pre-B Acute Lymphoblastic Leukemia Research Articles

C-Myc inhibition sensitizes pre-B ALL cells to the anti-tumor effect of vincristine by altering apoptosis and autophagy: Proposing a probable mechanism of action for 10058-F4

Unlike the broad spectrum efficacies in wiping the malignant cells out, application of vincristine (VCR) in acute lymphoblastic leukemia (ALL) therapeutic protocol is partially restricted due to its high frequent resistant rate. Although several mechanisms have been enumerated for VCR resistance, to the best of our knowledge, there is no report reflecting the suppressive effect of oncogenic pathways on VCR cytotoxicity in ALL. The results of the present study indicated that both pre-B ALL-derived REH and Nalm-6 cells were partly resistant to VCR, with this note that Nalm-6 cells displayed more resistant phenotype. More interestingly, we showed for the first time that among inhibitors of different signaling pathways including those targeting PI3K, ERK, and NF-κB, the enhancive effect of small molecule inhibitor of c-Myc 10058-F4 was more significant on VCR cytotoxicity. Inhibition of c-Myc in VCR-treated Nalm-6 cells promoted a caspase-3-dependent apoptosis not only through altering the balance between death promoters to death suppressors, but also via modulating the expression of autophagy-related genes. Noteworthy, favorable impact of 10058-F4 on VCR anti-leukemic effect was not restricted to the induction of cell death and this agent also reinforced VCR anti-proliferative effect through disturbing cell cycle progression and hampering the expression of Pin1 and hTERT. In conclusion, it seems that targeting c-Myc could produce a synergistic anti-cancer effect with VCR and provide a fundamental infrastructure for a promising approach in ALL.

Impact of Targeted Immunotherapies and Novel Cytogenetic and Clinical Risk Groups on Outcome after Allogeneic Hematopoietic Stem Cell Transplant (AlloHCT) for Acute Lymphoblastic Leukemia (ALL): The Mayo Clinic Cohort

Introduction Novel high-risk groups have been identified in adult ALL, including secondary (sALL) and Philadelphia-like ALL (Ph-like, based on CRLF2, IgH, ABL2, JAK2 and other tyrosine kinase translocations), and those with minimal residual disease >0.1% (MRD+) after induction therapy. Novel targeted therapies are now routinely incorporated into 1st line regimens, including tyrosine kinase inhibitors (BCR-ABL1-pos), rituximab (CD20+) and blinatumomab (Blina) for MRD+. The impact of these novel high risk groups and therapies after alloHCT is unknown; therefore, we evaluated their impact on overall survival (OS), relapse rate (REL), non-relapse mortality (NRM) and acute and chronic GVHD. Methods We evaluated pts receiving 1st Allo-HCT for ALL at Mayo Clinic (Rochester, Phoenix, and Jacksonville) from 2008-2018 for outcomes of interest, specifically the impact of novel therapies and risk groups. Associations of patient factors with outcomes were examined using univariable (UVA) and multivariable (MVA) Cox proportional hazards regression models, where the cause-specific hazard of the given outcome was modeled to account for the competing risk of death. Results We identified 261 consecutive AlloHCT recipients during the study period. Median age at transplant was 48 years (18-72) and 147 (56.3%) were male. The median comorbidity (HCT-CI) score was 2 (0-8). 213 pts (81.6%) had B-lineage ALL, of which 85 (32.6%) were BCR-ABL-pos, 17 (6.5%) Ph-like (identified by FISH), 16 (6.1%) hypoploidy/near triploidy (Hy/Tri), and 67 (25.7%) pre-B ALL NOS. The remaining 48 (18.4%) had T-ALL. 30 pts (11.5%) had sALL (i.e. prior chemo/radiotherapy for another malignancy). HyperCVAD was the most common 1st line regimen (68.2%). 243 (93.1%) pts achieved Complete Remission (CR1) after induction therapy, and 203 (77.8%) were in CR1 at the time of alloHCT. Blina was administered for MRD+ in 14 pts (5.4%), and for relapsed/refractory ALL (R/R) in 13 (27% of R/R pts), 7 of whom received Blina as initial therapy for R/R. Donors were matched unrelated in 149 (57.1%), matched related in 98 (37.5%), and haploidentical in 14 (5.4%). Peripheral blood (PB) grafts were used in 233 (89.3%). 103 (54.5%) were donor:recipient (D:R) sex-matched, and 86 D:R mismatched [47 (24.9%) M:F; and 39 (20.6%) F:M]. Myeloablative conditioning was used for the majority (78.5%) mostly with Cy/TBI (60.5%). Standard GVHD prophylaxis regimens were used. Outcomes Median follow-up after transplant was 22.4 months (0.5-135), and 51 (19.5%) had REL. The 1, 2 and 5-year survival rates were 71.9%, 64.9%, and 54.1%, respectively (Figure 1). Acute GVHD developed in 144 (55.2%) and chronic GVHD in 100 (38.3%). Ph-like ALL, Blina for MRD+, Blina for R/R, sALL and CD20-pos had no independent impact on OS. In contrast, age>60, Hy/Tri, and >CR1 at alloHCT were associated with worse OS in UVA, however, in MVA only pre-B ALL NOS was associated with better OS. Female:male D:R status was associated with inferior OS. Blina for R/R disease was associated with increased risk of REL in UVA [HR 5.26 95% CI (1.33, 20.00), p=0.017], whereas other novel high risk groups had no impact on REL. In contrast, T-ALL, Hy/Tri and >CR1 at AlloHCT were associated with increased REL in UVA, but only T-ALL and Hy/Tri continued to predict for increased REL in MVA. Secondary ALL was associated with increased NRM in UVA [HR 1.96 95% CI (1.07, 3.57), p=0.028], whereas other novel high risk groups had no impact on NRM. In contrast, age>60, >CR1 at AlloHCT and D:R sex mismatch were associated with higher NRM in UVA, but only sex mismatch and >CR1 at AlloHCT were associated with higher NRM in MVA. TBI use was associated with higher risk of acute GvHD (p=0.008) and ATG use with lower risk chronic GVHD (p<0.001). Similarly non-PB grafts were associated with a lower risk of chronic GVHD (p=0.005). Results for OS, REL, NRM, acute and chronic GVHD analysis are shown in Table 1. Conclusion Novel high risk groups (CD20+, Ph-like and sALL) do not appear to adversely impact OS after alloHCT, although sALL was associated with increased risk of NRM. Interestingly, pre-B-ALL NOS appear to be associated with favorable OS. Novel targeted therapies also do not independently predict outcome, with the exception of Blina for R/R ALL which may be associated with REL after subsequent alloHCT (a subgroup for whom novel maintenance strategies should be explored). Our analysis highlights the importance of allo-HCT for novel high risk ALL subgroups. Disclosures Patnaik: Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees. Kharfan-Dabaja:Daiichi Sankyo: Consultancy; Pharmacyclics: Consultancy. Foran:Agios: Honoraria, Research Funding.

