CRISPR-based gene editing enables FOXP3 gene repair in IPEX patient cells.

The prototypical genetic autoimmune disease is immune dysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome, a severe pediatric disease with limited treatment options. IPEX syndrome is caused by mutations in the forkhead box protein 3 (FOXP3) gene, which plays a critical role in immune regulation. As a monogenic disease, IPEX is an ideal candidate for a therapeutic approach in which autologous hematopoietic stem and progenitor (HSPC) cells or T cells are gene edited ex vivo and reinfused. Here, we describe a CRISPR-based gene correction permitting regulated expression of FOXP3 protein. We demonstrate that gene editing preserves HSPC differentiation potential, and that edited regulatory and effector T cells maintain their in vitro phenotype and function. Additionally, we show that this strategy is suitable for IPEX patient cells with diverse mutations. These results demonstrate the feasibility of gene correction, which will be instrumental for the development of therapeutic approaches for other genetic autoimmune diseases.

Population pharmacokinetics of treosulfan in paediatric patients undergoing hematopoietic stem cell transplantation.

AIMS: Treosulfan is an alkylating agent increasingly used prior to haematopoietic stem cell transplantation. The aim of this study was to develop a population pharmacokinetic (PK) model of treosulfan in paediatric haematopoietic stem cell transplantation recipients and to explore the effect of potential covariates on treosulfan PK. Also, a limited sampling model (LSM) will be developed to accurately predict treosulfan exposure suitable for a therapeutic drug monitoring setting. METHODS: In this multicentre study, 91 patients, receiving a total dose of 30, 36 or 42 g/m2 treosulfan, administered over 3 consecutive days, were enrolled. A population PK model was developed and demographic factors, as well as laboratory parameters, were included as potential covariates. In addition, a LSM was developed using data from 28 patients. RESULTS: A 2-compartment model with first order elimination best described the data. Bodyweight with allometric scaling and maturation function were identified as significant predictors of treosulfan clearance. Treosulfan clearance reaches 90% of adult values at 4 postnatal years. A model-based dosing table is presented to target an exposure of 1650 mg*h/L (population median) for different weight and age groups. Samples taken at 1.5, 4 and 7 hours after start of infusion resulted in the best limited sampling strategy. CONCLUSIONS: This study provides a treosulfan population PK model in children and captures the developmental changes in clearance. A 3-point LSM allows for accurate and precise estimation of treosulfan exposure.

Correction: NG2 antigen is involved in leukemia invasiveness and central nervous system infiltration in MLL-rearranged infant B-ALL.

The original version of this Article contained an error in the spelling of the author Juan Carlos Rodriguez-Manzaneque, which was incorrectly given as J Carlos Rodríguez-Manzaneque. This has now been corrected in both the PDF and HTML versions of the Article.

NG2 antigen is involved in leukemia invasiveness and central nervous system infiltration in MLL-rearranged infant B-ALL.

Mixed-lineage leukemia (MLL)-rearranged (MLLr) infant B-cell acute lymphoblastic leukemia (iMLLr-B-ALL) has a dismal prognosis and is associated with a pro-B/mixed phenotype, therapy refractoriness and frequent central nervous system (CNS) disease/relapse. Neuron-glial antigen 2 (NG2) is specifically expressed in MLLr leukemias and is used in leukemia immunophenotyping because of its predictive value for MLLr acute leukemias. NG2 is involved in melanoma metastasis and brain development; however, its role in MLL-mediated leukemogenesis remains elusive. Here we evaluated whether NG2 distinguishes leukemia-initiating/propagating cells (L-ICs) and/or CNS-infiltrating cells (CNS-ICs) in iMLLr-B-ALL. Clinical data from the Interfant cohort of iMLLr-B-ALL demonstrated that high NG2 expression associates with lower event-free survival, higher number of circulating blasts and more frequent CNS disease/relapse. Serial xenotransplantation of primary MLL-AF4+ leukemias indicated that NG2 is a malleable marker that does not enrich for L-IC or CNS-IC in iMLLr-B-All. However, NG2 expression was highly upregulated in blasts infiltrating extramedullar hematopoietic sites and CNS, and specific blockage of NG2 resulted in almost complete loss of engraftment. Indeed, gene expression profiling of primary blasts and primografts revealed a migratory signature of NG2+ blasts. This study provides new insights on the biology of NG2 in iMLLr-B-ALL and suggests NG2 as a potential therapeutic target to reduce the risk of CNS disease/relapse and to provide safer CNS-directed therapies for iMLLr-B-ALL.

