Mesenchymal stem cells-like as a prognostic biomarker in patients diagnosed with acute myeloid leukemia

Mesenchymal stem cells-like as a prognostic biomarker in patients diagnosed with acute myeloid leukemia | 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 Mesenchymal stem cells-like as a prognostic biomarker in patients diagnosed with acute myeloid leukemia Tamara Castaño-Bonilla, Daniel Láinez-González, Juana Serrano, and 17 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7035200/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Nov, 2025 Read the published version in Scientific Reports → Version 1 posted 11 You are reading this latest preprint version Abstract The bone marrow (BM) microenvironment plays a pivotal role in acute myeloid leukemia (AML) progression. Among its components, mesenchymal stem cells (MSCs) can exhibit tumor-modulating properties, yet their precise prognostic significance in AML remains unclear. To evaluate the prognostic impact of MSC-like (MSC-l) cells in adult AML patients treated with intensive chemotherapy. We retrospectively analyzed 65 adult AML patients (excluding acute promyelocytic leukemia) treated between 2017 and 2024 at four spanish institutions. MSC-l cells were identified in BM aspirates at the end of treatment by multiparameter flow cytometry as CD13bright/CD45low/CD34neg/CD117neg/CD11bneg/CD16neg/CD71neg/CD64neg. Patients were stratified based on MSC-l proportion using a cut-off of 0.265% (MSC-l HIGH patients ˃0265% and MSC-l LOW patients ≤ 0.265%). Survival outcomes were assessed using Kaplan-Meier and Cox regression analyses, adjusting for age and ELN 2017 risk classification. MSC-l HIGH patients showed significantly reduced overall survival (OS) (median OS: 0.66 years vs. not calculable; P < 0.001) and relapse-free survival (RFS) (median RFS: 1.27 vs. 1.49 years; P = 0.027) compared to MSC-l LOW patients. Stratified analyses confirmed this trend across ELN 2017 risk groups. Multivariate Cox regression identified MSC-l HIGH status as an independent predictor of worse OS (HR = 11.68, 95% CI: 3.80–35.9, P < 0.01) and RFS (HR = 3.38, 95% CI: 1.15–9.96, P = 0.03). A higher proportion of MSC-l cells at end of treatment is associated with inferior survival outcomes in AML, independent of ELN risk and age. These findings suggest that MSC-l quantification may provide additional prognostic value and warrant validation in prospective studies. Health sciences/Biomarkers Biological sciences/Cancer Health sciences/Oncology Biological sciences/Stem cells Acute myeloid leukemia mesenchymal stem cells prognostic biomarker multiparameter flow cytometry hematopoietic microenvironment Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Acute myeloid leukemia (AML) is a hematologic neoplasm characterized by the uncontrolled proliferation of abnormal myeloid progenitor cells 1 . AML arises from a series of genetic and epigenetic changes, primarily affecting hematopoietic stem and progenitor cells (HSPCs) 2 , 3 The pathogenesis of AML, as well as other hematological malignancies, depends not only on intrinsic tumoral factors but is also influenced by the bone marrow (BM) microenvironment 4 , 5 . The BM microenvironment includes a complex network of extracellular matrix proteins, soluble growth factors, cytokines, and distinct-though possibly overlapping- cellular niches, such as the endosteal (or osteoblastic) and vascular niches. Several studies have demonstrated that blast cells can manipulate the marrow microenvironment to create a permissive conditions that favors disease initiation, progression and therapeutic resistance 6 , 7 , 8 . Mesenchymal stem cells (MSCs) play a decisive role in this process, known as "stromal plasticity". MSCs are non-hematopoietic multipotent stem cells characterized by their self-renewal capacity, clonogenic efficiency and multilineage differentiation capacity 9 . Furthermore, MSCs may influence the development of various diseases, including hematological malignancies such as chronic myeloid leukemia, multiple myeloma, and myelodysplastic syndrome/AML 10,11,12 . However, despite extensive research over the past decade, it remains unclear whether MSCs have tumor-promoting or tumor-suppressive effects, and their precise role in AML is yet to be determined 13 , 14 . CD13, also known as aminopeptidase N, is a transmembrane metalloprotease widely recognized as a surface marker in flow cytometric analysis of MSCs and various progenitor cell populations. Its expression is commonly used as part of a panel of positive markers, including CD73 and CD90, to identify MSCs in accordance with the minimal criteria proposed by the International Society for Cellular Therapy (ISCT) 15 . Beyond its utility as a phenotypic marker, CD13 plays functional roles in cellular adhesion, migration, and differentiation. In flow cytometry, anti-CD13 monoclonal antibodies enable the reliable detection and isolation of MSCs based on differential surface expression, thereby supporting downstream applications in regenerative medicine and cellular therapies 16 . Recently, our group identified a CD13-bright cell population enriched for canonical MSC markers CD105 and CD90 in myelodysplastic syndromes (MDS) patients at diagnostic. Notably, these MSC-like cells were found to be a predictor of progression to AML 17 . In recent years, the introduction of new drugs into the AML therapeutic arsenal has improved the prognosis, but survival rates vary greatly depending on the characteristics of the disease, in particular genetic abnormalities included in the ELN risk score 2 . However, in every ELN risk group patients show differences in outcome and we should try to refine prognosis of patient as much as possible for appropriate consolidation treatment. In light of these considerations, in this manuscript we explore the potential of MSCs as a prognostic factor in intensively treated AML patients. Results 65 AML patients treated with intensive chemotherapy, showed MSCs measured by MFC at the end of treatment (post-induction treatment: induction-1 (n = 18) and induction-2 (4); post-consolidation treatment: consolidation-1 (n = 9), consolidation-2 (n = 5) and consolidation-3 (n = 5); allogeneic pre-transplantation (n = 2) and allogeneic post-transplantation (n = 22). The median age of this group was 58 years (range 18–77 years); 32 males and 33 females. The median proportion of MSCs was 0.10%. Table 1 shows the baseline characteristics of AML, the proportion of MSCs at the end of treatment, and the therapies administered to these 65 patients 2.1. Impact of MSCs determination at end of treatment We set 0.265% as the cut-off for MSC-l quantification based on the total analyzed BM cellularity (excluding debris). In the analysis of OS, the MSCs determination showed that 48 patients (74%) were MSCs-l LOW and 17 patients (26%) were MSCs-l HIGH . In the MSCs-l LOW group, the median OS was not calculable (NC), while in the MSCs-l HIGH group, the median OS was 0.66 years (95% CI: 0.28–1.04). When comparing the two subgroups using a log-rank test, MSCs-l HIGH patients had significantly reduced OS ( P < 0.001).The RFS analysis was performed on 51 patients: MSCs-l LOW (n = 40) and MSCs-l HIGH (n = 11). In the MSCs-l LOW group, the median RFS was 1.49 years (95% CI: 0.01–3.51), while in the MSCs-l HIGH group, the median RFS was 1.27 years (95% CI: 0.02–2.52). A log-rank test showed significantly reduced RFS in MSCs-l HIGH patients ( P = 0.027) (Fig. 1 ). A stratified analysis by the ELN 2017 genetic risk classification was performed. In the analysis of OS, the distribution of MSCs-l determination was as follows: 13 patients (20%) with MSCs-l LOW and 2 patients (3%) with MSCs-l HIGH in the favorable-risk group, 20 patients (31%) with MSCs-l LOW and 8 patients (12%) with MSCs-l HIGH in the intermediate-risk group, and 15 patients (23%) with MSCs-l LOW , and 7 patients (11%) with MSCs-l HIGH in the adverse-risk group. The OS in the favorable group was NC in MSCs-l LOW and 0.4 years (95% CI: NC) in MSCs-l HIGH . The OS in the intermediate group was 3.4 years (95% CI: NC) in MSCs-l LOW and 0.9 years (95% CI:0.3–1.4) in MSCs-l HIGH . The OS in the adverse group was 1.4 years (95% CI:0.1–2.6) in MSC-ls LOW and 0.4 years (95% CI:0.3–0.6) in MSCs-l HIGH . A log-rank test comparing subgroups according to genetic risk showed a reduced OS in MSCs-l HIGH patients ( P ≤ 0.001) (Fig. 2 ). In the analysis of RFS, the distribution was as follows: 12 patients (24%) with MSCs-l LOW and 2 patients (4%) with MSCs-l HIGH in the favorable-risk group, 17 patients (33%) with MSC-ls LOW and 5 patients (10%) with MSCs-l HIGH in the intermediate-risk group, and 11 patients (22%) with MSCs-l LOW , and 4 patients (8%) with MSCs-l HIGH in the adverse-risk group. The RFS in the favorable group was 1.47 years (95% CI: 0.01–3.42) in MSCs-l LOW and NC in MSCs-l HIGH (. The RFS in the intermediate group was 1.49 years (95% CI: 0.47–2.50) in MSCs-l LOW and 1.27 years (95% CI: NC) in MSCs-l HIGH . The RFS in the adverse group was not calculable in MSCs-l LOW and 0.18 years (95% CI: 0.01–1.21) in MSCs-l HIGH (Fig. 2 ). A log-rank test comparing subgroups according to genetic risk showed a reduction of RFS in MSCs-l HIGH patients ( P = 0.05). Cox regression analysis for OS prediction showed that age, ELN 2017 classification and the proportion of MSCs-l were independent predictor factors ( P = 0.02, HR:0.27, [95% CI: 0.09–0.79], P = 0.03, HR: NC, [95% CI: NC] and P < 0.01, HR:11.68, [95% CI: 3.80–35.9], respectively). Cox regression analysis for the RFS prediction showed that proportion of MSCs-l was an independent predictor factor ( P = 0.03, HR: 3.38, [95% CI: 1.15–9.96]. Age and ELN 2017 classification were not independent predictors factors ( P = 0.37, HR: 0.63, [95% CI: 0.23–1.72] and P = 0.87, HR: NC, [95% CI: NC], respectively). Proportional hazard assumptions were checked by adding the interaction of every variable with time in every analysis performed. 