Role of IGF2BP1 and Target Genes in ETV6-RUNX1 Positive B-Acute Lymphoblastic Leukemia

Acute Lymphoblastic Leukemia is the most prevalent hematological neoplasm in Indian children and around 85% are of B-cell origin (B-Acute Lymphoblastic Leukemia (B-ALL)). ETV6-RUNX1 (E/R) is one of the common translocations seen in B-ALL. We have identified Insulin like Growth Factor 2 mRNA Binding Protein 1 (IGF2BP1), an oncofetal protein, to be specifically overexpressed in bone marrow samples of E/R positive B-ALL patients. We have tried to characterize the role of this protein in B-ALL. We collected 193 new paediatric pre B-ALL bone marrow samples. Most of the patients were under 5 years of age (73%) and only 3% of patients were more than 10 years. 66% of patients had no known translocation. Among the ones with translocation, ETV6-RUNX1 and BCR-ABL translocation were the commonest (11% each). 10% were E2A-PBX positive and only 3% had an MLL translocation. The translocation profile was very different from that of previously published reports. E/R translocation generally portends a good prognosis. There was a significantly lower initial WBC count in E/R positive group when compared to the other groups. However, there was no significant difference in other blood parameters including initial Hemoglobin levels and platelet counts. Interestingly, the proportion of patients with a high number of blasts in peripheral blood on day 8 post chemotherapy was actually significantly higher in the E/R +ve group indicating that there might be a proportion of patients in the E/R +ve group with a worse prognosis. There was no difference in minimal residual disease between the E/R translocation positive and negative groups. Real time RT-qPCR analysis of IGF2BP1 and ETV6 mRNA expression in bone marrow samples revealed significantly higher expression of IGF2BP1 in E/R positive B-ALL compared to E/R negative B-ALL samples. ETV6 mRNA relative expression on the other hand was found to be significantly lower in the E/R positive group. This corresponds to existing data that there is usually loss of the other wild type ETV6 allele in these patients. We used data from multiple studies to narrow down on true targets of IGF2BP1 in B-ALL. We used a published eCLIP dataset of IGF2BP1 to identify cognate RNA targets. Over expression of these targets in E/R +ve samples was confirmed from the other previously published high throughput gene expression studies on B-ALL (MILE (microarray innovations in leukemia) study (n = 3242) and Palanichamy et al, 2016, J Clin Invest). We identified 13 such genes which were bound by IGF2BP1 and regulated by it in E/R +ve B-ALL. One of these targets, EGFL7, has previously been shown to play an important role in haematopoiesis and involved in Acute Myeloid Leukemia pathogenesis. c-MYC is a known target of IGF2BP1 and. being a proto oncogene has an important role in B-cell proliferation. However, the biological and clinical role of cMYC and EGFL7 genes had not been evaluated in ETV6-RUNX1 positive Pre B-ALL. Real time analysis of EGFL7 and cMYC expression in the patient bone marrow samples showed significantly altered expression in both groups. As expected, EGFL7 showed a significantly higher expression in E/R +ve samples. Unexpectedly, cMYC showed lower expression in E/R positive group. The expression of EGFL7 also showed a significant positive correlation with initial hemoglobin levels and a negative correlation with initial WBC counts. To study the effect of IGF2BP1 binding to the EGFL7 or c-MYC UTRs, they were cloned downstream of firefly luciferase and coexpressed with IGF2BP1 in HeLa cells. Interestingly, the dual luciferase assay revealed no stabilization of either of these UTRs by IGF2BP1. This reveals a much more complex regulation by IGF2BP1 of its targets which might involve RNA methylation. To utilize the expression of IGF2BP1 and its target genes as a diagnostic marker for the presence of the E/R translocation, ROC curves using FISH/PCR as the gold standard test were prepared. The AUC (Area under curve) for ROC of the expression of IGF2BP1, c-MYC and EGFL7 were 0.917, 0.7 and 0.78 respectively. We also analyzed ROC curves using various combinations of these genes. Gene expression of IGF2BP1 alone was the best indicator of the presence of the ETV6-RUNX1 translocation with the highest sensitivity and specificity. In conclusion, relative expression of IGF2BP1 can serve as an excellent diagnostic marker for the E/R translocation with more than 90% sensitivity and 85% specificity. Disclosures No relevant conflicts of interest to declare.

