantation, but experienced bone and bone marrow relapse three years post-transplantation. Flow cytometry of the bone marrow revealed mesenchymal transition in the tumor cells. The case harbored multiple gene mutations associated with poor prognosis in neuroblastoma. Following relapse, the patient has received two cycles of chemotherapy and achieved partial response. Conclusion Mesenchymal transition may be a key mechanism underlying late relapse in neuroblastoma. High-risk genetic background, insufficient intensity of induction therapy and conditioning regimen, lack of prophylactic radiotherapy, and absence of immunotherapy may contribute to the risk of long-term relapse after hematopoietic stem cell transplantation. Registration The treatment protocol for this patient was conducted under the clinical trial registered at ClinicalTrials.gov (Identifier: NCT05303727, First submitted :March 16, 2022). Neuroblastoma Allogeneic hematopoietic stem cell transplantation Drug resistance Mesenchymal transition Figures Figure 1 Figure 2 Figure 3 Background Neuroblastoma is the most common extracranial solid tumor in children 1 .Despite significant advances in multimodal therapies, including surgery, chemotherapy, radiotherapy, immunotherapy, and autologous or allogeneic hematopoietic stem cell transplantation, the long-term survival rate for patients with high-risk neuroblastoma remains unsatisfactory, particularly in cases of relapse after treatment, which carries a dismal prognosis.During development, multipotent precursor cells exhibit epigenetic heterogeneity that determines their differentiation at key lineage commitment points. This phenotypic plasticity contributes to substantial intratumoral heterogeneity in neuroblastoma, which is a major contributor to treatment resistance. MES-type neuroblastoma has been shown to possess enhanced resistance to therapy and increased migratory capacity, resulting in poorer outcomes. Allogeneic hematopoietic stem cell transplantation is theoretically capable of eliminating minimal residual disease through graft-versus-tumor (GvT) effects. However, late relapses still occur in some patients, suggesting that tumor cells may evade immune surveillance by altering their phenotype or remodeling the microenvironment. Here, we report a case of late relapse in a high-risk neuroblastoma patient after allo-HSCT, and by integrating clinical and molecular features, we explore the potential role of mesenchymal transition in mediating immune escape, aiming to provide new insights into the mechanisms of immune evasion and inform strategies for optimizing immunotherapeutic interventions. Case Presentation A 9-year-old boy was admitted to the hospital due to recurrent bilateral hip pain, limping, and fever. Initial investigations at a local hospital revealed elevated levels of vanillylmandelic acid (VMA): 316.5 ng/ml and neuron-specific enolase (NSE): 362 ng/L. Contrast-enhanced CT scans of the chest and abdomen indicated a soft tissue mass in the right adrenal gland with metastases to the T9 vertebra, sacrum, bilateral femoral head epiphyses, upper femur segments, and pelvic bones. A biopsy confirmed poorly differentiated neuroblastoma, and bone marrow smears indicated tumor cell infiltration. The diagnosis was stage M neuroblastoma with multiple lymph node, bone, and bone marrow metastases. Genetic testing did not detect SRD, KMT2A gene deletions, or MYCN amplification. The patient was treated according to the high-risk protocol with three cycles of CAV regimen (vincristine 1.5 mg/m² on day 1, epirubicin 25 mg/m² on days 1 and 2, cyclophosphamide 1.5 g/m² on days 1 and 2). This was followed by surgical resection of the right adrenal tumor and retroperitoneal lymph node dissection. Postoperative pathology confirmed neuroblastoma, and genetic testing revealed mutations in CDKN2A, CDKN2B, GATA3, GNA13, and PPM1D. From the fifth cycle, the patient received an arsenic-containing chemotherapy regimen (ATO + PVP: As₂O₃ 0.16 mg/kg on days 1–10, cisplatin 50 mg/m² on days 3–6, etoposide 200 mg/m² on days 3–5). After five cycles, NSE levels decreased to 14.3 ng/ml, VMA to 13.4 ng/ml, and bone marrow smears and flow cytometry were negative. Subsequently, the patient underwent two more chemotherapy regimens (sixth cycle: ATO + CAV; seventh cycle: ATO + PVP), achieving partial remission on PET-CT and complete remission in bone marrow. Radiotherapy was administered to the T12 vertebra to the sacrococcygeal region, pelvis, primary tumor bed in the right adrenal gland, and retroperitoneal lymph nodes (total dose 23.4 Gy/13 fractions). Following this, the patient underwent umbilical cord blood hematopoietic stem cell transplantation with a conditioning regimen of fludarabine, busulfan, cyclophosphamide, and topotecan. The donor was a male with 9/10 HLA match, blood type AB Rh(+), while the recipient's blood type was A Rh(+). The transplant contained 5.97 × 10⁷ TNC cells/kg and 4.18 × 10⁵ CD34 + cells/kg. Post-transplant, the patient developed severe chronic graft-versus-host disease (GVHD) involving the skin, gastrointestinal tract, and lungs, which improved with standard anti-rejection therapy. Due to financial constraints, the family