Hematopoietic stem cells in chronic myeloid leukemia (original) (raw)
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American Journal of Hematology, 2014
For decades now, it is well established that chronic myeloid leukemia (CML) is a hematopoietic stem cell (HPC) disorder. However, it remains to be determined whether BCR-ABL1 gene rearrangement occurs in a HPC or at an earlier stem cell and whether the degree of involvement of hematopoiesis by the BCR-ABL1 fusion gene relates to the response to therapy. Here, we have investigated by interphase fluorescence in situ hybridization (iFISH) the distribution of BCR-ABL1 fusion gene in FACS-sorted bone marrow (BM) populations of mesenchymal precursor cells (MPC) and other hematopoietic cell populations from 18 newly diagnosed CML patients. Overall, our results showed systematic involvement at relatively high percentages of BM maturing neutrophils (97% 6 15%), basophils (95% 6 12%), eosinophils (90% 6 8%), CD34 1 precursors cells (90% 6 7%), monocytes (84% 6 30%), nucleated red blood cells (87% 6 24%), and mast cells (77% 6 33%). By contrast, MPC (30% 6 34%), B-cells (15% 6 27%), T-lymphocytes (50% 6 26%), and NK-cells (35% 6 34%) were involved at lower percentages. In 8/18 CML patients, 2 tumor BCR-ABL1 1 subclones were detected by iFISH. Of note, all tumor cell subclones were systematically detected in CD34 1 cells, whereas MPC were only involved by the ancestral tumor cell subclone. In summary, here we confirm the presence at diagnosis of the BCR-ABL1 fusion gene in MPC, CD34 1 precursors, and other different BM hematopoietic myeloid cell lineages from CML patients, including also in a significant fraction of cases, a smaller percentage of T, B, and NK lymphocytes. Interestingly, involvement of MPC was restricted to the ancestral BCR-ABL1 1 subclone.
Leukemia, 1997
Philadelphia chromosome-positive (Ph + ) hemopoietic cells precan be mobilized, presumably from the bone marrow, into the dominate in patients with chronic myeloid leukemia (CML) in blood of CML patients following chemotherapy. The blood of chronic phase, but some Ph − presumably normal stem cells CML patients has also been used for autologous rescue followpersist in most patients. Ph − cells are relatively frequent, coming high-dose therapy and in some cases Ph chromosomepared to mature cell populations, in primitive hemopoietic cell negative recovery has been observed. 10 Some in vitro studies populations from CML patients. We have purified CD34 + cells from chronic phase CML blood and separated them into two have suggested that Ph − cells are restricted to the bone marrow fractions on the basis of adherence or non-adherence to tissue and do not circulate in CML patients; 11 others have indicated culture plastic. We also separated CD34 + CML cell populations that the long-term culture-initiating cell population in the into HLA-DR hi and HLA-DR lo fractions and CD38 hi and CD38 lo blood of CML patients is often predominantly, and sometimes fractions by flow cytometry. The CD34 + cells that adhered to exclusively, Ph − . 12 Moreover, the results of peripheral blood plastic were predominantly CD33 − , CD38 − and HLA − -DR; cells autografting indicate that Ph − , presumably normal, stem cells with these phenotypic properties were significantly rarer in the CD34 + non-adherent cell population (P = 0.008-0.02). circulate in CML patients in numbers that are sufficient to Expression of p210 BCR/ABL mRNA by adherent, non-adherrepopulate the hemopoietic system. 10 ent, HLA-DR hi and HLA-DR lo CD34 + cell subpopulations was The proportions of Ph chromosome-negative hemopoietic demonstrated by RT-PCR. Using fluorescence in situ hybridizcells are not uniform throughout all stages of hemopoietic cell ation (FISH) in conjunction with BCR and ABL probes we development. Rather, the Ph − cells appear to be more frequent detected Ph + and Ph − cells in both adherent and non-adherent in the primitive hemopoietic stem cell population implying CD34 + cell fractions of 15/15 patients studied and in the HLA-DR lo or CD38 lo sorted CD34 + cell fractions. The concentration that normal hemopoietic stem cell activity is suppressed In of Ph − cells in the adherent CD34 + cell fraction was three-fold CML. Most of these studies have been based on cell surface higher than in the non-adherent fraction (P = 0.001). Ph− adherantigen expression 13-17 leading to the possibility that benign ent cells were detected in untreated CML patients and as late and malignant cells in CML can be separated on the basis of as 6 years after diagnosis of CML in patients treated with hydtheir different phenotypes. 17 Leukemic progenitor cells in CML roxyurea (HU) or interferon-␣ (IFN-␣). We conclude that whilst also differ from normal cells in terms of their adhesive properappreciable numbers of Ph − primitive hemopoietic progenitors are present in the circulation in untreated patients and also in ties. 5,6 Therefore, it might be possible to exploit differential treated patients in late chronic phase, the majority of cells adhesion in the enrichment of Ph − progenitor cells in CML. expressing CD34 but not CD33, CD38 or HLA-DR antigens, are