Transcriptomic Profile of Bone Marrow-Derived Mesenchymal Stromal Cells of Pediatric Pre-B Acute Lymphoblastic Leukemia Patients and Healthy Donors after Interaction with Leukemic Cells

Background: Acute lymphoblastic leukemia (ALL) is a hematological malignancy characterized by the abnormal expansion of clonal lymphoblasts in the bone marrow (BM). The behavior of malignant lymphoblasts is not only dependent on their genomic abnormalities but also, on their complex and reciprocal relationships with their local niche. BM microenvironment is a dynamic network of growth factors, cytokines, and stromal cells, providing a permissive environment for leukemogenesis and progression. Despite some evidence about the implication of the bidirectional interaction between mesenchymal stromal cells (MSCs) and leukemic cell on growth and survival, it is still largely unknown. Although an increasing number of studies are reporting on the expression of specific genes in leukemic cells-interacting MSCs, gene expression profiling in cocultured MSCs (with respect to mono culture conditions) has not been done on a transcriptome-wide basis. Taking all this into consideration, we have established cocultures between BM-derived MSCs and the leukemic mononuclear cells and performed gene expression profile (GEP) studies on the MSC population to determine those deregulated genes due to the coculture condition with respect to MSCs in mono culture. Methods: Primary MSCs from BM samples of healthy donors (n=4) and pediatric pre-B ALL patients (n=4) were isolated and expanded and mononuclear cells were, also, isolated from patients peripheral blood. At passage 2, healthy/patient (h/p) MSCs were cocultured with the leukemic cells for 72 hours and subsequently subjected to GEP analyses. RNA isolated from h/pMSCs and biotinylated amplified RNA was synthesized. For the hybridization HG-U133 Plus, 2.0 GeneChip oligonucleotide arrays (Affymetrix) was used. Raw microarray data were processed into expression values through the Affy package in R. RMA (robust multi-array average) algorithm was used for preprocessing. The linear regression model package LIMMA (Linear Models for Microarray Data) was used to identify significant differential expression of genes (DEGs) compared to h/pMSCs after coculture with MSCs from the same origin in mono culture. Minimum 1.5 fold change and p-value < 0.001 were defined as the threshold. Functional enrichment, gene network and pathway analysis, were performed on selected sets of genes after differential expression analyses using two bioinformatics tools: DAVID Bioinformatics Resources 6.8 and WebGestalt 2019. Results: We identified 293 DEGs when analyzing pMSC samples vs. hMSC in coculture condition and 853 DEGs of pMSC vs. hMSC analyze in mono culture condition and only 15 DEGs common in both group. According to the result of functional enrichment analysis of pMSC vs. hMSC in coculture condition, 29 gene ontology (GO) term biological process including (top five) apoptotic process, axon guidance, regulation of JNK cascade, cell-cell adhesion and protein phosphorylation and 4 KEGG pathways of DEGs including axon guidance, FoxO signalling pathway, Wnt signalling pathway and signalling pathways regulating pluripotency of stem cells were determined. Comparing pMSC vs. hMSC in mono culture condition, 292 GO term biological process including extracellular matrix organization, skeletal system development, angiogenesis and signal transduction and 43 KEGG pathways of DEGs including pathways in cancer, TGFß signalling pathway, ECM-receptor interaction, p53 signalling pathway and PI3-Akt signalling pathway were determined.Five genes with upregulated expression after coculture (COL18A1, EFNA5, MAP3K8, CASP1, IGF1R) and after mono culture (MMP1, CXCL1, IL1B, PCDH10, CXCL6) were identified. Conclusions: Deregulated genes especially after coculture condition in axon guidance and ephrin receptor-mediated signalling pathway of which the importance is not yet understood in cancer microenvironment including EPHB1, ephrinA5, ephrinB3, p21 activated kinase 2 (PAK2), and ROBO3 showed highly differential expression pattern between pMSCs and hMSCs. This is the first report indicating the importance of axon guidance genes as a result of interaction between MSCs and leukemic cells. The elucidation of deregulated mechanisms and pathways provides new molecular insights to the contribution of these cells to ALL pathophysiology and understanding cross talk between these cells may lead to found new therapeutic targets. Disclosures No relevant conflicts of interest to declare.

