Early Hematopoietic Stem Cells Research Articles

Outcomes of Adolescents and Young Adults With Acute Myeloid Leukemia Treated in a Single Latin American Center

IntroductionThe outcomes for adolescents and young adults (AYAs) with acute myeloid leukemia (AML) have been poorly characterized in Hispanics in low- to middle-income countries. The results are influenced by biologic and socioeconomic factors. The clinical paths for AYA patients with AML are reported. Patients and MethodsA retrospective analysis of AYA and pediatric AML patients aged 1 to 39 years during 2003 to 2016 from a single reference center in Northeast Mexico treated with a 7+3 standard protocol was performed. The 5-year overall survival (OS) and event-free survival (EFS) were estimated using Kaplan-Meier analysis. The hazard ratios for relapse and death were estimated using a Cox regression model. The patients with promyelocytic leukemia were analyzed separately. ResultsThe study included 110 non-PML AML patients, 39 children and 71 AYAs. No difference in complete remission was found (P = .446), although the overall response rate was greater in the children (87.2% vs. 69% in AYAs; P = .034). The 5-year EFS rate was 33% for the children versus 9.3% in the AYAs at a median follow-up of 22 and 9 months, respectively (P = .008). The 5-year OS rate was 51% in the children and 22% in the AYAs (P = .001). Of the 44 AYAs with complete remission, 29 (65%) developed a relapse. Of the 39 children and 71 AYAs, 20 children (51.3%) and 21 AYAs (29.6%) underwent transplantation (P = .024). Patients with refractory disease had a 1-year OS rate of 14.4%. Older age (hazard ratio [HR], 2.55; P = .002) and white blood cell count > 50 × 109/L (HR, 1.79; P = .023) were significant for death, and transplantation was protective (HR, 0.57; P = .023). ConclusionLow EFS and OS rates were found for AML patients in the AYA group. To improve survival rates, intensified chemotherapy regimens and early hematopoietic stem cell transplantation are needed.

Effectiveness of early hematopoietic stem cell transplantation in preventing neurocognitive decline in mucopolysaccharidosis type II: A case series

The early progressive form of the X-linked disorder, Hunter syndrome or mucopolysaccharidosis type II (MPS II) (OMIM #309900), is characterized by cognitive decline, and pulmonary and cardiac complications that often cause death before 20 years of age. Deficiency of the lysosomal enzyme, iduronate-2-sulfatase (EC 3.1.6.13) results in deposition of the glycosaminoglycans, dermatan, and heparan sulfate in various tissues. In recent years, enzyme replacement therapy (ERT) has become the mainstay of treatment, but is expensive and ineffective in arresting cognitive decline. Hematopoietic stem cell transplantation (HSCT) also provides enzyme replacement, and may be effective in stabilizing neurocognitive function if initiated early, though data are limited. We present a case series of four patients who demonstrated neurocognitive stabilization with early HSCT. HSCT is a potentially underutilized treatment strategy for select groups of MPS II patients.

Effectiveness of Early Hematopoietic Stem Cell Transplantation in Preventing Neurocognitive Decline inMucopolysaccharidosis Type II: A Case Series.

The early progressive form of the X-linked disorder, Hunter syndrome or mucopolysaccharidosis type II (MPS II) (OMIM #309900), is characterized by cognitive decline, and pulmonary and cardiac complications that often cause death before 20years of age. Deficiency of the lysosomal enzyme, iduronate-2-sulfatase (EC 3.1.6.13) results in deposition of the glycosaminoglycans, dermatan, and heparan sulfate in various tissues. In recent years, enzyme replacement therapy (ERT) has become the mainstay of treatment, but is expensive and ineffective in arresting cognitive decline. Hematopoietic stem cell transplantation (HSCT) also provides enzyme replacement, and may be effective in stabilizing neurocognitive function if initiated early, though data are limited. We present a case series of four patients who demonstrated neurocognitive stabilization with early HSCT. HSCT is a potentially underutilized treatment strategy for select groups of MPS II patients.

