Exagamglogene Autotemcel for Transfusion-Dependent ß-Thalassemia.

BACKGROUND: Exagamglogene autotemcel (exa-cel) is a nonviral cell therapy designed to reactivate fetal hemoglobin synthesis through ex vivo clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 gene editing of the erythroid-specific enhancer region of BCL11A in autologous CD34+ hematopoietic stem and progenitor cells (HSPCs). METHODS: We conducted an open-label, single-group, phase 3 study of exa-cel in patients 12 to 35 years of age with transfusion-dependent ß-thalassemia and a ß0/ß0, ß0/ß0-like, or non-ß0/ß0-like genotype. CD34+ HSPCs were edited by means of CRISPR-Cas9 with a guide mRNA. Before the exa-cel infusion, patients underwent myeloablative conditioning with pharmacokinetically dose-adjusted busulfan. The primary end point was transfusion independence, defined as a weighted average hemoglobin level of 9 g per deciliter or higher without red-cell transfusion for at least 12 consecutive months. Total and fetal hemoglobin concentrations and safety were also assessed. RESULTS: A total of 52 patients with transfusion-dependent ß-thalassemia received exa-cel and were included in this prespecified interim analysis; the median follow-up was 20.4 months (range, 2.1 to 48.1). Neutrophils and platelets engrafted in each patient. Among the 35 patients with sufficient follow-up data for evaluation, transfusion independence occurred in 32 (91%; 95% confidence interval, 77 to 98; P<0.001 against the null hypothesis of a 50% response). During transfusion independence, the mean total hemoglobin level was 13.1 g per deciliter and the mean fetal hemoglobin level was 11.9 g per deciliter, and fetal hemoglobin had a pancellular distribution (≥94% of red cells). The safety profile of exa-cel was generally consistent with that of myeloablative busulfan conditioning and autologous HSPC transplantation. No deaths or cancers occurred. CONCLUSIONS: Treatment with exa-cel, preceded by myeloablation, resulted in transfusion independence in 91% of patients with transfusion-dependent ß-thalassemia. (Supported by Vertex Pharmaceuticals and CRISPR Therapeutics; CLIMB THAL-111 ClinicalTrials.gov number, NCT03655678.).

CRISPR-Cas9 Gene Editing for Sickle Cell Disease and ß-Thalassemia.

Transfusion-dependent ß-thalassemia (TDT) and sickle cell disease (SCD) are severe monogenic diseases with severe and potentially life-threatening manifestations. BCL11A is a transcription factor that represses γ-globin expression and fetal hemoglobin in erythroid cells. We performed electroporation of CD34+ hematopoietic stem and progenitor cells obtained from healthy donors, with CRISPR-Cas9 targeting the BCL11A erythroid-specific enhancer. Approximately 80% of the alleles at this locus were modified, with no evidence of off-target editing. After undergoing myeloablation, two patients - one with TDT and the other with SCD - received autologous CD34+ cells edited with CRISPR-Cas9 targeting the same BCL11A enhancer. More than a year later, both patients had high levels of allelic editing in bone marrow and blood, increases in fetal hemoglobin that were distributed pancellularly, transfusion independence, and (in the patient with SCD) elimination of vaso-occlusive episodes. (Funded by CRISPR Therapeutics and Vertex Pharmaceuticals; ClinicalTrials.gov numbers, NCT03655678 for CLIMB THAL-111 and NCT03745287 for CLIMB SCD-121.).

Immune reconstitution after transplantation of autologous peripheral stem cells in children: a comparison between CD34+ selected and nonmanipulated grafts.