Determinants of response and resistance to CAR T cell therapy.

The remarkable success of chimeric antigen receptor (CAR)-engineered T cells in pre-B cell acute lymphoblastic leukemia (ALL) and B cell lymphoma led to the approval of anti-CD19 CAR T cells as the first ever CAR T cell therapy in 2017. However, with the number of CAR T cell-treated patients increasing, observations of tumor escape and resistance to CAR T cell therapy with disease relapse are demonstrating the current limitations of this therapeutic modality. Mechanisms hampering CAR T cell efficiency include limited T cell persistence, caused for example by T cell exhaustion and activation-induced cell death (AICD), as well as therapy-related toxicity. Furthermore, the physical properties, antigen heterogeneity and immunosuppressive capacities of solid tumors have prevented the success of CAR T cells in these entities. Herein we review current obstacles of CAR T cell therapy and propose strategies in order to overcome these hurdles and expand CAR T cell therapy to a broader range of cancer patients.

Integrin Antibody Decreases Deformability of Patient-Derived Pre-B Acute Lymphocytic Leukemia Cells as Measured by High-Frequency Acoustic Tweezers.

This article reports a study of cell mechanics in patient-derived (primary) B-cell acute lymphocytic leukemia (ALL) cells treated with antibodies against integrins. Leukemia cell adhesion to stromal cells mediates chemotherapeutic drug resistance, also known as cell adhesion-mediated chemotherapeutic drug resistance. We have previously shown that antibodies against integrin α4 and α6 adhesion molecules can de-adhere ALL cells from stromal cells or counter-receptors. Because drug-resistant cells are more deformable, as evaluated by single-beam acoustic tweezers, we hypothesized that changes in cell mechanics might contribute to the de-adhesive effect of integrin-targeting antibodies. In this study, the deformability of primary pre-B ALL cells was evaluated by single-beam acoustic tweezers after treatments with the de-adhering antibody Tysabri or P5G10 against integrin α4 and α6 adhesion molecules. We demonstrated that primary ALL cells treated with P5G10 expressed decreased deformability compared with immunoglobulin G1 -treated control cells (P < .05). Tysabri did not show an effect on deformability (P > .05). These results suggest that decreased deformability is associated with an integrin α6 blockade. Further assessments of the functional roles of deformability and integrin blockades in B-ALL cell drug resistance and deformability, respectively, are necessary.

DISSECTION OF THE PRE-B LEUKAEMIA BONE MARROW MICROENVIRONMENT

The microenvironments of leukaemia and cancer are critical for multiple stages of malignancies, and they are an attractive therapeutic target. Clinical studies in children diagnosed with acute lymphoblastic leukaemia (ALL) have clearly demonstrated defects in bone marrow microenvironment, yet little is known about the contribution of the normal bone marrow cells during the development of the disease, progression and relapse. It remains unclear whether the clinical symptoms of increased bone fragility and reduced bone mineral density should be treated at diagnosis, and whether such treatments would have any impact on the leukaemia progression. To elucidate the influence of ALL on haematopoiesis and the microenvironment, we studied the development of pre-B cell ALL (pre-B ALL) in an immunocompetent BCR-ABL1+ model. We found that haematopoiesis was perturbed, B lymphopoiesis was impaired, collagen production was reduced, and the number of osteoblastic cells was decreased in the bone marrow microenvironment. Moreover, the leukaemia-bearing mice exhibited severe bone loss during leukaemogenesis, which is consistent with clinical data in paediatric ALL. We showed that leukaemia cells produce receptor activator of nuclear factor κB ligand (RANKL), and observed osteoclast-mediated bone resorption. We demonstrated that inhibition of osteoclasts with zoledronic acid in leukaemia mice significantly reduced disease burden and prolonged survival. Our study reveals the impact of pre-B ALL development on haematopoiesis, bones and bone marrow microenvironment. It provides evidence for targeting leukaemia-induced bone loss as a novel therapeutic strategy for patients. 1