Zoledronic acid boosts γδ T-cell activity in children receiving αß+ T and CD19+ cell-depleted grafts from an HLA-haplo-identical donor.

We demonstrated that γδ T cells of patients given HLA-haploidentical HSCT after removal of αß+ T cells and CD19+ B cells are endowed with the capacity of killing leukemia cells after ex vivo treatment with zoledronic acid (ZOL). Thus, we tested the hypothesis that infusion of ZOL in patients receiving this type of graft may enhance γδ T-cell cytotoxic activity against leukemia cells. ZOL was infused every 28 d in 43 patients; most were treated at least twice. γδ T cells before and after ZOL treatments were studied in 33 of these 43 patients, till at least 7 mo after HSCT by high-resolution mass spectrometry, flow-cytometry, and degranulation assay. An induction of Vδ2-cell differentiation, paralleled by increased cytotoxicity of both Vδ1 and Vδ2 cells against primary leukemia blasts was associated with ZOL treatment. Cytotoxic activity was further increased in Vδ2 cells, but not in Vδ1 lymphocytes in those patients given more than one treatment. Proteomic analysis of γδ T cells purified from patients showed upregulation of proteins involved in activation processes and immune response, paralleled by downregulation of proteins involved in proliferation. Moreover, a proteomic signature was identified for each ZOL treatment. Patients given three or more ZOL infusions had a better probability of survival in comparison to those given one or two treatments (86% vs. 54%, respectively, p = 0.008). Our data indicate that ZOL infusion in pediatric recipients of αß T- and B-cell-depleted HLA-haploidentical HSCT promotes γδ T-cell differentiation and cytotoxicity and may influence the outcome of patients.

Remestemcel-L for the treatment of graft versus host disease.

INTRODUCTION: Remestemcel-L, a third-party, off-the-shelf preparation of bone-marrow derived mesenchymal stromal cells (MSCs), has been developed for experimental use in acute graft-versus-host disease (aGvHD) and other immune-mediated conditions. Several preclinical and clinical studies have indeed suggested the potential of human mesenchymal stromal cells (MSCs) as an effective treatment for steroid-refractory aGvHD. However, an unambiguous demonstration of efficacy is still lacking. Areas covered: This review critically examines the biologic rationale supporting MSCs use in aGvHD and analyzes the results of published clinical trials in this setting, with a particular focus on the potential benefits and drawbacks of Remestemcel-L. For this purpose, a systematic literature search was performed in PubMed using the following keywords: 'mesenchymal stromal cells', 'mesenchymal progenitor cells', 'multipotent stromal cells', 'mesenchymal cells', 'MSC', 'Remestemcel-L', 'Prochymal', and 'graft-versus-host disease' or 'GvHD'. Expert commentary: Remestemcel-L represents a promising alternative to second-line immunosuppressive agents for the treatment of steroid-refractory aGvHD. Despite the safety and the favorable risk/benefit profile of this cell product, which has been demonstrated in several phase I-II studies, large and prospective randomized trials are required to confirm its efficacy in aGvHD and to define the optimal schedule of administration in terms of infusion timing, cell dose and pharmacological synergism.

Infections by carbapenem-resistant Klebsiella pneumoniae in SCT recipients: a nationwide retrospective survey from Italy.

