Acute Lymphoblastic Leukemia Therapy Research Articles

Next-Generation Sequencing of Minimal Residual Disease for Predicting Relapse after Tisagenlecleucel in Children and Young Adults with Acute Lymphoblastic Leukemia.

We assessed minimal residual disease (MRD) detection and B-cell aplasia after tisagenlecleucel therapy for acute lymphoblastic leukemia (ALL) to define biomarkers predictive of relapse (N = 143). Next-generation sequencing (NGS) MRD detection >0 in bone marrow (BM) was highly associated with relapse. B-cell recovery [signifying loss of functional chimeric antigen receptor (CAR) T cells] within the first year of treatment was associated with a hazard ratio (HR) for relapse of 4.5 [95% confidence interval (CI), 2.03-9.97; P < 0.001]. Multivariate analysis at day 28 showed independent associations of BMNGS-MRD >0 (HR = 4.87; 95% CI, 2.18-10.8; P < 0.001) and B-cell recovery (HR = 3.33; 95% CI, 1.44-7.69; P = 0.005) with relapse. By 3 months, the BMNGS-MRD HR increased to 12 (95% CI, 2.87-50; P < 0.001), whereas B-cell recovery was not independently predictive (HR = 1.27; 95% CI, 0.33-4.79; P = 0.7). Relapses occurring with persistence of B-cell aplasia were largely CD19- (23/25: 88%). Detectable BMNGS-MRD reliably predicts risk with sufficient time to consider approaches to relapse prevention such as hematopoietic cell transplantation (HCT) or second CAR-T cell infusion. SIGNIFICANCE: Detectable disease by BMNGS-MRD with or without B-cell aplasia is highly predictive of relapse after tisagenlecleucel therapy for ALL. Clonotypic rearrangements used to follow NGS-MRD did not change after loss of CD19 or lineage switch. High-risk patients identified by these biomarkers may benefit from HCT or investigational cell therapies.See related commentary by Ghorashian and Bartram, p. 2.This article is highlighted in the In This Issue feature, p. 1.

Rare Sequences Make Sense of CAR T-cell Therapy Outcomes.

In this issue, Pulsipher and colleagues used next-generation sequencing to detect leukemia-specific sequences following tisagenlecleucel therapy of acute lymphoblastic leukemia. A challenge for the field currently is to identify which patients will have therapy failure and to do so early enough to allow planning for further treatment, for example, stem cell transplantation. Detection of disease below the standard detection level for this technique (less than one per million cells) at day 28 was associated with poorer outcomes and potentially therefore could be used to identify those that might benefit from adjunctive therapies.See related article by Pulsipher et al., p. 66.

Overexpression of the GR Riborepressor LncRNA GAS5 Results in Poor Treatment Response and Early Relapse in Childhood B-ALL.

Simple SummaryAlthough childhood acute lymphoblastic leukemia (chALL) management is considered as one of the success stories in modern clinical oncology, the increased incidence of relapse in high-risk patients and the severe toxicity/long-term health effects due to chemotherapy intensity highlight the need for further improvements in patients’ risk stratification and personalized management. Synthetic glucocorticoids (GCs) are the core agents in chALL chemotherapy, exerting their role through the nuclear glucocorticoid receptor (GR), while GAS5 lncRNA suppresses the GCs-GR axis through binding to GR’s DNA binding domain. The objective of the study was the evaluation of GAS5 prognostic utility in chALL. GAS5 overexpression was strongly associated with a higher risk for short-term relapse and poor treatment outcome, independently of patients’ clinicopathological data. Moreover, “GAS5-including” multivariate models resulted in superior risk stratification and clinical benefit for disease prognosis, supporting precision medicine decisions in chALL.Glucocorticoids (GCs) remain the cornerstone of childhood acute lymphoblastic leukemia (chALL) therapy, exerting their cytotoxic effects through binding and activating of the glucocorticoid receptor (GR). GAS5 lncRNA acts as a potent riborepressor of GR transcriptional activity, and thus targeting GAS5 in GC-treated chALL could provide further insights into GC resistance and support personalized treatment decisions. Herein, to study the clinical utility of GAS5 in chALL prognosis and chemotherapy response, GAS5 expression was quantified by RT-qPCR in bone marrow samples of chB-ALL patients at diagnosis (n = 164) and at end-of-induction (n = 109), treated with ALL-BFM protocol. Patients’ relapse and death were used as clinical end-points for survival analysis. Bootstrap analysis was performed for internal validation, and decision curve analysis assessed the clinical net benefit for chALL prognosis. Our findings demonstrated the elevated GAS5 levels in blasts of chALL patients compared to controls and the significantly higher risk for short-term relapse and poor treatment outcome of patients overexpressing GAS5, independently of their clinicopathological data. The unfavorable prognostic value of GAS5 overexpression was strongly validated in the high-risk/stem-cell transplantation subgroup. Finally, multivariate models incorporating GAS5 levels resulted in superior risk stratification and clinical benefit for chALL prognostication, supporting personalized prognosis and precision medicine decisions in chALL.