Methanolic Extract from Aerial Parts of Artemisia Annua L. Induces Cytotoxicity and Enhances Vincristine-Induced Anticancer Effect in Pre-B Acute Lymphoblastic Leukemia Cells

Background: Nowadays, remarkable attention has been drawn towards the effective therapeutic characteristic of natural products targeting cancerous cells. This study aimed to investigate the anti-cancer effect of Artemisia annua extract (AAE), a Chinese herbal medicine alone and in combination with a microtubule binding agent used in ALL treatment, vincristine (VCR), in B-Acute lymphoblastic leukemia (ALL) Nalm-6 and Reh cells. Materials and Methods: Cytotoxic activity of AAE and VCR was determined using MTT assay in Nalm-6, and Reh cell lines and synergism was evaluated using the CompuSyn software. Caspase 3 activity and Annexin/PI staining were performed for apoptosis assessment. The expression level of apoptosis-related genes, caspase 3, Bax and Bcl-2 were determined using real time-PCR. One-way ANOVA and post hoc Tukey multiple comparisons were used for statistical analysis. Results: Our findings revealed that a single administration of AAE exerted an anti-leukemic effect in both ALL-derived cells in a time- and dose-dependent manner. Interestingly, the growth inhibitory activity of the extract was more potentiated when combined with 0.1 and 1 nM VCR through caspase 3-dependent apoptosis. Moreover, real-time PCR analysis showed that VCR-induced cytotoxicity was augmented by AAE through alteration of Bax, and Bcl-2 mRNA expression. Conclusion: Overall, owing to the nontoxic nature of AAE and its explicit role in enhancing VCR effectiveness, our study provided new insight into the development of a novel combinatorial approach in ALL using natural herbs. The practical implication of the research requires further investigation through clinical trials, opening avenues for forthcoming treatment improvements.

FoxO3a Gene Down-regulation in Pathogenesis of Pediatric Acute Lymphoblastic Leukemia

Abstract Introduction: Acute lymphoblastic leukemia (ALL) is the most common malignancy found in the pediatrics with the peak prevalence between the ages of 2 and 5 years. The constitutive activation of PI3K/AKT pathway inhibits the tumor-suppressor role of FoxO3a (a member of the forkhead class O [FoxO] transcription factor family) in a variety of cancers and leads to tumorigenesis. This study aims to investigate the expression of FoxO3a in three different stages of pediatric ALL in mRNA level. Subjects and Methods: In this case-control study, 70 patients with childhood ALL and 70 healthy age- and gender-matched as the control group were enrolled. Real-time quantitative RT-polymerase chain reaction (qRT-PCR) was used to detect the mRNA expression level of FoxO3a in children with different stages of ALL and healthy children as a control group. Results: Data showed that the expression of FoxO3a mRNA was lower in newly diagnosed ALL patients compared to controls (P &lt; 0.0001), maintenance (P = 0.0342), and relapse (P = 0.0006) groups, while no difference was observed between other groups. In addition, T-ALL patients showed decreased expression of FoxO3a compared to Pre-B ALL ones (P &lt; 0.0001). Conclusion: The study results suggest that FoxO3a plays a tumor-suppressor role in ALL. Thus, its up-regulation seems to be a plausible therapeutic strategy for this type of tumor.

Variants in ARID5B gene are associated with the development of acute lymphoblastic leukemia in Mexican children.

A high impact of ARID5B SNPs on acute lymphoblastic leukemia (ALL) susceptibility has been described in Hispanic children; therefore, it is relevant to know if they influence the high incidence of childhood-ALL in Mexicans. Seven SNPs (rs10821936, rs10994982, rs7089424, rs2393732, rs2393782, rs2893881, rs4948488) of ARID5B were analyzed in 384 controls and 298 ALL children using genomic DNA and TaqMan probes. The SNPs were analyzed for deviation of Hardy-Weinberg equilibrium; Fisher's exact test was used to compare the genotypic and allelic frequencies between controls and patients. The association between SNPs and ALL susceptibility was calculated, and haplotype and ancestry analyses were conducted. All SNPs were associated with ALL, pre-B ALL, and hyperdiploid-ALL susceptibility (p < 0.05). No association with T-ALL and gene fusions was found (p > 0.05). The seven SNPs were associated with risk of pre-B ALL in younger children; however, rs2393732, rs2393782, rs2893881, and rs4948488 were not associated with susceptibility in older children and adolescents. The CAG haplotype (rs10821936, rs10994982, rs7089424) was strongly associated with ALL risk in our population (p < 0.00001). The frequency of all risk alleles in our ALL, pre-B, and hyperdiploid-ALL patients was higher than that in Hispanic children reported. This is the first report showing the association between rs2393732, rs2393782, and rs4948488 with pre-B hyperdiploid-ALL children. The G allele at rs2893881 confers major risk for pre-B hyperdiploid-ALL in Mexican (OR, 2.29) than in Hispanic children (OR, 1.71). The genetic background of our population could influence the susceptibility to ALL and explain its high incidence in Mexico.

