1:Josep Carreras Leukemia Research Institute. Campus Clinic. Barcelona. Spain.; 2:Centro de Investigación Biomédica en Red en Oncología (CIBER-ONC), ISCIII, Madrid, Spain.; 3:Red Española de Terapias Avanzadas (TERAV)-Instituto de Salud Carlos III (ISCIII) (RICORS, RD21/0017/0029), 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.; 6:Department of Paediatrics. Children's Hospital, John Radcliffe Hospital, and MRC WIMM, University of Oxford, Oxford, U.K.; 7:MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK.; 8:Centre for Computational Biology, MRC Weatherall Institute of Molecular Medicine (MRC WIMM), University of Oxford, Oxford, U.K.; 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), Instituto de Salud Carlos III, 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, U.K.; 16:Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS); 17:Immunology Department. Hospital Clinic of Barcelona.; 18:Department of Biomedicine. School of Medicine, University of Barcelona. Barcelona. Spain.; 19:Institució Catalana de Recerca i Estudis Avançats (ICREA). Barcelona. Spain.

CD19-directed immunotherapies have revolutionized the treatment of advanced B-ALL. Despite initial impressive rates of complete remission (CR) many patients ultimately relapse. The fact that B-ALL patients successfully treated with CD19-directed T-cells eventually relapse, coupled with the early onset of CD22 expression during B-cell development suggest that pre-existing CD34⁺CD22⁺CD19⁻ (pre)-leukemic cells could 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 BM samples of ~70% B-ALL patients, and their frequency increases 2-fold in B-ALL patients in CR after CD19-CAR T-cell therapy. Important, the median of CD34⁺CD19⁻CD22⁺ cells before treatment was 3-fold higher in those B-ALL patients who relapse after CD19-directed immunotherapy (median follow-up of 24 months). FISH analysis in flow-sorted populations and xenograft modeling revealed that CD34⁺CD19⁻CD22⁺ cells harbor the genetic abnormalities present at diagnosis and initiate leukemogenesis in NSG mice. We suggest that pre-leukemic CD34⁺CD19⁻CD22⁺ progenitors underlie phenotypic escape after CD19-directed immunotherapies. Our data reinforces the ongoing clinical studies aimed at simultaneously targeting CD19 and CD22 as a strategy to reduce CD19- relapses and encourages the implementation of such CD34/CD19/CD22 panel in flow cytometry clinical laboratories for initial diagnosis and subsequent monitoring of B-ALL patients during CD19-targeted therapy.