declined GD2 monoclonal antibody treatment post-transplant. Two years after transplantation, whole-body PET-CT showed no signs of tumor recurrence, and bone marrow smears and flow cytometry were negative. However, three years post-transplantation, the patient presented with fever and generalized bone pain. Investigations revealed elevated NSE levels of 308 ng/mL and VMA/Cr ratio of 23.72 ng/mL. MRI confirmed tumor metastasis to the T1 and T8 vertebrae and pelvic bones (Fig. 1 ). Bone marrow smears showed extensive neuroblastoma cell infiltration, accounting for approximately 80% of the bone marrow (Fig. 2 ). Chimerism analysis indicated 100% donor-derived bone marrow engraftment. The patient's bone marrow flow cytometry showed GD2+, CD45−, CD56+, CD81+, CD9+, CD90+, CD73+, CD133−, CD105−, CD43− (Fig. 3 ). The family declined further genetic testing of bone marrow tumor cells. The patient received two cycles of IVT chemotherapy (irinotecan, temozolomide, vincristine), achieving negative results on bone marrow smear and flow cytometry. The patient remains under ongoing treatment. Discussion and Conclusion Mesenchymal transition may represent a potential mechanism of immune escape in neuroblastoma following allogeneic transplantation In this case, bone marrow flow cytometry revealed that the abnormal cells expressed GD2⁺, CD56+, CD90+, and CD73+, while being negative for CD133 and CD105, strongly suggesting that the neuroblastoma may be in or have undergone a mesenchymal-like transition. Neuroblastoma is a heterogeneous solid tumor with high phenotypic plasticity, classically divided into two major subtypes based on transcriptional profiles: adrenergic (ADRN) and mesenchymal (MES) 5 .Immune cells within the tumor microenvironment significantly influence neuroblastoma phenotypic transition. Mesenchymal transition is closely associated with therapy resistance in neuroblastoma. Studies indicate that the development of cisplatin resistance coincides with epithelial–mesenchymal transition-like processes in vitro. Moreover, the mesenchymal state confers resistance to anti-GD2 antibody therapy by downregulating ST8SIA1 expression and enhances immune evasion from natural killer (NK) cell-mediated cytotoxicity, representing a major barrier to effective immunotherapy in neuroblastoma 6 , 7 .Furthermore, mesenchymal transition significantly enhances the invasive and metastatic potential of neuroblastoma. These characteristics enable residual MES-type clones to persist in a dormant state for extended periods and ultimately evade immune surveillance, leading to relapse even in the setting of full donor chimerism and robust immune reconstitution. Analysis of Risk Factors for Late Relapse in Neuroblastoma A comprehensive review of the treatment course suggests that the patient's relapse was not incidental but rather the result of multiple converging high-risk factors spanning disease onset, induction therapy, local control, and post-transplant maintenance, collectively creating a permissive "soil" for relapse. Although MYCN amplification was absent at diagnosis, the tumor harbored several genetic alterations associated with poor prognosis, including CDKN2A/B deletion, GATA3 mutation, and PPM1D mutation. Specifically, CDKN2A/B deletion impairs p16INK4a/p14ARF function, leading to disruption of the G1/S cell cycle checkpoint. GATA3 is a key transcription factor in neural crest development, and neuroblastomas exhibiting complete lack of GATA3 methylation and/or very high levels of GATA3 protein expression have been associated with poor prognosis 8 .PPM1D mutations confer a survival advantage to tumor cells by suppressing p53-mediated apoptotic responses 9 .These molecular abnormalities not only indicate an intrinsically aggressive primary tumor, but also suggest a heightened propensity for adaptive evolution under therapeutic pressure, facilitating the emergence of resistant clones. The patient received radiotherapy to the region from T12 to the sacrococcygeal area, pelvis, primary tumor bed, and retroperitoneal lymph nodes; however, the relapse occurred at the T1 and T8 vertebrae, which were outside the radiation field. Although no definitive lesions were detected by imaging at those sites at the time, neuroblastoma is highly prone to skip metastases in bone 10 .Whether prophylactic low-dose radiotherapy to the entire spine or whole skeletal system should be considered warrants re-evaluation 11 . Due to financial constraints, the family declined GD2 monoclonal antibody as post-transplant maintenance therapy. GD2 monoclonal antibody activates NK cells to mediate antibody-dependent cellular cytotoxicity (ADCC) against GD2-positive tumor cells and has been shown in multiple international clinical trials, including those by COG and SIOPEN, to significantly improve long-term survival in high-risk patients 12 .Although this patient received allo-HSCT, which theoretically confers a graft-versus-tumor effect, the donor-derived immune system may fail to precisely recognize and eliminate disseminated minimal residual disease in the absence of specific targeting guidance. Inadequate conditioning intensity may also represent a risk factor for late relapse in