Association between Cortisol and Infection Risk of Children with Acute Lymphoblastic Receiving Induction and Consolidation Chemotherapy in Dr. Soetomo General Hospital Surabaya

Background: Mortality due to Infection associated therapy in acute lymphoblastic leukemia (ALL) children remains high, although therapeutic success and survival rate are substantially improving.Methods: This study used a pre and post-test group design for children aged less than 18 years with ALL newly diagnosed and receiving chemotherapy in pediatric patient of Dr. Soetomo hospital Surabaya.Results: The study involved 25 subjects, 10 subjects were male, average age ranged from 3 to 9 years. The high risk-ALL subjects were 15 (60%), hyperleukocytosis 4 (16%) subjects and 7 (28%) subjects with leukopenia. Statistical analysis showed a significant difference in decreasing cortisol levels in week-4 compared to week-0 (p &lt;0.001) and an increased in cortisol levels between week-12 compared to week-10. The incidence of infection during induction phase is higher than the consolidation phase (40vs15). The relationship between the mean cortisol levels and the incidence of infection in general showed a significant difference in the induction phase of week-0 (p=0.029), week-4 (p=0.041), and week-6 (p=0.005).Conclusion: In the induction phase, there is an association between mean cortisol levels and the risk of infection, but there is no such association in the consolidation phase.

Genetic and Epigenetic Targeting Therapy for Pediatric Acute Lymphoblastic Leukemia.

Acute lymphoblastic leukemia is the most common malignancy in children and is characterized by numerous genetic and epigenetic abnormalities. Epigenetic mechanisms, including DNA methylations and histone modifications, result in the heritable silencing of genes without a change in their coding sequence. Emerging studies are increasing our understanding of the epigenetic role of leukemogenesis and have demonstrated the potential of DNA methylations and histone modifications as a biomarker for lineage and subtypes classification, predicting relapse, and disease progression in acute lymphoblastic leukemia. Epigenetic abnormalities are relatively reversible when treated with some small molecule-based agents compared to genetic alterations. In this review, we conclude the genetic and epigenetic characteristics in ALL and discuss the future role of DNA methylation and histone modifications in predicting relapse, finally focus on the individual and precision therapy targeting epigenetic alterations.

Antileukemic Effects of Anti-miR-146a, Anti-miR-155, Anti-miR-181a, and Prednisolone on Childhood Acute Lymphoblastic Leukemia.

Prednisolone has been used frequently in the treatment of acute lymphoblastic leukemia. However, to overcome the challenges of the treatment, the development of additional therapies is of great importance. Small, non-protein-coding RNAs, namely, microRNAs (miRNAs), are critical epigenetic regulators with physiological and pathological importance. This study is aimed at determining the effects of miR-146a, miR-155, and miR-181a inhibition with their corresponding anti-miRs on both leukemic and healthy cells, individually and with prednisolone. Leukemic (SUP-B15) and healthy B-lymphocyte (NCI-BL 2171) cell lines were used in this study. A total of 12 experimental groups included individual and combinational silenced ALL-associated miRNAs (hsa-miR-155, hsa-miR-146a, and hsa-miR-181a) and their combination with prednisolone. Cytotoxicity, proliferation, cell cycle, and apoptosis analyses were performed by using WST-1, trypan blue, APC-BrdU, Annexin V, and JC-1 methods in each study group, respectively. To control the effectiveness of anti-miR transfection and prednisolone application, miRNA expression analysis was performed from all groups. Anti-miR application was effective on the viability, proliferation, cell cycle, and apoptosis of leukemia cells, and this effect was increased with prednisolone administration. In addition, this activity was found to be very low on healthy cells. In conclusion, anti-miR applications may have the potential for clinical use of adjuvant to or as an alternative to conventional therapies for childhood acute lymphoblastic leukemia.

Characterization of a novel glucocorticoid-resistant human B-cell acute lymphoblastic leukemia cell line, with AMPK, mTOR and fatty acid synthesis pathway inhibition

