7/7/23 9:42 CD34+CD19−CD22+ B-cell progenitors may underlie phenotypic escape in patients treated with CD19-directed therapies - PMC Blood. 2022 Jul 7; 140(1): 38–44. PMCID: PMC9346957 doi: 10.1182/blood.2021014840: 10.1182/blood.2021014840 PMID: 35421218 CD34+CD19−CD22+ B-cell progenitors may underlie phenotypic escape in patients treated with CD19-directed therapies 13 Clara Bueno, Susana Barrera,2,4 Alex Bataller,1,5 Valentín Ortiz-Maldonado,5 Natalina Elliot,6 Sorcha O’Byrne,6 Guanlin Wang,7,8 Montse Rovira,8 Francisco Gutierrez-Agüera,1 Juan L. Trincado,1,3 María González-González,2,4 Mireia Morgades,9 Marc Sorigué,9,10 Paloma Bárcena,2,4 Samanta Romina Zanetti,1 Montse Torrebadell,1113 Nerea Vega-Garcia,11,12 Susana Rives,13,14 Mar Mallo,10 Francesc Sole,10 Adam J. Mead,6,13 Irene Roberts,6,7,15 Supat Thongjuea,6,7,14 Bethan Psaila,6,15 Manel Juan,3,16,17 Julio Delgado,2,3,5,16 Alvaro Urbano-Ispizúa,1,3,5,16 Josep María Ribera,3,9,10 Alberto Orfao,2,4 Anindita Roy,6,7,15 and Pablo Menendez 13,18,19 1 Josep Carreras Leukemia Research Institute, Campus Clinic, Barcelona, Spain; 2 Centro de Investigación Biomédica en Red en Oncología (CIBER-ONC) and 3 Red Española de Terapias Avanzadas (TERAV), Redes de Investigación Cooperativa Orientadas a Resultados en Salud (RICORS RD21/0017/0029), Instituto de Salud Carlos III (ISCIII), Madrid, Spain; 4 Cancer Research Center (IBMCC-CSIC/USAL-IBSAL), Cytometry Service (NUCLEUS) and Department of Medicine, University of Salamanca, Salamanca, Spain; 5 Department of Clinical Hematology, Hospital Clinic of Barcelona, Barcelona, Spain; 6 Department of Paediatrics, Children's Hospital, John Radcliffe Hospital, 7 MRC (Medical Research Council) Molecular Haematology Unit, and 8 Centre for Computational Biology, MRC Weatherall Institute of Molecular Medicine (WIMM), University of Oxford, Oxford, United Kingdom; 9 Clinical Hematology Department, ICO-Hospital Germans Trias i Pujol, Badalona, Spain; 10 Josep Carreras Leukaemia Research Institute (IJC), Campus ICO-Hospital Germans Trias i Pujol, Universitat Autònoma de Barcelona (UAB), Badalona, Spain; 11 Haematology Laboratory, Hospital Sant Joan de Déu, University of Barcelona, Barcelona, Spain; 12 Developmental Tumor Biology Group, Leukemia and Other Pediatric Hemopathies, Institut de Recerca Sant Joan de Déu, Barcelona, Spain; 13 Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), ISCIII, Madrid, Spain; 14 Department of Clinical Hematology, Hospital Sant Joan de Déu, Barcelona, Spain; 15 National Institute for Health Research (NIHR) Oxford Biomedical Research Centre, Oxford, United Kingdom; 16 Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain; 17 Immunology Department, Hospital Clinic of Barcelona, Barcelona, Spain; 18Department of Biomedicine, School of Medicine, University of Barcelona, Barcelona, Spain; and 19 Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain Corresponding author. Received 2021 Nov 17; Accepted 2022 Mar 13. Copyright © 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9346957/?report=printable 1/14 7/7/23 9:42 CD34+CD19−CD22+ B-cell progenitors may underlie phenotypic escape in patients treated with CD19-directed therapies - PMC Key Points Preleukemic CD34+CD19−CD22+ immature progenitors may underlie phenotypic escape in patients treated with CD19-directed immunotherapies. The study contributes to identifying patients with B-ALL at risk of failure of CD19-targeted therapy. Visual Abstract Abstract CD19-directed immunotherapies have revolutionized the treatment of advanced B-cell acute lymphoblastic leukemia (B-ALL). Despite initial impressive rates of complete remission (CR) many patients ultimately relapse. Patients with B-ALL successfully treated with CD19-directed T cells eventually relapse, which, coupled with the early onset of CD22 expression during B-cell development, suggests that preexisting CD34+CD22+CD19− (pre)-leukemic cells represent an “early progenitor origin-related” mechanism underlying phenotypic escape to CD19-directed immunotherapies. We demonstrate that CD22 expression