Infections by carbapenem-resistant Klebsiella pneumoniae (CRKp) represent a challenging problem after SCT. A retrospective survey (January 2010 to July 2013) involving 52 Italian centers was performed to assess the epidemiology and the prognostic factors of CRKp infections in auto- and allo-SCT. Cases of CRKp infection were reported in 53.4% of centers. CRKp infections were documented in 25 auto-SCTs and 87 allo-SCTs, with an incidence of 0.4% (from 0.1% in 2010 to 0.7% in 2013) and 2% (from 0.4% in 2010 to 2.9% in 2013), respectively. A CRKp colonization documented before or after transplant was followed by an infection in 25.8% of auto-SCT and 39.2% of allo-SCT patients. The infection-related mortality rates were 16% and 64.4%, respectively. A pre-transplant CRKp infection (hazard ratio (HR) 0.33, 95% confidence intervals (CIs) 0.15-0.74; P=0.007) and a not CRKp-targeted first-line treatment (HR 2.67, 95% CI 1.43-4.99; P=0.002) were independent factors associated with an increased mortality in allo-SCT patients who developed a CRKp infection. Our study shows challenging findings of CRKp infections in SCT patients in Italy particularly after allo-SCT. The detection of carriers and the definition of early therapeutic strategies represent critical aspects of the management of CRKp infections after SCT.

Outcome of children with high-risk acute myeloid leukemia given autologous or allogeneic hematopoietic cell transplantation in the aieop AML-2002/01 study.

We analyzed the outcome of 243 children with high-risk (HR) AML in first CR1 enrolled in the AIEOP-2002/01 protocol, who were given either allogeneic (ALLO; n=141) or autologous (AUTO; n=102) hematopoietic SCT (HSCT), depending on the availability of a HLA-compatible sibling. Infants, patients with AML-M7, or complex karyotype or those with FLT3-ITD, were eligible to be transplanted also from alternative donors. All patients received a myeloablative regimen combining busulfan, cyclophosphamide and melphalan; [corrected] AUTO-HSCT patients received BM cells in most cases, while in children given ALLO-HSCT stem cell source was BM in 96, peripheral blood in 19 and cord blood in 26. With a median follow-up of 57 months (range 12-130), the probability of disease-free survival (DFS) was 73% and 63% in patients given either ALLO- or AUTO-HSCT, respectively (P=NS). Although the cumulative incidence (CI) of relapse was lower in ALLO- than in AUTO-HSCT recipients (17% vs 28%, respectively; P=0.043), the CI of TRM was 7% in both groups. Patients transplanted with unrelated donor cord blood had a remarkable 92.3% 8-year DFS probability. Altogether, these data confirm that HSCT is a suitable option for preventing leukemia recurrence in HR children with CR1 AML.

Cytotoxic activity of the casein kinase 2 inhibitor CX-4945 against T-cell acute lymphoblastic leukemia: targeting the unfolded protein response signaling.

Constitutively active casein kinase 2 (CK2) signaling is a common feature of T-cell acute lymphoblastic leukemia (T-ALL). CK2 phosphorylates PTEN (phosphatase and tensin homolog) tumor suppressor, resulting in PTEN stabilization and functional inactivation. Downregulation of PTEN activity has an impact on PI3K/Akt/mTOR signaling, which is of fundamental importance for T-ALL cell survival. These observations lend compelling weight to the application of CK2 inhibitors in the therapy of T-ALL. Here, we have analyzed the therapeutic potential of CX-4945-a novel, highly specific, orally available, ATP-competitive inhibitor of CK2α. We show that CX-4945 treatment induced apoptosis in T-ALL cell lines and patient T lymphoblasts. CX-4945 downregulated PI3K/Akt/mTOR signaling in leukemic cells. Notably, CX-4945 affected the unfolded protein response (UPR), as demonstrated by a significant decrease in the levels of the main UPR regulator GRP78/BIP, and led to apoptosis via upregulation of the ER stress/UPR cell death mediators IRE1α and CHOP. In vivo administration of CX-4945 to a subcutaneous xenotransplant model of human T-ALL significantly delayed tumor growth. Our findings indicate that modulation of the ER stress/UPR signaling through CK2 inhibition could be exploited for inducing apoptosis in T-ALL cells and that CX-4945 may be an efficient treatment for those T-ALLs displaying upregulation of CK2α/PI3K/Akt/mTOR signaling.

Activity of the pan-class I phosphoinositide 3-kinase inhibitor NVP-BKM120 in T-cell acute lymphoblastic leukemia.