BackgroundAcquired glucocorticoid (GC) resistance remains the main obstacle in acute lymphoblastic leukemia (ALL) therapy. The aim of the present study was to establish a novel GC-resistant B-ALL cell line and investigate its biological characteristics.MethodsA cell culture technique was used to establish the GC-resistant cell line from the parental cell, NALM-6. Molecular and cellular biological techniques including flow cytometry, MTT assay, western blotting, DNA fingerprinting analysis and whole transcriptome sequencing (WTS) were used to characterize the GC-resistant cell lines. Nude mice were used for xenograft studies.ResultsThe GC-resistant cell line, NALM-6/HDR, was established by culturing NALM-6 cells under hypoxia for 5 weeks with a single dexamethasone (Dex) treatment. We subcloned the NALM-6/HDR cell lines, and got 6 monoclone Dex-resistant cell lines, NALM-6/HDR-C1, C3, C4, C5, C6 and C9 with resistance index (RI) ranging from 20,000–50,000. NALM-6/HDR and its monoclone cell line, NALM-6/HDR-C5, exhibited moderate (RI 5–15) to high resistance (RI > 20) to Ara-c; low or no cross-resistance to L-Asp, VCR, DNR, and MTX (RI < 5). STR analysis confirmed that NALM-6/HDR and NALM-6/H were all derived from NALM-6. All these cells derived from NALM-6 showed similar morphology, growth curves, immunophenotype, chromosomal karyotype and tumorigenicity. WTS analysis revealed that the main metabolic differences between NALM-6 or NALM-6/H (GC-sensitive) and NALM-6/HDR (GC-resistant) were lipid and carbohydrates metabolism. Western blotting analysis showed that NALM-6/HDR cells had a low expression of GR and p-GR. Moreover, AMPK, mTORC1, glycolysis and de novo fatty acid synthesis (FAS) pathway were inhibited in NALM-6/HDR when compared with NALM-6.ConclusionsNALM-6/HDR cell line may represent a subtype of B-ALL cells in patients who acquired GC and Ara-c resistance during the treatment. These patients may get little benefit from the available therapy target of AMPK, mTORC1, glycolysis and FAS pathway.

Engineering Novel CD19/CD22 Dual-Target CAR-T Cells for Improved Anti-Tumor Activity

Despite high remission rates following chimeric antigen receptor T cell (CAR-T) cell therapy in B-cell acute lymphoblastic leukemia (B-ALL), relapse due to loss of the targeted antigen is increasingly recognized as a mechanism of immune escape. We hypothesized that simultaneous targeting of CD19 and CD22 may improve the CAR-T effect. The in vitro and in vivo leukemia model was established, and the anti-tumor effects of BiCAR-T, CD19 CAR-T, CD22 CAR-T, and LoopCAR6 cells were observed. We found that the BiCAR-T cells showed significant cytotoxicity in vitro and in vivo. The CD19/CD22 bivalent CAR provides an opportunity to test whether simultaneous targeting may reduce the risk of antigen loss.

Engineering CD70-Directed CAR-NK Cells for the Treatment of Hematological and Solid Malignancies

Advances in cellular immunotherapy have led to multiple FDA approvals for autologous CAR-T cell therapies in acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphomas (NHL), and multiple myeloma (MM). While effective, autologous CAR-T therapies are limited by safety concerns, lack of scalability for patient derived starting material, and long vein-to-vein timelines. Allogeneic CAR-NK cell therapies have the potential to overcome these limitations by providing an off-the-shelf product capable of delivering clinical benefit without the safety and manufacturing challenges associated with CAR-T therapy. CAR-NK cell therapies are particularly attractive in AML as the inherent graft-versus-leukemia activity of NK cells can be effectively augmented by a CAR directed to an AML expressed antigen. CD70 expression is associated with several indications, including AML, NHL, and renal cell carcinoma (RCC), and it is an attractive target for CAR therapy in AML since it is highly expressed on leukemic stem cells and blasts and is absent in normal bone marrow hematopoietic stem cells. 1 While aberrant expression of CD70 is associated with several solid and hematological indications, its expression in normal tissue is restricted to immune cells including T, B, DC, and NK cells. 2 Here we demonstrate that CD70 is not expressed in resting peripheral blood NK cells but is strongly upregulated in response to NK cell activation by engineered feeder cells. Introduction of CARs targeting CD70 into activated NK cells leads to substantial reduction of NK cell expansion due to fratricide. While CD70 is expressed in activated NK cells, knockout (KO) of CD70 by CRISPR/Cas9 editing does not inhibit NK cell expansion nor impair endogenous cytotoxicity against K562 target cells. Using the non-viral TcBuster™ Transposon System (Bio-Techne), we were able to deliver transposons containing a CD70 CAR and an IL15 expression cassette while simultaneously knocking out CD70 by CRISPR/Cas9 in primary human peripheral blood NK cells. This single-step process resulted in >70% CAR integration/expression and >80% knockout of CD70. The resulting CD70 knockout CAR-NK cells were resistant to fratricide and expanded comparably to mock-engineered NK cells following feeder cell activation. The IL15 expression cassette enabled enhanced persistence of CAR-NK cells in vitro without exogenous cytokine support. In functional assays, CD70 KO NK cells engineered with the CD70 CAR and IL15 expression cassette mediated cytotoxicity against multiple CD70-positive tumor cell lines, expressed the degranulation marker CD107a (LAMP1), and expressed the cytokines IFNγ and TNFα. Overall, the results demonstrate the potential for targeting CD70 with CAR-NK cell therapy for the treatment of AML, RCC, and other CD70-positive malignancies while overcoming the risk posed by fratricide by engineering with a non-viral transposon delivery system in combination with CRISPR/Cas9 editing.1 Perna et al. 2017, Cancer Cell. 32:506-519.2 McEarchern et al. 2008, Clin Cancer Res. 14(23):7763-7772. DisclosuresChoi: Unum Therapeutics: Divested equity in a private or publicly-traded company in the past 24 months, Ended employment in the past 24 months. Walsh: Obsidian Therapeutics: Ended employment in the past 24 months. Khamhoung: Rubius Therapeutics, Inc.: Ended employment in the past 24 months. Johnson: Celsius Therapeutics: Current holder of stock options in a privately-held company, Ended employment in the past 24 months. Franco: KSQ Therapeutics: Current holder of individual stocks in a privately-held company, Ended employment in the past 24 months. Swiech: Agenus: Current holder of individual stocks in a privately-held company, Ended employment in the past 24 months; Unum Therapeutics: Divested equity in a private or publicly-traded company in the past 24 months, Ended employment in the past 24 months. Richardson: Novartis Pharma: Current equity holder in publicly-traded company; Obsidian Therapeutics: Current holder of stock options in a privately-held company, Ended employment in the past 24 months.

