Acute myeloid leukemia (AML) with t(8;21) or inv(16) have been recognized as unique entities within AML and are usually reported together as core binding factor AML (CBF-AML). However, there is ...considerable clinical and biological heterogeneity within this group of diseases, and relapse incidence reaches up to 40%. Moreover, translocations involving CBFs are not sufficient to induce AML on its own and the full spectrum of mutations coexisting with CBF translocations has not been elucidated. To address these issues, we performed extensive mutational analysis by high-throughput sequencing in 215 patients with CBF-AML enrolled in the Phase 3 Trial of Systematic Versus Response-adapted Timed-Sequential Induction in Patients With Core Binding Factor Acute Myeloid Leukemia and Treating Patients with Childhood Acute Myeloid Leukemia with Interleukin-2 trials (age, 1-60 years). Mutations in genes activating tyrosine kinase signaling (including KIT, N/KRAS, and FLT3) were frequent in both subtypes of CBF-AML. In contrast, mutations in genes that regulate chromatin conformation or encode members of the cohesin complex were observed with high frequencies in t(8;21) AML (42% and 18%, respectively), whereas they were nearly absent in inv(16) AML. High KIT mutant allele ratios defined a group of t(8;21) AML patients with poor prognosis, whereas high N/KRAS mutant allele ratios were associated with the lack of KIT or FLT3 mutations and a favorable outcome. In addition, mutations in epigenetic modifying or cohesin genes were associated with a poor prognosis in patients with tyrosine kinase pathway mutations, suggesting synergic cooperation between these events. These data suggest that diverse cooperating mutations may influence CBF-AML pathophysiology as well as clinical behavior and point to potential unique pathogenesis of t(8;21) vs inv(16) AML.
•Recurrent mutations in chromatin modifiers and cohesin were observed in t(8;21) AML, but not inv(16) AML.•t(8;21) AML patients with mutations in kinase signaling plus chromatin modifiers or cohesin members had the highest risk of relapse.
In vitro RBC production from stem cells could represent an alternative to classic transfusion products. Until now the clinical feasibility of this concept has not been demonstrated. We addressed the ...question of the capacity of cultured RBCs (cRBCs) to survive in humans. By using a culture protocol permitting erythroid differentiation from peripheral CD34+ HSC, we generated a homogeneous population of cRBC functional in terms of their deformability, enzyme content, capacity of their hemoglobin to fix/release oxygen, and expression of blood group antigens. We then demonstrated in the nonobese diabetes/severe combined immunodeficiency mouse that cRBC encountered in vivo the conditions necessary for their complete maturation. These data provided the rationale for injecting into one human a homogeneous sample of 1010 cRBCs generated under good manufacturing practice conditions and labeled with 51Cr. The level of these cells in the circulation 26 days after injection was between 41% and 63%, which compares favorably with the reported half-life of 28 ± 2 days for native RBCs. Their survival in vivo testifies globally to their quality and functionality. These data establish the proof of principle for transfusion of in vitro–generated RBCs and path the way toward new developments in transfusion medicine. This study is registered at http://www.clinicaltrials.gov as NCT0929266.
Mutations in receptor tyrosine kinase/RAS signaling pathway genes are frequent in core-binding factor (CBF) acute myeloid leukemias (AMLs), but their prognostic relevance is debated. A subset of CBF ...AML patients harbors several signaling gene mutations. Genotyping of colonies and of relapse samples indicates that these arise in independent clones, thus defining a process of clonal interference (or parallel evolution). Clonal interference is pervasive in cancers, but the mechanisms underlying this process remain unclear, and its prognostic impact remains unknown. We analyzed a cohort of 445 adult and pediatric patients with CBF AML treated with intensive chemotherapy and with deep sequencing of 6 signaling genes (KIT, NRAS, KRAS, FLT3, JAK2, CBL). A total of 152 (34%), 167 (38%), and 126 (28%) patients harbored no, a single, and multiple signaling clones (clonal interference), respectively. Clonal interference of signaling mutations was associated with older age (P = .004) and inv(16) subtype (P = .025) but not with white blood cell count or mutations in chromatin or cohesin genes. The median allele frequency of signaling mutations was 31% in patients with a single clone or clonal interference (P = .14). The repertoire of KIT, FLT3, and NRAS/KRAS variants differed between groups. Clonal interference did not affect complete remission rate or minimal residual disease after 1-2 courses, but it did convey inferior event-free survival (P < 10−4), whereas the presence of a single signaling clone did not (P = .44). This inferior outcome was independent of clinical parameters and of the presence of specific signaling clones. Our results suggest that specific clonal architectures can herald distinct prognoses in AML.
•Presence of ≥2 independent subclones in the receptor tyrosine kinase/RAS pathway, defining clonal interference, is found in 28% of CBF AMLs.•Clonal interference predicts shorter event-free survival independently of clinical variables and presence of specific signaling mutations.
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Ex vivo manufacture of red blood cells from stem cells is a potential means to ensure an adequate and safe supply of blood cell products. Advances in somatic cell reprogramming of human induced ...pluripotent stem cells have opened the door to generating specific cells for cell therapy. Human induced pluripotent stem cells represent a potentially unlimited source of stem cells for erythroid generation for transfusion medicine.
