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Blinatumomab is the first in its class bispecific T-cell engager monoclonal antibody, which binds to CD19 expressed on B-cells and CD3 expressed on T-cells, resulting in lysis of CD19-positive cells common in B-cell malignancies. Blinatumomab is Food and Drug Administration (FDA) approved for the treatment of adults and children with relapsed/refractory or minimal residual disease (MRD) positive precursor B-cell ALL (B-ALL). Despite impressive efficacy for the approved indications and favorable toxicity profile compared to standard-of-care chemotherapy, blinatumomab presents unique health-system challenges related to preparation, administration, toxicity monitoring and medication error prevention. Blinatumomab delivery also offers plethora of opportunities for interdisciplinary planning and collaboration. The purpose of this paper is to discuss practical considerations for safe blinatumomab delivery from the pharmacy and nursing perspectives. [ABSTRACT FROM AUTHOR]

Acute lymphoblastic leukemia (ALL) represents around 25% of adult acute leukemias. Despite the increasing improvement in the survival rate of ALL patients during the last decade, the heterogeneous clinical and molecular features of this malignancy still represent a major challenge for treatment and achieving better outcomes. To identify aberrantly expressed genes in bone marrow (BM) samples from adults with ALL, transcriptomic analysis was performed using Affymetrix Human Transcriptome Array 2.0 (HTA 2.0). Differentially expressed genes (DEGs) (±2-fold change, p-value < 0.05, and FDR < 0.05) were detected using the Transcriptome Analysis Console. Gene Ontology (GO), Database for Annotation, Visualization, and Integrated Discovery (DAVID), and Ingenuity Pathway Analysis (IPA) were employed to identify gene function and define the enriched pathways of DEGs. The protein–protein interactions (PPIs) of DEGs were constructed. A total of 871 genes were differentially expressed, and DNTT, MYB, EBF1, SOX4, and ERG were the top five up-regulated genes. Meanwhile, the top five down-regulated genes were PTGS2, PPBP, ADGRE3, LUCAT1, and VCAN. An association between ERG, CDK6, and SOX4 expression levels and the probability of relapse and death was observed. Regulation of the immune system, immune response, cellular response to stimulus, as well as apoptosis signaling, inflammation mediated by chemokines and cytokines, and T cell activation were among the most altered biological processes and pathways, respectively. Transcriptome analysis of ALL in adults reveals a group of genes consistently associated with hematological malignancies and underscores their relevance in the development of ALL in adults. [ABSTRACT FROM AUTHOR]

This paper chronicles a 2-year-old girl who presented with acute leukemia/lymphoma syndrome of the T cell immunophenotype. At this time, the cytogenetic analysis of her bone marrow cells showed a reciprocal translocation between the short arm of chromosome 12 and the long arm of chromosome 13, t(12;13)(p13;q14). The immunophenotyping of bone marrow blast cells by flow cytometry revealed a population of cells positive for CD56, CD117, CD45, partial CD33, partial HLA-DR, CD13, CD7, CD2 and CD5. Therefore, a diagnosis of acute leukemia with a mixed T cell/myeloid phenotype was made. The patient had a poor response to classic T cell acute lymphocytic leukemia/lymphoma therapy; thus, her treatment was changed to a myeloid leukemia protocol, which produced a good response. She underwent a successful cord blood transplantation from an unrelated HLA partially matched donor. The coexistence of these two phenotypes prompts questions about the existence of clonal instability, which might influence the choice of therapy. The rarity of the t(12;13)(p13;q14) and the coexistence of T cell/myeloid markers suggest a nonrandom association. To the best of our knowledge, this is the first reported case in which a cell clone bearing a t(12;13)(p13;q14) translocation in a mixed T cell/myeloid lesion was detected. Copyright © 2012 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Chimeric Antigen Receptor (CAR) T-cell therapy has demonstrated high rates of response in recurrent B-cell Acute Lymphoblastic Leukemia in children and young adults. Despite this success, a fraction of patients' experience relapse after treatment. Relapse is often preceded by recovery of healthy B cells, which suggests loss or dysfunction of CAR T-cells in bone marrow. This site is harder to access, and thus is not monitored as frequently as peripheral blood. Understanding the interplay between B cells, leukemic cells, and CAR T-cells in bone marrow is paramount in ascertaining the causes of lack of response. In this paper, we put forward a mathematical model representing the interaction between constantly renewing B cells, CAR T-cells, and leukemic cells in the bone marrow. Our model accounts for the maturation dynamics of B cells and incorporates effector and memory CAR T-cells. The model provides a plausible description of the dynamics of the various cellular compartments in bone marrow after CAR T infusion. After exploration of the parameter space, we found that the dynamics of CAR T product and disease were independent of the dose injected, initial B-cell load, and leukemia burden. We also show theoretically the importance of CAR T product attributes in determining therapy outcome, and have studied a variety of possible response scenarios, including second dosage schemes. We conclude by setting out ideas for the refinement of the model. [ABSTRACT FROM AUTHOR]