Clonal Hematopoiesis: Cell of Origin, Lineage Repartition and Dynamic Evolution during Chemotherapy

Background: Clonal hematopoiesis of indeterminate potential (CHIP) is defined by the presence of hematologic cancer associated mutations in the peripheral blood (PB) of at least 10% of elderly people without history of hematologic disorders (Genovese et al ., NEJM, 2014; Jaiswal et al ., NEJM, 2014). At present, caution is needed when predicting clinical consequences from CHIP in healthy people. An essential step towards a better understanding of CHIP requires identification of the cell of origin, clonal expansion patterns within the hematopoietic differentiation tree, and its dynamic behavior under stress scenarios (e.g. chemotherapy).Methods: PB and bone marrow (BM) samples were collected from 437 donors ≥ 55 years without known hematologic disease including a sub-cohort of 72 patients with newly diagnosed non-hematologic cancer requiring chemotherapy.Whole blood DNA was screened for CHIP with a 54 gene panel. A total of 63 PB and 9 BM samples were flow-sorted and variant allele frequencies (VAFs) were quantified in the hematopoietic fractions. In the cancer cohort, 32 clonal mutations were studied at 110 time points to investigate clonal dynamics under chemotherapy.Results: We identified 168 confirmed variants in 121 patients. 34 patients (28.1%) had 2 or more mutations (Fig. 1A). Presence of ≥ 2 mutations was significantly associated with peripheral artery disease (P=.002), diabetes (P=.04), and hyperlipoproteinaemia (P= .047). The most frequent combination was DNMT3A / TET2 (n=10) followed by DNMT3A/DNMT3A and TET2/TET2 in four cases each. TET2 mutations were significantly associated with DNMT3A (P=.015) and ASXL1 (P=.046) (Fig. 1B).Allelic burden of 91 mutations in 63 patients was determined in CD34+ progenitors, monocytes, granulocytes, NK-, B-, and T-cells (median VAFs: 5.1%, 7.1%, 6.3%, 6.0%, 1.9%, and 0.5%). B- and T-cells showed significantly lower VAFs when compared to WB or any other sorted cell fraction (P <.001 for each comparison). NK-cells showed significantly higher VAFs than T- and B-cells (P <.001), reaching comparable VAFs of myeloid cell fractions (Fig. 1C). Next, we compared mutation-specific effects on allelic burden within the cellular subfractions for DNMT3A, TET2, ASXL1, SF3B1, and TP53. No differences were observed except for a higher VAF in T-cells of DNMT3A -mutated individuals compared to other CHIP positive patients (P <.001), indicating an involvement of very early hematopoietic stem cells (HSCs).Next, we tracked individual mutations in flow-sorted stem and precursor cells in the BM of 9 CHIP patients (example in Fig. 1D). In all cases, we were able to identify the mutation in the Lin-CD34+CD38- HSC fraction. Although mutations showed different expansion profiles [expansion ratio (ER)=VAF(monocytes)/VAF(HSCs) or ER=VAF(granulocytes)/VAF(HSCs)], the biggest expansion proportion always occurred in the stem cell compartment indicative for early clonal dominance.In patients with more than one clonal mutation, the repartition of patient specific mutations showed similar patterns in most cases, suggesting that mutations were acquired within one clone. However, in some cases differential outgrowth of mutations was observed, indicating oligoclonality (examples in Fig. 1E).In the cancer cohort, CHIP was significantly associated with chemotherapy dose reduction due to hematotoxicity (P=.028). When following 32 mutations at 110 time points, we observed 3 different patterns of VAF dynamics: 1) increasing, 2) decreasing, and 3) no major changes (example for each in Fig. 1F). We categorized the 3 groups by defining a VAF change of at least +/- 50 % in ≥ 2 time points as cut-off. Only one of the 13 DNMT3A mutations showed VAF dynamics (1/13=7.7%), in the remaining 19 clonal mutations other than DNMT3A, VAF dynamics were observed in 13 clones (68.4% vs. 7.7%; P <.001). When excluding the 7 solely DNMT3A- mutated cases, we observed significantly lower hemoglobin levels prior to cycles 5 and 7 (P=.049 and P=.02) and an elevated red cell transfusion necessity (P=.013).Conclusion: CHIP derives from somatic mutations in Lin-CD34+CD38- HSCs and leads to preferential expansion in myeloid and NK-cell fractions. Clonal dynamics during chemotherapy lead to higher rates of red blood cell transfusions and dose reductions. Larger prospective studies in homogenously treated cancer patients are now warranted to verify the impact of CHIP during chemotherapy applications. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Reducing Gastrointestinal Toxicity Associated with Autologous Transplantation for Multiple Myeloma without Compromising Its Anti-Myeloma Effect