Sequential Blinatumomab with Reduced Intensity Chemotherapy in the Treatment of Older Adults with Newly Diagnosed Ph Negative B-Precursor Acute Lymphoblastic Leukemia - Interim Analysis of the Australasian Leukemia and Lymphoma Group ALL08 Study

Sequential Blinatumomab with Reduced Intensity Chemotherapy in the Treatment of Older Adults with Newly Diagnosed Ph Negative B-Precursor Acute Lymphoblastic Leukemia - Interim Analysis of the Australasian Leukemia and Lymphoma Group ALL08 Study

How Important Are in-Person Clinic Visits during Maintenance Therapy for Pediatric Acute Lymphoblastic Leukemia?

▪BackgroundAcute Lymphoblastic Leukemia (ALL) is the most common cancer seen in the pediatric age group. The treatment consists of an initial intensive phase of chemotherapy followed by a prolonged period of maintenance chemotherapy intended to reduce the risk of relapse. Children are commonly seen in clinic every 4-6 weeks for bloodwork and physical examination during the maintenance phase. The COVID-19 pandemic has prompted consideration of alternative means of providing medical care. The objective of this study was to determine the proportion of in-person clinic visits during ALL maintenance therapy for which the outcome of the physical examination resulted in a change of patient management.MethodsA retrospective chart review of children diagnosed with precursor-B ALL between January 2017 and December 2018, and who were in maintenance therapy between September 2019 and February 2020, was conducted. All routine maintenance visits were reviewed to identify new physical examination findings and patient outcomes and classified as either “could be managed virtually” or “essential in-person visit”. For the latter, a second classification was conducted to distinguish between visits necessitating a change of management versus not.ResultsEighty-five children were diagnosed with precursor B ALL and continued to maintenance treatment during the study period. 10 children were excluded as not meeting the inclusion criteria or not evaluable. Of the remaining 75 children, 54 were male (72%) and 21 female (28%). The median age at diagnosis was 4.83 years (0.73 - 14.8 years). 39 patients (52%) had standard risk ALL, 35 patients (46.7%) had high risk ALL and one patient had Infant ALL (1.3%) A total of 240 routine maintenance visits were included in the final analysis. An abnormal physical exam finding was noted in 20 visits (8.3%) and of these, new findings were noted in 14 (5.8%). 6 visits were classified as essential in-person visits (2 for new bruising, 1 for new limp, 1 for new lymphadenopathy, 1 for acute otitis media, and 1 for new wheezing). Among the 14 visits with new exam findings, only 5 had an impact on patient management and of these, only 2 (0.8%) were classified as obligate in-person visit for requiring immediate management (acute otitis media and wheezing).ConclusionOur results demonstrate that most in-person visits can be provided as virtual visits without affecting patient outcomes. The results of this study provide the foundation for a prospective study that will evaluate the benefits, risks and families' preferences associated with virtual visits and delineate the optimal frequency and timing of in-person clinic visits during ALL maintenance therapy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Heterozygous NUDT15 Gene Polymorphism Would Not Associate with the Severity of 6-Mercaptopurine Side Effects in Early Intensification Therapy for Childhood Acute Lymphoblastic Leukemia