Relapse is a major limitation of chimeric antigen receptor (CAR) T-cell therapy. Here, we speculated that decitabine (DAC) in combination with fludarabine and cyclophosphamide (FC) as a lymphodepletion regimen may improve the efficacy of CD19/CD22 CAR T-cell therapy. Fourteen of 26 patients with relapsed/refractory B cell acute lymphoblastic leukemia (r/r B-ALL) without remission before lymphodepletion treatment were treated with DAC (total dose 100 mg/m2 in 3 days) followed by the FC regimen (DAC group), while twelve patients received the FC regimen (CON group). On Day 28 after CAR T-cells infusion, no significant differences in complete remission (CR) and minimal residual disease negative CR rates were found between both groups. However, there were significant differences in overall survival (OS) and leukemia-free survival (LFS) between two groups: 3-year OS, 92.3% (DAC) versus 41.7% (CON), P = 0.005 and 3-year LFS, 92.9% (DAC) versus 27.3% (CON), P < 0.001. There was no significant difference in the incidence of cytokine release syndrome between both groups. Median time to platelet and neutrophil counts recovery was similar in both groups. All adverse events were reversible and manageable. In conclusion, DAC in combination with the FC lymphodepletion regimen may be a new treatment option that can improve the efficacy of CAR T-cell therapy in r/r B-ALL. [ABSTRACT FROM AUTHOR]

Background: Previous studies have revealed conflicting findings concerning the efficacy of radiotherapy (RT) and radiochemotherapy (RCT) in IE/IIE extranodal nasal-type natural killer/T cell lymphoma (ENKTL). In this study, we conducted a comprehensive meta-analysis to address this issue. Methods: We systematically searched PubMed, Cochrane Central Register of Controlled Trials (CENTRAL), EmBase, BISOS, Clinical Trials and some Chinese databases for relevant studies, and 2 prospective and 15 retrospective studies involving a total of 1595 patients met our inclusion criteria. Results: The meta-analysis showed no significant differences in complete remission (CR) [odds ratio (OR) 0.85, 95% confidence interval (CI) 0.42–1.72, p = 0.65], 5-year overall survival (OS) [hazard ratio (HR) 1.11, 95% CI 0.85–1.45, p = 0.43] and 5-year progression free survival (PFS) (HR 1.07, 95% CI 0.75–1.53, p = 0.70) in patients who received RT versus RCT. Furthermore, the addition of CT decreased neither systemic failure (SL) (OR 0.75, 95% CI 0.47–1.21, p = 0.24) nor locoregional failure (LF) (OR 1.17, 95% CI 0.68–2.01, p = 0. 57). Conclusions: RCT did not have an obvious advantage over RT for treating IE/IIE ENKTL. [ABSTRACT FROM AUTHOR]

Simple Summary: Acute lymphoblastic leukemia is the most common blood cancer in pediatric patients. Despite the enormous progress in ALL treatment, which is reflected by a high 5-year overall survival rate that reaches up to 96% in the most recent studies, there are still patients that cannot be saved. Treatment of ALL is based on conventional methods, including chemotherapy and radiotherapy. These methods carry with them the risk of very high toxicities. Severe complications related to conventional therapies decrease their effectiveness and can sometimes lead to death. Therefore, currently, numerous studies are being carried out on novel forms of treatment. In this work, classical methods of treatment have been summarized. Furthermore, novel treatment methods and the possibility of combining them with chemotherapy have been incorporated into the present work. Targeted treatment, CAR-T-cell therapy, and immunotherapy for ALL have been described. Treatment options for the relapse/chemoresistance ALL have been presented. Acute lymphoblastic leukemia is the most common blood cancer in pediatric patients. There has been enormous progress in ALL treatment in recent years, which is reflected by the increase in the 5-year OS from 57% in the 1970s to up to 96% in the most recent studies. ALL treatment is based primarily on conventional methods, which include chemotherapy and radiotherapy. Their main weakness is severe toxicity, which prompts dose reduction, decreases the effectiveness of the treatment, and, in some cases, can lead to death. Currently, numerous modifications in treatment regimens are applied in order to limit toxicities emerging from conventional approaches and improve outcomes. Hematological treatment of pediatric patients is reaching for more novel treatment options, such as targeted treatment, CAR-T-cells therapy, and immunotherapy. These methods are currently used in conjunction with chemotherapy. Nevertheless, the swift progress in their development and increasing efficacity can lead to applying those novel therapies as standalone therapeutic options for pediatric ALL. [ABSTRACT FROM AUTHOR]

Chimeric antigen receptor (CAR) T cell therapy is a potent new treatment option for relapsed or refractory hematologic malignancies. As the monitoring of CAR T cell kinetics can provide insights into the activity of the therapy, appropriate CAR T cell detection methods are essential. Here, we report on the comprehensive validation of a flow cytometric assay for peripheral blood CD19 CAR T cell detection. Further, a retrospective analysis (n = 30) of CAR T cell and B cell levels over time has been performed, and CAR T cell phenotypes have been characterized. Serial dilution experiments demonstrated precise and linear quantification down to 0.05% of T cells or 22 CAR T cell events. The calculated detection limit at 13 events was confirmed with CAR T cell negative control samples. Inter-method comparison with real-time PCR showed appreciable correlation. Stability testing revealed diminished CAR T cell values already one day after sample collection. While we found long-term CAR T cell detectability and B cell aplasia in most patients (12/17), some patients (5/17) experienced B cell recovery. In three of these patients the coexistence of CAR T cells and regenerating B cells was observed. Repeat CAR T cell infusions led to detectable but limited re-expansions. Comparison of CAR T cell subsets with their counterparts among all T cells showed a significantly higher percentage of effector memory T cells and a significantly lower percentage of naïve T cells and T EMRA cells among CAR T cells. In conclusion, flow cytometric CAR T cell detection is a reliable method to monitor CAR T cells if measurements start without delay and sufficient T cell counts are given. [ABSTRACT FROM AUTHOR]