Early autologous hematopoietic stem cell transplantation (auto-HCT) is recommended for transplant-eligible patients (pts) with newly diagnosed multiple myeloma (MM). However, gastrointestinal toxicities, i.e., oral mucositis (OM), nausea and diarrhea, are the major limitation to the use of auto-HCT especially in the elderly population which constitute a significant proportion of MM pts, where the median age at diagnosis is 68 years. There is an unmet need for measures to minimize non-hematological toxicities without compromising melphalan anti-myeloma efficacy; this could lead to expansion of transplant eligibility to older pts. Amifostine, a FDA-approved cytoprotective agent to prevent OM for Head and Neck cancer, may reduce HDM-associated GI toxicity. We conducted a case-control study comparing auto-HCT with or without amifostine for MM pts.Methodspre-transplant amifostine has been incorporated to standard protocol for all MM patients underwent auto-HCT at University Hospitals Cleveland Medical Center (UH) for the last decade. One hundred and seven pts treated at UH who received amifostine, from January 2007 to July 2014, were compared to 114 matched-control pts treated at MD Anderson Cancer Center (MDACC) without use of this agent. The institutional review boards at both institutions approved the study. Amifostine 740 mg/m2 was administered as a bolus infusion at 24 hours and 15 minutes before HDM. All pts received ice chips peri-melphalan infusion. Survival outcomes were measured from the date of auto-HCT to the date of disease relapse or progression, Survival distribution was estimated using Kaplan-Meier methods. The effect of treatment on OS and PFS was estimated using a Cox model after controlling for the effects of age, gender, and number of prior therapies, time from diagnosis to transplant, Eastern Cooperative Oncology Group (ECOG) performance status and number of infused CD34+ cells.ResultsPts' characteristics were similar in both groups.Median follow-up of surviving pts was 35 (range 2-100) months in both cohorts. Amifostine therapy was well tolerated without any serious adverse effects. There was no significant difference between all-grade OM, nausea or vomiting between the two cohorts. However, > grade II GI toxicities were significantly lower in the amifostine group as follows: OM: 27.1% vs 47.4% (P=0.002), diarrhea: 56.1% vs. 72.7% (P=0.006), nausea: 31.8% vs. 86.0% (P=0.0001) and vomiting: 18.7% vs. 52.6%, (P=0.0001) (Figure-1) (Table-1). Median time to platelet engraftment was similar between the two groups while neutrophil engraftment period was shorter with use of amifostine (10 vs. 11 days, P=0.011) (Table-2). Multivariable logistic regression showed that use of amifostine pre-transplant was associated with a significant decrease in OM, diarrhea, nausea and vomiting. Pts who received amifostine and melphalan developed grade ≥ II OM significantly less often than those given melphalan alone with odds ratio (OR) = 0.366 (95% CI: 0.196-0.685, P=0.001; Table 3). Female gender was associated with a significant increase in OM, nausea and vomiting. Female pts were more likely to develop grade ≥ II OM with OR of 1.967 (P=0.017). Similarly, an ECOG performance status (PS) of ≥ 2 was associated with a significant increase in OM and diarrhea. For every additional score of ECOG, the risk of having grade ≥ II OM increased 1.89 fold (p=0.015). Subgroup analysis of grade II and higher OM rates are shown in Table-4. Use of amifostine was associated with reduced grade II or higher GI toxicity after adjusting for the effects of age, gender, number of prior therapies, time from diagnosis to transplant, ECOG PS and infused CD34 cell dose. There was no detrimental effect of amifostine on progression-free or overall survival.ConclusionsOur analysis indicates that the use of two amifostine doses of 740 mg/m2 before auto-HCT is safe and associated with significant reduction in grade II and higher GI toxicities without any deleterious effect on engraftment or anti-myeloma efficacy. Amifostine use could conceivably allow further melphalan dose-intensification for pts with resistant or high-risk disease. Also, pre-treatment with amifostine potentially could expand the utilization of auto-HCT for modestly frail MM pts that might not be considered eligible for this treatment modality. The protective effect of amifostine should be confirmed in randomized trial. [Display omitted] DisclosuresMalek:Celgene: Speakers Bureau; Takeda: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi: Membership on an entity's Board of Directors or advisory committees. Cooper:Novartis: Research Funding. Caimi:Abbvie: Equity Ownership; Incyte: Equity Ownership; Celgene: Speakers Bureau; Seattle Genetics: Equity Ownership. De Lima:Pfizer: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Research Funding.