…, 2004
In order to develop murine models of leukemogenesis, a series of transgenic mice expressing BCR-ABL in different hematopoietic cell subsets were generated. Here we describe targeted expression of P210 BCR-ABL in stem and progenitor cells of murine bone marrow using the tet-off system. The transactivator protein tTA was placed under the control of the murine stem cell leukemia gene (SCL) 3' enhancer. Induction of BCR-ABL resulted in neutrophilia and leukocytosis, and the mice became moribund within 29 to 122 days. Necropsy of sick mice demonstrated splenomegaly, myeloid bone marrow hyperplasia, and extramedullary myeloid cell infiltration of multiple organs. BCR-ABL mRNA and protein were detectable in the affected organs. FACS analysis demonstrated a significant increase of mature and immature myeloid cells in bone marrow and spleen together with increased bilineal B220+/Mac-1+ cells in the bone marrow. tTA mRNA was expressed in FACS-sorted hematopoietic stem cells which were expanded 26-fold after BCR-ABL induction. 31% of the animals demonstrated a biphasic phenotype, consisting of neutrophilia and subsequent B-cell lymphoblastic disease, reminiscient of blast crisis. In summary, this phenotype recapitulates many characteristics of human chronic myeloid leukemia (CML) and may help elucidate basic leukemogenic mechanisms in CML stem cells during disease initiation and progression.
Leukemia, 2009
We found that composition of cell subsets within the CD34 þ cell population is markedly altered in chronic phase (CP) chronic myeloid leukemia (CML). Specifically, proportions and absolute cell counts of common myeloid progenitors (CMP) and megakaryocyte-erythrocyte progenitors (MEP) are significantly greater in comparison to normal bone marrow whereas absolute numbers of hematopoietic stem cells (HSC) are equal. To understand the basis for this, we performed gene expression profiling (Affymetrix HU-133A 2.0) of the distinct CD34 þ cell subsets from six patients with CP CML and five healthy donors. Euclidean distance analysis revealed a remarkable transcriptional similarity between the CML patients' HSC and normal progenitors, especially CMP. CP CML HSC were transcriptionally more similar to their progeny than normal HSC to theirs, suggesting a more mature phenotype. Hence, the greatest differences between CP CML patients and normal donors were apparent in HSC including downregulation of genes encoding adhesion molecules, transcription factors, regulators of stem-cell fate and inhibitors of cell proliferation in CP CML. Impaired adhesive and migratory capacities were functionally corroborated by fibronectin detachment analysis and transwell assays, respectively. Based on our findings we propose a loss of quiescence of the CML HSC on detachment from the niche leading to expansion of myeloid progenitors.
Leukemia stem cells in a genetically defined murine model of blast-crisis CML
Blood, 2007
Myeloid leukemia arises from leukemia stem cells (LSCs), which are resistant to standard chemotherapy agents and likely to be a major cause of drug-resistant disease and relapse. To investigate the in vivo properties of LSCs, we developed a mouse model in which the biologic features of human LSCs are closely mimicked. Primitive normal hematopoietic cells were modified to express the BCR/ABL and Nup98/HoxA9 translocation products, and a distinct LSC population, with the aberrant immunophenotype of lineage−, Kit+/−, Flt3+, Sca+, CD34+, and CD150−, was identified. In vivo studies were then performed to assess the response of LSCs to therapeutic insult. Treatment of animals with the ABL kinase inhibitor imatinib mesylate induced specific modulation of blasts and progenitor cells but not stem- cell populations, thereby recapitulating events inferred to occur in human chronic myelogenous leukemia (CML) patients. In addition, challenge of leukemic mice with total body irradiation was selec...