2.2. In vitro expansion, and osteogenic, adipogenic, and chondrogenic differentiation of MSCs In order to identify similarities between MSC-1 and previously described MSC-like cells (17) we conducted comparable sorting and culture experiments. MSC-1 cells exhibited similar enrichment of canonical CD105 and CD90 MSC markers and equivalent attachment potential to MSC-like cells (Fig. 3). Discussion This study shows that the proportion of MSCs-l at end of treatment has prognostic impact on survival and relapse in newly diagnosed AML patients treated with front-line intensive regimens. We analyzed the proportion of MSCs-l in 65 AML patients treated with intensive chemotherapy at end of treatment A cut-off of 0.265% for MSCs-l was used to assess the prognostic significance of MSCs-l proportion and its clinical applicability. Patients with a proportion of MSCs-l ≥ 0.265% regardless of their 2017 ELN genetic risk (favorable, intermediate or adverse risk) showed a significant reduction in both OS and RFS either in global and ELN-stratified analysis. In the multivariate analysis for OS including age, genetic risk according to the 2017 ELN guidelines, and categorized MSCs-l quantification all of them were identified as independent predictive factors in AML patients treated with intensive therapy. In the multivariate analysis for RFS, only the proportion of MSCs-l was found to be a significant independent predictor. These data show that this population of MSCs-l cells have a role in AML prognosis and more importantly add prognostic value to already established predictor factors such as age and ELN risk score. To date, no published studies in the literature have addressed the prognostic role of MSCs in AML patients. Our study did not fully meet the International Society for Cellular Therapy (ISCT) criteria for the definitive identification of mesenchymal stem cells (MSCs) within the bright CD13 compartment. Classical MSC markers—such as CD73, CD90, and CD105—were not included in our flow cytometry panel; however, the specificity and consistency of these markers are limited, as they are not exclusive to MSCs and display variable expression across individuals and disease states 15 , 17 . Despite the absence of these markers, the CD13bright cells, in the absence of other myeloid markers, exhibited key mesenchymal characteristics, including plastic adherence and in vitro expansion, although they failed to undergo trilineage differentiation into adipocytes, chondrocytes, and osteoblasts. This could be attributed to the limited number of cells available for functional assays and/or intrinsic alterations in pathological mesenchymal populations 18 , 19 , 20 . Flow cytometry analysis indicated that the CD13bright compartment also includes endothelial cells and fibroblasts, which can be distinguished, based on CD34 expression and CD13 mean fluorescence intensity (MFI), both of which are lower than in MSCs 21 . Fibroblasts, in particular, require enzymatic digestion for recovery and share similar CD13 MFI values with endothelial cells. In contrast, hematopoietic cells exhibit markedly lower or absent CD13 expression and lack the myeloid markers included in the panel, thereby supporting the exclusion of these lineages from the CD13bright gate 18 , 22 . Based on the above findings, we conclude that the analyzed population corresponds to MSCs. In summary, in our cohort of 65 intensively treated AML patients, we propose that the proportion of MSCs-l at the end of treatment may function as an independent prognostic factor. In particular, we demonstrate that the categorical variable MSC-l furthers improve ELN risk and age prognostication of OS, being the only independent factor for RFS. To strengthen the validity and statistical power of these findings, confirmation in a larger prospective cohort is warranted. If these results are validated, further studies will be necessary to confirm this population corresponds to MSC by differentiation assays, which could ultimately support their exploration as novel therapeutic targets in this aggressive and frequently relapsed disease. Material and Methods 5.1. Patients and samples The primary patient and disease characteristics were collected retrospectively, including: age, sex, cytomorphological assessment, AML diagnosis confirmation (according to the site´s routine practice), cytogenetics, molecular studies, description of the front-line treatment approach, disease response assessments, and disease follow-up 23 . Patients diagnosed with acute promyelocytic leukemia (APL) were excluded. We reviewed data collected from 65 intensively treated adult AML patients with available MSC data measured by flow cytometry (FCM) at the end of treatment. These patients were diagnosed between 2017 and 2024 at Hospital Universitario Fundación Jiménez Díaz, Hospital Universitario Rey Juan Carlos, Hospital Universitario General de Villalba, and Hospital Universitario Infanta Elena, all in Madrid (Spain). Written informed consent was obtained from all study participants. Approval of the study was obtained from the Clinical Research Ethics Committee of the Hospital Universitario Fundación Jiménez Díaz (TFG017-19_FJD). The study complied with the Declaration of Helsinki. 5.2. MSCs screening Multiparameter flow cytometry (MFC) immunophenotypic studies were performed on BM aspirate samples, processed under sterile conditions within 24 hours of collection. The pre-filtered bone marrow sample (EDTA) is incubated for 15 minutes with a combination of eight pre-selected monoclonal antibodies (MoAb) which were used to identify and characterize BM MSCs (Table 2). Erythrocytes were subsequently lysed with BD FACS™ solution for 10 minutes (BD Biosciences), followed by two washes with PBS (Inova Diagnostics). The FACS Canto II flow cytometer (Becton Dickinson Biosciences, San Jose, CA, USA) was used to acquire a minimum of 500,000 events per tube (two tubes analyzed). Infinicyt™ software (Cytognos SL, Salamanca, Spain) was used to analyze the main leukocyte subpopulations in the generated FCS files. Regarding MSCs gating strategy, a retrospective search for phenotypic information on MSCs was performed in the flow cytometry files of 65 AML patients. The phenotypic analysis of AML MSCs was adjusted from Muñiz et al 24 considering that CD105 and CD90 were not routinely included in our routine AML monitoring panel. A bright CD13-positive population was identified, which was negative for CD34, CD45, CD117, CD11b, CD16, CD71 and CD64. This population is referred to as the MSC-like (MSC-l) compartment (Fig. 4 ). Gates were set according to the appropriate isotype control, and the expression of each marker was reported as the percentage of positive cells of total BM nucleated cells, excluding debris 5.3. In vitro expansion of MSCs The same sorting strategy and culture methods described by the group (17) were employed based on the CD13 bright CD45 low/neg CD105 pos CD90 pos population, for further studies on MSC-like cells to confirm their MSC identity. A total of three samples were analyzed to isolate the MSC-like phenotype from erythrocyte-depleted, low-density bone marrow cells. Then, cells were stained for phycoerythrin (PE) anti-CD13 (clone WM15) (Beckton Dickinson), fluorescein (FITC) anti-CD45 (clone T29/33) (Dako), allophycocyanin (APC) anti-CD105 (clone 43/A3) (Beckton Dickinson) and violet 450 anti-CD90 (clone 5E10) (Beckton Dickinson). Cell sorting was conducted using a FACSMelody™ Cell Sorter (Becton Dickinson) equipped with 3 lasers and FACSChorus™ software. Subsequent flow cytometry analysis was performed using FlowJo v.10.9.0. 