Establishment of CMab-43, a Sensitive and Specific Anti-CD133 Monoclonal Antibody, for Immunohistochemistry.

CD133, also known as prominin-1, was first described as a cell surface marker on early progenitor and hematopoietic stem cells. It is a five-domain transmembrane protein composed of an N-terminal extracellular tail, two small cytoplasmic loops, two large extracellular loops containing seven potential glycosylation sites, and a short C-terminal intracellular tail. CD133 has been used as a marker to identify cancer stem cells derived from primary solid tumors and as a prognostic marker of gliomas. Herein, we developed novel anti-CD133 monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. We expressed the full length of CD133 in LN229 glioblastoma cells, immunized mice with LN229/CD133 cells, and performed the first screening using flow cytometry. After limiting dilution, we established 100 anti-CD133 mAbs, reacting with LN229/CD133 cells but not with LN229 cells. Subsequently, we performed the second and third screening with Western blot and immunohistochemical analyses, respectively. Among 100 mAbs, 11 strongly reacted with CD133 in Western blot analysis. One of 11 clones, CMab-43 (IgG2a, kappa), showed a sensitive and specific reaction against colon cancer cells, warranting the use of CMab-43 in detecting CD133 in pathological analyses of CD133-expressing cancers.

Gene editing rescue of a novel MPL mutant associated with congenital amegakaryocytic thrombocytopenia.

Thrombopoietin (Tpo) and its receptor (Mpl) are the principal regulators of early and late thrombopoiesis and hematopoietic stem cell maintenance. Mutations in MPL can drastically impair its function and be a contributing factor in multiple hematologic malignancies, including congenital amegakaryocytic thrombocytopenia (CAMT). CAMT is characterized by severe thrombocytopenia at birth, which progresses to bone marrow failure and pancytopenia. Here we report unique familial cases of CAMT that presented with a previously unreported MPL mutation: T814C (W272R) in the background of the activating MPL G117T (K39N or Baltimore) mutation. Confocal microscopy, proliferation and surface biotinylation assays, co-immunoprecipitation, and western blotting analysis were used to elucidate the function and trafficking of Mpl mutants. Results showed that Mpl protein bearing the W272R mutation, alone or together with the K39N mutation, lacks detectable surface expression while being strongly colocalized with the endoplasmic reticulum (ER) marker calreticulin. Both WT and K39N-mutated Mpl were found to be signaling competent, but single or double mutants bearing W272R were unresponsive to Tpo. Function of the deficient Mpl receptor could be rescued by using 2 separate approaches: (1) GRASP55 overexpression, which partially restored Tpo-induced signaling of mutant Mpl by activating an autophagy-dependent secretory pathway and thus forcing ER-trapped immature receptors to traffic to the cell surface; and (2) CRISPR-Cas9 gene editing used to repair MPL T814C mutation in transfected cell lines and primary umbilical cord blood-derived CD34+ cells. We demonstrate proof of principle for rescue of mutant Mpl function by using gene editing of primary hematopoietic stem cells, which indicates direct therapeutic applications for CAMT patients.

Neonatal umbilical cord blood transplantation halts skeletal disease progression in the murine model of MPS-I

Umbilical cord blood (UCB) is a promising source of stem cells to use in early haematopoietic stem cell transplantation (HSCT) approaches for several genetic diseases that can be diagnosed at birth. Mucopolysaccharidosis type I (MPS-I) is a progressive multi-system disorder caused by deficiency of lysosomal enzyme α-L-iduronidase, and patients treated with allogeneic HSCT at the onset have improved outcome, suggesting to administer such therapy as early as possible. Given that the best characterized MPS-I murine model is an immunocompetent mouse, we here developed a transplantation system based on murine UCB. With the final aim of testing the therapeutic efficacy of UCB in MPS-I mice transplanted at birth, we first defined the features of murine UCB cells and demonstrated that they are capable of multi-lineage haematopoietic repopulation of myeloablated adult mice similarly to bone marrow cells. We then assessed the effectiveness of murine UCB cells transplantation in busulfan-conditioned newborn MPS-I mice. Twenty weeks after treatment, iduronidase activity was increased in visceral organs of MPS-I animals, glycosaminoglycans storage was reduced, and skeletal phenotype was ameliorated. This study explores a potential therapy for MPS-I at a very early stage in life and represents a novel model to test UCB-based transplantation approaches for various diseases.