Despite high complete remission (CR) rates with frontline therapy, relapses are frequent in adults with T‐cell acute lymphoblastic leukemia (T‐ALL) with limited salvage options. We analyzed the outcomes and prognostic factors for CR to salvage therapy and overall survival (OS) of patients with R/R T‐ALL included in two prospective measurable residual disease‐oriented trials. Seventy‐five patients (70 relapsed, 5 refractory) were identified. Relapses occurred in bone marrow, isolated or combined in 50 patients, and in the central nervous system (CNS; isolated or combined) in 20. Second CR was attained in 30/75 patients (40%). Treatment with FLAG‐Ida and isolated CNS relapse were independently associated with a higher CR rate after first salvage therapy. The median OS was 6.2 (95% confidence interval [CI], 3.9–8.6) months, with a 4‐year OS probability of 18% (95% CI, 9%–27%). No differences in survival were observed according to the treatment with hematopoietic stem cell transplantation in patients in CR after first salvage therapy. Multivariable analysis showed a ≥12‐month interval between first CR and relapse, CR after first salvage therapy and isolated CNS relapse as favorable prognostic factors for OS with hazard ratios (HR) (95% CI) of 1.931 (1.109–3.362), 2.958 (1.640–5.334), and 2.976 (1.157–7.655), respectively. This study confirms the poor outcomes of adults with R/R T‐ALL among whom FLAG‐Ida was the best of the rescue therapies evaluated. Late relapse, CR after first rescue therapy and isolated CNS relapse showed prognostic impact on survival. More effective rescue therapies are needed in adults with R/R T‐ALL. [ABSTRACT FROM AUTHOR]

The article focuses on a paper which discusses the management of Down Syndrome-related leukemia. It notes the contribution of financial and socioeconomic factors on positive outcomes of the condition's treatment. Moreover, a chart is presented depicting the outcome of T lineage acute lymphoblastic leukemia (ALL) in India.

The B cell surface antigen CD19 is a target for treating B cell malignancies, such as B cell precursor acute lymphoblastic leukemia and B cell non-Hodgkin lymphoma. The BiTE® immuno-oncology platform includes blinatumomab, which is approved for relapsed/refractory B cell precursor acute lymphoblastic leukemia and B cell precursor acute lymphoblastic leukemia with minimal residual disease. Blinatumomab is also being evaluated in combination with other agents (tyrosine kinase inhibitors, checkpoint inhibitors, and chemotherapy) in various treatment settings, including frontline protocols. An extended half-life BiTE molecule is also under investigation. Patients receiving blinatumomab may experience cytokine release syndrome and neurotoxicity; however, these events may be less frequent and severe than in patients receiving other CD19-targeted immunotherapies, such as chimeric antigen receptor T cell therapy. We review BiTE technology for treating malignancies that express CD19, analyzing the benefits and limitations of this bispecific T cell engager platform from clinical experience with blinatumomab. [ABSTRACT FROM AUTHOR]

Use of adoptive T-cell therapy modified with chimeric antigen receptor (CAR-T) has revolutionized treatment of patients with relapsed/refractory (r/r) B-cell acute lymphoblastic leukemia (B-ALL). CAR-T cells directed against CD19 antigen have produced response rates as high as 90% in clinical trials for r/r B-ALL. Despite high rates of complete remissions, the durability of responses has been sub-optimal with frequent relapses, especially in adult B-ALL population. Systemic toxicities from CAR-T therapy and standardization of toxicities grading and management is another major hurdle in the development of CAR-T field. In this review, we discuss the latest evidence of CAR-T therapy in B-ALL, potential mechanisms of relapse and barriers to CAR-T cell therapy in B-ALL. We also debate the role of allogeneic hematopoietic stem cell transplant (allo-HCT) post CAR-T therapy. [ABSTRACT FROM AUTHOR]