precedes CD19 expression during B- cell development. CD34+CD19−CD22+ cells are found in diagnostic and relapsed bone marrow samples of ∼70% of patients with B-ALL, and their frequency increases twofold in patients with B-ALL in CR after CD19 CAR T-cell therapy. The median of CD34+CD19−CD22+ cells before treatment was threefold higher in patients in whom B-ALL relapsed after CD19-directed im‐ munotherapy (median follow-up, 24 months). Fluorescence in situ hybridization analysis in flow-sorted cell populations and xenograft modeling revealed that CD34+CD19−CD22+ cells harbor the genetic abnormalities present at diagnosis and initiate leukemogenesis in vivo. Our data suggest that preleukemic CD34+CD19−CD22+ progenitors underlie phenotypic escape af‐ ter CD19-directed immunotherapies and reinforce ongoing clinical studies aimed at CD19/CD22 dual targeting as a strategy for reducing CD19− relapses. The implementation of CD34/CD19/CD22 immunophenotyping in clinical laboratories for initial diagnosis and subse‐ quent monitoring of patients with B-ALL during CD19-targeted therapy is encouraged. Introduction Both bispecific antibody-based (blinatumomab) and cell-based (chimeric antigen receptor [CAR]), CD19-directed immunotherapies have been transformational in the treatment of re‐ lapsed or refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL).1 Despite high rates of complete response (CR), many patients eventually relapse over time.2 On the one hand, re‐ lapses that retain CD19 expression (CD19+ relapses) may be observed and are commonly as‐ sociated with poor T-cell function or loss of CAR T cells, most likely because of T-cell intrinsic immune-regulatory exhaustion signals.3-5 On the other hand, relapses where the disease re‐ curs with loss of CD19 expression (CD19− relapses) have also emerged as a major clinical chal‐ lenge. Mechanisms of lineage switch or restrained membrane expression of CD19 (ie, splice variants or truncating mutations in the CD19 gene) underlie the loss of CD19, which repre‐ sents a widely accepted immune escape mechanism to CD19-directed immunotherapies.6-12 The intrinsic properties of leukemic cells and the tumor microenvironment surely contribute to the mechanisms underlying resistance to CD19-directed immunotherapy pressure.13 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9346957/?report=printable 2/14 7/7/23 9:42 CD34+CD19−CD22+ B-cell progenitors may underlie phenotypic escape in patients treated with CD19-directed therapies - PMC Normal human B-cell development occurs through multistep commitment to B cells from hematopoietic stem/progenitor cells (HSPCs),14,15 which is characterized by serial acquisition of B-cell–associated cell surface antigens, with CD19 being the definitive marker of B-cell com‐ mitment, almost always strongly expressed on B-ALL cells.16 Human fetal B-lymphoid develop‐ ment has been recently described with CD34+CD19+CD10− PreProB progenitors identified as the earliest B-committed progenitors (Figure 1A). However, the panspecific B-cell surface pro‐ tein CD22 is considered to be a very early marker for B-cell commitment and its expression precedes that of CD19.17 In fact, dual targeting of CD19 and CD22 is being clinically investi‐ gated as a strategy to overcome single target-associated immune escape.18,19 Relapse of patients with B-ALL who had achieved CR after successful anti-CD19–directed CAR T-cell therapy coupled to the very early onset of CD22 expression during B-cell development prompted us to hypothesize that preexisting CD22+CD19− (pre)-leukemic cells may represent an early/immature progenitor origin-related mechanism that underlies phenotypic escape to CD19-directed immunotherapies. In fact, an elegant single-cell RNA-sequencing (scRNA-seq) study profiled leukemic cells from 1 patient with B-ALL who experienced CD19− B-ALL relapse after CD19-directed CAR T-cell therapy and showed that CD19− leukemic cells were already present before CAR T-cell therapy, suggesting that relapse emerges from the selection of rare CD19− B-ALL clones.20 In this study, using