Liver disease predicts mortality in patients with X-linked immunodeficiency with hyper-IgM but can be prevented by early hematopoietic stem cell transplantation

Liver disease predicts mortality in patients with X-linked immunodeficiency with hyper-IgM but can be prevented by early hematopoietic stem cell transplantation

Advances on relationship between Homebox gene and acute leukemia

In recent years, the study has found that homeobox gene plays a significance role not only in regulating and controlling hematopoietic stem cells/progenitor cells, and its expression level has a close relationship of the occurrence, development and prognosis of acute leukemia (AL). At present, the researchers believe that the homeobox gene is not only related to the function of early hematopoietic stem cells, but also participates in the differentiation and orientation of hematopoietic cells in late stage, and its abnormal expression can lead to AL. However its related regulatory mechanisms to AL has not been fully disclosed.The authors review the researches of the relationship between homeobox gene and AL, in order to finding new molecular targets for diagnosis, gene therapy and prognosis evaluation of AL. Key words: Homebox gene; Acute leukemia; HOX gene

Impact of the c-MybE308G mutation on mouse myelopoiesis and dendritic cell development.

Booreana mice carrying the c-Myb308G point mutation were analyzed to determine changes in early hematopoiesis in the bone marrow and among mature cells in the periphery. This point mutation led to increased numbers of early hematopoietic stem and progenitor cells (HSPCs), with a subsequent reduction in the development of B cells, erythroid cells, and neutrophils, and increased numbers of myeloid cells and granulocytes. Myelopoiesis was further investigated by way of particular subsets affected. A specific question addressed whether booreana mice contained increased numbers of dendritic-like cells (L-DC subset) recently identified in the spleen, since L-DCs arise in vitro by direct differentiation from HSPCs co-cultured over splenic stroma. The non-lethal c-Myb mutation in booreana mice was associated with significantly lower representation of splenic CD8- conventional dendritic cells (cDCs), inflammatory monocytes, and neutrophils compared to wild-type mice. This result confirmed the bone marrow origin of progenitors for these subsets since c-Myb is essential for their development. Production of L-DCs and resident monocytes was not affected by the c-MybE308G mutation. These subsets may derive from different progenitors than those in bone marrow, and are potentially established in the spleen during embryogenesis. An alternative explanation may be needed for why there was no change in CD8+ cDCs in booreana spleen since these cells are known to derive from common dendritic progenitors in bone marrow.

Aplastic anemia and clonal evolution: germline and somatic genetics

Clonal progression to myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML) remains a dreaded complication for a subset of patients with bone marrow failure (BMF). Recognizing risk factors for the development of MDS or AML would inform individualized treatment decisions and identify patients who may benefit from early or upfront hematopoietic stem cell transplantation. Now that next-generation DNA sequencing is available in the clinical laboratory, research has focused on the implications of germline and somatic mutations for diagnosing and monitoring patients with BMF. Most germline genetic BMF disorders are characterized by a high propensity to develop MDS or AML. Recent studies of somatic variants in marrow failure revealed a high frequency of clonal hematopoiesis with the acquisition of mutations in genes associated with MDS or AML. The evaluation and implications of germline and somatic mutations for the development of clonal disorders in patients with BMF and challenges of clinical genetic testing will be summarized in this paper based on the reports from the 58th American Society of Hematology (ASH) Annual Meeting. Key words: Bone marrow failure; Anemia, aplastic; Myelodysplastic syndrome; Leukemia, myeloid, acute; Germline genetics; Somatic genetics; Clonal progression; Next generation sequencing; American Society of Hematology Annual Meeting

Current Status of Dedicator of Cytokinesis-Associated Immunodeficiency: DOCK8 and DOCK2.

DOCK8 deficiency is an autosomal recessive combined immunodeficiency disease associated with elevated IgE, atopy, recurrent sinopulmonary and cutaneous viral infections, and malignancy. The DOCK8 protein is critical for cytoskeletal organization, and deficiency impairs dendritic cell transmigration, T-cell survival, and NK cell cytotoxicity. Early hematopoietic stem cell transplantation is gaining prominence as a definitive treatment given the potential for severe complications and mortality in this disease. Recently, DOCK2 deficiency has been identified in several patients with early-onset invasive bacterial and viral infections.