this patient. The regimen used—fludarabine, busulfan, cyclophosphamide, and topotecan—is classified as non-myeloablative to moderately intensified. While this approach has lower toxicity and facilitates umbilical cord blood engraftment, its anti-tumor efficacy is likely inferior compared to myeloablative regimens incorporating total body irradiation (TBI) or higher-dose busulfan 13 .Multiple studies have shown that patients receiving TBI-based conditioning regimens have lower relapse rates and higher event-free survival 14 .Moreover, umbilical cord blood grafts are inherently associated with delayed engraftment and higher infection risk; when combined with a conditioning regimen of suboptimal anti-tumor activity, eradication of occult extramedullary disease, particularly outside the central nervous system, may be inadequate. For patients at high risk of relapse, future strategies should prioritize donors with higher HLA match and adequate cell dose, along with more potent conditioning regimens to enhance curative potential. Overall, we report a case of late relapse in a high-risk neuroblastoma patient after allo-HSCT, with flow cytometry evidence of mesenchymal transition in recurrent tumor cells. This finding supports the hypothesis that phenotypic switching to a MES state may serve as a key mechanism of immune evasion, enabling tumor escape despite full donor chimerism and intact graft-versus-tumor potential. The presence of high-risk genomic alterations (e.g., CDKN2A/B deletion, PPM1D mutation), suboptimal induction therapy, lack of radiotherapy to eventual relapse sites, and absence of post-transplant immunotherapy likely contributed to minimal residual disease persistence and clonal evolution. Our case highlights mesenchymal transition as a potential therapeutic barrier and underscores the need for early intervention strategies targeting phenotypic plasticity and enhanced immune surveillance to prevent late relapse in high-risk neuroblastoma. Abbreviations Allo-HSCT allogeneic hematopoietic stem cell transplantation GvT graft-versus-tumor VMA vanillylmandelic acid NSE neuron-specific enolase NK cell natural killer cell ATO arsenic-containing ADCC antibody-dependent cellular cytotoxicity TBI total body irradiation Declarations Ethical approval The study protocol was approved by the ethics committee of Sun Yat-sen Memorial Hospital and conformed to the principles of the Declaration of Helsinki. The treatment protocol for this patient was conducted under the clinical trial registered at ClinicalTrials.gov (Identifier: NCT05303727, First submitted :March 16, 2022). Consent for publication Consent for publication have be obtained from legal guardian. Data availability The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request Consent for publication Parental written informed consent on behalf of the patient was obtained for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request. All authors have viewed and agreed to the submission. Competing interests The authors declare no competing interests. Funding There was no funding source for this report. Author Contributions Administrative support was provided by Yang Li and Ke Huang. Material preparation and data collection were performed by Han Hu, Liping Zhan, Xilin Xiong, Wenxia Wang and Honggui Xu . Interpretation were performed by HanHu and Xiaomin Peng. The first draft of the manuscript was written by Han Hu. All the authors commented on previous versions of the manuscript. All the authors read and approved the final manuscript. Acknowledgements Not applicable. References Tas ML, et al. Neuroblastoma between 1990 and 2014 in the Netherlands: Increased incidence and improved survival of high-risk neuroblastoma. Eur J Cancer. 2020;124:47–55. Zhao X, Xu Z, Feng X. Clinical characteristics and prognoses in pediatric neuroblastoma with bone or liver metastasis: data from the SEER 2010–2019. BMC Pediatr. 2024;24:162. Lu Q-Y, et al. [Treatment of stage IV neuroblastoma with allogeneic hematopoietic stem cell transplantation in children]. Zhongguo Dang Dai Er Ke Za Zhi Chin J Contemp Pediatr. 2008;10:464–6. Tang S-Q, Huang D-S, Wang J-W, Feng C, Yang G. [Long-term effect of high dose chemotherapy combined with stem cell transplantation on stage IV neuroblastoma in children]. Zhongguo Dang Dai Er Ke Za Zhi Chin J Contemp Pediatr. 2006;8:93–6. Zhang A, Aslam H, Sharma N, Warmflash A, Fakhouri WD. Conservation of Epithelial-to-Mesenchymal Transition Process in Neural Crest Cells and Metastatic Cancer. Cells Tissues Organs. 2021;210:151–72. Piskareva O, et al. The development of cisplatin resistance in neuroblastoma is accompanied by epithelial to mesenchymal transition in vitro. Cancer Lett. 2015;364:142–55. Gautier M, Thirant C, Delattre O. Janoueix-Lerosey, I. Plasticity in Neuroblastoma Cell Identity Defines a Noradrenergic-to-Mesenchymal Transition (NMT). Cancers. 