Chimeric antigen receptor (CAR)–engineered T‐cell therapy is becoming one of the most promising approaches in the treatment of cancer. On June 28, 2018, the Committee for Advanced Therapies (CAT) and the Committee for Medicinal Products for Human Use (CHMP) of the European Medicines Agency adopted a positive opinion, recommending the granting of a marketing authorization for the medicinal product Kymriah for pediatric and young adult patients up to 25 years of age with B‐cell acute lymphoblastic leukemia (ALL) that is refractory, in relapse after transplant, or in second or later relapse and for adult patients with relapsed or refractory diffuse large B‐cell lymphoma (DLBCL) after two or more lines of systemic therapy. Kymriah became one of the first European Union–approved CAR T therapies. The active substance of Kymriah is tisagenlecleucel, an autologous, immunocellular cancer therapy that involves reprogramming the patient's own T cells to identify and eliminate CD19‐expressing cells. This is achieved by addition of a transgene encoding a CAR. The benefit of Kymriah was its ability to achieve remission with a significant duration in patients with ALL and an objective response with a significant duration in patients with DLBCL. The most common hematological toxicity was cytopenia in both patients with ALL and those with DLBCL. Nonhematological side effects in patients with ALL were cytokine release syndrome (CRS), infections, secondary hypogammaglobulinemia due to B‐cell aplasia, pyrexia, and decreased appetite. The most common nonhematological side effects in patients with DLBCL were CRS, infections, pyrexia, diarrhea, nausea, hypotension, and fatigue. Kymriah also received an orphan designation on April 29, 2014, following a positive recommendation by the Committee for Orphan Medicinal Products (COMP). Maintenance of the orphan designation was recommended at the time of marketing authorization as the COMP considered the product was of significant benefit for patients with both conditions. Implications for Practice: Chimeric antigen receptor (CAR)–engineered T‐cell therapy is becoming the most promising approach in cancer treatment, involving reprogramming the patient's own T cells with a CAR‐encoding transgene to identify and eliminate cancer‐specific surface antigen–expressing cells. On June 28, 2018, Kymriah became one of the first EMA approved CAR T therapies. CAR T technology seems highly promising for diseases with single genetic/protein alterations; however, for more complex diseases there will be challenges to target clonal variability within the tumor type or clonal evolution during disease progression. Products with a lesser toxicity profile or more risk‐minimization tools are also anticipated. The EMA summarizes the reasons behind marketing authorization of the medicinal product Kymriah, one of the first European Union‐approved CAR T therapies, for B cell acute lymphoblastic leukemia and diffuse large B cell lymphoma. [ABSTRACT FROM AUTHOR]

By increasing disease‐free survival and offering the potential for long‐term cure, chimeric antigen receptor (CAR) T‐cell therapy has dramatically expanded therapeutic options among those with high‐risk B‐cell malignancies. As CAR T‐cell utilization evolves however, novel challenges are generated. These include determining how to optimally integrate CAR T cells into standard of care and overcoming mechanisms of resistance to CAR T‐cell therapy, such as evolutionary stress induced on cancer cells leading to immunophenotypic changes that allow leukemia to evade this targeted therapy. Compounding these challenges are the limited ability to determine differences between various CAR T‐cell constructs, understanding the generalizability of trial outcomes from multiple sites utilizing unique CAR manufacturing strategies, and comparing distinct criteria for toxicity grading while defining optimal management. Additionally, as understanding of CAR behavior in humans has developed, strategies have appropriately evolved to proactively mitigate toxicities. These challenges offer complimentary insights and guide next steps to enhance the efficacy of this novel therapeutic modality. With a focus on B‐cell malignancies as the paradigm for effective CAR T‐cell therapy, this review describes advances in the field as well as current challenges and future directions. [ABSTRACT FROM AUTHOR]

Leukemia relapse is the main cause for mortality after allogeneic stem cell transplantation (allo-SCT). Donor-derived allo-immune responses eliminate the residual host hematopoiesis and protect against relapse. Cytomegalovirus (CMV) reactivation (CMV-R) after allo-SCT may trigger anti-leukemic effects. The impact of CMV-specific CD8+ T-cells (CMV-CTLs) on the outcome after allo-SCT is currently unknown. Here, we studied the relationship between CMV-CTLs, overall T-cell reconstitution and relapse incidence in 103 patients with acute leukemia (n = 91) or myelodysplastic syndrome (n = 12) following CMV-seropositive recipient/donor (R+/D+) allo-SCT. Patients were subdivided based on the presence or absence of CMV-CTLs at 3 months after allo-SCT. Presence of CMV-CTLs was associated with preceding CMV-R and a fast T-cell reconstitution. Univariate analysis showed a significantly lower 1-, 2- and 5-year cumulative incidence of relapse (CIR) in patients with CMV-CTLs compared to those without CMV-CTLs. Multivariable regression analysis of the outcome performed with other relevant parameters chosen from univariate analysis revealed that presence of CMV-CTLs and chronic graft-versus-host disease (cGvHD) were the only independent factors associated with a low CIR. Onset of relapse was significantly later in patients with CMV-CTLs (median 489 days) than in in those without (median 152 days, p = 0.041) during a five-year follow-up. Presence of CMV-CTLs was associated with a lower incidence of early relapses (1 and 2-years), while cGvHD lead to a lower incidence of late relapses (2 to 5-years). In conclusion, our data show that CMV-CTLs indicate a functional immune-reconstitution protective against early relapse. [ABSTRACT FROM AUTHOR]

