CD34⁺CD19⁻CD22⁺ (pre)-leukemic progenitors are present in patients with B-ALL, harbor the genetic abnormalities present at disease presentation, and initiate leukemogenesis in NSG mice, phenocopying the primary 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 abnormality 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 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.