BACKGROUND AND AIMS: High-dose chemotherapy (HDC) followed by autologous stem cell transplantation (ASCT) improves the prognosis in pediatric patients with several solid tumors and lymphomas. Little is known about the reconstitution of the immune system after ASCT and the influence of CD34+ cell selection on the reconstitution in pediatric patients. METHODS: Between 1990 and 2001, 94 pediatric patients with solid tumors and lymphomas received autologous CD34+ selected or unmanipulated peripheral stem cells after HDC. CD34+ selection was carried out with magnetic microbeads. The absolute numbers of T cells, B cells and natural killer (NK) cells were measured and compared in both groups at various time points post-transplant. RESULTS: Recovery of T cells was significantly faster in the unmanipulated group at day 30, with no significant difference later on. Reconstitution of B and NK cells was similar in both groups without significant differences at any time. The CD34+-selected group was divided into patients receiving less or more than 5.385 × 106/kg CD34+ cells. Patients in the CD34+ high-dose group displayed significantly faster reconstitutions of neutrophiles and lymphocyte subsets than the CD34+ low-dose group. CONCLUSIONS: Engraftment and reconstitution of leukocytes, B cells and NK cells after transplantation of CD34+ selected stem cells were comparable to that in patients receiving unmanipulated grafts. T-cell recovery was faster in the unmanipulated group only within the first month. However, this delay could be compensated by transplantation of >5.385 × 106 CD34+ cells/kg. Especially for patients receiving immunotherapy after HDC large numbers of immune effector cells such as NK and T cells are necessary to mediate antibody-dependent cellular cytotoxicity. Therefore, in patients receiving autologous CD34+-selected grafts, our data emphasize the need to administer high stem cell counts.

Low leukemia burden improves blinatumomab efficacy in patients with relapsed/refractory B-cell acute lymphoblastic leukemia.

BACKGROUND: A lower baseline bone marrow blast percentage (bBMB%) is associated with better outcomes in patients with B-cell acute lymphoblastic leukemia (B-ALL) receiving blinatumomab. The objective of this analysis was to investigate the association between bBMB% and treatment outcomes in relapsed/refractory (R/R) B-ALL. METHODS: Data from five trials of blinatumomab for R/R B-ALL were pooled for analyses. Patients were placed in one of three groups: group 1, ≥50% bBMBs; group 2, ≥25% to <50% bBMBs; group 3, ≥5% to <25% bBMBs. Response and survival outcomes were compared between groups. RESULTS: Data from 683 patients (166 pediatric, 517 adult) were analyzed. Collectively, patients in groups 2 and 3 had significantly higher odds of achieving a complete remission (CR) (odds ratio [OR], 3.50 [95% confidence interval (CI), 2.23-5.48] and 3.93 [95% CI, 2.50-6.18], respectively; p < .001) and minimal/measurable residual disease response (OR, 2.61 and 3.37, respectively; p < .001) when compared with group 1 (reference). Groups 2 and 3 had a 37% and 46% reduction in the risk of death (hazard ratio [HR], 0.63 and 0.54, respectively; p < .001) and a 41% and 43% reduction in the risk of an event (relapse or death) (HR, 0.59 and 0.57, respectively; p < .001) compared with group 1. No significant differences in response or survival outcomes were observed between groups 2 and 3. Seven of nine patients whose bBMB% was lowered to <50% with dexamethasone achieved CR with blinatumomab. CONCLUSION: Any bBMB% <50% was associated with improved efficacy following blinatumomab treatment for R/R B-ALL.

Th1 cytokines in pediatric acute lymphoblastic leukemia.

Immune milieus play an important role in various types of cancer. The present study focuses on the effect of Th1 cytokines on pediatric acute lymphoblastic leukemia (ALL). The reaction of ALL cell lines and patient-derived xenografts (PDX) to the most important Th1 cytokines TNF-α (tumor necrosis factor alpha) and IFN-γ (interferon gamma) is analyzed and correlated with the respective cytokine receptors and the intracellular signaling molecules. ALL cell lines and ALL PDX display a great heterogeneity in cell death after incubation with TNF-α and IFN-γ. Several samples show a dose-dependent and additive induction of cell death by both cytokines; others do not react at all or even display an increased viability. Apoptosis is the main type of cell death induced by Th1 cytokines in ALL cells. Over all leukemia cells analyzed, IFN-γ receptor (IFNGR) shows a higher expression than both TNF-receptors, resulting in higher phosphorylation of STAT1 (signal transducer and activator of transcription) compared to phosphorylation of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B-cells) in the TNF pathway. The activation of STAT1 correlates with the amount of cell death after stimulation with Th1 cytokines. TNF-α and IFN-γ lead to heterogeneous reactions in ALL cell lines and ALL PDX but are able to induce cell death by apoptosis in the majority of ALL blasts. The correlation of a high expression of IFNGR and following activation of STAT1 with cell death indicates an important role for IFN-γ signaling in this setting.

Automated production of specific T cells for treatment of refractory viral infections after allogeneic stem cell transplantation.