CD19 chimeric antigen receptor (CAR) T cell therapy has shown impressive results in treating acute lymphoblastic leukemia (B-ALL), chronic lymphoblastic leukemia (B-CLL), and B-cell non-Hodgkin lymphoma (B-NHL) over the past few years. Meanwhile, the cytokine release syndrome (CRS), which could be moderate or even life-threatening, has emerged as the most significant adverse effect in the clinical course of this novel targeting immunotherapy. In this systematic review, we analyzed the incidence of severe CRS in 19 clinical trials selected from studies published between 2010 and 2017. The pooled severe CRS proportion was 29.3% (95% confidence interval [CI] 12.3-49.1%) in B-ALL, 38.8% (95%CI 12.9-67.6%) in B-CLL, and 19.8% (95%CI 4.2-40.8%) in B-NHL. In the univariate meta regression analysis, the proliferation of CD19-CAR-T cell in vivo was correlated with the severe CRS. Specifically, total infusion cell dose contributed to the severe CRS occurring in B-ALL patients but not in B-CLL or B-NHL patients. Tumor burden was strongly associated with the severity of CRS in B-ALL. Besides, post-HSCT CD19 CAR-T cell infusion represented lower severe CRS incidence. Further investigations into the risk factors of CRS in B-CLL and B-NHL are needed. [ABSTRACT FROM AUTHOR]

Introduction: Acute lymphoblastic leukemia (ALL) is the most common cancer diagnosed in children. The precise mechanism behind the relapse in this disease is not clearly known. One possible mechanism could be the accumulation of immunosuppressive cells, including myeloid-derived suppressor cells (MDSCs) and T regulatory cells (Tregs) which we and others have reported to mediate suppression of anti-tumor immune responses. Aim: In this study, we aimed to analyze the numbers of these cells in a population of B-ALL pediatric patients. Methods: Peripheral blood samples withdrawn from B-ALL pediatric patients (n = 45 before, during and after the induction phase of chemotherapy. Using multi parametric flow cytometric analysis. MDSCs were identified as Lin-HLA-DR-CD33+CD11b+; and Treg cells were defined as CD4+CD25+CD127-/low. Results: Early diagnosed B-ALL patients showed significant increases in the numbers of MDSCs and Tregs as compared to healthy volunteers. During induction of chemotherapy, however, the patients showed higher and lower numbers of MDSCs and Treg cells, respectively as compared to early diagnosed patients (i.e., before chemotherapy). After induction of chemotherapy, the numbers of MDSCs and Treg cells showed higher increases and decreases, respectively as compared to the numbers in patients during chemotherapy. Conclusion: Our results indicate that B-ALL patients harbor high numbers of both MDSCs and Tregs cells. This pilot study opens a new avenue to investigate the mechanism mediating the emergence of these cells on larger number of B-ALL patients at different treatment stages. [ABSTRACT FROM AUTHOR]

Opinion Statement: Adoptive transfer of autologous T cells engineered to express a chimeric antigen receptor (CAR) represents a powerful targeted immunotherapy that has shown great promise in some of the most refractory leukemias. CAR-modified T cells directed against CD19 have led the way, setting a high standard with remission rates as high as 90 % in clinical trials for relapsed/refractory acute lymphoblastic leukemia (ALL). Yet, the first demonstration of efficacy was in another disease, chronic lymphocytic leukemia (CLL), in which CD19-targeted CAR T cells eradicated bulky, highly refractory disease. Despite early encouraging results, clinical trials in CLL have yielded lower response rates, revealing disease-specific differences in response in this form of immunotherapy. Ongoing research focused on identifying and overcoming these limitations, promises to improve response rates. Beyond the induction of remission, the transformative impact of engineered T cell therapy lies in its potential for long-term disease control. With longer follow-up and durable T cell persistence now reported, we are closer to answering the question of whether sustained remissions are possible with CAR T cell monotherapy. As might be expected with a highly effective therapy using a single mechanism of action, escape pathways have emerged. Combinatorial approaches are needed to anticipate and prevent this mode of relapse. Lastly, toxicity management is vital to ensure the safety of this exciting cancer immunotherapy. [ABSTRACT FROM AUTHOR]

The molecular determinants that render specific populations of normal cells susceptible to oncogenic reprogramming into self-renewing cancer stem cells are poorly understood. Here, we exploit T-cell acute lymphoblastic leukemia (T-ALL) as a model to define the critical initiating events in this disease. First, thymocytes that are reprogrammed by the SCL and LMO1 oncogenic transcription factors into self-renewing pre-leukemic stem cells (pre-LSCs) remain non-malignant, as evidenced by their capacities to generate functional T cells. Second, we provide strong genetic evidence that SCL directly interacts with LMO1 to activate the transcription of a self-renewal program coordinated by LYL1. Moreover, LYL1 can substitute for SCL to reprogram thymocytes in concert with LMO1. In contrast, inhibition of E2A was not sufficient to substitute for SCL, indicating that thymocyte reprogramming requires transcription activation by SCL-LMO1. Third, only a specific subset of normal thymic cells, known as DN3 thymocytes, is susceptible to reprogramming. This is because physiological NOTCH1 signals are highest in DN3 cells compared to other thymocyte subsets. Consistent with this, overexpression of a ligand-independent hyperactive NOTCH1 allele in all immature thymocytes is sufficient to sensitize them to SCL-LMO1, thereby increasing the pool of self-renewing cells. Surprisingly, hyperactive NOTCH1 cannot reprogram thymocytes on its own, despite the fact that NOTCH1 is activated by gain of function mutations in more than 55% of T-ALL cases. Rather, elevating NOTCH1 triggers a parallel pathway involving Hes1 and Myc that dramatically enhances the activity of SCL-LMO1 We conclude that the acquisition of self-renewal and the genesis of pre-LSCs from thymocytes with a finite lifespan represent a critical first event in T-ALL. Finally, LYL1 and LMO1 or LMO2 are co-expressed in most human T-ALL samples, except the cortical T subtype. We therefore anticipate that the self-renewal network described here may be relevant to a majority of human T-ALL. [ABSTRACT FROM AUTHOR]