2021;13:2904. Almutairi B, et al. Epigenetic deregulation of GATA3 in neuroblastoma is associated with increased GATA3 protein expression and with poor outcomes. Sci Rep. 2019;9:18934. Lu Z-W, et al. Silencing of PPM1D inhibits cell proliferation and invasion through the p38 MAPK and p53 signaling pathway in papillary thyroid carcinoma. Oncol Rep. 2020;43:783–94. Granchi D, et al. Neuroblastoma and bone metastases: clinical significance and prognostic value of Dickkopf 1 plasma levels. Bone. 2011;48:152–9. Sibley GS, et al. Patterns of failure following total body irradiation and bone marrow transplantation with or without a radiotherapy boost for advanced neuroblastoma. Int J Radiat Oncol Biol Phys. 1995;32:1127–35. Sait S, Modak S. Anti-GD2 immunotherapy for neuroblastoma. Expert Rev Anticancer Ther. 2017;17:889–904. Styczynski J, et al. Acute Lymphoblastic Leukemia in Children: Better Transplant Outcomes After Total Body Irradiation-based Conditioning. Vivo Athens Greece. 2021;35:3315–20. Khimani F et al. Impact of Total Body Irradiation-Based Myeloablative Conditioning Regimens in Patients with Acute Lymphoblastic Leukemia Undergoing Allogeneic Hematopoietic Stem Cell Transplantation: Systematic Review and Meta-Analysis. Transplant. Cell. Ther. 27, 620.e1-620.e9 (2021). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 18 May, 2026 Reviewers agreed at journal 11 May, 2026 Reviewers invited by journal 05 Apr, 2026 Editor assigned by journal 03 Apr, 2026 Submission checks completed at journal 31 Mar, 2026 First submitted to journal 31 Mar, 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-9280514","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Case Report","associatedPublications":[],"authors":[{"id":619355647,"identity":"d55f5c84-d9e2-4b6d-92ea-eda118364d2f","order_by":0,"name":"han hu","email":"","orcid":"","institution":"Sun Yat-sen Memorial Hospital","correspondingAuthor":false,"prefix":"","firstName":"han","middleName":"","lastName":"hu","suffix":""},{"id":619355648,"identity":"37339935-a999-4726-aa78-54d83ceb3353","order_by":1,"name":"Liping Zhan","email":"","orcid":"","institution":"Sun Yat-sen Memorial 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16:46:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":74674,"visible":true,"origin":"","legend":"\u003cp\u003eAbnormal signal intensity of the metastatic tumors of the T1 and T8 vertebral bodies\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-9280514/v1/bb26743b2205a10b5b1e67fa.png"},{"id":106725972,"identity":"4933098d-71dd-420b-8091-db34d1b6e4b5","added_by":"auto","created_at":"2026-04-12 18:34:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":268997,"visible":true,"origin":"","legend":"\u003cp\u003eNeuroblastoma cells were observed in the bone marrow smear.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-9280514/v1/efa0aca4fca252d72f3d4fdc.png"},{"id":106725129,"identity":"b016285a-4a83-464a-b103-d8e29460d4e0","added_by":"auto","created_at":"2026-04-12 18:31:30","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":953190,"visible":true,"origin":"","legend":"\u003cp\u003eFlow cytometry analysis showed positive expression of CD90 and CD73\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-9280514/v1/359ca86297c3b4620c7e1296.png"},{"id":106727487,"identity":"d2fe48ce-71a1-4288-89e0-890d296b9b64","added_by":"auto","created_at":"2026-04-12 18:39:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1728061,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9280514/v1/e4a6d670-a8ae-4bd3-83bd-fa28405f501e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mesenchymal Transition as a Potential Mechanism of Late Immune Evasion in Neuroblastoma: A Case of Late Relapse after Allogeneic Hematopoietic Stem Cell Transplantation","fulltext":[{"header":"Background","content":"\u003cp\u003eNeuroblastoma is the most common extracranial solid tumor in children\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e.Despite significant advances in multimodal therapies, including surgery, chemotherapy, radiotherapy, immunotherapy, and autologous or allogeneic hematopoietic stem cell transplantation, the long-term survival rate for patients with high-risk neuroblastoma remains unsatisfactory, particularly in cases of relapse after treatment, which carries a dismal prognosis.During development, multipotent precursor cells exhibit epigenetic heterogeneity that determines their differentiation at key lineage commitment points. This phenotypic plasticity contributes to substantial intratumoral heterogeneity in neuroblastoma, which is a major contributor to treatment resistance. MES-type neuroblastoma has been shown to possess enhanced resistance to therapy and increased migratory capacity, resulting in poorer outcomes. Allogeneic hematopoietic stem cell transplantation is theoretically capable of eliminating minimal residual disease through graft-versus-tumor (GvT) effects. However, late relapses still occur in some patients, suggesting that tumor cells may evade immune surveillance by altering their phenotype or remodeling the microenvironment. Here, we report a case of late relapse in a high-risk neuroblastoma patient after allo-HSCT, and by integrating clinical and molecular features, we explore the potential role of mesenchymal transition in mediating immune escape, aiming to provide new insights into the mechanisms of immune evasion and inform strategies for optimizing immunotherapeutic interventions.