5.4. Statistical analysis Overall survival (OS) was defined as the time from the date of AML diagnosis to the date of death due to any cause. Relapse free survival (RFS) was calculated from the date of achieving complete response (CR) or CR with incomplete hematologic recovery (CRi) until the date of relapse or death from any cause. CR and CRi were defined according to the current 2017 ELN guidelines 25 . Groups for comparison were established based on MSC quantification, with an arbitrary cut-off determined according to its sensitivity and specificity. Kaplan-Meier and log-ranks test were used to compare OS and RFS between groups. Intensively-treated patients were analyzed globally and in a stratified analysis by the ELN 2017 genetic risk classification. Multivariate Cox regression analysis was used to predict OS and RFS, with MSC quantification (categorized as explained above), age (categorized in < 60 years or ≥ 60 years) and the ELN 2017 genetic risk model as covariates. Statistical analyses were perfomed using SPSS 19.0 (IBM, Arkmo0.0n, NY). The confidence interval was set at 95% and the P -values were bilateral, considered significant if ≤ 0.05. Declarations Acknowledgments T.C. is a PhD candidate at the Universidad Autónoma de Madrid (UAM), and this work is submitted in partial fulfillment of the requirements for the doctoral degree. The authors gratefully acknowledge the technical assistance of Raquel Gonzalo and Susana Castañón in performing the experiments with mesenchymal stem cells (MSCs) using multiparametric flow cytometry (MFC). We also extend our sincere gratitude to the research team at the Hospital Universitario Fundación Jiménez Díaz for their contributions to the expansion, culture, and differentiation of MSCs into various cell subtypes. Author Contributions: Formal analysis : Raquel Mata, Carlos Blas, Cristina Serrano, Juan Manuel Alonso Domínguez, Juana Serrano, Gonzalo Castellanos, Raquel Capellán, Rocío Salgado, Laura Pardo, Laura Solán, Alvaro V. Arriero, Belén Rosado, Daniel Laínez, María Yuste, Pilar Beltran, Eva Oliva, Pilar Llamas,Rocío Olivera y Mireia Atance. Investigation : Tamara Castaño-Bonilla. Methodology: Raquel Mata, Carlos Blas, Cristina Serrano, Juan Manuel Alonso Domínguez, Juana Serrano, Gonzalo Castellanos, Raquel Capellán, Rocío Salgado, Laura Pardo, Laura Solán, Alvaro V. Arriero, Belén Rosado, Daniel Laínez, María Yuste, Pilar Beltran, Eva Oliva, Pilar Llamas,Rocío Olivera y Mireia Atance. Supervision : Juana Serrano, Daniel Lainez, Raquel Mata, Cristina Serrano, Mireia Atance, Alberto Lázaro, Pilar Llamas, Juan M. Alonso-Dominguez. Visualisation : Tamara Castaño Bonilla, Juan Manuel Alonso Dominguez y Daniel Láinez González. Data curation: Raquel Mata, Carlos Blas, Cristina Serrano, Juan Manuel Alonso Domínguez, Juana Serrano, Gonzalo Castellanos, Raquel Capellán, Rocío Salgado, Laura Pardo, Laura Solán, Alvaro V. Arriero, Belén Rosado, Daniel Laínez, María Yuste, Pilar Beltran, Eva Oliva, Pilar Llamas,Rocío Olivera y Mireia Atance. Writing—original draft , Tamara Castaño. Writing—review and editing , Raquel Mata, Carlos Blas, Cristina Serrano, Juan Manuel Alonso Domínguez, Juana Serrano, Gonzalo Castellanos, Raquel Capellán, Rocío Salgado, Laura Pardo, Laura Solán, Alvaro V. Arriero, Belén Rosado, Daniel Laínez, María Yuste, Pilar Beltran, Eva Oliva, Pilar Llamas,Rocío Olivera y Mireia Atance. Funding This research received no external funding. Conflict of Interest Statement Juan Manuel Alonso Domínguez has received honoraria as a member of the advisory board for Astellas and Servier. Ethics Statement All participants provided written informed consent. The study protocol was approved by the Clinical Research Ethics Committee of the Hospital Universitario Fundación Jiménez Díaz (TFG017-19_FJD) and was conducted in accordance with the Declaration of Helsinki. Data availability The datasets used and/or analysed during the current study available from the corresponding author on reasonable request References Shimony, S., Stahl, M. & Stone, R. M. Acute myeloid leukemia: 2023 update on diagnosis, risk-stratification, and management. Am. J. Hematol. 98 , 502–526 (2023). Döhner, H. et al. Diagnosis and management of AML in adults: 2022 recommendations from an international expert panel on behalf of the ELN. Blood 140 , 1345–1377 (2022). Prada-Arismendy, J., Arroyave, J. C. & Röthlisberger, S. Molecular biomarkers in acute myeloid leukemia. Blood Rev. 31 , 63–76 (2017). Pittenger, M. F. et al. Mesenchymal stem cell perspective: cell biology to clinical progress. npj Regen Med. 4 , 1–15 (2019). Jovic, D. et al. A Brief Overview of Global Trends in MSC-Based Cell Therapy. Stem Cell. Rev. Rep. 18 , 1525–1545 (2022). Shafat, M. S., Gnaneswaran, B., Bowles, K. M. & Rushworth, S. A. The bone marrow microenvironment - Home of the leukemic blasts. Blood Rev. 31 , 277–286 (2017). Menter, T. & Tzankov, A. Tumor Microenvironment in Acute Myeloid Leukemia: Adjusting Niches. Front. Immunol. 13 , 811144 (2022). Pimenta, D. B. et al. The Bone Marrow Microenvironment Mechanisms in Acute Myeloid Leukemia. Frontiers Cell. Dev. Biology 9 , (2021). Tan, L., Liu, X., Dou, H. & Hou, Y. Characteristics and regulation of mesenchymal stem cell plasticity by the microenvironment — specific factors involved in the regulation of MSC plasticity. Genes Dis. 9 , 296–309 (2020). Garcia-Gomez, A., Sanchez-Guijo, F., Cañizo, D., Miguel, M. C. S., Garayoa, M. & J. F. & Multiple myeloma mesenchymal stromal cells: Contribution to myeloma bone disease and therapeutics. World J. Stem Cells . 6 , 322–343 (2014). Chandia, M. et al. Involvement of primary mesenchymal precursors and hematopoietic bone marrow cells from chronic myeloid leukemia patients by BCR-ABL1 fusion gene. Am. J. Hematol. 89 , 288–294 (2014). Lopez-Villar, O. et al. Both expanded and uncultured mesenchymal stem cells from MDS patients are genomically abnormal, showing a specific genetic profile for the 5q- syndrome. Leukemia 23 , 664–672 (2009). Lee, M. W. et al. Mesenchymal stem cells in suppression or progression of hematologic malignancy: current status and challenges. Leukemia 33 , 597–611 (2019). Galland, S. & Stamenkovic, I. Mesenchymal stromal cells in cancer: a review of their immunomodulatory functions and dual effects on tumor progression. J. Pathol. 250 , 555–572 (2020). Dominici, M. et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 8 , 315–317 (2006). Rahman, M. M. et al. CD13 promotes mesenchymal stem cell-mediated regeneration of ischemic muscle. Front Physiol 4 , (2014). Viswanathan, S. et al. Mesenchymal stem versus stromal cells: International Society for Cell & Gene Therapy (ISCT®) Mesenchymal Stromal Cell committee position statement on nomenclature. Cytotherapy 21 , 1019–1024 (2019). Muñiz, C. et al. Ex vivo identification and characterization of a population of CD13(high) CD105(+) CD45(-) mesenchymal stem cells in human bone marrow. Stem Cell. Res. Ther. 6 , 169 (2015). Phinney, D. G. & Prockop, D. J. Concise review: mesenchymal stem/multipotent stromal cells: the state of transdifferentiation and modes of tissue repair–current views. Stem Cells . 25 , 2896–2902 (2007). Hoogduijn, M. J. et al. The immunomodulatory properties of mesenchymal stem cells and their use for immunotherapy. Int. Immunopharmacol. 10 , 1496–1500 (2010). Li, H., Ghazanfari, R., Zacharaki, D., Lim, H. C. & Scheding, S. Isolation and characterization of primary bone marrow mesenchymal stromal cells. Ann. N Y Acad. Sci. 1370 , 109–118 (2016). Fonseca, L. N. et al. Cell surface markers for mesenchymal stem cells related to the skeletal system: A scoping review. Heliyon 9 , e13464 (2023). Castaño-Bonilla, T. et al. No Evidence that CD33 rs12459419 Polymorphism Predicts Gemtuzumab Ozogamicin Response in Consolidation Treatment of Acute Myeloid Leukemia Patients: Experience of the PETHEMA Group. Dis Markers 3132941 (2022). (2022). Muñiz, C. et al. Ex vivo identification and characterization of a population of CD13high CD105 + CD45 – mesenchymal stem cells in human bone marrow. Stem Cell Res. Ther. 6 , 169 (2015). Döhner, H. et al. Diagnosis and management of AML in adults: 2017 ELN recommendations from an international expert panel. Blood 129 , 424–447 (2017). Tables Tables 1 and 2 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Tables.docx Cite Share Download PDF Status: Published Journal Publication published 28 Nov, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 25 Jul, 2025 Reviews received at journal 24 Jul, 2025 Reviewers agreed at journal 21 Jul, 2025 Reviewers agreed at journal 17 Jul, 2025 Reviews received at journal 16 Jul, 2025 Reviewers agreed at journal 15 Jul, 2025 Reviewers invited by journal 14 Jul, 2025 Editor assigned by journal 14 Jul, 2025 Editor invited by journal 07 Jul, 2025 Submission checks completed at journal 07 Jul, 2025 First submitted to journal 03 Jul, 2025 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-7035200","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":486137729,"identity":"2de1c328-fedc-4e9c-9b24-c27e2e144577","order_by":0,"name":"Tamara Castaño-Bonilla","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA70lEQVRIiWNgGAWjYAgAG37GhgT8SngQTGYQkSbZSKqWw5INDAS02LOfTvxcUcMgz99//uDngorzEsztCYwPf+CzhSd3s+SZYwyGM24kM0vPOHNbgrHnAbMxDz4tDLkbJBvYgI65wcwgzdt2u45xRgKbNF6/8L/d/LPhH0OC/PnDzL95285JALWw/8TrMIncbZKNbQwJBgeS2YC2HABpYWPA67Abb7dZNvZJGG68kWxmzXMmGeiXh83S+LSw9+duvtnwzUZe7vzBx7d5KuwkDNuTD37E5zAokEAwDRsYGwhrQAHyJKofBaNgFIyC4Q8AV65JUkY0DQAAAAAASUVORK5CYII=","orcid":"","institution":"Hospital Universitario Fundación Jiménez Díaz","correspondingAuthor":true,"prefix":"","firstName":"Tamara","middleName":"","lastName":"Castaño-Bonilla","suffix":""},{"id":486137730,"identity":"cb088e65-57a8-42d3-956a-a80138452963","order_by":1,"name":"Daniel