Bispecific ligand-directed toxin targeting CD22 and CD19 (DT2219) for refractory B-cell malignancies: Results of phase I-II trial.

e19066 Background: The novel bispecific toxin DT2219 consists of a fusion between the catalytic domain of diphtheria toxin (DT) and single-chain variable fragments (scFV) of antibodies targeting human CD19 and CD22. Methods: The primary objective of Phase I Dose was to determine DT2219 dose which is well tolerated on an 8 dose schedule (days 1, 3, 5, 8 and day 15, 17, 19 and 22). Phase II Extension aimed to establish a preliminary estimate of overall response at day 29 in patients stratified to Arm A (relapsed/refractory B-cell lymphoma) and Arm B (B-cell leukemia). Baseline serum level of neutralizing antibody (NA) against diphteria toxin below 50% was required for eligibility. Results: Eighteen patients (NHL n = 12; ALL n = 6) at median age 65 years (range 50-83 yrs) with relapse/refractory disease and confirmed expression of CD19 and/or CD22 were enrolled. Treatment was well tolerated at 60 mcg/kg x 8 doses and the most common adverse events included capillary leak sy, elevated AST/ALT, low albumin, weight gain and leukopenia. All were Gr 1-2 and resolved after 3-5 days allowing day 15 DT2219 administration. There were no neutropenic fever or immune mediated adverse events. Four patients experienced dose limiting toxicity at dose 80 μg/kg/day: Gr 4 capillary leak syndrome (n = 1), Gr 3 LFTs abnormalities (n = 2) and Gr 4 thrombocytopenia &gt; 7 days duration (n = 1). Thirteen patients were evaluable for response and 3 experienced objective clinical benefit. One patient with primary refractory pre-B acute lymphoblastic leukemia achieved complete remission after 1st cycle. Two patients with transformed lymphoma demonstrated transient tumor shrinkage, however, DT2219 therapy was discontinued due to DLT and increased NA titer after 1st cycle (pre C1 28%, pre C2 108%). Correlative studies showed a low incidence of neutralizing antibody in NHL patients recently exposed to Rituximab. Conclusions: We have established the biologically active dose of DT2219 at 60 μg/kg/day ×8 doses. Further development of DT2219 includes combination with rituximab (to reduce NA formation) and synergistic combinations against B-cell malignancies. Clinical trial information: NCT02370160.

O-GlcNAcylation of c-Myc dynamically regulates pre-B cell proliferation

Abstract O-GlcNAc modification regulates the activity of hundreds of nucleocytoplasmic proteins involved in a wide variety of cellular processes such as gene expression, signaling and cell growth. However, the mechanism underlying the regulation of B cell development and function by O-GlcNAcylation is largely unknown. Here, we demonstrate that changes in cellular O-GlcNAc levels significantly affect the growth of large pre-B cells transiently expressing pre-B cell receptors on their surface membrane, which are known to rapidly proliferate for the expansion of functional clones that express successfully rearranged heavy chains during early B cell development. Our study first show that overall O-GlcNAc levels in these proliferative pre-B cells are highly induced compared to pro-B cells, depending on pre-B cell receptor expression. Mechanistically, inhibition of O-GlcNAcylation in pre-B cells markedly decreases c-Myc expression, which results in downregulation of cell proliferation through the blocking of E- and A-type cyclin expressions. In addition, restriction of glucose uptake in pre-B cells also reduces cellular O-GlcNAc levels as well as c-Myc expression and subsequently decreases cell proliferation. Furthermore, the population of large pre-B cells in the bone marrow is significantly reduced by the administration of O-GlcNAc inhibitor to mice. Thus, our study strongly suggests that O-GlcNAcylation dependent expression of c-Myc is a new component in regulation of pre-B cell proliferation and also a potential therapeutic target for the treatment of pre-B cell acute lymphoblastic leukemia.

