MLL-AF4 patient CD34+CD38−CD19+CD33− cells, which were found to contain the MLL rearrangement, also showed clonal bands (supplemental Table 5).

Disruption of SIRPα signaling in macrophages eliminates human acute myeloid leukemia stem cells in xenografts. (A-B,D) Sequential immunoperoxidase/APAAP staining; no counterstaining. In contrast, human CD19+ B cells in recipients of CD34+CD38−CD19−CD33− cells (case 3 recipient N-1/N-2, case 6 recipient N-2, case 15 recipient N-3, and case 17 recipient N-1/N-2 in supplemental Table 5) showed polyclonal IGH rearrangement patterns comparable to those of human B cells in normal CB HSC recipients (CB HSC-engrafted recipient-1/2 in supplemental Table 5) that exhibit physiological diversity of IGH rearrangements (case 3 shown as an example in Figure 4B). Yuki Aoki, Takashi Watanabe, Yoriko Saito, Yoko Kuroki, Atsushi Hijikata, Masatoshi Takagi, Daisuke Tomizawa, Mariko Eguchi, Minenori Eguchi-Ishimae, Akiko Kaneko, Rintaro Ono, Kaori Sato, Nahoko Suzuki, Saera Fujiki, Katsuyoshi Koh, Eiichi Ishii, Leonard D. Shultz, Osamu Ohara, Shuki Mizutani, Fumihiko Ishikawa; Identification of CD34+ and CD34− leukemia-initiating cells in MLL-rearranged human acute lymphoblastic leukemia. CD90 was negative in all cases (data not shown). In addition, MLL-AF9 CD34+CD38−CD19−CD33− cells generated CD10−CD20+ mature B cells without MLL rearrangement in the spleen (0 of 50 cells examined) and DP and SP T cells in the thymus (case 16 shown as an example in supplemental Figure 4). In 9 of 41 (21.9%) samples (Table 1; supplemental Table 1), a double CD34low and CD34bright cell population was observed (Figure 2E). The latter pattern of engraftment was characterized by areas of bone marrow infiltrated by human NPMc+ AML cells adjacent to areas of normal human trilineage hematopoiesis (Table 2; Figure 5).

Immunophenotyping was performed using the following antibodies: peridinin chlorophyll protein complex-conjugated anti-CD45 (CD45-peridinin chlorophyll protein complex), fluorescein isothiocyanate (FITC)–conjugated anti-CD34 (CD34−FITC), phycoerythrin (PE)–conjugated, or allophycocyanin (APC)–conjugated anti-CD38 (CD38-PE, CD38-APC), CD33-APC, and CD123-PE (Becton Dickinson [BD] Biosciences) or phycoerythrin-Texas Red (ECD)–conjugated anti-CD45 (CD45-ECD), CD34−FITC, phycoerythrin-cyanin 5 (PC5)–conjugated anti-CD38 (CD38-PC5), CD33-PE, phycoerythrin-cyanin 7 (PC7)–conjugated anti-CD19 (CD19-PC7), CD11b-FITC, CD90-FITC, and CD90-PC5 (Beckman Coulter). In 1 leukopenic patient (patient 19), only 0.1 × 106 CD34+ bone marrow cells (99.7% pure) could be recovered, which were not enough for WB analysis. Despite significant improvement in overall clinical outcomes of pediatric ALL in the last few decades, the prognosis in MLL-rearranged ALL has remained poor.