bulk and scRNA-seq on multiple cell populations across fetal human B-cell development and immunophenotyping characterization of healthy fetal, neonatal, and postna‐ tal CD34+progenitors, we demonstrate that CD22 precedes CD19 in normal B-cell development and that CD34+CD19−CD22+ early/immature progenitors are found in most patients with re‐ lapsed B-ALL. A trend toward an increased frequency of these cells before treatment was ob‐ served in patients with R/R B-ALL who had a relapse after CD19-directed immunotherapy. Fluorescence in situ hybridization (FISH) studies in flow-sorted cell populations, together with in vivo xenograft modeling, revealed that CD34+CD19−CD22+ cells harbor the genetic abnor‐ malities present at diagnosis and initiate leukemogenesis in vivo, phenocopying the primary di‐ agnostic B-ALL. We suggest that preleukemic CD34+CD19−CD22+ immature progenitors under‐ lie phenotypic escape in patients treated with CD19-directed immunotherapies. Our study helps to identify patients at risk of CD19-targeted therapy failure. Study design Detailed information on B-ALL and healthy primary samples, clinical correlations, bulk RNA- seq, scRNA-seq, immunophenotyping, FISH on flow-sorted CD34+ subpopulations, in vivo xenografts, targeted next-generation sequencing, and optical genome mapping (OGM) can be found in the supplemental Methods (available on the Blood Web site). The study was approved by the institutional review board of the Ethics Committee on Clinical Research of the Clinic Hospital of Barcelona (HCB/2013/868) and the Barcelona Biomedical Research Park Ethics Committee on Animal Experimentation (HRH-17-0029-P1). Ethics regard‐ ing use of human fetal samples were also covered (18/NE/0290 and 18/LO/0822). https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9346957/?report=printable 3/14 7/7/23 9:42 CD34+CD19−CD22+ B-cell progenitors may underlie phenotypic escape in patients treated with CD19-directed therapies - PMC Results and discussion CD22 expression precedes CD19 in normal B-cell development To define the earliest HSPC fractions that express CD19 and CD22, both at gene and protein level, we examined specific HSPC populations from second-trimester fetal bone marrow (FBM) (Figure 1A) by bulk and scRNA-seq and by multiparametric flow cytometry. There was weak expression of CD19 on cells of the lymphoid-primed multipotent progenitor (%s) and the early lymphoid progenitor (ELP) stages, but its expression progressively increased with B-cell com‐ mitment in PreProB and ProB progenitors. However, CD22 was readily expressed at the LMPP stage and was robustly upregulated in ELPs, which showed CD22 expression levels similar to those of PreProB and ProB progenitors (Figure 1A-B). This finding was confirmed by flow cy‐ tometry where CD22 expression was positive in 1.5% ± 1.2% of HSCs/MPPs, 9% ± 5.4% of LMPPs, and 77% ± 8.9% of ELPs (Figure 1C). Using our own scRNA-seq transcriptomic dataset,21 CD22 expression was confirmed in Lin−CD19−CD34+ fetal liver (FL)- and FBM-HSPCs at a single-cell level, with 2% of FL and 8% of FBM Lin−CD19−CD34+ cells expressing CD22, re‐ spectively (Figure 1D-E). In addition, ∼13% of FL- and FBM-CD34+ HSPCs and 3% to 5% of postnatal (cord blood and adult BM)–derived CD34+ cells are CD19−CD22+, as confirmed by flow cytometry (Figure 1F). These data confirm that CD22 precedes CD19 in normal human B- cell development in very early lymphoid/B progenitors. CD34+CD19−CD22+ (pre)-leukemic progenitors are present in patients with B-ALL, harbor the diagnostic genetic abnormalities, and initiate leukemogenesis in vivo phenocopying the disease The relapse of patients with B-ALL who had achieved CR after successful anti-CD19–targeted immunotherapy and our data showing the very early onset of CD22 expression during B-cell development suggest that preexisting CD34+CD19−CD22+ (pre)-leukemic cells represent early lymphoid/B progenitors underlying the phenotypic escape of B-ALL in response to CD19-di‐ rected immunotherapies. Accordingly, Rabilloud et al have very recently shown in a patient