Blinatumomab as a bridge to trasplantation in refractory Philadelphia chromosome negative b-cell acute lymphoblastic leukemia: a case report

Blinatumomab is a first class bispecific T-cell engager that has been shown to achieve negative minimal residual disease in patients with relapsed or refractory pre-B acute lymphoblastic leukemia after conventional chemotherapy. Nevertheless, there is little evidence about its role as an off label enhancer of cytological remission prior to stem cell transplantation (SCT). We describe the case of a patient with an excellent performance status who was allowed to undergo alogenic SCT after a single blinatumomab cycle, as well as the management of adverse events and the observed results.

SUN-276 Acute Pancreatitis and Diabetic Ketoacidosis in an 8-Year-Old Following Induction Therapy for Pre-B Acute Lymphocytic Leukemia

Introduction Peg-Asparaginase and Dexamethasone are both used in the induction therapy for pre-B Acute Lymphocytic Leukemia (ALL) and have overlapping adverse effects. Peg-Asparaginase is an enzyme that causes depletion of asparagine which is an essential amino acid for leukemia cells. Dexamethasone is a long-acting glucocorticoid with a 30-fold increase in anti-inflammatory activity relative to Hydrocortisone. Hyperglycemia and acute pancreatitis are both adverse reactions of Peg-asparaginase and glucocorticoids. Clinical case An 8-year-old male with obesity presented with acute pancreatitis and diabetic ketoacidosis (DKA) following induction therapy for pre-B ALL. The patient was euglycemic at the time of diagnosis. On day 1 induction chemotherapy was administered which included IV vincristine, IT cytarabine and PO dexamethasone (4 mg BID daily). On day 2, patient developed hyperglycemia (167 mg/dL). On day 4 he received IV Peg-Asparaginase (3,800 IU). On day 8 patient returned with back pain, fatigue, polydipsia, polyuria, and Kussmaul breathing. Labs revealed hyperglycemia (1,118 mg/dL), metabolic acidosis with an elevated anion gap (pH 7.14, pCO2 < 17 mmHg, HCO3 4.6 mmol/L, anion gap 36), elevated lipase (2,256 IU/L), mild transaminitis, and elevated creatinine and BUN. Pancreatic ultrasound was consistent with pancreatitis. The patient was admitted to the pediatric ICU for insulin drip and management of pancreatitis. On day 10 he was transitioned to SQ glargine insulin. Chemotherapy was restarted with dexamethasone and vincristine. Patient had persistent hyperglycemia requiring lispro insulin to cover meals and to correct hyperglycemia. His glucose normalized on this treatment and insulin was tapered down to be discontinued 9 days after initially started. His HbA1c was 7.5% (normal <5.6%) and he had negative T1DM-related antibodies. A CT of the abdomen showed pancreatic inflammation but no fluid collection or necrosis. At the time of discharge, patient was euglycemic. In the following months, in the absence of glucocorticoids and asparaginase, the patient had multiple admissions for recurrent pancreatitis with development of a pseudocyst. No further episodes of hyperglycemia were documented. Conclusion DKA can develop as side effect of treatment with Peg-Asparaginase and glucocorticoids. Patients treated with these medications need to be closely monitored to identify and treat hyperglycemia and pancreatitis as early as it develops.

IKZF1 Gene Deletion in Pediatric Patients Diagnosed with Acute Lymphoblastic Leukemia in Mexico

The IKZF1 gene is formed by 8 exons and encodes IKAROS, a transcription factor that regulates the expression of genes that control cell cycle progression and cell survival. In general, 15-20% of the patients with preB acute lymphoblastic leukemia (preB ALL) harbor IKZF1 deletions, and the frequency of these deletions increases in BCR-ABL1 or Ph-like subgroups. These deletions have been associated with poor treatment response and the risk of relapse. The aim of this descriptive study was to determine the frequency of IKZF1 deletions and the success of an induction therapy response in Mexican pediatric patients diagnosed with preB ALL in 2 hospitals from 2017 to August 2018. Thirty-six bone marrow samples from patients at the Instituto Nacional de Pediatría in Mexico City and the Centro Estatal de Cancerología in Tepic were analyzed. The IKZF1 deletion was identified by MLPA using the SALSA MLPA P335 ALL-IKZF1 probemix. Deletions of at least 1 IKZF1 exon were observed in 7/34 samples (20.6%): 3 with 1 exon deleted; 1 with 2 exons, 1 with 5 exons, 1 with 6 exons, and 1 patient with a complete IKZF1 deletion. This study was descriptive in nature; we calculated the frequency of the IKZF1 gene deletion in a Mexican pediatric population with preB ALL as 20.6%.

Cell circuits and niches controlling B cell development.