Constitutively active phosphoinositide 3-kinase (PI3K) signaling is a common feature of T-cell acute lymphoblastic leukemia (T-ALL), where it upregulates cell proliferation, survival and drug resistance. These observations lend compelling weight to the application of PI3K inhibitors in the therapy of T-ALL. Here, we have analyzed the therapeutic potential of the pan-PI3K inhibitor NVP-BKM120 (BKM120), an orally bioavailable 2,6-dimorpholino pyrimidine derivative, which has entered clinical trials for solid tumors, on both T-ALL cell lines and patient samples. BKM120 treatment resulted in G2/M phase cell cycle arrest and apoptosis, being cytotoxic to a panel of T-ALL cell lines and patient T lymphoblasts, and promoting a dose- and time-dependent dephosphorylation of Akt and S6RP. BKM120 maintained its pro-apoptotic activity against Jurkat cells even when cocultured with MS-5 stromal cells, which mimic the bone marrow microenvironment. Remarkably, BKM120 synergized with chemotherapeutic agents currently used for treating T-ALL patients. Moreover, in vivo administration of BKM120 to a subcutaneous xenotransplant model of human T-ALL significantly delayed tumor growth, thus prolonging survival time. Taken together, our findings indicate that BKM120, either alone or in combination with chemotherapeutic drugs, may be an efficient treatment for T-ALLs that have aberrant upregulation of the PI3K signaling pathway.

Validation of flow cytometric phospho-STAT5 as a diagnostic tool for juvenile myelomonocytic leukemia.

To diagnose juvenile myelomonocytic leukemia (JMML) is sometimes challenging, because around 10% of patients lack molecular abnormalities affecting Ras-MAPK (mitogen-activated protein kinase) pathway and other diseases such as cytomegalovirus infection can mimic clinical signs of JMML. In order to validate a phospho-specific flow cytometry assay assessing phospho-signal transducer and activator of transcription factor 5 (p-STAT5) as a new diagnostic tool for JMML, we examined 22 samples from children with JMML and 47 controls. CD33+/CD34+ cells from 22 patients with JMML showed hyperphosphorylation of STAT5 induced by sub-saturating doses of granulocyte-macrophage colony-stimulating factor (GM-CSF). Using a training set of samples (11 JMML and 23 controls), we identified a threshold for p-STAT5-positive after stimulation with 0.1 ng/ml GM-CSF (17.17%) that discriminates JMML from controls. This threshold was validated in an independent series (11 JMML, 24 controls and 7 cases with diseases other than JMML) where we demonstrated that patients with JMML could be distinguished from other subjects with a sensitivity of 91% (confidence interval (CI) 59-100%) and a specificity of 87% (CI 70-96%). Positive and negative predictive values were 71% (CI 42-92%) and 96% (CI 82-100%), respectively. In conclusion, flow cytometric p-STAT5 profiling is a reliable diagnostic tool for identifying patients with JMML and can contribute to consistency of current diagnostic criteria.

Absence of IL-12Rß2 in CD33(+)CD38(+) pediatric acute myeloid leukemia cells favours progression in NOD/SCID/IL2RγC-deficient mice.

Childhood acute myeloid leukemia (AML) is a hematological malignancy in which tumor burden is continuously replenished by leukemic-initiating cells (ICs), which proliferate slowly and are refractory to chemotherapeutic agents. We investigated whether interleukin (IL)-12, an immuno-modulatory cytokine with anti-tumor activity, may target AML blasts (CD45(+)CD33(+)) and populations known to contain leukemia ICs (that is, CD34(+)CD38(-), CD33(+)CD38(+) and CD44(+)CD38(-) cells). We demonstrate for the first time that: i) AML blasts and their CD34(+)CD38(-), CD33(+)CD38(+), CD44(+)CD38(-) subsets express the heterodimeric IL-12 receptor (IL-12R), ii) AML cells injected subcutaneously into NOD/SCID/Il2rg(-/-) (NSG) mice developed a localized tumor mass containing leukemic ICs and blasts that were virtually eliminated by IL-12 treatment, iii) AML cells injected intravenously into NSG mice engrafted within the first month in the spleen, but not in bone marrow or peripheral blood. At this time, IL-12 dramatically dampened AML CD45(+)CD33(+), CD34(+)CD38(-), CD33(+)CD38(+) and CD44(+)CD38(-) populations, only sparing residual CD33(+)CD38(+) cells that did not express IL-12Rß2. From 30 to 60 days after the initial inoculum, these IL-12-unresponsive cells expanded and metastasized in both control and IL-12-treated NSG mice. Our data indicate that the absence of IL-12Rß2 in pediatric AML cells favours leukemia progression in NOD/SCID/IL2Rγc-deficient mice.