with B-ALL that CD19− leukemic cells are already present before CAR T-cell therapy, suggesting that relapse emerges from the selection of rare CD19− B-ALL clones.20 We next investigated the presence of CD34+CD19−CD22+ cells in nonmatched BM samples from 237 patients with B- ALL. CD34+CD19−CD22+ early lymphoid/B progenitors were found in 72% (mean, 2.1% ± 7.6%), 92% (mean, 6.1% ± 17%), and 66% (mean, 1.6% ± 3.4%) of diagnostic, CR, and re‐ lapsed B-ALL BM samples, respectively (Table 1; Figure 2A). This CD34+CD19−CD22+ popula‐ tion was similarly represented among the major cytogenetic/molecular groups (data not shown). In addition, the frequency of CD34+CD19−CD22+ cells was 1.7-fold higher (3.8 ± 2.9 vs 2.1 ± 1.8; P = .016) in BM on day +30 than before CAR T-cell infusion, in 15 of 20 (75%) pa‐ tients with B-ALL who achieved CR after CD19-CAR T-cell therapy (Figure 2B). This results is indicative of a relative increase in the proportion of CD34+CD19−CD22+ cells as a result of the targeted elimination of CD19+ blasts. We next analyzed 53 patients with R/R B-ALL treated with CD19-directed immunotherapy (n = 37 CD19− CAR T-cells; n = 16 blinatumomab) to as‐ sess the impact of CD34+CD19−CD22+ cells on disease relapse risk within a median follow-up of 24 months (Table 2). The median of CD34+CD19−CD22+ cells before treatment was three‐ fold higher in patients with B-ALL who had a relapse after CD19-directed immunotherapy (1% https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9346957/?report=printable 4/14 7/7/23 9:42 CD34+CD19−CD22+ B-cell progenitors may underlie phenotypic escape in patients treated with CD19-directed therapies - PMC vs 0.3%; P > .05; Figure 2C). Furthermore, the cumulative incidence of relapse was analyzed by splitting our cohort into 2 groups, according to the frequency of CD34+CD19−CD22+ cells. Although not statistically significant, a trend toward a higher cumulative incidence of relapse was found in patients with a frequency of CD34+CD19−CD22+ cells above the mean (supple‐ mental Figure 1), suggesting that the presence of CD34+CD19−CD22+ cells negatively impacts relapse-free survival. This premise must be validated on a larger, more homogeneous series of patients. We next assessed whether CD34+CD19−CD22+ cells were normal hematopoietic progenitors or harbored the molecular oncogenic lesions observed at disease presentation. CD34+CD19−CD22+ and CD34+CD19+CD22+ cells from 3 patients with B-ALL harboring differ‐ ent genetic lesions (11q23, t(8;14), MLL+) were purified by fluorescence-activated cell sorting at disease presentation, immediately fixed, and subjected to interphase FISH analysis with com‐ mercial chromosomal probes routinely used in diagnostic laboratories. CD34+CD19−CD22+ cells from all 3 patients harbored the molecular lesion (15%, 32%, and 93%, respectively; Figure 2D-E). These data strongly support previous studies demonstrating that the initiating molecular lesions in B-ALL arise very early during B-cell development, in pre-VDJ progenitors that are likely to be CD19−.24-26 The cell of origin and the leukemia-initiating cell may represent different entities. We finally wanted to assess whether these CD34+CD19−CD22+ (pre)-leukemic progenitors can initiate leukemia in vivo. CD34+CD19−CD22+ or CD34+CD19+CD22+ cells (1.3 × 105) from pooled sam‐ ples (because of the rarity of these CD34+CD19−2CD22+ cells) from CD19 CAR T-cell–treated patients with B-ALL were flow sorted and transplanted into sublethally irradiated NSG mice by intraosseous transplantation (Figure 2F). Strikingly, both CD34+CD19−CD22+ and CD34+CD19+CD22+ populations initiated leukemia in vivo that phenocopied the primary diag‐ nostic B-ALL (CD45+CD19+CD22+; Figure 2G). The leukemic identity of the human graft from CD34+CD19−CD22+ cells was confirmed by detecting the pathogenic alterations found at diag‐ nostic by targeted next-generation sequencing and optical genome mapping (Figure 2H). This, together with the B-lymphoid–skewed graft in the absence of a myeloid graft (Figure 2G), con‐ firms that the leukemic graft