BackgroundIn the treatment of childhood acute lymphoblastic leukemia (ALL), 6-mercaptopurine (6-MP) is essential for early intensification and maintenance therapy. Recently, it has been reported that a gene polymorphism of nucleoside diphosphate-linked moiety X-type motif 15 (NUDT15) rs116855232 is associated with the 6-MP induced severe myelotoxicity. Since the NUDT15 rs116855232 polymorphism is relatively common in East Asian and Hispanic populations, it is important to evaluate the association between the polymorphism and 6-MP for determining the dose of effective therapy and avoiding the side effect in Japan. However, there are few reports on the association between NUDT15 polymorphisms and the therapeutic efficacy or side effects of 6-MP in the early intensification; most previous reports focused on the maintenance therapy so far. The purpose of this study is to clarify the association between NUDT15 polymorphism and the actual treatment status with 6-MP or its side effects, therefore to contribute to the effective and safe treatment of childhood ALL in Japan.MethodsTwenty-four patients with ALL who received early intensification therapy according to the JPLSG ALL-B12 or JPLSG ALL-T11 protocol at the Department of Pediatrics, Hokkaido University Hospital, between April 2013 and May 2021 were included in the study. We retrospectively collected the clinical and laboratory data from the clinical records. And we also performed the sequence analysis of the exon 1 and 3 in NUDT15 geneResultsGenetic analysis of NUDT15 showed no nucleotide changes other than rs116855232. Twenty patients were CC (wild-type), 4 patients were CT (heterozygous-variant), and 0 patients were TT (homozygous-variant). Of the 4 patients with heterozygous-variant, one patient received a reduced dose of 6-MP because of early onset myelotoxicity during early intensification therapy. In this patient, the total dose of 6-MP was 70% of the standard dose. All the 24 patients showed myelotoxicity and hepatotoxicity during early intensification therapy. Correlations between NUDT15 haplotype and side effects as myelotoxicity and hepatotoxicity were not significant.DiscussionIn the heterozygous-variant of NUDT15 rs116855232, the incidence and degree of myelotoxicity and hepatotoxicity during early intensification therapy did not differ from the wild-type. The international consortium (Clinical Pharmacogenetics Implementation Consortium Guideline, 2018) recommends that the dose of 6-MP should be reduced to 10mg/m2/day in patients with homozygous variant of NUDT15, however, the reduction of the starting dose of 6-MP in those with heterozygous variant is not recommended. Of note, these recommendations were made based on the data in the maintenance therapy. Our study suggests that the reduction of the starting dose may not be necessary in children with heterozygous variant. The NUDT15 rs116855232 polymorphism is common in Japan, with 10% of heterozygotes and 1.1% of homozygotes. In future study, a larger study on the association between NUDT15 polymorphism and treatment outcome including side effects in the early intensification is needed in East Asia to confirm the results of our study. DisclosuresNo relevant conflicts of interest to declare.

High Mobility Group A1 (HMGA1) Epigenetic Regulators Induce ETV5 Networks in Relapsed B-Cell Leukemia and Provide Novel Therapeutic Targets

Introduction: Despite advances in therapy for B-cell acute lymphoblastic leukemia (B-ALL), relapsed disease remains the leading cause of death in children with cancer. The gene encoding the High Mobility Group A1 (HMGA1) chromatin regulator is highly expressed in stem cells and diverse malignancies where high levels portend poor outcomes. We discovered that transgenic mice misexpressing Hmga1 in lymphoid cells develop leukemic transformation by amplifying transcriptional networks involved in stem cell function, proliferation, and inflammation (Hillion et al, Cancer Res 2008, Schuldenfrei et al, BMC Genomics 2011, Xian et al, Nature Commun 2017). In pediatric B-ALL (pB-ALL), HMGA1 is overexpressed with highest levels in blasts from early relapse (Roy et al, Leuk Lymphoma 2013). Together, these findings suggest that HMGA1 is required for leukemogenesis and drives relapse through epigenetic reprogramming. We therefore sought to: 1) test the hypothesis that HMGA1 is required for leukemogenesis and relapse in pB-ALL, and, 2) elucidate targetable mechanisms mediated by HMGA1.Methods: To elucidate the function of HMGA1 and downstream targets, we employed CRISPR/Cas9 gene inactivation and lentiviral-mediated gene silencing via delivery of short hairpin RNA (shRNA) targeting 2 sequences per gene in cell lines from relapsed pB-ALL, including REH, which harbor the TEL-AML1 fusion, and 697, which harbor the E2A-PBX1 fusion. We assessed leukemia phenotypes in vitro and leukemic engraftment in vivo. To dissect molecular mechanisms, we performed RNA sequencing (RNAseq) and applied in silico pathway analysis. To validate these pathways in human pB-ALL, we assessed gene expression and clinical outcomes in independent cohorts. The Broad Institute Connectivity Map (CMAP) was applied to identify drugs to target HMGA1 networks.Results:HMGA1 is overexpressed in pB-ALL in independent cohorts with highest levels at relapse. Decreasing HMGA1 expression via CRISPR/Cas9 inactivation or shRNA-mediated gene silencing in relapsed pB-ALL cell lines (REH, 697) disrupts proliferation, decreases the frequency of cells in S phase concurrent with increases in G0/G1, enhances apoptosis, and impairs clonogenicity. To assess HMGA1 function in vivo, we compared leukemogenesis following tail vein injection of pB-ALL cell lines with or without HMGA1 depletion in immunodeficient mice (NOD/SCID/IL2 receptor gamma null). Survival was prolonged in mice injected with either pB-ALL cell line (REH, 697) after HMGA1 depletion. Further, leukemic cells that ultimately engraft show increased HMGA1 expression relative to the pool of injected cells with HMGA1 silencing, suggesting that escape from HMGA1 silencing was required for engraftment. RNAseq revealed transcriptional networks governed by HMGA1 that regulate proliferation (G2M checkpoint, E2F), RAS/ERK signaling, hematopoietic stem cells, and ETV5 (ETS variant 5 transcription factor) targets. Given its association with aggressive ALL harboring the BCR-ABL fusion, we focused on the ETV5 gene. CRISPR/Cas9 inactivation or gene silencing of ETV5 in relapsed pB-ALL cell lines (REH, 697) decreases proliferation and clonogenicity in vitro, while delaying leukemogenesis in vivo. Further, restoring ETV5 expression in pB-ALL cell lines with HMGA1 silencing partially rescues anti-leukemogenic effects of HMGA1 depletion. Mechanistically, HMGA1 binds to AT-rich regions within the ETV5 promoter (-0.7 kb and -0.2 kb) and recruits active histone marks (H3K27Ac, H3K4me3, H3K4me1) to induce ETV5. Epigenetic drugs predicted to target HMGA1-ETV5 networks synergize with HMGA1 silencing in cytotoxicity assays with pB-ALL cell lines. Most importantly, HMGA1 and ETV5 are co-expressed and up-regulated in primary blasts from children with pB-ALL with highest levels at relapse, thus underscoring the significance of this pathway in relapsed pediatric B-ALL.Conclusions: We discovered a previously unknown epigenetic program whereby HMGA1 up-regulates ETV5 networks by binding to chromatin and recruiting active histone marks to the ETV5 promoter. Both HMGA1 and ETV5 are up-regulated at relapse. Finally, the HMGA1-ETV5 axis can be targeted by epigenetic drugs (HDAC inhibitors) that synergize with HMGA1 depletion. Our findings reveal the HMGA1-ETV5 axis as a key molecular switch in relapsed pB-ALL and rational therapeutic target to treat or prevent relapse. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Omission of High-Dose Cytarabine during Consolidation Therapy of Ph-Positive ALL Patients Treated with Nilotinib and Low-Intensity Chemotherapy Results in an Increased Risk of Relapses Despite Non-Inferior Levels of Late BCR-ABL1 MRD Response. First Results of the Randomized Graaph-2014 Study