Co-infusion of ex vivo-expanded, parental MSCs prevents life-threatening acute GVHD, but does not reduce the risk of graft failure in pediatric patients undergoing allogeneic umbilical cord blood transplantation.

When compared with BMT, umbilical cord blood transplantation (UCBT) is associated with a lower rate of engraftment and delayed hematological/immunological recovery. This leads to increased risk of TRM in the early post transplantation period due to infection. Acute GVHD, although occurring less frequently in UCBT compared with BMT, is also significantly associated with increased rate of early TRM. BM MSCs are known to support normal in vivo hematopoiesis, and co-transplantation of MSCs has been shown to enhance engraftment of human cord blood hematopoietic cells in nonobese diabetic/SCID mice. In 13 children with hematological disorders (median age 2 years) undergoing UCBT, we co-transplanted paternal, HLA-disparate MSCs with the aim of improving hematological recovery and reducing rejection. We observed no differences in hematological recovery or rejection rates compared with 39 matched historical controls, most of whom received G-CSF after UCBT. However, the rate of grade III and IV acute GVHD was significantly decreased in the study cohort when compared with controls (P=0.05), thus resulting in reduced early TRM. Although these data do not support the use of MSCs in UCBT to support hematopoietic engraftment, they suggest that MSCs, possibly because of their immunosuppressive effect, may abrogate life-threatening acute GVHD and reduce early TRM.

B lymphocyte subsets and their functional activity in the early months of life.

In the present study we evaluated B-cell subsets and their functional development in 74 newborns from birth to 6 months of life. Moreover, we evaluated natural antibody production in vitro. The results documented a predominance of naive B-lymphocytes at all time-points evaluated, decreasing from birth to 6 months (p=0.009). The percentages of CD27+IgD+ and CD27+IgDneg memory B-cells were very low at birth and significantly increased only at 6 months (p=0.02 and p less than 0.001, respectively). We found a significant increase only in in vitro stimulated IgG production at 6 months as compared to birth (p less than 0.001). Moreover, a lower secretion of anti-Pn IgM antibodies up to 6 months of age, as compared to controls was observed. Our results underline that the susceptibility and severe course of infection in the neonate can be attributed, at least in part, to the lack of pre-existing immunological memory and competent adaptive immunity.

The role of killer immunoglobulin-like receptor haplotypes on the outcome of unrelated donor haematopoietic SCT for thalassaemia.

Recent insight into the pathophysiology of acute GVHD after allogeneic haematopoietic SCT has led to a growing interest in the role of natural killer (NK) cells. NK cell cytotoxicity is mainly regulated by the interaction of activating and inhibitory killer immunoglobulin-like receptors (KIRs) with their respective ligands. To investigate the impact of KIRs and their ligands on haematopoietic SCT outcome, we performed a retrospective study of 78 transfusion-dependent thalassaemia patients (median age 10 years, range 1-29 years) transplanted from an unrelated donor selected using high-resolution molecular typing for both class I and II loci after a myeloablative conditioning regimen. GVHD prophylaxis consisted of CsA, short-term MTX and anti-thymocyte globulin in all patients. We found that patients transplanted from donors homozygous for KIR haplotype A had a greater risk of developing grade II-IV acute GVHD compared with those transplanted from a donor carrying at least one B haplotype (hazard ratio=4.5, 99% confidence interval=1.2-17.1, P=0.003). Our study suggests that KIR genotyping of donor and recipient pairs could contribute to the identification of patients at high risk for developing severe complications of haematopoietic SCT and thus may help with the choice of intensity of GVHD prophylaxis.