For the last two decades, measurable residual disease (MRD) has become one of the most powerful independent prognostic factors in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, the effect of therapy on the bone marrow (BM) microenvironment and its potential relationship with the MRD status and disease free survival (DFS) still remain to be investigated. Here we analyzed the distribution of mesenchymal stem cells (MSC) and endothelial cells (EC) in the BM of treated BCP-ALL patients, and its relationship with the BM MRD status and patient outcome. For this purpose, the BM MRD status and EC/MSC regeneration profile were analyzed by multiparameter flow cytometry (MFC) in 16 control BM (10 children; 6 adults) and 1204 BM samples from 347 children and 100 adult BCP-ALL patients studied at diagnosis (129 children; 100 adults) and follow-up (824 childhood samples; 151 adult samples). Patients were grouped into a discovery cohort (116 pediatric BCP-ALL patients; 338 samples) and two validation cohorts (74 pediatric BCP-ALL, 211 samples; and 74 adult BCP-ALL patients; 134 samples). Stromal cells (i.e., EC and MSC) were detected at relatively low frequencies in all control BM (16/16; 100%) and in most BCP-ALL follow-up samples (874/975; 90%), while they were undetected in BCP-ALL BM at diagnosis. In control BM samples, the overall percentage of EC plus MSC was higher in children than adults (p = 0.011), but with a similar EC/MSC ratio in both groups. According to the MRD status similar frequencies of both types of BM stromal cells were detected in BCP-ALL BM studied at different time points during the follow-up. Univariate analysis (including all relevant prognostic factors together with the percentage of stromal cells) performed in the discovery cohort was used to select covariates for a multivariate Cox regression model for predicting patient DFS. Of note, an increased percentage of EC (>32%) within the BCP-ALL BM stromal cell compartment at day +78 of therapy emerged as an independent unfavorable prognostic factor for DFS in childhood BCP-ALL in the discovery cohort—hazard ratio (95% confidence interval) of 2.50 (1−9.66); p = 0.05—together with the BM MRD status (p = 0.031). Further investigation of the predictive value of the combination of these two variables (%EC within stromal cells and MRD status at day +78) allowed classification of BCP-ALL into three risk groups with median DFS of: 3.9, 3.1 and 1.1 years, respectively (p = 0.001). These results were confirmed in two validation cohorts of childhood BCP-ALL (n = 74) (p = 0.001) and adult BCP-ALL (n = 40) (p = 0.004) treated at different centers. In summary, our findings suggest that an imbalanced EC/MSC ratio in BM at day +78 of therapy is associated with a shorter DFS of BCP-ALL patients, independently of their MRD status. Further prospective studies are needed to better understand the pathogenic mechanisms involved.

Center of Biostatistics for Clinical Epidemiology Department of Health Science University of Milano Bicocca 20126 Milano Italy

Childhood Leukaemia Investigation Prague Department of Paediatric Haematology and Oncology 2nd Faculty of Medicine Charles University University Hospital Motol 15006 Prague Czech Republic

Cytometry Service Instituto de Puericultura e Pediatria Martagão Gesteira Rio de Janeiro 21941 912 Brazil

Department of Immunohematology and Blood Transfusion Leiden University Medical Center 2333 ZA Leiden The Netherlands

Department of Immunology Erasmus MC University Medical Center Rotterdam Dr Molewaterplein 80 3015 CN Rotterdam The Netherlands

Department of Microbiology and Immunology Medical University of Silesia in Katowice 41 808 Zabrze Poland

Department of Pediatric Hematology and Oncology Medical University of Silesia in Katowice 41 800 Zabrze Poland

Department of Pediatric Hematology University Hospital Schleswig Holstein Campus Kiel 24105 Kiel Germany

Institut Català d'Oncologia Hospital Germans Trias 1 Pujol Josep Carreras Research Institute Badalona Universitat Autònoma de Barcelona 08193 Barcelona Spain

Internal Medicine Postgraduate Program Faculty of Medicine Federal University of Rio de Janeiro Rio de Janeiro 21941 617 Brazil

M Tettamanti Foundation Research Center Department of Pediatrics University of Milano Bicocca 20900 Monza Italy

Pediatric Hematology Oncology Unit Department of Pediatrics University of Milano Bicocca MBBM Foundation ASST Monza 20900 Monza Italy

Princess Maxima Center for Pediatric Oncology 3584 CS Utrecht The Netherlands

Translational and Clinical Research Program Centro de Investigación del Cáncer and IBMCC 37007 Salamanca Spain

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