Therapy-resistant viral reactivations contribute significantly to mortality after hematopoietic stem cell transplantation. Adoptive cellular therapy with virus-specific T cells (VST) has shown efficacy in various single-center trials. However, the scalability of this therapy is hampered by laborious production methods. In this study we describe the in-house production of VST in a closed system (CliniMACS Prodigy® system, Miltenyi Biotec). In addition, we report the efficacy in 26 patients with viral disease following hematopoietic stem cell transplantation in a retrospective analysis (adenovirus, n=7; cytomegalovirus, n=8; Epstein-Barr virus, n=4; multi-viral, n=7). The production of VST was successful in 100% of cases. The safety profile of VST therapy was favorable (n=2 grade 3 and n=1 grade 4 adverse events; all three were reversible). A response was seen in 20 of 26 patients (77%). Responding patients had a significantly better overall survival than patients who did not respond (P<0.001). Virus-specific symptoms were reduced or resolved in 47% of patients. The overall survival of the whole cohort was 28% after 6 months. This study shows the feasibility of automated VST production and safety of application. The scalability of the CliniMACS Prodigy® device increases the accessibility of VST treatment.

Transferring measurable residual disease measurement in pediatric acute lymphoblastic leukemia from quantitative real-time PCR to digital droplet PCR.

BACKGROUND: Since the measurement of measurable residual disease (MRD) is part of clinical routine examination for children affected with acute lymphoblastic leukemia (ALL), continuous efforts are made to improve its method, applicability and accuracy. Whereas quantitative real-time polymerase chain reaction (qPCR) is considered as the gold standard for MRD detection and endowed with international guidelines for implementation and evaluation, these do not yet exist for digital droplet PCR (ddPCR). However, advantages are seen in droplet partitioning for MRD measurement to allow absolute quantification without depending on reference samples. METHODS: In this study, 17 MRD targets of nine patients with childhood B-ALL were analyzed with qPCR and ddPCR, respectively. All patients were assigned to high risk group and had hematopoietic stem cell transplantation and CD19 antibody therapy for relapse prevention. Starting with the sequences and guidelines of qPCR and optimizing the protocol for ddPCR, the MRD targets could also be measured precisely with this novel method, using the same primer and probe sets as for qPCR. RESULTS: The already established MRD protocol of qPCR could be transferred to ddPCR and all 17 MRD targets were measured in dilution series reaching comparable Limit of detection levels with both PCR methods. CONCLUSIONS: With a given qPCR protocol and some experience in conventional MRD monitoring, it is conceivable to transfer the procedure of MRD measurement to ddPCR technology. Our data is in line with other studies which are summarized and discussed here as well to facilitate the transfer of MRD diagnostics to ddPCR.

Protective T cell receptor identification for orthotopic reprogramming of immunity in refractory virus infections.

Viral infections cause life-threatening disease in immunocompromised patients and especially following transplantation. T cell receptor (TCR) engineering redirects specificity and can bring significant progress to emerging adoptive T cell transfer (ACT) approaches. T cell epitopes are well described, although knowledge is limited on which TCRs mediate protective immunity. In this study, refractory adenovirus (AdV) infection after hematopoietic stem cell transplantation (HSCT) was treated with ACT of highly purified Hexon5-specific T cells using peptide major histocompatibility complex (pMHC)-Streptamers against the immunodominant human leukocyte antigen (HLA)-A∗0101-restricted peptide LTDLGQNLLY. AdV was successfully controlled through this oligoclonal ACT. Novel protective TCRs were isolated ex vivo and preclinically engineered into the TCR locus of allogeneic third-party primary T cells by CRISPR-Cas9-mediated orthotopic TCR replacement. Both TCR knockout and targeted integration of the new TCR in one single engineering step led to physiological expression of the transgenic TCR. Reprogrammed TCR-edited T cells showed strong virus-specific functionality such as cytokine release, effector marker upregulation, and proliferation capacity, as well as cytotoxicity against LTDLGQNLLY-presenting and AdV-infected targets. In conclusion, ex vivo isolated TCRs with clinical proven protection through ACT could be redirected into T cells from naive third-party donors. This approach ensures that transgenic TCRs are protective with potential off-the-shelf use and widened applicability of ACT to various refractory emerging viral infections.

