Erythropoiesis has been extensively studied using in vitro and in vivo animal models. Despite this, there is still limited data about the gene expression profiles (GEP) of primary (ex vivo) normal... Show moreErythropoiesis has been extensively studied using in vitro and in vivo animal models. Despite this, there is still limited data about the gene expression profiles (GEP) of primary (ex vivo) normal human bone marrow (BM) erythroid maturation. We investigated the GEP of nucleated red blood cell (NRBC) precursors during normal human BM erythropoiesis. Three maturation-associated populations of NRBC were identified and purified from (fresh) normal human BM by flow cytometry and the GEP of each purified cell population directly analyzed using DNA oligonucleotide microarrays. Overall, 6569 genes (19% of the genes investigated) were expressed in >= 1 stage of BM erythropoiesis at stable (e.g., genes involved in DNA process, cell signaling, protein organization and hemoglobin production) or variable amounts (e.g., genes related to cell differentiation, apoptosis, metabolism), the latter showing a tendency to either decrease from stage 1 to 3 (genes associated with regulation of erythroid differentiation and survival, e.g., SPI1, STAT5A) or increase from stage 2 to stage 3 (genes associated with autophagy, erythroid functions such as heme production, e.g., ALAS1, ALAS2), iron metabolism (e.g., ISCA1, SLC11A2), protection from oxidative stress (e.g., UCP2, PARK7), and NRBC enucleation (e.g., ID2, RB1). Interestingly, genes involved in apoptosis (e.g., CASP8, P2RX1) and immune response (e.g., FOXO3, TRAF6) were also unregulated in the last stage (stage 3) of maturation of NRBC precursors. Our results confirm and extend on previous observations and providing a frame of reference for better understanding the critical steps of human erythroid maturation and its potential alteration in patients with different clonal and non-clonal erythropoietic disorders. Show less
Dasilva-Freire, N.; Mayado, A.; Teodosio, C.; Jara-Acevedo, M.; Alvarez-Twose, I.; Matito, A.; ... ; Orfao, A. 2019
Despite recent therapeutic advances, systemic mastocytosis (SM) remains an incurable disease due to limited complete remission (CR) rates even after novel therapies. To date, no study has evaluated... Show moreDespite recent therapeutic advances, systemic mastocytosis (SM) remains an incurable disease due to limited complete remission (CR) rates even after novel therapies. To date, no study has evaluated the expression on SM bone marrow mast cells (BMMC) of large panel of cell surface suitable for antibody-targeted therapy. In this study, we analyzed the expression profile of six cell-surface proteins for which antibody-based therapies are available, on BMMC from 166 SM patients vs. 40 controls. Overall, variable patterns of expression for the markers evaluated were observed among SM BMMC. Thus, CD22, CD30, and CD123, while expressed on BMMC from patients within every subtype of SM, showed highly variable patterns with a significant fraction of negative cases among advanced SM (aggressive SM (ASM), ASM with an associated clonal non-MC lineage disease (ASM-AHN) and MC leukemia (MCL)), 36%, 46%, and 39%, respectively. In turn, CD25 and Fc epsilon RI were found to be expressed in most cases (89% and 92%) in virtually all BMMC (median: 92% and 95%) from both indolent and advanced SM, but with lower/absent levels in a significant fraction of MC leukemia (MCL) and both in MCL and well-differentiated SM (WDSM) patients, respectively. In contrast, CD33 was the only marker expressed on all BMMC from every SM patient. Thus, CD33 emerges as the best potentially targetable cell-surface membrane marker in SM, particularly in advanced SM. Show less
Systemic mastocytosis (SM) is a highly heterogeneous disease with indolent and aggressive forms, with the mechanisms leading to malignant transformation still remaining to be elucidated. Here, we... Show moreSystemic mastocytosis (SM) is a highly heterogeneous disease with indolent and aggressive forms, with the mechanisms leading to malignant transformation still remaining to be elucidated. Here, we investigated the presence and frequency of genetic variants in 34 SM patients with multilineal KIT D816V mutations. Initial screening was performed by targeted sequencing of 410 genes in DNA extracted from purified bone marrow cells and hair from 12 patients with nonadvanced SM and 8 patients with advanced SM, followed by whole-genome sequencing (WGS) in 4 cases. Somatic mutations were further investigated in another 14 patients with advanced SM. Despite the fact that no common mutation other than KIT D816V was found in WGS analyses, targeted next-generation sequencing identified 67 nonsynonymous genetic variants involving 39 genes. Half of the mutations were somatic (mostly multilineal), whereas the other half were germline variants. The presence of >= 1 multilineal somatic mutation involving genes other than KIT D816V, >= 3 germline variants, and >= 1 multilineal mutation in the SRSF2, ASXL1, RUNX1, and/or EZH2 genes (S/A/R/E genes), in addition to skin lesions, splenomegaly, thrombocytopenia, low hemoglobin levels, and increased alkaline phosphatase and beta 2-microglobulin serum levels, were associated with a poorer patient outcome. However, the presence of >= 1 multilineal mutation, particularly involving S/A/R/E genes, was the only independent predictor for progression-free survival and overall survival in our cohort. Show less
Mayado, A.; Teodosio, C.; Dasilva-Freire, N.; Jara-Acevedo, M.; Garcia-Montero, A.C.; Alvarez-Twose, I.; ... ; Orfao, A. 2018
BackgroundRecent studies show that most systemic mastocytosis (SM) patients, including indolent SM (ISM) with (ISMs+) and without skin lesions (ISMs-), carry the KIT D816V mutation in PB leukocytes... Show moreBackgroundRecent studies show that most systemic mastocytosis (SM) patients, including indolent SM (ISM) with (ISMs+) and without skin lesions (ISMs-), carry the KIT D816V mutation in PB leukocytes. We investigated the potential association between the degree of involvement of BM hematopoiesis by the KIT D816V mutation and the distribution of different maturation-associated compartments of bone marrow (BM) and peripheral blood (PB) CD34(+) hematopoietic precursors (HPC) in ISM and identified the specific PB cell compartments that carry this mutation.MethodsThe distribution of different maturation-associated subsets of BM and PB CD34(+) HPC from 64 newly diagnosed (KIT-mutated) ISM patients and 14 healthy controls was analyzed by flow cytometry. In 18 patients, distinct FACS-purified PB cell compartments were also investigated for the KIT mutation.ResultsISM patients showed higher percentages of both BM and PB MC-committed CD34(+) HPC vs controls, particularly among ISM cases with MC-restricted KIT mutation (ISMMC); this was associated with progressive blockade of maturation of CD34(+) HPC to the neutrophil lineage from ISMMC to multilineage KIT-mutated cases (ISMML). Regarding the frequency of KIT-mutated cases and cell populations in PB, variable patterns were observed, the percentage of KIT-mutated PB CD34(+) HPC, eosinophils, neutrophils, monocytes and T cells increasing from ISMs-(MC) and ISMs+(MC) to ISMML patients.ConclusionThe presence of the KIT D816V mutation in PB of ISM patients is associated with (early) involvement of circulating CD34(+) HPC and multiple myeloid cell subpopulations, KIT-mutated PB CD34(+) HPC potentially contributing to early dissemination of the disease. Show less