Láinez-González","email":"","orcid":"","institution":"Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM)","correspondingAuthor":false,"prefix":"","firstName":"Daniel","middleName":"","lastName":"Láinez-González","suffix":""},{"id":486137731,"identity":"0151873c-7a73-4ae8-9244-a8b08144d400","order_by":2,"name":"Juana Serrano","email":"","orcid":"","institution":"Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM)","correspondingAuthor":false,"prefix":"","firstName":"Juana","middleName":"","lastName":"Serrano","suffix":""},{"id":486137732,"identity":"54b75c88-b020-41f3-8f62-fabbd48927c3","order_by":3,"name":"Mireia Atance","email":"","orcid":"","institution":"Hospital Universitario Fundación Jiménez Díaz","correspondingAuthor":false,"prefix":"","firstName":"Mireia","middleName":"","lastName":"Atance","suffix":""},{"id":486137733,"identity":"6f9668e4-f28f-47d8-be1c-ec7abc015e0f","order_by":4,"name":"Raquel Mata","email":"","orcid":"","institution":"Hospital Universitario Fundación Jiménez Díaz","correspondingAuthor":false,"prefix":"","firstName":"Raquel","middleName":"","lastName":"Mata","suffix":""},{"id":486137734,"identity":"9f8320d7-cf44-4ff4-ade3-270d2f623d4e","order_by":5,"name":"Cristina Serrano","email":"","orcid":"","institution":"Hospital Universitario Fundación Jiménez Díaz","correspondingAuthor":false,"prefix":"","firstName":"Cristina","middleName":"","lastName":"Serrano","suffix":""},{"id":486137735,"identity":"ad46ee94-3e85-42c3-9787-a70fb12bf04c","order_by":6,"name":"Rocío Olivera","email":"","orcid":"","institution":"Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM)","correspondingAuthor":false,"prefix":"","firstName":"Rocío","middleName":"","lastName":"Olivera","suffix":""},{"id":486137736,"identity":"2c1f78ca-4116-4e6a-8cf8-1453a1eb035c","order_by":7,"name":"Rocío Salgado","email":"","orcid":"","institution":"Hospital Universitario Fundación Jiménez Díaz","correspondingAuthor":false,"prefix":"","firstName":"Rocío","middleName":"","lastName":"Salgado","suffix":""},{"id":486137737,"identity":"b00cef37-8923-4f26-8dc7-8c9d70b474d2","order_by":8,"name":"Raquel Capellan","email":"","orcid":"","institution":"Hospital Universitario Fundación Jiménez Díaz","correspondingAuthor":false,"prefix":"","firstName":"Raquel","middleName":"","lastName":"Capellan","suffix":""},{"id":486137738,"identity":"6fe7fbe2-73d9-4806-8e89-fbed53294f97","order_by":9,"name":"Alberto Lázaro","email":"","orcid":"","institution":"Hospital Universitario Fundación Jiménez Díaz","correspondingAuthor":false,"prefix":"","firstName":"Alberto","middleName":"","lastName":"Lázaro","suffix":""},{"id":486137739,"identity":"a7e8b35e-9006-479d-967d-9dba84d1cd59","order_by":10,"name":"Gonzalo Castellanos","email":"","orcid":"","institution":"Hospital Universitario Fundación Jiménez Díaz","correspondingAuthor":false,"prefix":"","firstName":"Gonzalo","middleName":"","lastName":"Castellanos","suffix":""},{"id":486137740,"identity":"13b21e5b-230f-407e-9b1d-0190ed1bb3da","order_by":11,"name":"Carlos Blas","email":"","orcid":"","institution":"Hospital Universitario Fundación Jiménez Díaz","correspondingAuthor":false,"prefix":"","firstName":"Carlos","middleName":"","lastName":"Blas","suffix":""},{"id":486137741,"identity":"7cc674b2-770b-481f-bff1-29bff335ec2a","order_by":12,"name":"Laura Pardo","email":"","orcid":"","institution":"Hospital Universitario Fundación Jiménez Díaz","correspondingAuthor":false,"prefix":"","firstName":"Laura","middleName":"","lastName":"Pardo","suffix":""},{"id":486137742,"identity":"5259d4f9-4000-4515-95ce-63e3f6b7a2d1","order_by":13,"name":"Laura Solán","email":"","orcid":"","institution":"Hospital Universitario Fundación Jiménez Díaz","correspondingAuthor":false,"prefix":"","firstName":"Laura","middleName":"","lastName":"Solán","suffix":""},{"id":486137743,"identity":"b2acc3d5-3264-4fca-843f-8ebade556d37","order_by":14,"name":"Belén Rosado","email":"","orcid":"","institution":"Hospital Universitario Rey Juan Carlos","correspondingAuthor":false,"prefix":"","firstName":"Belén","middleName":"","lastName":"Rosado","suffix":""},{"id":486137744,"identity":"adc0c95f-4e47-4416-b8cf-8588b645da7f","order_by":15,"name":"María Yuste","email":"","orcid":"","institution":"Hospital Universitario General de Villalba","correspondingAuthor":false,"prefix":"","firstName":"María","middleName":"","lastName":"Yuste","suffix":""},{"id":486137745,"identity":"17cc9669-e806-4775-9923-239ba9765d44","order_by":16,"name":"Pilar Beltran","email":"","orcid":"","institution":"Hospital Universitario Infanta Elena","correspondingAuthor":false,"prefix":"","firstName":"Pilar","middleName":"","lastName":"Beltran","suffix":""},{"id":486137746,"identity":"bf51bebc-6764-40e9-bb12-7e88d3c7521f","order_by":17,"name":"Eva Oliva","email":"","orcid":"","institution":"Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM)","correspondingAuthor":false,"prefix":"","firstName":"Eva","middleName":"","lastName":"Oliva","suffix":""},{"id":486137747,"identity":"58ec89f8-a7ae-4e6e-adeb-e1482bfcfda0","order_by":18,"name":"Pilar Llamas","email":"","orcid":"","institution":"Hospital Universitario Fundación Jiménez Díaz","correspondingAuthor":false,"prefix":"","firstName":"Pilar","middleName":"","lastName":"Llamas","suffix":""},{"id":486137748,"identity":"972e088b-1285-4803-b007-bcfa758f6494","order_by":19,"name":"Juan M. Alonso-Dominguez","email":"","orcid":"","institution":"Hospital Universitario Fundación Jiménez Díaz","correspondingAuthor":false,"prefix":"","firstName":"Juan","middleName":"M.","lastName":"Alonso-Dominguez","suffix":""}],"badges":[],"createdAt":"2025-07-03 07:08:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7035200/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7035200/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-26852-x","type":"published","date":"2025-11-28T15:58:29+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":87270198,"identity":"175cad93-562c-4ac9-a389-cee6525c7a72","added_by":"auto","created_at":"2025-07-22 08:18:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":33749,"visible":true,"origin":"","legend":"\u003cp\u003eClinical outcome stratified according to proportion of MSCs at end of treatment for all patients treated with intensive chemotherapy (MSCs \u0026lt;0.265%, n=48 and MSCs≥0.265%=17) (A) Overall survival. (B) Relapse free survival. Abbreviations: MSCs, mesenchymal cells\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7035200/v1/8872ae2d4e54ba8b2302824c.png"},{"id":87272333,"identity":"b85f6f5c-e6b6-46dc-9255-3c2f129b1b05","added_by":"auto","created_at":"2025-07-22 08:26:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":54509,"visible":true,"origin":"","legend":"\u003cp\u003eClinical outcome stratified according to proportion of MSCs at end of treatment for all patients treated with intensive chemotherapy\u003cdel\u003e \u003c/del\u003e. (A-C) OS according to proportion of MSCs and 2017 ELN genetic risk (A: favorable risk, MSCs\u0026lt;0.265%, n=13 and MSCs≥0.0265%, n=2; B: intermediate risk, MSCs\u0026lt;0.265%, n= 20 and MSCs≥0.0265%, n=8; C: adverse risk, MSCs\u0026lt;0.265%, n=15 and MSCs≥0.0265% n=7). (D-F) RFS according to proportion of MSCs and 2017 ELN genetic risk (D: favorable risk, MSCs\u0026lt;0.265%, n=13 and MSCs≥0.0265%, n=2, E: intermediate risk, MSCs\u0026lt;0.265%, n= 20 and MSCs≥0.0265%, n=8 and F: adverse risk, MSCs\u0026lt;0.265%, n=15 and MSCs≥0.0265% n=7).Abbreviations: MSCs, mesenchymal cells\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7035200/v1/0dd632f10194cbc76d59db82.png"},{"id":87270201,"identity":"2325f780-89d1-4168-823e-5166ab11e90b","added_by":"auto","created_at":"2025-07-22 08:18:26","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":105299,"visible":true,"origin":"","legend":"\u003cp\u003eGating strategy for MSC-like cell sorting. (A) MSC-like cells expressing CD13\u003csup\u003ebright\u003c/sup\u003eCD45\u003csup\u003elow/neg\u003c/sup\u003eCD105\u003csup\u003epos\u003c/sup\u003eCD90\u003csup\u003epos\u003c/sup\u003e surface markers were isolated from low-density bone marrow samples of AML patients. (B) Brightfield images of culture growth of\u0026nbsp; CD13\u003csup\u003ebright\u003c/sup\u003eCD45\u003csup\u003elow/neg\u003c/sup\u003e CD105\u003csup\u003epos\u003c/sup\u003e and CD90\u003csup\u003epos\u003c/sup\u003e MSC-like cells. The white arrows highlight cells displaying characteristic MSC morphologies, such as elongated spindle shapes and cuboidal or polygonal forms, demonstrating successful culture and growth of MSC-like cells. Scale bar 200 um.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7035200/v1/2601a6e0a96698ea47c1083a.png"},{"id":87270207,"identity":"7b00ee39-3d22-492c-9ecd-85d4c2364212","added_by":"auto","created_at":"2025-07-22 08:18:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":278226,"visible":true,"origin":"","legend":"\u003cp\u003eGating strategy to identify the mesenchymal cells (pink cells)\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7035200/v1/080eeea6a8c4f1e0a719bad7.png"},{"id":97178612,"identity":"b02c0188-7020-4931-9f04-e8e4bd725a60","added_by":"auto","created_at":"2025-12-01 16:11:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1218250,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7035200/v1/00101b8c-af07-48aa-9091-4bd2d45fa414.pdf"},{"id":87270199,"identity":"3530c2d9-93c1-43bb-9a5a-c0dc29a6af5e","added_by":"auto","created_at":"2025-07-22 08:18:26","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":205381,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-7035200/v1/04dfe84664c5c0cbf56a8b90.