Definitive evidence that CD34+ cells belong to the leukemic clone and contain the LICs mainly apply to NPM1-mutated AML cases containing more than 1% of CD34+ cells. Evaluated by genomic DNA fragment analysis. Because down-regulation of CD34 at both RNA7,8  and protein1  level is a unique characteristic of NPM1-mutated AML, we monitored CD34 expression after engraftment of purified hCD34+ leukemic cells in immunocompromised mice. In normal CB, the frequency of normal CD34+CD38− hematopoietic stem/progenitor cells expressing these molecules was low, whereas CD34−CD19+ B cells express these antigens. CD34+CD38−CD19−CD33− cells in MLL ALL are enriched for normal hematopoietic stem/progenitor cells. Until the discovery of NPM1 mutation,1  the genetic and functional characterization of CD34+/CD38− cells in AML with normal karyotype was difficult because of the lack of reliable molecular markers. Mice were originally obtained from The Jackson Laboratory. Almost all CD34- patients (22/24) had P-glycoprotein activity lower than the median of normal bone marrow (nBM) CD34+ cells. Patients were from the Institutes of Hematology of the Universities of Perugia, Bari, and Catania (Italy), and Dresden (Germany). Moreover, immunohistochemistry frequently showed dot-like CD34 positivity in cytoplasm, which could reflect CD34 internalization and down-regulation. and V.P. From primary CD34+CD38+CD19+ cell recipient BM, 1000 CD34+CD38+CD19+ cells sorted from fraction 1 (Fr.1; Figure 2B) initiated leukemia in secondary recipients, demonstrated by engraftment in the PB, BM, and spleen. Panels A, B, and C are as designated in Figure 1. In MLL-ENL+ patient samples, there were 2 distinct CD34 expression patterns (cases 18 and 20 shown as examples in Figure 2D).

Expression of cytoplasmic NPM1 in CD34+ cells (both CD38− and CD38+ subsets) was also found by flow cytometry (Figure 3E-F). However, there is experimental evidence that, in some AML cases37,38  (including NPM1-mutated AML21 ), also the CD34− population may contain the LICs. 14, 15 Engraftment capability of AML cells has been also associated with prognosis. Most or all of CD34+/CD38− cells were also CD123+ in 21 of 24 samples and CD33+ (generally at high intensity) in 14 of 15 samples (eg, patient 18 of Figure 2A; supplemental Table 2). Search for other works by this author on: Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype. CD34+ leukemic cell (brown) from the same case show nucleus-restricted positivity for C23/nucleolin (blue; D inset). In MLL-AF4+ ALL, dominant IGH clones detected in the donor (patient) cells were also present in the recipient BM CD34+CD38+CD19+ and CD34−CD19+ cells (indicated in red in supplemental Table 5). Leukemic engraftment of CD34+ cells from 10 NPM1-mutated AML in immunocompromised mice. Although LICs developed leukemic cells with clonal immunoglobulin heavy-chain (IGH) rearrangement in vivo, CD34+CD38−CD19−CD33− cells repopulated recipient bone marrow and spleen with B cells, showing broad polyclonal IGH rearrangement and recipient thymus with CD4+ single positive (SP), CD8+ SP, and CD4+CD8+ double-positive (DP) T cells.