\u003c/p\u003e"},{"header":"Case Presentation","content":"\u003cp\u003eA 9-year-old boy was admitted to the hospital due to recurrent bilateral hip pain, limping, and fever. Initial investigations at a local hospital revealed elevated levels of vanillylmandelic acid (VMA): 316.5 ng/ml and neuron-specific enolase (NSE): 362 ng/L. Contrast-enhanced CT scans of the chest and abdomen indicated a soft tissue mass in the right adrenal gland with metastases to the T9 vertebra, sacrum, bilateral femoral head epiphyses, upper femur segments, and pelvic bones. A biopsy confirmed poorly differentiated neuroblastoma, and bone marrow smears indicated tumor cell infiltration. The diagnosis was stage M neuroblastoma with multiple lymph node, bone, and bone marrow metastases. Genetic testing did not detect SRD, KMT2A gene deletions, or MYCN amplification.\u003c/p\u003e \u003cp\u003eThe patient was treated according to the high-risk protocol with three cycles of CAV regimen (vincristine 1.5 mg/m² on day 1, epirubicin 25 mg/m² on days 1 and 2, cyclophosphamide 1.5 g/m² on days 1 and 2). This was followed by surgical resection of the right adrenal tumor and retroperitoneal lymph node dissection. Postoperative pathology confirmed neuroblastoma, and genetic testing revealed mutations in CDKN2A, CDKN2B, GATA3, GNA13, and PPM1D. From the fifth cycle, the patient received an arsenic-containing chemotherapy regimen (ATO + PVP: As₂O₃ 0.16 mg/kg on days 1–10, cisplatin 50 mg/m² on days 3–6, etoposide 200 mg/m² on days 3–5). After five cycles, NSE levels decreased to 14.3 ng/ml, VMA to 13.4 ng/ml, and bone marrow smears and flow cytometry were negative.\u003c/p\u003e \u003cp\u003eSubsequently, the patient underwent two more chemotherapy regimens (sixth cycle: ATO + CAV; seventh cycle: ATO + PVP), achieving partial remission on PET-CT and complete remission in bone marrow. Radiotherapy was administered to the T12 vertebra to the sacrococcygeal region, pelvis, primary tumor bed in the right adrenal gland, and retroperitoneal lymph nodes (total dose 23.4 Gy/13 fractions). Following this, the patient underwent umbilical cord blood hematopoietic stem cell transplantation with a conditioning regimen of fludarabine, busulfan, cyclophosphamide, and topotecan. The donor was a male with 9/10 HLA match, blood type AB Rh(+), while the recipient's blood type was A Rh(+). The transplant contained 5.97 × 10⁷ TNC cells/kg and 4.18 × 10⁵ CD34 + cells/kg. Post-transplant, the patient developed severe chronic graft-versus-host disease (GVHD) involving the skin, gastrointestinal tract, and lungs, which improved with standard anti-rejection therapy. Due to financial constraints, the family declined GD2 monoclonal antibody treatment post-transplant.\u003c/p\u003e \u003cp\u003eTwo years after transplantation, whole-body PET-CT showed no signs of tumor recurrence, and bone marrow smears and flow cytometry were negative. However, three years post-transplantation, the patient presented with fever and generalized bone pain. Investigations revealed elevated NSE levels of 308 ng/mL and VMA/Cr ratio of 23.72 ng/mL. MRI confirmed tumor metastasis to the T1 and T8 vertebrae and pelvic bones (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Bone marrow smears showed extensive neuroblastoma cell infiltration, accounting for approximately 80% of the bone marrow (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Chimerism analysis indicated 100% donor-derived bone marrow engraftment. The patient's bone marrow flow cytometry showed GD2+, CD45−, CD56+, CD81+, CD9+, CD90+, CD73+, CD133−, CD105−, CD43− (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). The family declined further genetic testing of bone marrow tumor cells. The patient received two cycles of IVT chemotherapy (irinotecan, temozolomide, vincristine), achieving negative results on bone marrow smear and flow cytometry. The patient remains under ongoing treatment.\u003c/p\u003e "},{"header":"Discussion and Conclusion","content":"\u003ch2\u003eMesenchymal transition may represent a potential mechanism of immune escape in neuroblastoma following allogeneic transplantation\u003c/h2\u003e\u003cp\u003eIn this case, bone marrow flow cytometry revealed that the abnormal cells expressed GD2⁺, CD56+, CD90+, and CD73+, while being negative for CD133 and CD105, strongly suggesting that the neuroblastoma may be in or have undergone a mesenchymal-like transition. Neuroblastoma is a heterogeneous solid tumor with high phenotypic plasticity, classically divided into two major subtypes based on transcriptional profiles: adrenergic (ADRN) and mesenchymal (MES)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.Immune cells within the tumor microenvironment significantly influence neuroblastoma phenotypic transition. Mesenchymal transition is closely associated with therapy resistance in neuroblastoma. Studies indicate that the development of cisplatin resistance coincides with epithelial–mesenchymal transition-like processes in vitro. Moreover, the mesenchymal state confers resistance to anti-GD2 antibody therapy by downregulating ST8SIA1 expression and enhances immune evasion from natural killer (NK) cell-mediated cytotoxicity, representing a major barrier to effective immunotherapy in neuroblastoma\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.Furthermore, mesenchymal transition significantly enhances the invasive and metastatic potential of neuroblastoma. These characteristics enable residual MES-type clones to persist in a dormant state for extended periods and ultimately evade immune surveillance, leading to relapse even in the setting of full donor chimerism and robust immune reconstitution.