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mesenchymal stem cells-like as a prognostic biomarker in patients diagnosed with acute myeloid leukemia","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAcute myeloid leukemia (AML) is a hematologic neoplasm characterized by the uncontrolled proliferation of abnormal myeloid progenitor cells\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. AML arises from a series of genetic and epigenetic changes, primarily affecting hematopoietic stem and progenitor cells (HSPCs)\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eThe pathogenesis of AML, as well as other hematological malignancies, depends not only on intrinsic tumoral factors but is also influenced by the bone marrow (BM) microenvironment\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. The BM microenvironment includes a complex network of extracellular matrix proteins, soluble growth factors, cytokines, and distinct-though possibly overlapping- cellular niches, such as the endosteal (or osteoblastic) and vascular niches. Several studies have demonstrated that blast cells can manipulate the marrow microenvironment to create a permissive conditions that favors disease initiation, progression and therapeutic resistance\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Mesenchymal stem cells (MSCs) play a decisive role in this process, known as \"stromal plasticity\". MSCs are non-hematopoietic multipotent stem cells characterized by their self-renewal capacity, clonogenic efficiency and multilineage differentiation capacity\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Furthermore, MSCs may influence the development of various diseases, including hematological malignancies such as chronic myeloid leukemia, multiple myeloma, and myelodysplastic syndrome/AML\u003csup\u003e10,11,12\u003c/sup\u003e. However, despite extensive research over the past decade, it remains unclear whether MSCs have tumor-promoting or tumor-suppressive effects, and their precise role in AML is yet to be determined \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eCD13, also known as aminopeptidase N, is a transmembrane metalloprotease widely recognized as a surface marker in flow cytometric analysis of MSCs and various progenitor cell populations. Its expression is commonly used as part of a panel of positive markers, including CD73 and CD90, to identify MSCs in accordance with the minimal criteria proposed by the International Society for Cellular Therapy (ISCT)\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Beyond its utility as a phenotypic marker, CD13 plays functional roles in cellular adhesion, migration, and differentiation. In flow cytometry, anti-CD13 monoclonal antibodies enable the reliable detection and isolation of MSCs based on differential surface expression, thereby supporting downstream applications in regenerative medicine and cellular therapies\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Recently, our group identified a CD13-bright cell population enriched for canonical MSC markers CD105 and CD90 in myelodysplastic syndromes (MDS) patients at diagnostic. Notably, these MSC-like cells were found to be a predictor of progression to AML\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn recent years, the introduction of new drugs into the AML therapeutic arsenal has improved the prognosis, but survival rates vary greatly depending on the characteristics of the disease, in particular genetic abnormalities included in the ELN risk score\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. However, in every ELN risk group patients show differences in outcome and we should try to refine prognosis of patient as much as possible for appropriate consolidation treatment. In light of these considerations, in this manuscript we explore the potential of MSCs as a prognostic factor in intensively treated AML patients.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e65 AML patients treated with intensive chemotherapy, showed MSCs measured by MFC at the end of treatment (post-induction treatment: induction-1 (n\u0026thinsp;=\u0026thinsp;18) and induction-2 (4); post-consolidation treatment: consolidation-1 (n\u0026thinsp;=\u0026thinsp;9), consolidation-2 (n\u0026thinsp;=\u0026thinsp;5) and consolidation-3 (n\u0026thinsp;=\u0026thinsp;5); allogeneic pre-transplantation (n\u0026thinsp;=\u0026thinsp;2) and allogeneic post-transplantation (n\u0026thinsp;=\u0026thinsp;22). The median age of this group was 58 years (range 18\u0026ndash;77 years); 32 males and 33 females. The median proportion of MSCs was 0.10%. Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the baseline characteristics of AML, the proportion of MSCs at the end of treatment, and the therapies administered to these 65 patients\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Impact of MSCs determination at end of treatment\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eWe set 0.265% as the cut-off for MSC-l quantification based on the total analyzed BM cellularity (excluding debris). In the analysis of OS, the MSCs determination showed that 48 patients (74%) were MSCs-l\u003csup\u003eLOW\u003c/sup\u003e and 17 patients (26%) were MSCs-l\u003csup\u003eHIGH\u003c/sup\u003e. In the MSCs-l\u003csup\u003eLOW\u003c/sup\u003e group, the median OS was not calculable (NC), while in the MSCs-l\u003csup\u003eHIGH\u003c/sup\u003e group, the median OS was 0.66 years (95% CI: 0.28\u0026ndash;1.04). When comparing the two subgroups using a log-rank test, MSCs-l\u003csup\u003eHIGH\u003c/sup\u003e patients had significantly reduced OS (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).The RFS analysis was performed on 51 patients: MSCs-l\u003csup\u003eLOW\u003c/sup\u003e (n\u0026thinsp;=\u0026thinsp;40) and MSCs-l\u003csup\u003eHIGH\u003c/sup\u003e (n\u0026thinsp;=\u0026thinsp;11). In the MSCs-l\u003csup\u003eLOW\u003c/sup\u003e group, the median RFS was 1.49 years (95% CI: 0.01\u0026ndash;3.51), while in the MSCs-l\u003csup\u003eHIGH\u003c/sup\u003e group, the median RFS was 1.27 years (95% CI: 0.02\u0026ndash;2.52). A log-rank test showed significantly reduced RFS in MSCs-l\u003csup\u003eHIGH\u003c/sup\u003e patients (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.027) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eA stratified analysis by the ELN 2017 genetic risk classification was performed. In the analysis of OS, the distribution of MSCs-l determination was as follows: 13 patients (20%) with MSCs-l\u003csup\u003eLOW\u003c/sup\u003e and 2 patients (3%) with MSCs-l\u003csup\u003eHIGH\u003c/sup\u003e in the favorable-risk group, 20 patients (31%) with MSCs-l\u003csup\u003eLOW\u003c/sup\u003e and 8 patients (12%) with MSCs-l\u003csup\u003eHIGH\u003c/sup\u003e in the intermediate-risk group, and 15 patients (23%) with MSCs-l\u003csup\u003eLOW\u003c/sup\u003e, and 7 patients (11%) with MSCs-l\u003csup\u003eHIGH\u003c/sup\u003e in the adverse-risk group. The OS in the favorable group was NC in MSCs-l\u003csup\u003eLOW\u003c/sup\u003e and 0.4 years (95% CI: NC) in MSCs-l\u003csup\u003eHIGH\u003c/sup\u003e. The OS in the intermediate group was 3.4 years (95% CI: NC) in MSCs-l\u003csup\u003eLOW\u003c/sup\u003e and 0.9 years (95% CI:0.3\u0026ndash;1.4) in MSCs-l\u003csup\u003eHIGH\u003c/sup\u003e. The OS in the adverse group was 1.4 years (95% CI:0.1\u0026ndash;2.6) in MSC-ls\u003csup\u003eLOW\u003c/sup\u003e and 0.4 years (95% CI:0.3\u0026ndash;0.6) in MSCs-l\u003csup\u003eHIGH\u003c/sup\u003e. A log-rank test comparing subgroups according to genetic risk showed a reduced OS in MSCs-l\u003csup\u003eHIGH\u003c/sup\u003e patients (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn the analysis of RFS, the distribution was as follows: 12 patients (24%) with MSCs-l\u003csup\u003eLOW\u003c/sup\u003e and 2 patients (4%) with MSCs-l\u003csup\u003eHIGH\u003c/sup\u003e in the favorable-risk group, 17 patients (33%) with MSC-ls\u003csup\u003eLOW\u003c/sup\u003e and 5 patients (10%) with MSCs-l\u003csup\u003eHIGH\u003c/sup\u003e in the intermediate-risk group, and 11 patients (22%) with MSCs-l\u003csup\u003eLOW\u003c/sup\u003e, and 4 patients (8%) with MSCs-l\u003csup\u003eHIGH\u003c/sup\u003e in the adverse-risk group. The RFS in the favorable group was 1.47 years (95% CI: 0.01\u0026ndash;3.42) in MSCs-l\u003csup\u003eLOW\u003c/sup\u003e and NC in MSCs-l\u003csup\u003eHIGH\u003c/sup\u003e (. The RFS in the intermediate group was 1.49 years (95% CI: 0.47\u0026ndash;2.50) in MSCs-l\u003csup\u003eLOW\u003c/sup\u003e and 1.27 years (95% CI: NC) in MSCs-l\u003csup\u003eHIGH\u003c/sup\u003e. The RFS in the adverse group was not calculable in MSCs-l\u003csup\u003eLOW\u003c/sup\u003e