Inoculation of CD34− fraction (≥ 2 × 106 cells) from 3 cases (Table 4: patients 22, 27, and 28) resulted into marrow engraftment by hCD45+/hCD33+ cells (Figure 6A), which, in tissue sections, consisted of 2 populations (Figure 6B-D): (1) myeloperoxidase-positive cells located close to bone trabeculae that exhibited weak cytoplasmic NPM1 positivity (not shown); and (2) mature CD68+ histiocytes that were located in the central area of bone marrow. However, definitive conclusions on this issue can only be drawn from the study of additional cases, possibly searching for the NPM1 mutant by single-cell polymerase chain reaction analysis. CD34 and CD38 expression patterns in CD45+ leukocytes obtained from MLL-rearranged ALL patients are summarized in Figure 1D. and B.F. had the original idea for the study, designed experiments, and wrote the paper; V.P. These findings clearly show that the CD34+ fraction from NPM1-mutated AML with CD34+ cells representing more than 1% of the bulk cell population is mutated both at gene and protein level, thus indicating it belongs to the leukemic clone. We found that expression profiles clustered by patient not by surface phenotype, indicating that LIC gene expression signatures are relatively patient specific (supplemental Figure 6C). In search of tumor suppressing functions of menin. Our findings suggest the CD34− phenotype of NPM1-mutated AML developing in mice may be the result of CD34 down-regulation. Asterisk in panel B inset and in panel C indicates bone area. Summary of serial transplantations experiments. Positive marrow samples (> 0.1% hCD45+ cells) were further analyzed by immunophenotyping, immunohistochemistry, WB, and molecular analysis for NPM1-mutated protein and/or gene to confirm and characterize the type of engraftment. CD34− cells used for isotype controls were incubated for 20 minutes in 50 μL FITC-murine IgG (diluted 1:100; Coulter, Hialeah, FL) and 50 μL PE-murine IgG (diluted 1:50, Coulter). Cooperative activation of Hoxa and Pbx1-related genes in murine myeloid leukaemias. FLT3-ITD showed the highest prevalence in the CD34+/CD38− cells (Figure 2Di), whereas, in the more differentiated cells (CD34+/CD38+), the FLT3-ITD mut/wt ratio was significantly lower (Figure 2Dii) and, in the bulk leukemic population, FLT3-ITD was only present in a small fraction of cells (Figure 2Diii). M.P.M. AML engraftment in the NOD/SCID assay reflects the outcome of AML: implications for our understanding of the heterogeneity of AML. Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine. (E-F) Flow cytometric detection of NPM-cytoplasmic expression (patient 25) in CD34+ and CD34− blasts (E), and after additional gating on CD34+/CD38− or CD34+/CD38+ cells, respectively (F). Interestingly, starting from CD34+ cells with a purity of 98.3% and 99.9%, respectively, we observed a progressive reduction of the percentage of CD34+ cells within the leukemic bulk with time (eg, patient 17, from 52% at 9 weeks to 35% at 12 weeks and 14% at 20 weeks; Figure 5G-I; Table 2) and outgrowth of leukemia from 15.8% to 52% and 80% at correspondent time points (Table 2). (B-D) Tibia paraffin sections showing massive marrow infiltration by a double population: (1) mononuclear cells close to bone trabeculae (B double arrows; inset, from a different section), which are MPO+ and PGM1(CD68)+ (C-D double arrows); (2) mature histiocytes located in the central area of bone marrow (B single arrow), which are MPO− and PGM1+ (C-D single arrows). SATB1 is also involved in normal T cell immunity. Then we asked whether CD34+NPM1-mutated AML cells expressed aberrantly nucleophosmin in their cytoplasm, which is the most distinctive functional consequence of NPM1 mutations.33-36.

(A) Hematoxylin-eosin. This strategy was chosen to minimize the possibility that sorter errors in samples in which most of the cells were leukemic would lead to false-positive results. Gene expression profiling-based dissection of MLL translocated and MLL germline acute lymphoblastic leukemia in infants. Notably, transplanted hCD34+ cells gave rise to an NPM1-mutated AML mainly consisting of CD34− cells. In Ebf1 knockout mice, accumulation of early lymphoid progenitors that lack B-lineage priming but have T- and myeloid lineage potential occurs while mature B cells are completely absent.42,43  Mice transgenic for Zfp521, a murine homolog for ZNF521, were also reported to develop B-lineage ALL.44, Genes upregulated in MLL-ENL LICs compared with MLL-AF4 and MLL-AF9 LICs included SIRPA and SATB1. Translocation of the mixed-lineage leukemia (MLL) gene with AF4, AF9, or ENL results in acute leukemia with both lymphoid and myeloid involvement. AML development was usually seen later than normal human hematopoietic engraftment (Table 2). In 4 cases, the clonotypic leukemic Vδ2-Dδ3 rearrangement was detected in the CD34+CD38− population, indicating that the putative normal HSC population also contained leukemic cells. H3K79 methylation profiles define murine and human MLL-AF4 leukemias. We gratefully acknowledge Lora Barsky and Felix Burotto for their technical expertise in flow cytometry. Such a model would predict that leukemic progenitors would be present in both the CD34+CD38+ and CD34+CD38− populations. As previously reported by Taussig et al,21  we also found that CD34− cells from a few NPM1-mutated AML patients had significant LIC potential in immunocompromised mice. (A-C) Representative flow cytometry plots showing expression of CD34, CD38, CD19, and CD33 in cells derived from infant ALL patients with (A) MLL-AF4, (B) MLL-AF9, and (C) MLL-ENL translocations. The SIRP family of receptors and immune regulation. In MLL-AF9 case 14, CD24 was differentially expressed between HSC/HPC and LIC populations (supplemental Figure 7A).


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