Adoptive transfer of T cells expressing a CD19-specific chimeric antigen receptor (CAR) is being evaluated in multiple clinical trials. Our current approach to adoptive immunotherapy is based on a second generation CAR (designated CD19RCD28) that signals through a CD28 and CD3-ζ endodomain. T cells are electroporated with DNA plasmids from the Sleeping Beauty (SB) transposon/transposase system to express this CAR. Stable integrants of genetically modified T cells can then be retrieved when co-cultured with designer artificial antigen presenting cells (aAPC) in the presence of interleukin (IL)-2 and 21. Here, we reveal how the platform technologies of SB-mediated transposition and CAR-dependent propagation on aAPC were adapted for human application. Indeed, we have initiated clinical trials in patients with high-risk B-lineage malignancies undergoing autologous and allogeneic hematopoietic stem-cell transplantation (HSCT). We describe the process to manufacture clinical grade CD19-specific T cells derived from healthy donors. Three validation runs were completed in compliance with current good manufacturing practice for Phase I/II trials demonstrating that by 28 days of co-culture on γ-irradiated aAPC ∼1010 T cells were produced of which >95% expressed CAR. These genetically modified and propagated T cells met all quality control testing and release criteria in support of infusion. [ABSTRACT FROM AUTHOR]

: The Notch pathway can have both oncogenic and tumor suppressor roles, depending on cell context. For example, Notch signaling promotes T cell differentiation and is leukemogenic in T cells, whereas it inhibits early B cell differentiation and acts as a tumor suppressor in B cell leukemia where it induces growth arrest and apoptosis. The regulatory mechanisms that contribute to these opposing roles are not understood. Aberrant promoter DNA methylation and histone modifications are associated with silencing of tumor suppressor genes and have been implicated in leukemogenesis. Using methylated CpG island amplification (MCA)/DNA promoter microarray, we identified Notch3 and Hes5 as hypermethylated in human B cell acute lymphoblastic leukemia (ALL). We investigated the methylation status of other Notch pathway genes by bisulfite pyrosequencing. Notch3, JAG1, Hes2, Hes4 and Hes5 were frequently hypermethylated in B leukemia cell lines and primary B-ALL, in contrast to T-ALL cell lines and patient samples. Aberrant methylation of Notch3 and Hes5 in B-ALL was associated with gene silencing and was accompanied by decrease of H3K4 trimethylation and H3K9 acetylation and gain of H3K9 trimethylation and H3K27 trimethylation. 5-aza-2′-deoxycytidine treatment restored Hes5 expression and decreased promoter hypermethylation in most leukemia cell lines and primary B-ALL samples. Restoration of Hes5 expression by lentiviral transduction resulted in growth arrest and apoptosis in Hes5 negative B-ALL cells but not in Hes5 expressing T-ALL cells. These data suggest that epigenetic modifications are implicated in silencing of tumor suppressor of Notch/Hes pathway in B-ALL. [ABSTRACT FROM AUTHOR]

Although many adults with B cell acute lymphoblastic leukemia (B-ALL) are induced into remission, most will relapse, underscoring the dire need for novel therapies for this disease. We developed murine CD19-specific chimeric antigen receptors (CARs) and an immunocompetent mouse model of B-ALL that recapitulates the disease at genetic, cellular, and pathologic levels. Mouse T cells transduced with an all-murine CD3ζ/CD28-based CAR that is equivalent to the one being used in our clinical trials, eradicate B-ALL in mice and mediate long-term B cell aplasias. In this model, we find that increasing conditioning chemotherapy increases tumor eradication, B cell aplasia, and CAR-modified T cell persistence. Quantification of recipient B lineage cells allowed us to estimate an in vivo effector to endogenous target ratio for B cell aplasia maintenance. In mice exhibiting a dramatic B cell reduction we identified a small population of progenitor B cells in the bone marrow that may serve as a reservoir for long-term CAR-modified T cell stimulation. Lastly, we determine that infusion of CD8+ CAR-modified T cells alone is sufficient to maintain long-term B cell eradication. The mouse model we report here should prove valuable for investigating CAR-based and other therapies for adult B-ALL. [ABSTRACT FROM AUTHOR]

Tcf1 is known to function as a transcriptional activator of Wnt-induced proliferation during T cell development in the thymus. Evidence for an additional contrasting role for Tcf1 as a T-cell specific tumor suppressor gene is now presented. [ABSTRACT FROM AUTHOR]