and 0.18 years (95% CI: 0.01\u0026ndash;1.21) in MSCs-l\u003csup\u003eHIGH\u003c/sup\u003e(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). A log-rank test comparing subgroups according to genetic risk showed a reduction of RFS in MSCs-l\u003csup\u003eHIGH\u003c/sup\u003e patients (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.05). Cox regression analysis for OS prediction showed that age, ELN 2017 classification and the proportion of MSCs-l were independent predictor factors (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.02, HR:0.27, [95% CI: 0.09\u0026ndash;0.79], \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.03, HR: NC, [95% CI: NC] and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, HR:11.68, [95% CI: 3.80\u0026ndash;35.9], respectively). Cox regression analysis for the RFS prediction showed that proportion of MSCs-l was an independent predictor factor (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.03, HR: 3.38, [95% CI: 1.15\u0026ndash;9.96]. Age and ELN 2017 classification were not independent predictors factors (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.37, HR: 0.63, [95% CI: 0.23\u0026ndash;1.72] and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.87, HR: NC, [95% CI: NC], respectively). Proportional hazard assumptions were checked by adding the interaction of every variable with time in every analysis performed.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. In vitro expansion, and osteogenic, adipogenic, and chondrogenic differentiation of MSCs\u003c/h2\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn order to identify similarities between MSC-1 and previously described MSC-like cells (17) we conducted comparable sorting and culture experiments. MSC-1 cells exhibited similar enrichment of canonical CD105 and CD90 MSC markers and equivalent attachment potential to MSC-like cells (Fig.\u0026nbsp;3).\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study shows that the proportion of MSCs-l at end of treatment has prognostic impact on survival and relapse in newly diagnosed AML patients treated with front-line intensive regimens. We analyzed the proportion of MSCs-l in 65 AML patients treated with intensive chemotherapy at end of treatment A cut-off of 0.265% for MSCs-l was used to assess the prognostic significance of MSCs-l proportion and its clinical applicability. Patients with a proportion of MSCs-l\u0026thinsp;\u0026ge;\u0026thinsp;0.265% regardless of their 2017 ELN genetic risk (favorable, intermediate or adverse risk) showed a significant reduction in both OS and RFS either in global and ELN-stratified analysis. In the multivariate analysis for OS including age, genetic risk according to the 2017 ELN guidelines, and categorized MSCs-l quantification all of them were identified as independent predictive factors in AML patients treated with intensive therapy. In the multivariate analysis for RFS, only the proportion of MSCs-l was found to be a significant independent predictor. These data show that this population of MSCs-l cells have a role in AML prognosis and more importantly add prognostic value to already established predictor factors such as age and ELN risk score. To date, no published studies in the literature have addressed the prognostic role of MSCs in AML patients.\u003c/p\u003e\u003cp\u003eOur study did not fully meet the International Society for Cellular Therapy (ISCT) criteria for the definitive identification of mesenchymal stem cells (MSCs) within the bright CD13 compartment. Classical MSC markers\u0026mdash;such as CD73, CD90, and CD105\u0026mdash;were not included in our flow cytometry panel; however, the specificity and consistency of these markers are limited, as they are not exclusive to MSCs and display variable expression across individuals and disease states \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Despite the absence of these markers, the CD13bright cells, in the absence of other myeloid markers, exhibited key mesenchymal characteristics, including plastic adherence and in vitro expansion, although they failed to undergo trilineage differentiation into adipocytes, chondrocytes, and osteoblasts. This could be attributed to the limited number of cells available for functional assays and/or intrinsic alterations in pathological mesenchymal populations\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Flow cytometry analysis indicated that the CD13bright compartment also includes endothelial cells and fibroblasts, which can be distinguished, based on CD34 expression and CD13 mean fluorescence intensity (MFI), both of which are lower than in MSCs\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Fibroblasts, in particular, require enzymatic digestion for recovery and share similar CD13 MFI values with endothelial cells. In contrast, hematopoietic cells exhibit markedly lower or absent CD13 expression and lack the myeloid markers included in the panel, thereby supporting the exclusion of these lineages from the CD13bright gate\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Based on the above findings, we conclude that the analyzed population corresponds to MSCs.\u003c/p\u003e\u003cp\u003eIn summary, in our cohort of 65 intensively treated AML patients, we propose that the proportion of MSCs-l at the end of treatment may function as an independent prognostic factor. In particular, we demonstrate that the categorical variable MSC-l furthers improve ELN risk and age prognostication of OS, being the only independent factor for RFS. To strengthen the validity and statistical power of these findings, confirmation in a larger prospective cohort is warranted. If these results are validated, further studies will be necessary to confirm this population corresponds to MSC by differentiation assays, which could ultimately support their exploration as novel therapeutic targets in this aggressive and frequently relapsed disease.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e5.1. Patients and samples\u003c/h2\u003e\u003cp\u003eThe primary patient and disease characteristics were collected retrospectively, including: age, sex, cytomorphological assessment, AML diagnosis confirmation (according to the site\u0026acute;s routine practice), cytogenetics, molecular studies, description of the front-line treatment approach, disease response assessments, and disease follow-up\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Patients diagnosed with acute promyelocytic leukemia (APL) were excluded. We reviewed data collected from 65 intensively treated adult AML patients with available MSC data measured by flow cytometry (FCM) at the end of treatment. These patients were diagnosed between 2017 and 2024 at Hospital Universitario Fundaci\u0026oacute;n Jim\u0026eacute;nez D\u0026iacute;az, Hospital Universitario Rey Juan Carlos, Hospital Universitario General de Villalba, and Hospital Universitario Infanta Elena, all in Madrid (Spain).\u003c/p\u003e\u003cp\u003e Written informed consent was obtained from all study participants. Approval of the study was obtained from the Clinical Research Ethics Committee of the Hospital Universitario Fundaci\u0026oacute;n Jim\u0026eacute;nez D\u0026iacute;az (TFG017-19_FJD). The study complied with the Declaration of Helsinki.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e5.2. MSCs screening\u003c/h2\u003e\u003cp\u003eMultiparameter flow cytometry (MFC) immunophenotypic studies were performed on BM aspirate samples, processed under sterile conditions within 24 hours of collection. The pre-filtered bone marrow sample (EDTA) is incubated for 15 minutes with a combination of eight pre-selected monoclonal antibodies (MoAb) which were used to identify and characterize BM MSCs (Table\u0026nbsp;2). Erythrocytes were subsequently lysed with BD FACS\u0026trade; solution for 10 minutes (BD Biosciences), followed by two washes with PBS (Inova Diagnostics). The FACS Canto II flow cytometer (Becton Dickinson Biosciences, San Jose, CA, USA) was used to acquire a minimum of 500,000 events per tube (two tubes analyzed). Infinicyt\u0026trade; software (Cytognos SL, Salamanca, Spain) was used to analyze the main leukocyte subpopulations in the generated FCS files. Regarding MSCs gating strategy, a retrospective search for phenotypic information on MSCs was performed in the flow cytometry files of 65 AML patients. The phenotypic analysis of AML MSCs was adjusted from Mu\u0026ntilde;iz et al\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e considering that CD105 and CD90 were not routinely included in our routine AML monitoring panel. A bright CD13-positive population was identified, which was negative for CD34, CD45, CD117, CD11b, CD16, CD71 and CD64. This population is referred to as the MSC-like (MSC-l) compartment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Gates were set according to the appropriate isotype control, and the expression of each marker was reported as the percentage of positive cells of total BM nucleated cells, excluding debris\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e5.3. In vitro expansion of MSCs\u003c/h2\u003e\u003cp\u003eThe same sorting strategy and culture methods described by the group (17) were employed based on the CD13\u003csup\u003ebright\u003c/sup\u003eCD45\u003csup\u003elow/neg\u003c/sup\u003eCD105\u003csup\u003epos\u003c/sup\u003eCD90\u003csup\u003epos\u003c/sup\u003e population, for further studies on MSC-like cells to confirm their MSC identity. A total of three samples were analyzed to isolate the MSC-like phenotype from erythrocyte-depleted, low-density bone marrow cells. Then, cells were stained for phycoerythrin (PE) anti-CD13 (clone WM15) (Beckton Dickinson), fluorescein (FITC) anti-CD45 (clone T29/33) (Dako), allophycocyanin (APC) anti-CD105 (clone 43/A3) (Beckton Dickinson) and violet 450 anti-CD90 (clone 5E10) (Beckton Dickinson). Cell sorting was conducted using a FACSMelody\u0026trade; Cell Sorter (Becton Dickinson) equipped with 3 lasers and FACSChorus\u0026trade; software. Subsequent flow cytometry analysis was performed using FlowJo v.10.9.0.