stems from a (pre)-leukemic B-cell clone and not from residual healthy HSPCs. We report a possible “phenotypic escape” mechanism of resistance to CD19-directed im‐ munotherapies. We describe the existence of (pre)-leukemic CD34+CD22+CD19− immature B- cell progenitors that are not recognized by CD19-directed CAR T cells. This phenotypic escape mechanism differs from, but does not necessarily lead to, “immune escape” mechanisms driven by alternative splicing or CD19 mutations, resulting in CD19 downmodulation as a response to the antigenic pressure by CD19-directed T-cell therapies. Our data reinforce the ongoing thera‐ peutic clinical studies aimed at simultaneously targeting CD19 and CD22 as a strategy for re‐ ducing the likelihood of CD19− relapses and encourage the implementation of CD34/CD19/CD22 panels in flow cytometry diagnostic laboratories for initial diagnosis and follow-up monitoring of patients with B-ALL during CD19-targeted therapy. Supplementary Material The online version of this article contains a data supplement. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9346957/?report=printable 5/14 7/7/23 9:42 CD34+CD19−CD22+ B-cell progenitors may underlie phenotypic escape in patients treated with CD19-directed therapies - PMC View large: Download slide: Download PPT: Download PPT: Supplement File 1: Acknowledgments The authors thank CERCA/Generalitat de Catalunya and Fundació Josep Carreras-Obra Social la Caixa for core support. The work in P.M. and C.B.’s Laboratory was supported by the European Research Council (CoG-2014-646903, PoC-2018-811220) and the Spanish Ministry of Economy and Competitiveness (SAF2016-80481R, PID2019-108160RB-I00) (P.M.); the ISCIII (ISCIII/FEDER, PI17/01028 and PI20/00822), the Spanish Association against Cancer (AECC), and the FERO Foundation (C.B.). P.M. and J.M.R. acknowledge the support of ISCIII-RICORS within the Next Generation EU program (Plan de Recuperació n, Transformació n y Resiliencia). N.E. is supported by Cancer Research UK and a Children and Young People’s Cancer Innovation Award (DRCPGM\100058). A.R. is supported by a Wellcome Trust Clinical Research Career Development Fellowship (216632/Z/19/Z). A.O. was supported by ISCIII (PI19/011183). F.S. was supported by AGAUR/Generalitat de Catalunya (SGR288). The single-cell transcriptomic analysis was supported by MRC Discovery award MRCDA 0816-11 and the MRC WIMM Single Cell Facility and MRC-funded Oxford Consortium for Single-Cell Biology (MR/M00919X/1) pro‐ vided assistance. The human fetal tissue was provided by the Joint MRC/Wellcome Trust (grant MR/R006237/1) Human Developmental Biology Resource (http://hdbr.org). Footnotes All the transcriptomic data have already been published, and the data have been deposited in the National Center for Biotechnology Information’s Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/) with the following accession numbers: 10× single-cell data: GSE1552592521; FBMA-seq data: GSE122982.27 Original data are available upon reasonable request. All data relevant to the study are included in the article or up- loaded as supplemental information. The online version of this article contains a data supplement. There is a Blood Commentary on this article in this issue. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9346957/?report=printable 6/14 7/7/23 9:42 CD34+CD19−CD22+ B-cell progenitors may underlie phenotypic escape in patients treated with CD19-directed therapies - PMC Authorship Contribution: C.B. conceived the study, designed and performed the experiments, analyzed and interpreted the data, prepared the figures, wrote the manuscript, and financially supported the study; S.B., F.G.-A., J.L.T., M.G., P.B., S.R.Z., N.V., M. Mallo, F.S., N.E., and S.O. performed the experi‐ ments and analyzed the data; G.W., B.P., and S.T. performed the computational analysis and de‐ signed the pipelines for the transcriptomic analysis; A.B. provided the clinical data and ana‐ lyzed the data; V.O.-M., M.R., M. Morgades, M.S., M.T., S.R., M.J., J.D., A.U.