Epitope detection in monocytes (EDIM) for liquid biopsy including identification of GD2 in childhood neuroblastoma-a pilot study.

BACKGROUND: Neuroblastoma (NB) is the most common paediatric extracranial solid malignancy. We analysed the role of the epitope detection in monocytes (EDIM) technique for liquid biopsy in NB patients. METHODS: Tumour epitopes transketolase-like 1 (TKTL1), Apo10 (DNaseX) and GD2 were assessed: expression levels in seven NB tumour samples and five NB cell lines were analysed using RT-PCR and flow cytometry. LAN-1 cells were co-cultured with blood and assessed using EDIM. Peripheral blood macrophages of patients with neuroblastoma (n = 38) and healthy individuals (control group, n = 37) were labelled (CD14+/CD16+) and assessed for TKTL1, Apo10 and GD2 using the EDIM technology. RESULTS: mRNA expression of TKTL1 and DNaseX/Apo10 was elevated in 6/7 NB samples. Spike experiments showed upregulation of TKTL1, Apo10 and GD2 in LAN-1 cells following co-culturing with blood. TKTL1 and Apo10 were present in macrophages of 36/38 patients, and GD2 in 15/19 patients. The 37 control samples were all negative. EDIM expression scores of the three epitopes allowed differentiation between NB patients and healthy individuals. CONCLUSIONS: The EDIM test might serve as a non-invasive tool for liquid biopsy in children suffering from NB. Future studies are necessary for assessing risk stratification, tumour biology, treatment monitoring, and early detection of tumour relapses.

Immunological recovery following HLA-matched CD3+ TCR αß+/CD19+ depleted hematopoietic stem cell transplantation in children.

BACKGROUND: Allogeneic hematopoietic stem cell transplantation (HSCT) is a potentially curative option for children with both malignant and nonmalignant diseases. T-cell depletion techniques may result in reduced transplant-related mortality compared with unmanipulated grafts due to a lower incidence of GvHD. METHODS: Immune recovery and outcome were analyzed in a cohort of 23 patients with malignant and nonmalignant diseases who received CD3+TCRαß+ T- and B-cell-depleted allografts from matched donors after reduced-intensity or myeloablative conditioning. The median number of CD34+, CD3+TCRαß+, and CD19+B-cells infused was 12.7 × 106 /kg, 16.8 × 103 /kg, and 96 × 103 /kg bodyweight. RESULTS: With a median follow-up of 36 (range 1-73) months, overall survival and disease-free survival at 3 years were 65.2% and 60.8%. Eight patients died, six due to the underlying disease and two of extended visceral cGvHD. Immune reconstitution, disease-free, and overall survivals were similar compared with a historical cohort of 23 patients transplanted with matched unmanipulated bone marrow. A significant lower rate of higher grade (III-IV) aGvHD was observed in the manipulated HSCT group (8.7% vs. 26%; p = 0.001), whereas the incidence of cGvHD was equal. CONCLUSIONS: Our data suggest that this graft manipulation strategy could be a safe and effective alternative to conventional HSCT techniques in matched donors.

Introducing isotonic fluids into pediatric oncology.

Objective: Hypotonic fluids are commonly used in pediatric oncology despite evidence that these fluids can lead to hospital-acquired hyponatremia. This practice is most likely due to lack of data evaluating risks and benefits of isotonic fluids in pediatric oncology. To address this issue, our study investigates the effects of exchanging hypotonic fluids with isotonic fluids in a large pediatric oncology unit. Study Design: Prevalence of laboratory disorders before and after the change to balanced, isotonic fluids for all patients are compared in this retrospective analysis. Disturbances in electrolyte levels, fluid-, acid-base balance and kidney function were examined. Results: The rate of hyponatremia was reduced using isotonic fluids. There were no hypernatremic events. Volume overload might increase the use of furosemide when using isotonic fluids. Potassium and bivalent cation levels increased. The risk of acidosis is greatly reduced, whereas alkalosis was more frequent due to furosemide use. The rate of acute kidney injury did not increase. Conclusion: Using isotonic fluids for hyper-hydration in pediatric oncology lead to a modest reduction of hospital-acquired hyponatremia without causing hypernatremia, but the effects on fluid balance need further investigation. The additional intake of bivalent cations and buffering anions in balanced fluids has measurable effects.