The Omission of High-Dose Cytarabine during Consolidation Therapy of Ph-Positive ALL Patients Treated with Nilotinib and Low-Intensity Chemotherapy Results in an Increased Risk of Relapses Despite Non-Inferior Levels of Late BCR-ABL1 MRD Response. First Results of the Randomized Graaph-2014 Study

A Meta-Analysis of Hypersensitivity to Pegylated Escherichia coli Asparaginase Used As First-Line Treatment in Contemporary Pediatric Acute Lymphoblastic Leukemia Protocols

PURPOSEAsparaginase is a key component of acute lymphoblastic leukemia (ALL) therapy. Hypersensitivity reactions challenge its use and occur frequently (30-75%) after native Escherichia. Coli (E.coli) asparaginase (Appel et al., 2008; Muller et al., 2001; Panosyan et al., 2004; Silverman et al., 2001). The international ALL Ponte di Legno Toxicity Work Group (PTWG) classifies hypersensitivity to asparaginase as (i) allergy in case of symptoms of allergy (always associated with undetectable asparaginase activity levels), (ii) allergic-like reactions in case of symptoms without inactivation, and (iii) silent inactivation (SI) with inactivation of asparaginase activity, but without hypersensitivity symptoms (Schmiegelow et al., 2016). Allergic-like reactions and SI can only be diagnosed with monitoring of asparaginase activity levels. A meta-analysis was performed based on data from the PTWG to estimate the incidence of hypersensitivity and risk factors for hypersensitivity to asparaginase in ALL protocols using pegylated E.coli asparaginase (PEGasparaginase) as first line of treatment.PATIENTS AND METHODSQuestionnaires were sent to all members of the PTWG. Information on protocol level regarding PEGasparaginase dose, dosing regimen (e.g. dosing frequency, total number of doses, PEGasparaginase-free intervals(s)), administration route, total induction and post-induction hypersensitivity rates per protocol and per risk group and use of therapeutic drug monitoring (TDM). To facilitate comparison between protocols with and without TDM, we defined allergic reactions as the sum of allergies and allergic-like reactions. Silent inactivation was analyzed separately.RESULTSA total of 5880 patients with newly diagnosed ALL, aged 1 to 24 years old, were enrolled in seven different upfront ALL protocols using PEGasparaginase as first-line treatment. The overall incidence of allergic reactions along with 95% confidence interval (CI) were 9% [6%; 13%], 2% [1%; 3%] and 8% [5%; 11%] in the overall protocol, induction and post-induction, respectively (Figure 1). Severity of allergic reactions were described, according to the CTCAE version 3.0 or 4.03, per protocol. 47% of the reactions were classified as grade 3/4.Univariate meta-regression analysis showed a positive association between the incidence of allergic reactions and number of PEGasparaginase-free intervals (P=0.005). High risk group stratification (P<0.001), post-induction treatment phase (P<0.001) and start of PEGasparaginase treatment in post-induction (P=0.006) were also associated with a higher incidence of allergic reactions. Route of administration (IV (8.9%, range 8.6-10.5%) versus IM (6.5%, range 5.5-14.8%)) did not significantly influence risk of hypersensitivity. Number of doses, duration of first PEGasparaginase-free interval and dosage did not significantly influence risk of hypersensitivity. Multivariate meta-regression analysis showed a positive association between the incidence of allergic reactions and the number of PEGasparaginase-free intervals (P=0.006) and start of PEGasparaginase in the post-induction treatment phase (P=0.02).Two out of seven study groups reported an incidence of allergic reactions of 1.6-2.0%, which was 9-16% of all hypersensitivity. Three out of seven study groups reported an incidence of SI of 3.7-4.1%, which was 23-29% of all hypersensitivity reactions.All protocols prescribed a switch to Erwinia asparaginase in case of clinical hypersensitivity and/or SI. 308 out of 348 (89%) of the patients with hypersensitivity to PEGasparaginase received Erwinia asparaginase. 19 out of these 308 (6%) exposed patients had an allergic reaction to Erwinia asparaginase, of which 7 out of 19 (37%) were grade 3/4.CONCLUSIONThe incidence of allergic reactions is lower in protocols using PEGasparaginase as first-line treatment compared to that reported for native E.coli asparaginase or PEGasparaginase after native E.coli asparaginase. Post-induction phase, a higher number of PEGasparaginase-free intervals, and initiation of PEGasparaginase in post-induction phase are risk factors for allergic reactions. These results are important for planning of PEGasparaginase administrations in future frontline therapy. [Display omitted] DisclosuresAlbertsen: Erytech: Honoraria, Speakers Bureau; Servier: Speakers Bureau; BKA: Other: Sponsor of the investigator-initiated trial NOR-GRASPALL2016.