Leukemia, 1999
In this study the ability of malignant and normal progenitors in peripheral blood (PB) and bone marrow (BM) of CML patients in chronic phase to proliferate and produce mature progeny after transplantation into hereditary immunodeficient (SCID and NOD/SCID) mice was examined. Engraftment in NOD/SCID mice preconditioned by total body irradiation (TBI) alone was 10-fold higher than in SCID mice preconditioned by macrophage depletion and TBI, demonstrating that NOD/SCID mice are more suitable for engraftment of chronic phase CML cells. Low-density cells at cell doses of 10-30 × 10 6 and purified CD34 ؉ cells at doses of approximately 0.2 × 10 6 engrafted NOD/SCID mice, with levels of 2 to 20% CD45 ؉ cells with production of mono-
Blood, 2012
BCR-ABL expression in leukemic progenitors and primitive stem cells of patients with chronic myeloid leukemia Kumari and colleagues have recently reported a detailed analysis of BCR-ABL expression in CFU-Cs (colony forming units in culture) of patients with chronic myeloid leukemia (CML). 1 Using quantitative reverse-transcription PCR on individual Ph1 hematopoietic colonies, they demonstrated that CFU-Cs from patients in major molecular response (MMR) displayed lower BCR-ABL mRNA expression than CFU-Cs from patients at diagnosis. In addition, the authors observed a large variability of BCR-ABL/ABL ratios in homogeneous subpopulations of CFU-Cs. These interesting findings were evaluated in the progenitor compartment and not in the more primitive stem cell compartment, including long-term-culture initiating cell (LTC-IC)-derived CFU-Cs. We have previously analyzed BCR-ABL-expressing leukemic progenitors and stem cells, either in the context of imatinib resistance 2 or that of sustained undetectable molecular residual disease (UMRD). 3 In the light of the data presented by Kumari et al, we wished to examine the BCR-ABL mRNA levels in individual progenitors and LTC-IC derived CFU-Cs from our experiments. In the first study of a patient with imatinib-resistant CML, 2 BCR-ABL/ABL ratios in CFU-Cs and LTC-IC-derived CFU-Cs were found to be similar, with a median value of 52.1% and 51% respectively, comparable with the ratio (68%) measured in peripheral blood (Figure 1). In the second work, 3 we have analyzed the BCR-ABL mRNA expression in 6 patients with sustained UMRD (Ͼ 3 years). We have detected persistent BCR-ABL-expressing stem cells not only in the patient on targeted therapy, but also in the other 5 patients, in whom IFN-␣ or imatinib treatments were discontinued for 2-13 years. The analysis of BCR-ABL/ABL ratios in hematopoietic colonies showed a large expression variability from one CFU-C to another, in accordance with Kumari et al. 1 Moreover, a significant difference was observed between the amount of BCR-ABL mRNA transcript in CFU-Cs and LTC-IC-derived progeny in the context of UMRD (Figure 1). Interestingly, primitive stem cells expressed significantly less BCR-ABL mRNA than committed progenitors (median BCR-ABL/ ABL ratios of 0.7% and 12%, respectively). The persistence of primitive leukemic stem cells in CML patients with UMRD has been recently documented, 3,4 demonstrating that, despite a major success in the eradication of bulk leukemic cells, imatinib therapy does not fully eradicate leukemic stem cells. Several biologic explanations for the intrinsic refractoriness of CML stem cells against imatinib have been proposed, 5,6 but a mechanism involving low BCR-ABL expression in these primary cells had not been suggested before. The variable BCR-ABL transcription by Ph1 progenitors has previously been described in patients with chronic phase CML. 7,8 Kumari and coworkers observed a lower BCR-ABL expression in individual hematopoietic progenitor colonies from patients with MMR, and suggested a relationship between this phenomenon and the persistence of leukemic stem cells resistant to imatinib. Our data, based on patients with sustained UMRD, show that low amounts of BCR-2964