Aplastic anemia and clonal evolution: germ line and somatic genetics.

Clonal progression to myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) remains a dreaded complication for a subset of patients with bone marrow failure (BMF). Recognizing risk factors for the development of MDS or AML would inform individualized treatment decisions and identify patients who may benefit from early or upfront hematopoietic stem cell transplantation. Now that next-generation DNA sequencing is available in the clinical laboratory, research has focused on the implications of germ line and somatic mutations for diagnosing and monitoring patients with BMF. Most germ line genetic BMF disorders are characterized by a high propensity to develop MDS or AML. Many affected patients lack the physical stigmata traditionally associated with the inherited marrow failure syndromes. Although any single inherited marrow failure disorder is rare, multiplexed genetic sequencing that allows simultaneous evaluation of marrow failure genes en masse demonstrated that, as a group, these inherited disorders compose a significant subset (5% to 10%) of patients with BMF. Early diagnosis of a germ line genetic marrow failure disorder allows individualized monitoring and tailored therapy. Recent studies of somatic variants in marrow failure revealed a high frequency of clonal hematopoiesis with the acquisition of mutations in genes associated with MDS or AML. Investigation of somatic mutations in marrow failure revealed important insights into the mechanisms promoting clonal disease but also raised additional questions. This review will focus on the evaluation and implications of germ line and somatic mutations for the development of clonal disorders in patients with BMF. Challenges and limitations of clinical genetic testing will be explored.

High Disease-Free and Overall Survival Rate Following Allogeneic Hematopoietic Stem Cell Transplantation for FLT3-Mutated Acute Myeloid Leukemia Even in Non-Remission Status

[Background] FMS like tyrosine kinase 3 (FLT3) mutations occur in about 30% of patients with acute myeloid leukemia (AML). Patients with FLT3-mutated AML have a poor prognosis and are referred for early allogeneic hematopoietic stem cell transplantation (allo-HSCT) in first complete remission (CR1). So far, there is still limited data available on allo-HSCT for FLT3-mutated AML in non-remission status.[Objective and method] To assess the clinical features and outcome of allo-HSCT for FLT3-mutated AML, we retrospectively analyzed patients underwent first allo-HSCT for FLT3-mutated AML excluding acute promyelocytic leukemia (FAB M3) from January 2011 to March 2016.[Result] During the study period, 332 patients received first allo-HSCT for AML in our institute. One hundred and thirty-eight were tested for the presence of FLT3-mutation and 35 showed positive results and were subjected to the analysis. The median follow-up day of survivors was 602 (101-1867). The median age of the patients was 55 years (range, 21-72). Twenty-one patients had de novo AML, 12 had AML with myelodysplasia related changes, and 2 had therapy related AML. Eighteen had normal karyotype, 4 had complex, and 13 had others. Seven were in remission (5 in CR1, and 2 in CR2), and 28 were in non-remission (8 in primary induction failure, 13 in relapse 1, and 7 in chemo naïve status). Twenty-nine patients used unrelated cord blood, 2 did unrelated bone marrow, and 4 did related peripheral blood stem cell as grafts. All but 1 received myeloablative pretransplant conditionings. At 2 years after transplantation, overall survival (OS), disease free survival (DFS), relapse rate (RR), and non-relapse mortality (NRM) of whole studied population were 65.9%, 50.2%, 28.4%, and 21.4%, respectively. Among those in non-remission before transplantation, OS, DFS, RR, and NRM at 2 years post-transplant were 62.2% (Figure 1A), 49.9%(Figure 1B), 24.3%, and 25.8%, respectively. Only younger age (<55 years) was the factor associated with better OS with statistical significance in multivariate analysis.[Conclusion] Our data indicated that allo-HSCT could overcome the poor prognosis of FLT3-mutated AML even for those in non-remission status, despite the profound chemo-resistant character of FLT3-mutated AML. [Display omitted] [Display omitted] DisclosuresIzutsu:Abbvie: Research Funding; Gilead: Research Funding; Celgene: Research Funding; Janssen Pharmaceutical K.K.: Honoraria; Eisai: Honoraria; Kyowa Hakko Kirin: Honoraria; Chugai Pharmaceutical: Honoraria, Research Funding; Takeda Pharmaceutical: Honoraria; Mundipharma KK: Research Funding.