Predictive role of endothelial cell activation in cytokine release syndrome after chimeric antigen receptor T cell therapy for acute lymphoblastic leukaemia.

CD19‐target chimeric antigen receptor (CAR)‐T cell therapy is highly effective for relapsed/refractory (R/R) acute lymphoblastic leukaemia (ALL), but is often complicated by cytokine release syndrome (CRS), which is potentially life‐threatening. Endothelial cells are the core regulator of CRS in many infectious diseases and may also play a key role after CAR‐T cell therapy. We provided a detailed clinical, laboratory description and endothelial cell activation biomarkers in patients with CRS. Endothelial cell activation was associated with occurrence, development and severity of CRS, manifested by decreased serum angiopoietin (Ang)‐1 levels and increased levels of von Willebrand Factor (VWF), Ang‐2, Ang‐2:Ang‐1, sE‐selectin, soluble intercellular adhesion molecule (sICAM‐1) and soluble vascular cell adhesion molecule (sVCAM)‐1. Besides, the endothelial activation was correlated with the hepatic, kidney and hematopoietic dysfunction in CRS patients. After infusion of CAR‐T cells, we detected changes of endothelial activation‐related biomarkers within 36 hours in patients who subsequently developed CRS, especially severe CRS. Using top tree models, we could predict which patients would develop CRS, especially severe CRS, or identify the severity of CRS by certain biomarkers of endothelial activation. These data provide a new idea and will help identify new targets for early intervention and prevention of CRS.

Effect of genetic polymorphism of TPMT and NUDT15 on the tolerance of 6-mercaptopurine therapy in adult acute lymphoblastic leukemia

目的探讨TPMT*2 rs1800462、TPMT*3B rs1800460、TPMT*3C rs1142345和NUDT15 rs116855232基因多态性对成人急性淋巴细胞白血病（ALL）患者6-巯基嘌呤（6-MP）耐受性的影响。方法选取2015年9月至2019年12月于中国医学科学院血液病医院确诊并接受环磷酰胺、阿糖胞苷和6-MP（CAM方案）治疗的216例成人ALL患者，利用Taqman SNP基因分型方法检测患者的TPMT、NUDT15基因型。结合临床资料，分析基因多态性对ALL治疗中含6-MP方案耐受性的影响。结果216例患者中B-ALL 185例（85.65％）、T-ALL 31例（14.35％）。TPMT*2 rs1800462 CC型216例（100.00％）；TPMT*3B rs1800460 CC型216例（100.00％）；TPMT*3C rs1142345 TT型209例（96.76％）、TC型7例（3.24％），等位基因突变频率为1.62％。NUDT15 rs116855232 CC型166例（76.85％）、CT型48例（22.22％）、TT型2例（0.93％），等位基因突变频率为12.04％。TPMT*3C rs1142345突变型组（TC+CC型）悬浮红细胞的输注量少于野生型组（CC型）（P＝0.036）；TPMT*3C rs1142345突变型组出现肝损害（天冬氨酸转氨酶升高）的风险高于野生型组（OR＝9.559, 95％CI 1.135～80.475, P＝0.038）。NUDT15 rs116855232突变型组（CT+TT型）WBC<1×109/L和中性粒细胞绝对计数<0.5×109/L的持续时间均长于野生型组（CC型）（P＝0.005，P＝0.007）；突变型组单采血小板的输注量多于野生型组（P＝0.014）。结论TMPT、NUDT15基因多态性可以影响成人ALL患者对6-MP的耐受性。治疗前检测患者基因型，进而优化6-MP使用剂量，有助于缩短骨髓抑制时间和减少血制品输注量。临床试验注册：中国临床试验注册中心（ChiCTR-TNC-09000397）