Preclinical Evaluation of CRISPR-Edited CAR-NK-92 Cells for Off-the-Shelf Treatment of AML and B-ALL.

Acute myeloid leukemia (AML) and B-cell acute lymphocytic leukemia (B-ALL) are severe blood malignancies affecting both adults and children. Chimeric antigen receptor (CAR)-based immunotherapies have proven highly efficacious in the treatment of leukemia. However, the challenge of the immune escape of cancer cells remains. The development of more affordable and ready-to-use therapies is essential in view of the costly and time-consuming preparation of primary cell-based treatments. In order to promote the antitumor function against AML and B-ALL, we transduced NK-92 cells with CD276-CAR or CD19-CAR constructs. We also attempted to enhance cytotoxicity by a gene knockout of three different inhibitory checkpoints in NK cell function (CBLB, NKG2A, TIGIT) with CRISPR-Cas9 technology. The antileukemic activity of the generated cell lines was tested with calcein and luciferase-based cytotoxicity assays in various leukemia cell lines. Both CAR-NK-92 exhibited targeted cytotoxicity and a significant boost in antileukemic function in comparison to parental NK-92. CRISPR-Cas9 knock-outs did not improve B-ALL cytotoxicity. However, triple knock-out CD276-CAR-NK-92 cells, as well as CBLB or TIGIT knock-out NK-92 cells, showed significantly enhanced cytotoxicity against U-937 or U-937 CD19/tag AML cell lines. These results indicate that the CD19-CAR and CD276-CAR-NK-92 cell lines' cytotoxic performance is suitable for leukemia killing, making them promising off-the-shelf therapeutic candidates. The knock-out of CBLB and TIGIT in NK-92 and CD276-CAR-NK-92 should be further investigated for the treatment of AML.

LMO2 activation by deacetylation is indispensable for hematopoiesis and T-ALL leukemogenesis.

Hematopoietic transcription factor LIM domain only 2 (LMO2), a member of the TAL1 transcriptional complex, plays an essential role during early hematopoiesis and is frequently activated in T-cell acute lymphoblastic leukemia (T-ALL) patients. Here, we demonstrate that LMO2 is activated by deacetylation on lysine 74 and 78 via the nicotinamide phosphoribosyltransferase (NAMPT)/sirtuin 2 (SIRT2) pathway. LMO2 deacetylation enables LMO2 to interact with LIM domain binding 1 and activate the TAL1 complex. NAMPT/SIRT2-mediated activation of LMO2 by deacetylation appears to be important for hematopoietic differentiation of induced pluripotent stem cells and blood formation in zebrafish embryos. In T-ALL, deacetylated LMO2 induces expression of TAL1 complex target genes HHEX and NKX3.1 as well as LMO2 autoregulation. Consistent with this, inhibition of NAMPT or SIRT2 suppressed the in vitro growth and in vivo engraftment of T-ALL cells via diminished LMO2 deacetylation. This new molecular mechanism may provide new therapeutic possibilities in T-ALL and may contribute to the development of new methods for in vitro generation of blood cells.

Arginase 1+ IL-10+ polymorphonuclear myeloid-derived suppressor cells are elevated in patients with active pemphigus and correlate with an increased Th2/Th1 response.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells, which are characterized by their capability to suppress T-cell responses. While MDSCs have been traditionally associated with cancer diseases, their role as regulators of autoimmune diseases is emerging. Pemphigus is a chronic autoimmune blistering skin disease characterized by dysregulated T-cell responses and autoantibody production. The role of MDSCs in pemphigus disease has not been defined yet. The aim of this study was to characterize MDSCs in pemphigus patients and to dissect their relationship with CD4+ T-cell subsets and clinical disease assessments. For this purpose, we performed a cross-sectional analysis of 20 patients with pemphigus. Our results indicate that a population of CD66b+ CD11b+ polymorphonuclear-like MDSCs (PMN-MDSCs) is expanded in the peripheral blood mononuclear cell fraction of pemphigus patients compared to age-matched healthy donors. These PMN-MDSCs have the capability of suppressing allogeneic T-cell proliferation in vitro and show increased expression of characteristic effector molecules such as arginase I and interleukin-10. We further demonstrate that PMN-MDSCs are especially expanded in patients with active pemphigus, but not in patients in remission. Moreover, MDSC frequencies correlate with an increased Th2/Th1 cell ratio. In conclusion, the identification of a functional PMN-MDSC population suggests a possible role of these cells as regulators of Th cell responses in pemphigus.