We characterized the erythroid differentiation and maturation of human induced pluripotent stem cell lines obtained from human fetal (IMR90) and adult fibroblasts (FD-136) compared to those of a human embryonic stem cell line (H1). Our protocol comprises two steps: (i) differentiation of human induced pluripotent stem cells by formation of embryoid bodies with indispensable conditioning in the presence of cytokines and human plasma to obtain early erythroid commitment, and (ii) differentiation/maturation to the stage of cultured red blood cells in the presence of cytokines. The protocol dispenses with major constraints such as an obligatory passage through a hematopoietic progenitor, co-culture on a cellular stroma and use of proteins of animal origin.
We report for the first time the complete differentiation of human induced pluripotent stem cells into definitive erythrocytes capable of maturation up to enucleated red blood cells containing fetal hemoglobin in a functional tetrameric form.
Red blood cells generated from human induced pluripotent stem cells pave the way for future development of allogeneic transfusion products. This could be done by banking a very limited number of red cell phenotype combinations enabling the safe transfusion of a great number of immunized patients.
We describe here the large-scale ex vivo production of mature human red blood cells (RBCs) from hematopoietic stem cells of diverse origins. By mimicking the marrow microenvironment through the ...application of cytokines and coculture on stromal cells, we coupled substantial amplification of CD34(+) stem cells (up to 1.95 x 10(6)-fold) with 100% terminal differentiation into fully mature, functional RBCs. These cells survived in nonobese diabetic/severe combined immunodeficient mice, as do native RBCs. Our system for producing 'cultured RBCs' lends itself to a fundamental analysis of erythropoiesis and provides a simple in vitro model for studying important human viral or parasitic infections that target erythroid cells. Further development of large-scale production of cultured RBCs will have implications for gene therapy, blood transfusion and tropical medicine.
Human induced pluripotent stem cells offer perspectives for cell therapy and research models for diseases. We applied this approach to the normal and pathological erythroid differentiation model by ...establishing induced pluripotent stem cells from normal and homozygous sickle cell disease donors.
We addressed the question as to whether these cells can reach complete erythroid terminal maturation notably with a complete switch from fetal to adult hemoglobin. Sickle cell disease induced pluripotent stem cells were differentiated in vitro into red blood cells and characterized for their terminal maturation in terms of hemoglobin content, oxygen transport capacity, deformability, sickling and adherence. Nucleated erythroblast populations generated from normal and pathological induced pluripotent stem cells were then injected into non-obese diabetic severe combined immunodeficiency mice to follow the in vivo hemoglobin maturation.
We observed that in vitro erythroid differentiation results in predominance of fetal hemoglobin which rescues the functionality of red blood cells in the pathological model of sickle cell disease. We observed, in vivo, the switch from fetal to adult hemoglobin after infusion of nucleated erythroid precursors derived from either normal or pathological induced pluripotent stem cells into mice.
These results demonstrate that human induced pluripotent stem cells: i) can achieve complete terminal erythroid maturation, in vitro in terms of nucleus expulsion and in vivo in terms of hemoglobin maturation; and ii) open the way to generation of functionally corrected red blood cells from sickle cell disease induced pluripotent stem cells, without any genetic modification or drug treatment.
Risk stratification in childhood T-cell acute lymphoblastic leukemia (T-ALL) is mainly based on minimal residual disease (MRD) quantification. Whether oncogenetic mutation profiles can improve the ...discrimination of MRD-defined risk categories was unknown. Two hundred and twenty FRALLE2000T-treated patients were tested retrospectively for NOTCH1/FBXW7/RAS and PTEN alterations. Patients with NOTCH1/FBXW7 (N/F) mutations and RAS/PTEN (R/P) germ line (GL) were classified as oncogenetic low risk (gLoR; n = 111), whereas those with N/F GL and R/P GL mutations or N/F and R/P mutations were classified as high risk (gHiR; n = 109). Day 35 MRD status was available for 191 patients. Five-year cumulative incidence of relapse (CIR) and disease-free survival were 36% and 60% for gHiR patients and 11% and 89% for gLoR patients, respectively. Importantly, among the 60% of patients with MRD <10−4, 5-year CIR was 29% for gHiR patients and 4% for gLoR patients. Based on multivariable Cox models and stepwise selection, the 3 most discriminating variables were the oncogenetic classifier, MRD, and white blood cell (WBC) count. Patients harboring a WBC count ≥200 × 109/L, gHiR classifier, and MRD ≥10−4 demonstrated a 5-year CIR of 46%, whereas the 58 patients (30%) with a WBC count <200 × 109/L, gLoR classifier, and MRD <10−4 had a very low risk of relapse, with a 5-year CIR of only 2%. In childhood T-ALL, the N/F/R/P mutation profile is an independent predictor of relapse. When combined with MRD and a WBC count ≥200 × 109/L, it identifies a significant subgroup of patients with a low risk of relapse.
•In pediatric T-ALL, oncogenetic markers, MRD, and WBC count are independent predictors of outcome.•These factors should be used together for individual treatment stratification.
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