Biomarkers and Cognitive Function in Children and Adolescents During Maintenance Therapy for Leukemia.

To explore the relationship between biomarkers of oxidative stress and inflmmation in cerebrospinal fluid (CSF) and cognitive function in children receiving maintenance therapy for acute lymphocytic leukemia (ALL). 30 participants aged 4-17 years receiving ALL maintenance therapy at two pediatric cancer centers in the United States. F2-isoprostane (F2-ISoP) and interleukin-8 (IL-8) were evaluated in CSF samples, and cognitive function measures were completed during the first and last cycles of ALL maintenance. The Flanker Inhibitory Control and Attention Test (Flanker) and Dimensional Change Card Sort were completed during the last cycle. During maintenance therapy, IL-8 decreased, and parent reports of children's cognitive function improved. Higher IL-8 was associated with better parent reports of children's cognitive function at each timepoint. Higher F2-ISoP levels were associated with lower Flanker scores. F2-ISoP may be a useful biomarker in evaluating cognitive dysfunction in children with ALL and merits further investigation.

The urinary excretion of epigenetically modified DNA as a marker of pediatric ALL status and chemotherapy response

The active DNA demethylation process may be linked to aberrant methylation and may be involved in leukemogenesis. We investigated the role of epigenetic DNA modifications in childhood acute lymphoblastic leukemia (ALL) diagnostics and therapy monitoring. We analyzed the levels of 5-methyl-2′-deoxycytidine (5-mdC) oxidation products in the cellular DNA and urine of children with ALL (at diagnosis and during chemotherapy, n = 55) using two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry (2D UPLC–MS/MS). Moreover, the expression of Ten Eleven Translocation enzymes (TETs) at the mRNA and protein levels was determined. Additionally, the ascorbate level in the blood plasma was analyzed. Before treatment, the ALL patients had profoundly higher levels of the analyzed modified DNA in their urine than the controls. After chemotherapy, we observed a statistically significant decrease in active demethylation products in urine, with a final level similar to the level characteristic of healthy children. The level of 5-hmdC in the DNA of the leukocytes in blood of the patient group was significantly lower than that of the control group. Our data suggest that urinary excretion of epigenetic DNA modification may be a marker of pediatric ALL status and a reliable marker of chemotherapy response.

Plasma Distribution of Methotrexate and Its Polyglutamates in Pediatric Acute Lymphoblastic Leukemia: Preliminary Insights.

High-dose methotrexate (HD-MTX) is the mainstream therapy of current acute lymphoblastic leukemia (ALL) regimens, but frequent intra- and interindividual differences in the clinical response to HD-MTX lead to chemotherapeutic interruption or discontinuation. The exact mechanism of transport across the cell membrane and the disposition of active methotrexate metabolites-methotrexate polyglutamates (MTXPGs)-are not well described in the literature. The aim of this study was to gain more insight into the plasma distribution of methotrexate and MTXPGs in pediatric patients with ALL and to clarify the obscure pathways of MTXPGs. We prospectively measured the concentrations of MTXPG1-7 in plasma samples from three male pediatric patients treated with HD-MTX and leucovorin rescue according to the IC-BFM 2009 protocol using liquid chromatography-mass spectrometry (LC-MS). Blood samples were obtained at 24, 36, 42, and 48 h after the start of HD-MTX treatment. Noticeable plasma concentrations of MTXPGs with a 2.2-fold interpatient variability were detected. The highest interindividual variability in total plasma MTXPG concentration was observed at 36h, and ranged from 13.78 to 30.82 μmol/L. Among all patients, the predominant polyglutamate types in relation to the total plasma MTXPG concentration at each time point were MTXPG3 (16.71-30.02%) and MTXPG5 (26.23-38.60%), while MTXPG7 was the least abundant MTXPG (3.22-5.02%). The presence of MTXPGs in plasma of patients with ALL could be related to the action of ABC efflux transporters on blood cells and hepatocytes resulting from the administration of high doses of methotrexate. This study may not draw definitive conclusions, but it does reduce uncertainty about the dynamics of methotrexate and its active metabolites, which may be of vital importance for achieving a clinical response.