Nuclear NFATc1 Causes Development of Fms-like Receptor Tyrosine Kinase 3 Receptor (FLT3) Internal Tandem Duplication Mutation-Positive (FLT3-ITD) Acute Myeloid Leukemia (AML) and Mediates Drug Resistance

Abstract Background: Although an internal tandem duplication (ITD) of the Fms-like receptor tyrosine kinase 3 receptor (FLT3) confers a very poor prognosis in acute myeloid leukemia (AML), it is insufficient to cause AML, suggesting important cooperating genetic events. NFATc1 is a member of the nuclear factor of activated T-cells (NFAT) transcription factor family, whose activation is dependent on nuclear translocation. NFATc1 is well known for its central role in regulating T-cell development and function, but was also shown to contribute to neoplastic transformation in diverse types of cancers. We have recently demonstrated that NFATc1 is overexpressed in primary FLT3-ITD+ AML causing resistance to the FLT3-kinase inhibitor sorafenib in vitro. Here we asked, whether NFATc1 induces AML transformation and drug resistance when expressed in the context of FLT3-ITD+ in vivo. Methods: Using the stem cell like (SCL)-promoter, Cre-recombinase inducible expression of a constitutively nuclear NFATc1 protein (cnNFATc1) was targeted to the early hematopoietic stem cell compartment. The SCL-Cre/cnNFATc1 mice were subsequently crossed with transgenic FLT3-ITD mice to yield Scl-Cre+/-/cnNFATc1+/+/FLT3-ITD+/+ mice (FCN+/+), which co-express cnNFAT and FLT3-ITD in the stem cell compartment. Results: Whereas FLT3-ITD mice developed a non-lethal, mild myeloproliferative disease, co-expression of FLT3-ITD with cnNFATc1 led to rapidly fatal AML. The median survival of FCN mice was only four months (range, 2 to 8 ). In median, FCN mice had 15-fold (range, 10 to 20) increased white blood cell count, a high proportion of circulating peripheral blasts, severe anemia and thrombocytopenia. There was also a significant B-cell developmental block. As opposed to F+/+ animals, FCN+/+ mice revealed a severe spleno- and hepatomegaly secondary to a gross leukemic infiltration, which disrupted the normal anatomical architecture of the involved organs. Fluorescence activating cell sorting and colony forming unit (CFU) assays demonstrated that cnNFATc1, when expressed in the context of FLT3-ITD, leads to a significant expansion of the lin-Sca+c-kit+ (LSK) stem cell-, progenitor cell (LK)-, the Gr-1-/CD11b+ monocytic and Gr-1low/CD11b+ immature myeloid compartments in bone marrow and spleen. FCN+/+-AML was polyclonal, and re-transplantable in secondary wild-type recipient mice. In vivo, cnNFATc1 caused not only potent FLT3-ITD inhibitor resistance to sorafenib and to the more FLT3-ITD-selective compound quizartinib, but also to chemotherapy. Comparative mRNA-sequencing of sorted LK and LSK compartments in wild-type, F+/+, C+/- N+/+, and FCN+/+ mice will reveal the genetic basis of the biological cooperativity between NFATc1 and FLT3-ITD in stem and progenitor cell compartments. Consistent with the murine data, a data set of AML patients (n=163) revealed that NFATc1 overexpression alone or when combined with FLT3-overexpression was associated with a significantly decreased overall survival (hazard ratio [HR] 1.91, CI 1.29-2.18, p=0.001 versus HR 2.57, CI 1.62-4.08, p&lt;0.0001). Conclusions: These results provide for the first time evidence for an important role of nuclear NFATc1 overexpression in the molecular pathogenesis of FLT3-ITD positive AML and the induction of drug resistance. These data also underscore that the mutational landscape in AML insufficiently describes AML biology. Disclosures No relevant conflicts of interest to declare.

Chronic granulomatous disease: Clinical, functional, molecular, and genetic studies. The Israeli experience with 84 patients.