Tumor escape mechanisms in leukemia are not well defined. To dissect immunological mechanisms responsible for immune tolerance toward leukemia, we established a murine model system allowing clonotypic analysis of leukemia-specific CD4 T cells recognizing ovalbumin (OVA). Upon i.v. injection of genetically engineered leukemia cells, dendritic cells (DCs) engulfed, processed and presented OVA to OVA-specific CD4 T cells. Consequently, leukemia-specific T cells were primed in vivo as shown by expression of activation markers and proliferative responses. However, in spite of detectable CD4 T cell responses in vitro and in vivo, no effective anti-leukemia immunity was established. In contrast, adoptively transferred DO11.10 T cells that were primed ex vivo mediated effective antitumor immunity. Furthermore, ex vivo primed DO11.10 T cells showed high expression of Th1 cytokines (interferon-γ, tumor necrosis factor-α and interleukin-2) whereas in vivo primed OVA-specific CD4 T cells showed incomplete differentiation (proliferation without cytokine production). We conclude that activated T cells lacking effector function develop through incomplete differentiation in leukemia-bearing mice. Thus, priming conditions of leukemia-specific CD4 T cells critically determines the balance between immunity or tolerance toward leukemia.Leukemia (2008) 22, 2070–2079; doi:10.1038/leu.2008.193; published online 17 July 2008 [ABSTRACT FROM AUTHOR]

Amplification of the NUP214-ABL1 oncogene can be detected in patients with T cell acute lymphoblastic leukemia (T-ALL). We screened 29 patients with T cell malignancies for the expression of NUP214-ABL1 by reverse transcription-polymerase chain reaction (RT-PCR). NUP214-ABL1 was detected in three (10%) patients. These results were confirmed by fluorescence in situ hybridization techniques. We also studied the activity of imatinib, nilotinib and dasatinib against the human NUP214-ABL1-positive cell lines PEER and BE-13. All three tyrosine kinase inhibitors decreased the viability of PEER and BE-13 cells, but nilotinib and dasatinib had >1-log lower IC(50) values than imatinib (P<0.001). In contrast, the NUP214-ABL-negative T-ALL cell line Jurkat, was remarkably resistant to tyrosine kinase inhibition. The inhibition of cellular proliferation was associated with time-dependent induction of apoptosis and inhibition of ABL, CrKL and STAT5 phosphorylation. Moreover, dasatinib was active in a NUP214-ABL1-positive leukemia xenograft murine model and in marrow lymphoblasts from a patient with NUP214-ABL1-positive T-ALL. On the basis of these results, ABL1 kinase inhibitors warrant clinical investigation in patients with NUP214-ABL1-positive T-cell malignancies. [ABSTRACT FROM AUTHOR]

We retrospectively evaluated reticulin fiber density (RFD) in 166 diagnostic bone marrow (BM) biopsies and 62 biopsies obtained at treatment day 29 from children with acute lymphoblastic leukemia (ALL). Patients with B-cell precursor (BCP)-ALL showed higher RFD as compared to patients with T-cell ALL (P<0.001). RFD correlated negatively with white blood cell count (P=0.008) in BCP-ALL patients. Patients with high-hyperdiploid ALL (51–61 chromosomes), no high-risk criteria and low RFD showed a favorable outcome when compared to similar patients with high RFD (P=0.002). In BCP-ALL patients, RFD at diagnosis correlated to the levels of minimal residual disease (MRD) analyzed by flow cytometry on treatment day 29 (P=0.001). Accordingly, patients with MRD10−4 presented higher RFD at diagnosis compared to patients with MRD<10−4 (P=0.003). BCP-ALL patients with low RFD at diagnosis and a rapid reduction of RFD on day 29 had a favorable outcome compared to patients with the same baseline RFD level at diagnosis but a slow RFD reduction (P=0.041). To our knowledge, these findings are novel and may indicate BM fibrosis as a new valuable prognostic marker in childhood ALL. Expanded use of BM biopsy both at diagnosis and during follow-up is suggested.Leukemia (2008) 22, 504–510; doi:10.1038/sj.leu.2405072; published online 20 December 2007 [ABSTRACT FROM AUTHOR]

The cell cycle inhibitor p16(INK4A) is frequently inactivated in acute lymphoblastic T-cell leukemia (T-ALL). We analyzed mechanisms and consequences of p16(INK4A) reconstitution in T-ALL cells lacking this tumor suppressor. CCRF-CEM cells with tetracycline-regulated p16(INK4A) expression underwent stable G1-phase cell cycle arrest for 72 h followed by massive apoptosis. p16(INK4A) expression caused pRB hypophosphorylation and repression of certain E2F target genes. Interestingly, cyclin E and c-Myc were not affected, suggesting pRB/E2F-independent expression of these E2F targets. Cyclin E/CDK2, however, was inactive due to stabilization and redistribution of p27(Kip1) from CDK4/CDK6 to CDK2. Analyses of c-Myc target genes suggested that c-Myc was transcriptionally inactive, which correlated with hypophosphorylation of the c-Myc inhibitor p107. Thus, p16(INK4A), although unable to repress the expression of deregulated cyclin E and c-Myc, functionally inactivated these potential oncogenes. p16(INK4A)-arrested cells showed morphologic changes, induction of T-cell-specific surface markers and repression of telomerase activity, suggesting differentiation. Moreover, p16(INK4A) reconstitution was associated with increased cellular volume, normal protein synthesis rates and elevated ATP levels. Taken together, p16(INK4A) reconstitution in p16(INK4A)-deficient T-ALL cells induced cell cycle arrest in the presence of cyclin E and c-Myc expression, uncoupled growth from cell cycle progression and caused a sequential process of growth, differentiation and apoptosis. [ABSTRACT FROM AUTHOR]