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e5.4. Statistical analysis\u003c/h2\u003e\u003cp\u003eOverall survival (OS) was defined as the time from the date of AML diagnosis to the date of death due to any cause. Relapse free survival (RFS) was calculated from the date of achieving complete response (CR) or CR with incomplete hematologic recovery (CRi) until the date of relapse or death from any cause. CR and CRi were defined according to the current 2017 ELN guidelines\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Groups for comparison were established based on MSC quantification, with an arbitrary cut-off determined according to its sensitivity and specificity. Kaplan-Meier and log-ranks test were used to compare OS and RFS between groups. Intensively-treated patients were analyzed globally and in a stratified analysis by the ELN 2017 genetic risk classification.\u003c/p\u003e\u003cp\u003eMultivariate Cox regression analysis was used to predict OS and RFS, with MSC quantification (categorized as explained above), age (categorized in \u0026lt;\u0026thinsp;60 years or \u0026ge;\u0026thinsp;60 years) and the ELN 2017 genetic risk model as covariates. Statistical analyses were perfomed using SPSS 19.0 (IBM, Arkmo0.0n, NY). The confidence interval was set at 95% and the \u003cem\u003eP\u003c/em\u003e-values were bilateral, considered significant if\u0026thinsp;\u0026le;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eT.C. is a PhD candidate at the Universidad Aut\u0026oacute;noma de Madrid (UAM), and this work is submitted in partial fulfillment of the requirements for the doctoral degree. The authors gratefully acknowledge the technical assistance of Raquel Gonzalo and Susana Casta\u0026ntilde;\u0026oacute;n in performing the experiments with mesenchymal stem cells (MSCs) using multiparametric flow cytometry (MFC). We also extend our sincere gratitude to the research team at the Hospital Universitario Fundaci\u0026oacute;n Jim\u0026eacute;nez D\u0026iacute;az for their contributions to the expansion, culture, and differentiation of MSCs into various cell subtypes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFormal analysis\u003c/strong\u003e: Raquel Mata, Carlos Blas, Cristina Serrano, Juan Manuel Alonso Dom\u0026iacute;nguez, Juana Serrano, Gonzalo Castellanos, Raquel Capell\u0026aacute;n, Roc\u0026iacute;o Salgado, Laura Pardo, Laura Sol\u0026aacute;n, Alvaro V. Arriero, Bel\u0026eacute;n Rosado, Daniel La\u0026iacute;nez, Mar\u0026iacute;a Yuste, Pilar Beltran, Eva Oliva, Pilar Llamas,Roc\u0026iacute;o Olivera y Mireia Atance. \u003cstrong\u003eInvestigation\u003c/strong\u003e: Tamara Casta\u0026ntilde;o-Bonilla. \u003cstrong\u003eMethodology:\u003c/strong\u003e Raquel Mata, Carlos Blas, Cristina Serrano, Juan Manuel Alonso Dom\u0026iacute;nguez, Juana Serrano, Gonzalo Castellanos, Raquel Capell\u0026aacute;n, Roc\u0026iacute;o Salgado, Laura Pardo, Laura Sol\u0026aacute;n, Alvaro V. Arriero, Bel\u0026eacute;n Rosado, Daniel La\u0026iacute;nez, Mar\u0026iacute;a Yuste, Pilar Beltran, Eva Oliva, Pilar Llamas,Roc\u0026iacute;o Olivera y Mireia Atance. \u003cstrong\u003eSupervision\u003c/strong\u003e: Juana Serrano, Daniel Lainez, Raquel Mata, Cristina Serrano, Mireia Atance, Alberto L\u0026aacute;zaro, Pilar Llamas, Juan M. Alonso-Dominguez. \u003cstrong\u003eVisualisation\u003c/strong\u003e: Tamara Casta\u0026ntilde;o Bonilla, Juan Manuel Alonso Dominguez y Daniel L\u0026aacute;inez Gonz\u0026aacute;lez. \u003cstrong\u003eData curation:\u003c/strong\u003e Raquel Mata, Carlos Blas, Cristina Serrano, Juan Manuel Alonso Dom\u0026iacute;nguez, Juana Serrano, Gonzalo Castellanos, Raquel Capell\u0026aacute;n, Roc\u0026iacute;o Salgado, Laura Pardo, Laura Sol\u0026aacute;n, Alvaro V. Arriero, Bel\u0026eacute;n Rosado, Daniel La\u0026iacute;nez, Mar\u0026iacute;a Yuste, Pilar Beltran, Eva Oliva, Pilar Llamas,Roc\u0026iacute;o Olivera y Mireia Atance. \u0026nbsp;\u003cstrong\u003eWriting\u0026mdash;original draft\u003c/strong\u003e, Tamara Casta\u0026ntilde;o. \u003cstrong\u003eWriting\u0026mdash;review and editing\u003c/strong\u003e, Raquel Mata, Carlos Blas, Cristina Serrano, Juan Manuel Alonso Dom\u0026iacute;nguez, Juana Serrano, Gonzalo Castellanos, Raquel Capell\u0026aacute;n, Roc\u0026iacute;o Salgado, Laura Pardo, Laura Sol\u0026aacute;n, Alvaro V. Arriero, Bel\u0026eacute;n Rosado, Daniel La\u0026iacute;nez, Mar\u0026iacute;a Yuste, Pilar Beltran, Eva Oliva, Pilar Llamas,Roc\u0026iacute;o Olivera y Mireia Atance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research received no external funding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJuan Manuel Alonso Dom\u0026iacute;nguez has received honoraria as a member of the advisory board for Astellas and Servier.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll participants provided written informed consent. The study protocol was approved by the Clinical Research Ethics Committee of the Hospital Universitario Fundaci\u0026oacute;n Jim\u0026eacute;nez D\u0026iacute;az (TFG017-19_FJD) and was conducted in accordance with the Declaration of Helsinki.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study available from the corresponding author on reasonable request\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eShimony, S., Stahl, M. \u0026amp; Stone, R. M. Acute myeloid leukemia: 2023 update on diagnosis, risk-stratification, and management. \u003cem\u003eAm. J. Hematol.\u003c/em\u003e \u003cb\u003e98\u003c/b\u003e, 502\u0026ndash;526 (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eD\u0026ouml;hner, H. et al. Diagnosis and management of AML in adults: 2022 recommendations from an international expert panel on behalf of the ELN. \u003cem\u003eBlood\u003c/em\u003e \u003cb\u003e140\u003c/b\u003e, 1345\u0026ndash;1377 (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePrada-Arismendy, J., Arroyave, J. C. \u0026amp; R\u0026ouml;thlisberger, S. Molecular biomarkers in acute myeloid leukemia. \u003cem\u003eBlood Rev.\u003c/em\u003e \u003cb\u003e31\u003c/b\u003e, 63\u0026ndash;76 (2017).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePittenger, M. F. et al. Mesenchymal stem cell perspective: cell biology to clinical progress. \u003cem\u003enpj Regen Med.\u003c/em\u003e \u003cb\u003e4\u003c/b\u003e, 1\u0026ndash;15 (2019).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJovic, D. et al. A Brief Overview of Global Trends in MSC-Based Cell Therapy. \u003cem\u003eStem Cell. Rev. Rep.\u003c/em\u003e \u003cb\u003e18\u003c/b\u003e, 1525\u0026ndash;1545 (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShafat, M. S., Gnaneswaran, B., Bowles, K. M. \u0026amp; Rushworth, S. A. The bone marrow microenvironment - Home of the leukemic blasts. \u003cem\u003eBlood Rev.\u003c/em\u003e \u003cb\u003e31\u003c/b\u003e, 277\u0026ndash;286 (2017).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMenter, T. \u0026amp; Tzankov, A. Tumor Microenvironment in Acute Myeloid Leukemia: Adjusting Niches. \u003cem\u003eFront. Immunol.\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, 811144 (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePimenta, D. B. et al. The Bone Marrow Microenvironment Mechanisms in Acute Myeloid Leukemia. \u003cem\u003eFrontiers Cell. Dev. Biology\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e, (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTan, L., Liu, X., Dou, H. \u0026amp; Hou, Y. Characteristics and regulation of mesenchymal stem cell plasticity by the microenvironment \u0026mdash; specific factors involved in the regulation of MSC plasticity. \u003cem\u003eGenes Dis.\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e, 296\u0026ndash;309 (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGarcia-Gomez, A., Sanchez-Guijo, F., Ca\u0026ntilde;izo, D., Miguel, M. C. S., Garayoa, M. \u0026amp; J. F. \u0026amp; Multiple myeloma mesenchymal stromal cells: Contribution to myeloma bone disease and therapeutics. \u003cem\u003eWorld J. Stem Cells\u003c/em\u003e. \u003cb\u003e6\u003c/b\u003e, 322\u0026ndash;343 (2014).