\u003c/p\u003e\n\u003ch3\u003eAnalysis of Risk Factors for Late Relapse in Neuroblastoma\u003c/h3\u003e\n\u003cp\u003eA comprehensive review of the treatment course suggests that the patient's relapse was not incidental but rather the result of multiple converging high-risk factors spanning disease onset, induction therapy, local control, and post-transplant maintenance, collectively creating a permissive \"soil\" for relapse. Although MYCN amplification was absent at diagnosis, the tumor harbored several genetic alterations associated with poor prognosis, including CDKN2A/B deletion, GATA3 mutation, and PPM1D mutation. Specifically, CDKN2A/B deletion impairs p16INK4a/p14ARF function, leading to disruption of the G1/S cell cycle checkpoint. GATA3 is a key transcription factor in neural crest development, and neuroblastomas exhibiting complete lack of GATA3 methylation and/or very high levels of GATA3 protein expression have been associated with poor prognosis\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.PPM1D mutations confer a survival advantage to tumor cells by suppressing p53-mediated apoptotic responses\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.These molecular abnormalities not only indicate an intrinsically aggressive primary tumor, but also suggest a heightened propensity for adaptive evolution under therapeutic pressure, facilitating the emergence of resistant clones.\u003c/p\u003e \u003cp\u003eThe patient received radiotherapy to the region from T12 to the sacrococcygeal area, pelvis, primary tumor bed, and retroperitoneal lymph nodes; however, the relapse occurred at the T1 and T8 vertebrae, which were outside the radiation field. Although no definitive lesions were detected by imaging at those sites at the time, neuroblastoma is highly prone to skip metastases in bone\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.Whether prophylactic low-dose radiotherapy to the entire spine or whole skeletal system should be considered warrants re-evaluation\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDue to financial constraints, the family declined GD2 monoclonal antibody as post-transplant maintenance therapy. GD2 monoclonal antibody activates NK cells to mediate antibody-dependent cellular cytotoxicity (ADCC) against GD2-positive tumor cells and has been shown in multiple international clinical trials, including those by COG and SIOPEN, to significantly improve long-term survival in high-risk patients\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.Although this patient received allo-HSCT, which theoretically confers a graft-versus-tumor effect, the donor-derived immune system may fail to precisely recognize and eliminate disseminated minimal residual disease in the absence of specific targeting guidance.\u003c/p\u003e \u003cp\u003eInadequate conditioning intensity may also represent a risk factor for late relapse in this patient. The regimen used\u0026mdash;fludarabine, busulfan, cyclophosphamide, and topotecan\u0026mdash;is classified as non-myeloablative to moderately intensified. While this approach has lower toxicity and facilitates umbilical cord blood engraftment, its anti-tumor efficacy is likely inferior compared to myeloablative regimens incorporating total body irradiation (TBI) or higher-dose busulfan\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.Multiple studies have shown that patients receiving TBI-based conditioning regimens have lower relapse rates and higher event-free survival\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.Moreover, umbilical cord blood grafts are inherently associated with delayed engraftment and higher infection risk; when combined with a conditioning regimen of suboptimal anti-tumor activity, eradication of occult extramedullary disease, particularly outside the central nervous system, may be inadequate. For patients at high risk of relapse, future strategies should prioritize donors with higher HLA match and adequate cell dose, along with more potent conditioning regimens to enhance curative potential.\u003c/p\u003e \u003cp\u003eOverall, we report a case of late relapse in a high-risk neuroblastoma patient after allo-HSCT, with flow cytometry evidence of mesenchymal transition in recurrent tumor cells. This finding supports the hypothesis that phenotypic switching to a MES state may serve as a key mechanism of immune evasion, enabling tumor escape despite full donor chimerism and intact graft-versus-tumor potential. The presence of high-risk genomic alterations (e.g., CDKN2A/B deletion, PPM1D mutation), suboptimal induction therapy, lack of radiotherapy to eventual relapse sites, and absence of post-transplant immunotherapy likely contributed to minimal residual disease persistence and clonal evolution. Our case highlights mesenchymal transition as a potential therapeutic barrier and underscores the need for early intervention strategies targeting phenotypic plasticity and enhanced immune surveillance to prevent late relapse in high-risk neuroblastoma.