Chronic granulomatous disease (CGD) is an innate immunodeficiency with a genetic defect of the nicotinamide adenosine dinucleotide phosphate, reduced, oxidase components. This leads to decreased reactive oxygen species (ROS) production, which renders patients susceptible to life-threatening infections. Over the course of 30 years, we diagnosed CGD in 84 patients from 61 families using functional, molecular, and genetic studies. The incidence of CGD in Israel is 1.05 per 100,000 live-births in the Jewish population and 1.49 in the Israeli Arab population. We diagnosed 52 patients (62%) with autosomal recessive inheritance (AR-CGD) and 32 (38%) with X-linked recessive inheritance (XLR-CGD). Consanguinity was detected in 64% of AR-CGD families (14% in Jews and 50% in Israeli Arabs). We found 36 different mutations (23 in XLR-CGD and 13 in AR-CGD patients), 15 of which were new. The clinical spectrum of CGD varied from mild to severe disease in both XLR and AR forms, although the AR subtype is generally milder. Further, residual ROS production correlated with milder clinical expression, better prognosis and improved overall survival. Patients with recurrent pyogenic infections developed fibrosis and hyperinflammatory states with granuloma formation. The management of CGD has progressed substantially in recent years, evolving from a fatal disease of early childhood to one of long-term survival. Our present cohort displays an encouraging 81% overall long term survival. Early hematopoietic stem cell transplantation is advisable before tissue damage is irreversible. Successful transplantation was performed in 18/21 patients. Therapeutic gene modification could become an alternative cure for CGD. Am. J. Hematol. 92:28-36, 2017. © 2016 Wiley Periodicals, Inc.

P187 Hematopoietic stem cell transplantation in a patient with griscelli syndrome type 2: case report

Griscelli syndrome type 2 (GS-2) is a rare disorder belonging to the group of immune dysregulation. It is an autosomal recessive disease characterized by partial albinism with variable immunodeficiency. Grayish-silver hair with large and clumped melanosomes in hair shafts are diagnostic. The most frequent complication that leads to mortality includes lymphohistiocytic proliferation in several organs including the brain, but an early hematopoietic stem cell transplantation (HSCT) is the definitive treatment, therefore early recognition and treatment of GS-2 is critical. We present the case of a girl with GS-2 in which HSCT was successful.

Identification of factors promoting ex vivo maintenance of mouse hematopoietic stem cells by long-term single-cell quantification

AbstractThe maintenance of hematopoietic stem cells (HSCs) during ex vivo culture is an important prerequisite for their therapeutic manipulation. However, despite intense research, culture conditions for robust maintenance of HSCs are still missing. Cultured HSCs are quickly lost, preventing their improved analysis and manipulation. Identification of novel factors supporting HSC ex vivo maintenance is therefore necessary. Coculture with the AFT024 stroma cell line is capable of maintaining HSCs ex vivo long-term, but the responsible molecular players remain unknown. Here, we use continuous long-term single-cell observation to identify the HSC behavioral signature under supportive or nonsupportive stroma cocultures. We report early HSC survival as a major characteristic of HSC-maintaining conditions. Behavioral screening after manipulation of candidate molecules revealed that the extracellular matrix protein dermatopontin (Dpt) is involved in HSC maintenance. DPT knockdown in supportive stroma impaired HSC survival, whereas ectopic expression of the Dpt gene or protein in nonsupportive conditions restored HSC survival. Supplementing defined stroma- and serum-free culture conditions with recombinant DPT protein improved HSC clonogenicity. These findings illustrate a previously uncharacterized role of Dpt in maintaining HSCs ex vivo.

Bacteremia During Early Post-allogeneic Hematopoietic Stem Cell Transplantation Period: A Single Center Experience.

Bacteremia is a significant complication of allogeneic hematopoietic stem cell transplantation (HSCT). We aimed to study bacteremia occurring during early post-transplant period at Bone Marrow Transplantation Unit of Ain Shams University regarding its risk factors and impact on survival. Patients performing allogeneic HSCT were followed up for occurrence of bacteremia. Survival status was assessed at 180days post-transplant. Bacteremia occurred in 53.3% of patients. On univariate analysis, CD34 +ve cell dose (P=0.004), duration of neutropenia (P=0.018), time interval between day of stem cell infusion and day of neutrophil engraftment (P=0.043) and>1 apheresis days (P=0.040) were associated with higher rates of bacteremia. On multivariate analysis, CD34 +ve cell dose (P=0.002) and apheresis day number (P=0.038) remained significant. There was significant difference between patients who developed bacteremia and those who did not regarding overall survival (OS) (P=0.042). Patients developing bacteremia caused by Gram negative bacteria (GNB) had lower OS than Gram positive bacteria (GPB) (P<0.001). In conclusion, stem cell dose and apheresis day number influence bacteremia risk. Also, Gram negative bacteremia has negative impact on allogeneic transplant recipient survival rates.