-I., J.M.R., and A.O. pro‐ vided the human primary samples and the clinical data; A.J.M. and I.R provided the financial supported for the study (MR/M00919X/1); A.R. performed the experiments, prepared the fig‐ ures, and wrote the manuscript; P.M., conceived the study, designed the experiments, inter‐ preted data, wrote the manuscript, and financially supported the study; and all authors agreed to publish the manuscript. Conflict-of-interest disclosure: P.M. is a cofounder of the spinoff One Chain Immunotherapeutics. S.O. is now an employee of Becton Dickinson (BD). Her contributions to the work were made before her employment at BD commenced. The remaining authors de‐ clare no competing financial interests. 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Factors associated with the clinical outcome of patients with relapsed/refractory CD19+ acute lymphoblastic leukemia treated with ARI-0001 CAR T19-cell therapy. J Immunother of Cancer 2021;9:e003644. [PMCID: PMC8671976] [PubMed: 34907029] https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9346957/?report=printable 8/14 7/7/23 9:42 CD34+CD19−CD22+ B-cell progenitors may underlie phenotypic escape in patients treated with CD19-directed therapies - PMC 24. Agraz-Doblas A, Bueno C, Bashford-Rogers R, et al.. Unraveling the cellular origin and clinical prognostic markers of infant B-cell acute lymphoblastic leukemia using genome-wide analysis. Haematologica. 2019;104(6):1176-1188. [PMCID: PMC6545849] [PubMed: 30679323] 25. Bueno C, Tejedor JR, Bashford-Rogers R, et al.. Natural history and cell of origin of TC F3-ZN F384 and PTPN11 mutations in monozygotic twins with concordant BCP-ALL. Blood. 2019;134(11):900-905. [PubMed: 31221673] 26. Menendez P, Catalina P, Rodríguez R, et al.. Bone marrow mesenchymal stem cells from infants with MLL-AF4+ acute leukemia harbor and express the MLL-AF4 fusion gene. J Exp Med. 2009;206(13):3131-3141. [PMCID: PMC2806455] [PubMed: 19995953] 27. O’Byrne S, Elliott N, Sobhan R, et al.. Discovery of a CD10-negative B-progenitor in human fetal life identifies unique ontogeny-related developmental programs. Blood. 2018;134(13)1069-1071. [PubMed: 31383639] https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9346957/?report=printable 9/14 7/7/23 9:42 CD34+CD19−CD22+ B-cell progenitors may underlie phenotypic escape in patients treated with CD19-directed therapies - PMC Figures and Tables https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9346957/?report=printable 10/14 7/7/23 9:42 CD34+CD19−CD22+ B-cell progenitors may underlie phenotypic escape in patients treated with CD19-directed therapies - PMC Figure 1. CD22 expression precedes CD19 expression during normal human B-cell differentiation. (A) Cartoon depicting the immunophenotypes used to flow sort the indicated HSPC populations across human fetal B-cell differentiation. Lineage markers: Lin: CD2/3/14/16/19/56/235a; Lin2: CD2/3/14/16/56/235a. (B) Bulk RNA-seq–normalized gene expression (transcripts per million [TPM]) of CD19 (left) and CD22 (right) in flow-sorted hematopoietic stem cells (HSCs), multipotent progenitors (MPPs), lymphoid-primed multipotent progenitors (LMPPs), early lymphoid progeni‐ tors (ELPs), Pre-ProB progenitors, ProB progenitors, and PreB cells from human FBM (n = 3). (C) Percentage of CD22+ cells within these FBM-derived HSPC populations analyzed by flow cytometry. (D) scRNA-seq UMAP plots showing CD22 expression within flow-sorted Lin−CD19−CD34+ cells from matched human FL (left) and human FBM (right) (n = 2; total, 30 000 cells). (E) Violin plots showing the log normalized median expression levels of CD22 in CD19−Lin−CD34+ cells from human FL and FBM. (F) Frequency of CD34+CD19−CD22+ cells in healthy FL (n = 8), FBM (n = 9), neonatal CB (n = 10), and adult BM (n = 13). Red horizontal lines represent the median. Inset: a representative FACS plot of CD22 and CD19 within the gated CD34+ cells. Blue cells are non-B-progenitor cells (mainly myeloid). Red cells are CD19+CD22+ bona fide B-cell progenitors. Green cells are early CD22+CD19− B-cell progenitors. CB, cord blood; FACS, fluorescence activated cell sorting; UMAP, Uniform Manifold Approximation and Projection. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9346957/?report=printable 11/14 7/7/23 9:42 CD34+CD19−CD22+ B-cell progenitors may underlie phenotypic escape in patients treated with CD19-directed therapies - PMC Table 1. Clinical characteristics of patients with B-ALL according to flow cytometry immunophenotyping Age, y (range) 40 (17-60) Sex, % Male 50.7 Female 49.3 Genetics, n t(9;22)+ 26 Aneuploid 24 TCF3* 12 Normal karyotype 18 AML1 Gain 10 t(14q32)(IGH) 8 MLL† 6 TELdel 5 del(9) 4 t(12;21)+ 5 t(9;12)+ 3 CRLF2mut 2 t(4;17)+ 1 t(12;13)+ 1 t(2;4)+ 1 t(X;10)+ 1 RB/p53mut 1 Failed karyotype 20 NA 89 N = 237. NA, not available. * Deletion/translocation. † Translocation/gain. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9346957/?report=printable 12/14 7/7/23 9:42 CD34+CD19−CD22+ B-cell progenitors may underlie phenotypic escape in patients treated with CD19-directed therapies - PMC Figure 2. CD34 +CD19 −CD22 + (pre)-leukemic progenitors are present in patients with B-ALL, harbor the genetic abnor‐ malities present at disease presentation, and initiate leukemogenesis in NSG mice, phenocopying the pri‐ mary B-ALL sample. (A) Frequency of CD34+CD19−CD22+ cells in nonmatched samples from patients with B-ALL at diagnosis (n = 159), CR (n = 63), and relapse (n = 15). The black horizontal line represents the mean. FACS analysis (top) of 3 representative patients with a high proportion (left), low proportion (middle), and absence (right) of CD34+CD19−CD22+ cells. (B) Frequency of CD34+CD19−CD22+ cells in 20 patients with B-ALL before (pre) and after (post, BM day +30) treatment with CD19-directed CAR T-cells. Top panel shows representative FACS analysis of CD22 and CD19 within the gated CD34+ cells from patients with B-ALL before and after CD19-directed CAR T-cell infusion. Orange dots identify CD34+CD19−CD22+ cells. (C) Frequency of CD34+CD19−CD22+ cells in 53 patients with R/R B-ALL treated with CD19-directed immunotherapy (n = 37 CD19−CAR T-cells; n = 16 blinatumomab) who did or did not have a relapse after a median follow-up of 24 months. (D) Representative FISH analysis performed in flow-sorted CD34+CD19- CD22+ and CD34+CD19+CD22+ cells from 3 patients with B-ALL revealing the presence of the diagnostic genetic abnor‐ mality in preleukemic CD34+CD19−CD22+ progenitors. (E) Summary of the FISH analysis in diagnosed whole-BM and flow-sorted CD34+CD19−CD22+ and CD34+CD19+CD22+ cells. (F) Scheme of the experimental plan designed to study the ability of the preleukemic CD34+CD19−CD22+ progenitors and the bona fide CD34+CD19+CD22+ blasts to initiate B- ALL in vivo in NSG mice. Leukemic onset was evaluated every other week by PB bleeding. When leukemia was evident, mice were euthanized and the leukemic cells were characterized by FACS immunophenotyping and by NGSeq/Bionano technology to confirm the presence of the patient-specific genetic/molecular diagnostic alterations. (G) Representative https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9346957/?report=printable 13/14 7/7/23 9:42 CD34+CD19−CD22+ B-cell progenitors may underlie phenotypic escape in patients treated with CD19-directed therapies - PMC B-ALL engraftment in mouse transplant recipients of CD34+CD19−CD22+ or CD34+CD19+CD22+ B-ALL cells. Blue cells represent the CD45+HLA−ABC+ human graft. (H) Targeted NGSseq (left) and circus plot from Bionano (right) confirming the identity of the B-ALL graft from CD34+CD19−CD22+ cells. NGSeq, next generation sequencing. Table 2. Clinical features and type of CD19-directed immunotherapy received by the 53 patients with R/R B-ALL and used for clinicobiological correlations Group n Adult vs Pediatric Pediatric* 15 Adult 38 CD19-directed immunotherapy Blinatumomab 16 Kymriah 14 ARI001† 23 Clinical outcome Relapse 27 No relapse 26 Adapted from Ortiz-Maldonado et al. 22,23 * Age < 18 years. † Academic local CD19 CAR phase 1/2 trial (completed). https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9346957/?report=printable 14/14