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAllo-HSCT \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eallogeneic hematopoietic stem cell transplantation\u003c/p\u003e\n\u003cp\u003eGvT graft-versus-tumor\u003c/p\u003e\n\u003cp\u003eVMA vanillylmandelic acid\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNSE neuron-specific enolase\u003c/p\u003e\n\u003cp\u003eNK cell natural killer cell\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eATO arsenic-containing \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eADCC antibody-dependent cellular cytotoxicity\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTBI total body irradiation \u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study protocol was approved by the ethics committee of Sun Yat-sen Memorial Hospital and conformed to the principles of the Declaration of Helsinki. The treatment protocol for this patient was conducted under the clinical trial registered at ClinicalTrials.gov (Identifier: NCT05303727, First submitted :March 16, 2022).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConsent for publication have be obtained from legal guardian.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eParental written informed consent on behalf of the patient was obtained for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request. All authors have viewed and agreed to the submission.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere was no funding source for this report.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdministrative support was provided by Yang Li and Ke Huang. Material preparation and data collection were performed by Han Hu, Liping Zhan, Xilin Xiong, Wenxia Wang and Honggui Xu . Interpretation were performed by HanHu and Xiaomin Peng. The first draft of the manuscript was written by Han Hu. All the authors commented on previous versions of the manuscript. All the authors read and approved the final manuscript.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eTas ML, et al. Neuroblastoma between 1990 and 2014 in the Netherlands: Increased incidence and improved survival of high-risk neuroblastoma. Eur J Cancer. 2020;124:47\u0026ndash;55.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao X, Xu Z, Feng X. Clinical characteristics and prognoses in pediatric neuroblastoma with bone or liver metastasis: data from the SEER 2010\u0026ndash;2019. BMC Pediatr. 2024;24:162.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLu Q-Y, et al. [Treatment of stage IV neuroblastoma with allogeneic hematopoietic stem cell transplantation in children]. Zhongguo Dang Dai Er Ke Za Zhi Chin J Contemp Pediatr. 2008;10:464\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTang S-Q, Huang D-S, Wang J-W, Feng C, Yang G. [Long-term effect of high dose chemotherapy combined with stem cell transplantation on stage IV neuroblastoma in children]. Zhongguo Dang Dai Er Ke Za Zhi Chin J Contemp Pediatr. 2006;8:93\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang A, Aslam H, Sharma N, Warmflash A, Fakhouri WD. Conservation of Epithelial-to-Mesenchymal Transition Process in Neural Crest Cells and Metastatic Cancer. Cells Tissues Organs. 2021;210:151\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePiskareva O, et al. The development of cisplatin resistance in neuroblastoma is accompanied by epithelial to mesenchymal transition in vitro. Cancer Lett. 2015;364:142\u0026ndash;55.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGautier M, Thirant C, Delattre O. Janoueix-Lerosey, I. Plasticity in Neuroblastoma Cell Identity Defines a Noradrenergic-to-Mesenchymal Transition (NMT). Cancers. 2021;13:2904.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlmutairi B, et al. Epigenetic deregulation of GATA3 in neuroblastoma is associated with increased GATA3 protein expression and with poor outcomes. Sci Rep. 2019;9:18934.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLu Z-W, et al. Silencing of PPM1D inhibits cell proliferation and invasion through the p38 MAPK and p53 signaling pathway in papillary thyroid carcinoma. Oncol Rep. 2020;43:783\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGranchi D, et al. Neuroblastoma and bone metastases: clinical significance and prognostic value of Dickkopf 1 plasma levels. Bone. 2011;48:152\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSibley GS, et al. Patterns of failure following total body irradiation and bone marrow transplantation with or without a radiotherapy boost for advanced neuroblastoma. Int J Radiat Oncol Biol Phys. 1995;32:1127\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSait S, Modak S. Anti-GD2 immunotherapy for neuroblastoma. Expert Rev Anticancer Ther. 2017;17:889\u0026ndash;904.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStyczynski J, et al. Acute Lymphoblastic Leukemia in Children: Better Transplant Outcomes After Total Body Irradiation-based Conditioning. Vivo Athens Greece. 