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChandia, M. et al. Involvement of primary mesenchymal precursors and hematopoietic bone marrow cells from chronic myeloid leukemia patients by BCR-ABL1 fusion gene. \u003cem\u003eAm. J. Hematol.\u003c/em\u003e \u003cb\u003e89\u003c/b\u003e, 288\u0026ndash;294 (2014).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLopez-Villar, O. et al. Both expanded and uncultured mesenchymal stem cells from MDS patients are genomically abnormal, showing a specific genetic profile for the 5q- syndrome. \u003cem\u003eLeukemia\u003c/em\u003e \u003cb\u003e23\u003c/b\u003e, 664\u0026ndash;672 (2009).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLee, M. W. et al. Mesenchymal stem cells in suppression or progression of hematologic malignancy: current status and challenges. \u003cem\u003eLeukemia\u003c/em\u003e \u003cb\u003e33\u003c/b\u003e, 597\u0026ndash;611 (2019).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGalland, S. \u0026amp; Stamenkovic, I. Mesenchymal stromal cells in cancer: a review of their immunomodulatory functions and dual effects on tumor progression. \u003cem\u003eJ. Pathol.\u003c/em\u003e \u003cb\u003e250\u003c/b\u003e, 555\u0026ndash;572 (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDominici, M. et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. \u003cem\u003eCytotherapy\u003c/em\u003e \u003cb\u003e8\u003c/b\u003e, 315\u0026ndash;317 (2006).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRahman, M. M. et al. CD13 promotes mesenchymal stem cell-mediated regeneration of ischemic muscle. \u003cem\u003eFront Physiol\u003c/em\u003e \u003cb\u003e4\u003c/b\u003e, (2014).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eViswanathan, S. et al. Mesenchymal stem versus stromal cells: International Society for Cell \u0026amp; Gene Therapy (ISCT\u0026reg;) Mesenchymal Stromal Cell committee position statement on nomenclature. \u003cem\u003eCytotherapy\u003c/em\u003e \u003cb\u003e21\u003c/b\u003e, 1019\u0026ndash;1024 (2019).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMu\u0026ntilde;iz, C. et al. Ex vivo identification and characterization of a population of CD13(high) CD105(+) CD45(-) mesenchymal stem cells in human bone marrow. \u003cem\u003eStem Cell. Res. Ther.\u003c/em\u003e \u003cb\u003e6\u003c/b\u003e, 169 (2015).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePhinney, D. G. \u0026amp; Prockop, D. J. Concise review: mesenchymal stem/multipotent stromal cells: the state of transdifferentiation and modes of tissue repair\u0026ndash;current views. \u003cem\u003eStem Cells\u003c/em\u003e. \u003cb\u003e25\u003c/b\u003e, 2896\u0026ndash;2902 (2007).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHoogduijn, M. J. et al. The immunomodulatory properties of mesenchymal stem cells and their use for immunotherapy. \u003cem\u003eInt. Immunopharmacol.\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e, 1496\u0026ndash;1500 (2010).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi, H., Ghazanfari, R., Zacharaki, D., Lim, H. C. \u0026amp; Scheding, S. Isolation and characterization of primary bone marrow mesenchymal stromal cells. \u003cem\u003eAnn. N Y Acad. Sci.\u003c/em\u003e \u003cb\u003e1370\u003c/b\u003e, 109\u0026ndash;118 (2016).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFonseca, L. N. et al. Cell surface markers for mesenchymal stem cells related to the skeletal system: A scoping review. \u003cem\u003eHeliyon\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e, e13464 (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCasta\u0026ntilde;o-Bonilla, T. et al. No Evidence that CD33 rs12459419 Polymorphism Predicts Gemtuzumab Ozogamicin Response in Consolidation Treatment of Acute Myeloid Leukemia Patients: Experience of the PETHEMA Group. \u003cem\u003eDis Markers\u003c/em\u003e 3132941 (2022). (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMu\u0026ntilde;iz, C. et al. Ex vivo identification and characterization of a population of CD13high CD105\u0026thinsp;+\u0026thinsp;CD45\u0026thinsp;\u0026ndash;\u0026thinsp;mesenchymal stem cells in human bone marrow. \u003cem\u003eStem Cell Res. Ther.\u003c/em\u003e \u003cb\u003e6\u003c/b\u003e, 169 (2015).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eD\u0026ouml;hner, H. et al. Diagnosis and management of AML in adults: 2017 ELN recommendations from an international expert panel. \u003cem\u003eBlood\u003c/em\u003e \u003cb\u003e129\u003c/b\u003e, 424\u0026ndash;447 (2017).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"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":"Acute myeloid leukemia, mesenchymal stem cells, prognostic biomarker, multiparameter flow cytometry, hematopoietic microenvironment","lastPublishedDoi":"10.21203/rs.3.rs-7035200/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7035200/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe bone marrow (BM) microenvironment plays a pivotal role in acute myeloid leukemia (AML) progression. Among its components, mesenchymal stem cells (MSCs) can exhibit tumor-modulating properties, yet their precise prognostic significance in AML remains unclear. To evaluate the prognostic impact of MSC-like (MSC-l) cells in adult AML patients treated with intensive chemotherapy. We retrospectively analyzed 65 adult AML patients (excluding acute promyelocytic leukemia) treated between 2017 and 2024 at four spanish institutions. MSC-l cells were identified in BM aspirates at the end of treatment by multiparameter flow cytometry as CD13bright/CD45low/CD34neg/CD117neg/CD11bneg/CD16neg/CD71neg/CD64neg. Patients were stratified based on MSC-l proportion using a cut-off of 0.265% (MSC-l\u003csup\u003eHIGH\u003c/sup\u003e patients ˃0265% and MSC-l\u003csup\u003eLOW\u003c/sup\u003e patients\u0026thinsp;\u0026le;\u0026thinsp;0.265%). Survival outcomes were assessed using Kaplan-Meier and Cox regression analyses, adjusting for age and ELN 2017 risk classification. MSC-l\u003csup\u003eHIGH\u003c/sup\u003e patients showed significantly reduced overall survival (OS) (median OS: 0.66 years \u003cem\u003evs.\u003c/em\u003e not calculable; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and relapse-free survival (RFS) (median RFS: 1.27 \u003cem\u003evs.\u003c/em\u003e 1.49 years; P\u0026thinsp;=\u0026thinsp;0.027) compared to MSC-l\u003csup\u003eLOW\u003c/sup\u003e patients. Stratified analyses confirmed this trend across ELN 2017 risk groups. Multivariate Cox regression identified MSC-l\u003csup\u003eHIGH\u003c/sup\u003e status as an independent predictor of worse OS (HR\u0026thinsp;=\u0026thinsp;11.68, 95% CI: 3.80\u0026ndash;35.9, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and RFS (HR\u0026thinsp;=\u0026thinsp;3.38, 95% CI: 1.15\u0026ndash;9.96, P\u0026thinsp;=\u0026thinsp;0.03). A higher proportion of MSC-l cells at end of treatment is associated with inferior survival outcomes in AML, independent of ELN risk and age. These findings suggest that MSC-l quantification may provide additional prognostic value and warrant validation in prospective studies.\u003c/p\u003e","manuscriptTitle":"Mesenchymal stem cells-like as a prognostic biomarker in patients diagnosed with acute myeloid leukemia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-22 08:18:21","doi":"10.21203/rs.3.rs-7035200/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-25T06:56:23+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-24T19:01:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"35223331735848311946683190165206371814","date":"2025-07-21T13:48:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"3788598868400379839403255937718547449","date":"2025-07-17T04:31:46+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-16T06:48:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"286736706152944982731122127368190819792","date":"2025-07-15T04:40:14+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-15T03:57:38+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-15T03:56:03+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-07-07T20:14:50+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-07T05:44:15+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-07-03T07:02:12+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":"5dcf1032-6686-425a-b2ed-6ba99ec16009","owner":[],"postedDate":"July 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":51606078,"name":"Health sciences/Biomarkers"},{"id":51606079,"name":"Biological sciences/Cancer"},{"id":51606080,"name":"Health sciences/Oncology"},{"id":51606081,"name":"Biological sciences/Stem cells"}],"tags":[],"updatedAt":"2025-12-01T16:04:21+00:00","versionOfRecord":{"articleIdentity":"rs-7035200","link":"https://doi.org/10.1038/s41598-025-26852-x","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-11-28 15:58:29","publishedOnDateReadable":"November 28th, 2025"},"versionCreatedAt":"2025-07-22 08:18:21","video":"","vorDoi":"10.1038/s41598-025-26852-x","vorDoiUrl":"https://doi.org/10.1038/s41598-025-26852-x","workflowStages":[]},"version":"v1","identity":"rs-7035200","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7035200","identity":"rs-7035200","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}