Blinatumomab in pediatric patients with relapsed/refractory B-cell precursor acute lymphoblastic leukemia.

OBJECTIVE: Pediatric patients with relapsed or refractory acute lymphoblastic leukemia have a poor prognosis. We here assess the response rates, adverse events, and long-term follow-up of pediatric patients with relapsed/refractory acute lymphoblastic leukemia receiving blinatumomab. METHODS: Retrospective analysis of a single-center experience with blinatumomab in 38 patients over a period of 10 years. RESULTS: The median age at onset of therapy was 10 years (1-21 years). Seventy-one percent of patients had undergone at least one hematopoietic stem cell transplantation (HSCT) prior to treatment with blinatumomab. We observed a response to blinatumomab in 13/38 patients (34%). The predominant side effect was febrile reactions, nearly half of the patients developed a cytokine release syndrome. Eight events of neurotoxicity were registered over the 78 cycles (15%). To date, nine patients (24%) are alive and in complete molecular remission. All survivors underwent haploidentical HSCT after treatment with blinatumomab. CONCLUSIONS: Despite heavy pretreatment of most of our patients, severe adverse events were rare and response rates encouraging. Blinatumomab is a valuable bridging salvage therapy for relapsed or refractory patients to a second or even third HSCT.

A case series of children and young people admitted to a tertiary care hospital in Germany with COVID-19.

BACKGROUND: While our knowledge about COVID-19 in adults has rapidly increased, data on the course of disease and outcome in children with different comorbidities is still limited. METHODS: Prospective, observational study at a tertiary care children's hospital in southern Germany. Clinical and virology data from all paediatric patients admitted with SARS-CoV-2 infection at our hospital were prospectively assessed. RESULTS: Between March and November 2020, 14 patients were admitted with COVID-19. One patient was admitted a second time with COVID-19 6 months after initial disease. Among seven patients with severe underlying comorbidities, three developed multisystem inflammatory syndrome (MIS-C), two were admitted to the paediatric intensive care unit. One patient needed invasive ventilation. Another patient died shortly after discharge of COVID-19-related complications. CONCLUSIONS: While COVID-19 generally causes mild disease in children, severe respiratory illness and MIS-C occur, in some cases with fatal outcome. Children with underlying diseases might be at special risk for severe disease.

Mesenchymal Stem Cell Therapy for Severe COVID-19 ARDS.

BACKGROUND: The COVID-19 pandemic reached Germany in spring 2020. No proven treatment for SARS-CoV-2 was available at that time, especially for severe COVID-19-induced ARDS. We determined whether the infusion of mesenchymal stromal cells (MSCs) would help to improve pulmonary function and overall outcome in patients with severe COVID-19 ARDS. We offered MSC infusion as an extended indication to all critically ill COVID-19 patients with a Horovitz index <100. We treated 5 out of 23 patients with severe COVID-19 ARDS with an infusion of MSCs. One million MSCs/kg body weight was infused over 30 minutes, and the process was repeated in 3 patients twice and in 2 patients 3 times. RESULT: Four out of 5 MSC-treated patients compared to 50% of control patients (9 out of 18) received ECMO support (80%). The MSC group showed a higher Murray score on admission than control patients, reflecting more severe pulmonary compromise (3.5 ± 0.2 versus 2.8 ± 0.3). MSC infusion was safe and well tolerated. The MSC group had a significantly higher Horovitz score on discharge than the control group. Compared to controls, patients with MSC treatment showed a significantly lower Murray score upon discharge than controls. In the MSC group, 4 out of 5 patients (80%) survived to discharge and exhibited good pulmonary function, whereas only 8 out of 18 patients (45%) in the control group survived to discharge. CONCLUSION: MSC infusion is a safe treatment for COVID-19 ARDS that improves pulmonary function and overall outcome in this patient population.

Arsenic trioxide in pediatric cancer - a case series and review of literature.