Acute lymphoblastic leukemia (ALL) is the most frequent malignancy of childhood. Although therapeutical advances have been achieved, some ALL subgroups still fare poorly. CD1d is a monomorphic molecule that provides a suitable target for immunotherapy in view of the characterization of a glycolipid,a-galactosylceramide (a-GalCer), capable of being presented to CD1d-restricted T cells with cytotoxic potential. We investigated CD1d expression in 80 pediatric B-cell precursor (BCP) ALL cases defined according to immunophenotype, cytogenetic features and age at onset. CD1d was detected on ALL cells in 15%of the patients. CD1d+ ALLs were significantly associated with infant leukemia, pro-B phenotype and mixed-lineage leukemia (MLL)/AF4 gene rearrangement. Accordingly, overall survival of patients with CD1d+ ALL was significantly shorter. CD1d+ leukemic blasts were able to presenta-GalCer via CD1d to cytotoxic CD1d-restricted T cells, which induced apoptosis of ALL cells that was inhibited by mAb to CD1d. CD1d+ blasts loaded witha-GalCer elicited cytokine secretion by CD1d-restricted T cells. Analysis of bone marrow (BM) cells derived from normal donors revealed that CD19+/CD1d+ cells were mostly mature B lymphocytes. However, a minority of BCPs expressed CD1d. Thus, expression of CD1d in ALL cases heralds an adverse prognosis but may provide a therapeutic tool.Leukemia (2005) 19, 551-556. doi:10.1038/sj.leu.2403671 Published online 3 March 2005 [ABSTRACT FROM AUTHOR]

Background: Recent evidence suggests that resistance to CD19 chimeric antigen receptor (CAR)-modified T cell therapy may be due to the presence of CD19 isoforms that lose binding to the single-chain variable fragment (scFv) in current use. As such, further investigation of CARs recognize different epitopes of CD19 antigen may be necessary. Methods: We generated a new CD19 CAR T (HI19α-4-1BB-ζ CAR T, or CNCT19) that includes an scFv that interacts with an epitope of the human CD19 antigen that can be distinguished from that recognized by the current FMC63 clone. A pilot study was undertaken to assess the safety and feasibility of CNCT19-based therapy in both pediatric and adult patients with relapsed/refractory acute lymphoblastic leukemia (R/R B-ALL). Results: Data from our study suggested that 90% of the 20 patients treated with infusions of CNCT19 cells reached complete remission or complete remission with incomplete count recovery (CR/CRi) within 28 days. The CR/CRi rate was 82% when we took into account the fully enrolled 22 patients in an intention-to-treat analysis. Of note, extramedullary leukemia disease of two relapsed patients disappeared completely after CNCT19 cell infusion. After a median follow-up of 10.09 months (range, 0.49–24.02 months), the median overall survival and relapse-free survival for the 20 patients treated with CNCT19 cells was 12.91 months (95% confidence interval [CI], 7.74–18.08 months) and 6.93 months (95% CI, 3.13–10.73 months), respectively. Differences with respect to immune profiles associated with a long-term response following CAR T cell therapy were also addressed. Our results revealed that a relatively low percentage of CD8+ naïve T cells was an independent factor associated with a shorter period of relapse-free survival (p = 0.012, 95% CI, 0.017–0.601). Conclusions: The results presented in this study indicate that CNCT19 cells have potent anti-leukemic activities in patients with R/R B-ALL. Furthermore, our findings suggest that the percentage of CD8+ naïve T cells may be a useful biomarker to predict the long-term prognosis for patients undergoing CAR T cell therapy. Trial registration: ClinicalTrials.gov: NCT02975687; registered 29 November, 2016. https://clinicaltrials.gov/ct2/keydates/NCT02975687 [ABSTRACT FROM AUTHOR]

Perforin plays a key role in the cytotoxicity of natural killer and cytotoxic T cells. Genetic mutations in the perforin gene (PRF1) give rise to approximately 30% cases of familial hemophagocytic lymphohistiocytosis. A frequent polymorphism, A91V (C to T transition at position 272), may impair processing of perforin protein to the active form, and has been suggested to increase susceptibility to childhood acute lymphoblastic leukemia (ALL). To investigate the role of A91V in ALL, we genotyped 2272 children with de novo ALL registered on the Pediatric Oncology Group ALL Classification study P9900 and 655 normal controls. Allele frequencies in the controls showed a very low frequency of the variant allele in blacks, 0.7% compared to 4% in white controls. In light of this, analysis was restricted to a comparison of white cases and controls only. Overall genotype frequencies were similar in white ALL cases and normal white controls (P=0.58), indicating that in contrast to the previous report, A91V polymorphism is not associated with increased risk of childhood ALL. PRF1 A91V frequency was significantly increased in children with BCR-ABL positive ALL (24 vs 8.5%; P=0.0048); however, this observation includes a relatively small number of cases and needs further exploration. [ABSTRACT FROM AUTHOR]