2021;35:3315\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhimani F et al. Impact of Total Body Irradiation-Based Myeloablative Conditioning Regimens in Patients with Acute Lymphoblastic Leukemia Undergoing Allogeneic Hematopoietic Stem Cell Transplantation: Systematic Review and Meta-Analysis. \u003cem\u003eTransplant. Cell. Ther.\u003c/em\u003e 27, 620.e1-620.e9 (2021).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"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":"world-journal-of-surgical-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wjso","sideBox":"Learn more about [World Journal of Surgical Oncology](http://wjso.biomedcentral.com)","snPcode":"12957","submissionUrl":"https://submission.nature.com/new-submission/12957/3","title":"World Journal of Surgical Oncology","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Neuroblastoma, Allogeneic hematopoietic stem cell transplantation, Drug resistance, Mesenchymal transition","lastPublishedDoi":"10.21203/rs.3.rs-9280514/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9280514/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNeuroblastoma is the most common extracranial solid malignant tumor in children, and recurrence is not uncommon in high-risk cases, with immune evasion considered a key mechanism underlying relapse and drug resistance. Neural crest progenitor cells differentiate into adrenergic or mesenchymal phenotypes. MES-type neuroblastoma exhibits greater resistance to therapy and enhanced migratory capacity, leading to poorer prognosis. This report describes a child with high-risk neuroblastoma who experienced late relapse after allogeneic hematopoietic stem cell transplantation (allo-HSCT), with flow cytometry indicating mesenchymal transition in the recurrent tumor cells. By integrating clinical course and molecular characteristics, we explore the potential role of mesenchymal transition in mediating immune escape, aiming to provide new insights into the mechanisms of immune evasion in neuroblastoma and inform optimization of immunotherapeutic strategies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCase Presentation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe present a patient with stage M neuroblastoma who achieved complete remission after surgery, chemotherapy, radiotherapy, and allogeneic hematopoietic stem cell transplantation, but experienced bone and bone marrow relapse three years post-transplantation. Flow cytometry of the bone marrow revealed mesenchymal transition in the tumor cells. The case harbored multiple gene mutations associated with poor prognosis in neuroblastoma. Following relapse, the patient has received two cycles of chemotherapy and achieved partial response.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMesenchymal transition may be a key mechanism underlying late relapse in neuroblastoma. High-risk genetic background, insufficient intensity of induction therapy and conditioning regimen, lack of prophylactic radiotherapy, and absence of immunotherapy may contribute to the risk of long-term relapse after hematopoietic stem cell transplantation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRegistration\u003c/strong\u003e The treatment protocol for this patient was conducted under the clinical trial registered at ClinicalTrials.gov (Identifier: NCT05303727, First submitted :March 16, 2022).\u003c/p\u003e","manuscriptTitle":"Mesenchymal Transition as a Potential Mechanism of Late Immune Evasion in Neuroblastoma: A Case of Late Relapse after Allogeneic Hematopoietic Stem Cell Transplantation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-09 16:46:03","doi":"10.21203/rs.3.rs-9280514/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-18T06:12:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"204429336951940999998358001058547610162","date":"2026-05-11T06:54:45+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-05T20:42:54+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-04T03:24:56+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-01T00:07:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"World Journal of Surgical Oncology","date":"2026-03-31T13:07:42+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"world-journal-of-surgical-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wjso","sideBox":"Learn more about [World Journal of Surgical Oncology](http://wjso.biomedcentral.com)","snPcode":"12957","submissionUrl":"https://submission.nature.com/new-submission/12957/3","title":"World Journal of Surgical Oncology","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1b1a1a86-090e-4f40-8c7e-74a3c887ecef","owner":[],"postedDate":"April 9th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-18T06:12:03+00:00","index":150,"fulltext":""},{"type":"reviewerAgreed","content":"204429336951940999998358001058547610162","date":"2026-05-11T06:54:45+00:00","index":126,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-09T16:46:03+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-09 16:46:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9280514","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9280514","identity":"rs-9280514","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}