Arsenic trioxide (ATO) has become an established component of treatment protocols for acute promyelocytic leukemia (APL) with excellent efficacy and no relevant sustained toxicity. Part of its action has been attributed to the inhibition of Hedgehog signaling (Hh) which enables a possible therapeutic approach as many pediatric tumor entities have been associated with increased Hh activity. We retrospectively analyzed 31 patients with refractory and relapsed pediatric cancer who were treated with ATO at the University Children's Hospital of Tuebingen. Additionally a literature review on the clinical and preclinical use of ATO in pediatric cancer treatment was performed.ATO alone as well as combinations with other drugs have proven effective in vitro and in mouse models of various pediatric malignancies. However, only few data on the clinical use of ATO in pediatric patients besides APL exist. In our patient sample, ATO was overall well tolerated in the treatment of various pediatric cancers, even in combination with other cytostatic drugs. Due to distinct tumor entities, differently progressed disease stages and varying co-medication, no clear statement can be made regarding the efficacy of ATO treatment. However, patients with proven Hh activation in molecular tumor profiling surpassed all other patients, who received ATO in an experimental treatment setting, in terms of survival. As molecular profiling of tumors increases and enhanced Hh activity can be detected at an early stage, ATO might expand its clinical use to other pediatric malignancies beyond APL depending on further clinical studies.

Comparative analysis of lentiviral gene transfer approaches designed to promote fetal hemoglobin production for the treatment of ß-hemoglobinopathies.

ß-Hemoglobinopathies are among the most common single-gene disorders and are caused by different mutations in the ß-globin gene. Recent curative therapeutic approaches for these disorders utilize lentiviral vectors (LVs) to introduce a functional copy of the ß-globin gene into the patient's hematopoietic stem cells. Alternatively, fetal hemoglobin (HbF) can reduce or even prevent the symptoms of disease when expressed in adults. Thus, induction of HbF by means of LVs and other molecular approaches has become an alternative treatment of ß-hemoglobinopathies. Here, we performed a head-to-head comparative analysis of HbF-inducing LVs encoding for: 1) IGF2BP1, 2) miRNA-embedded shRNA (shmiR) sequences specific for the γ-globin repressor protein BCL11A, and 3) γ-globin gene. Furthermore, two novel baboon envelope proteins (BaEV)-LVs were compared to the commonly used vesicular-stomatitis-virus glycoprotein (VSV-G)-LVs. Therapeutic levels of HbF were achieved for all VSV-G-LV approaches, from a therapeutic level of 20% using γ-globin LVs to 50% for both IGF2BP1 and BCL11A-shmiR LVs. Contrarily, BaEV-LVs conferred lower HbF expression with a peak level of 13%, however, this could still ameliorate symptoms of disease. From this thorough comparative analysis of independent HbF-inducing LV strategies, we conclude that HbF-inducing VSV-G-LVs represent a promising alternative to ß-globin gene addition for patients with ß-hemoglobinopathies.

The potential role of γδ T cells after allogeneic HCT for leukemia.

Allogeneic hematopoetic stem cell transplantation (HCT) offers an option for patients with hematologic malignancies, in whom conventional standard therapies failed or are not effective enough to cure the disease. Successful HCT can restore functional hematopoiesis and immune function, and the new donor-derived immune system can exert a graft-versus-leukemia (GVL) effect. However, allogenic HCT can also be associated with serious risks for transplantation-related morbidities or mortalities such as graft-versus-host disease (GVHD) or life-threatening infectious complications. GVHD is caused by alloreactive T lymphocytes, which express the αß T-cell receptor, whereas lymphocytes expressing the γδ T-cell receptor are not alloreactive and do not induce GVHD but can exhibit potent antileukemia and anti-infectious activities. Therefore, γδ T cells are becoming increasingly interesting in allogeneic HCT, and clinical strategies to exploit the full function of these lymphocytes have been and are being developed. Such strategies comprise the in vivo activation of γδ T cells or subsets after HCT by certain drugs or antibodies or the ex vivo expansion and manipulation of either patient-derived or donor-derived γδ T cells and their subsets and the adoptive transfer of the ex vivo-activated lymphocytes. On the basis of the absence of dysregulated alloreactivity, such approaches could induce potent GVL effects in the absence of GVHD. The introduction of large-scale clinical methods to enrich, isolate, expand, and manipulate γδ T cells will facilitate future clinical studies that aim to exploit the full function of these beneficial nonalloreactive lymphocytes.