Studies over the last decade uncovered overlapping niches for hematopoietic stem cells (HSCs), multipotent progenitor cells, common lymphoid progenitors, and early B cell progenitors. HSC and lymphoid niches are predominantly composed by mesenchymal progenitor cells (MPCs) and by a small subset of endothelial cells. Niche cells create specialized microenvironments through the concomitant production of short-range acting cell-fate determining cytokines such as interleukin (IL)-7 and stem cell factor and the potent chemoattractant C-X-C motif chemokine ligand 12. This type of cellular organization allows for the cross-talk between hematopoietic stem and progenitor cells with niche cells, such that niche cell activity can be regulated by the quality and quantity of hematopoietic progenitors being produced. For example, preleukemic B cell progenitors and preB acute lymphoblastic leukemias interact directly with MPCs, and downregulate IL-7 expression and the production of non-leukemic lymphoid cells. In this review, we discuss a novel model of B cell development that is centered on cellular circuits formed between B cell progenitors and lymphopoietic niches.

Suppression of c-Myc using 10058-F4 exerts caspase-3-dependent apoptosis and intensifies the antileukemic effect of vincristine in pre-B acute lymphoblastic leukemia cells.

Despite an old history behind the identification of the leading role of c-Myc in leukemogenesis, the road to constructing a therapeutic perspective for this molecule in acute lymphoblastic leukemia (ALL) is yet mesmerizing. This study was designed to provide a better outlook for the anticancer property of 10058-F4, an appealing inhibitor of c-Myc, in pre-B ALL cell lines either in the context of monotherapy or in combination with chemotherapeutic drugs. Our results declared that abrogation of c-Myc decreased the proliferative capacity of pre-B ALL-derived cells through halting the transition of the cells from G1 phase, and reducing the replicative potential of both REH and Nalm-6 cells, at least partly, through c-Myc-mediated suppression of human telomerase reverse transcriptase. Moreover, 10058-F4 potently induced a caspase-3-dependent apoptosis in pre-B ALL cells via shifting the balance between pro- and anti-apoptotic target genes. Although the inhibition of PI3Kδ using Idelalisib upregulated the messenger RNAexpression of autophagy-related genes in 10058-F4-treated cells, treatment with autophagy inhibitor chloroquine decreased viability of the cells, either as a single agent or in combination with Idelalisib and/or 10058-F4; suggesting that the activation of autophagy in pre-B ALL cells could blunt apoptotic events and attenuate anticancer effect of both c-Myc and PI3K inhibitors. Finally, the results of our synergistic experiments delineated that 10058-F4 produced a synergistic effect with vincristine and provided an enhanced therapeutic efficacy in ALL cells, highlighting that c-Myc oncoprotein could be a bona fide target for the treatment of ALL.

Mechanisms of resistance to CAR T cell therapy.

The successes with chimeric antigen receptor (CAR) T cell therapy in early clinical trials involving patients with pre-B cell acute lymphoblastic leukaemia (ALL) or B cell lymphomas have revolutionized anticancer therapy, providing a potentially curative option for patients who are refractory to standard treatments. These trials resulted in rapid FDA approvals of anti-CD19 CAR T cell products for both ALL and certain types of B cell lymphoma - the first approved gene therapies in the USA. However, growing experience with these agents has revealed that remissions will be brief in a substantial number of patients owing to poor CAR T cell persistence and/or cancer cell resistance resulting from antigen loss or modulation. Furthermore, the initial experience with CAR T cells has highlighted challenges associated with manufacturing a patient-specific therapy. Understanding the limitations of CAR T cell therapy will be critical to realizing the full potential of this novel treatment approach. Herein, we discuss the factors that can preclude durable remissions following CAR T cell therapy, with a primary focus on the resistance mechanisms that underlie disease relapse. We also provide an overview of potential strategies to overcome these obstacles in an effort to more effectively incorporate this unique therapeutic strategy into standard treatment paradigms.

Blinatumomab As a Successful Bridge to Hematopoietic Stem Cell Transplant in a Down Syndrome Patient with Relapsed B-Precursor Acute Lymphoblastic Leukemia

Introduction Children with Down syndrome (DS) carry a 20-fold higher risk of developing acute lymphoblastic leukemia (ALL) than other children and have increased rates of treatment-related mortality (TRM) and relapse. The prognosis for relapsed DS-ALL patients is grim with long-term survival rates of 20%. Blinatumomab, a CD19/CD3 bispecific antibody, is FDA approved for relapsed pediatric pre-B ALL, and recently approved for patients with minimal residual disease (MRD). However there are few reports of the use of blinatumomab in DS-ALL patients, and no reports of blinatumomab as a bridge to stem cell transplant (SCT) in this population. Case Description A 19 year old male with DS presented with thrombocytopenia and was diagnosed with pre-B ALL. Upfront therapy included fractionated cyclophosphamide, vincristine, doxorubicin, dexamethasone, pegaspargase, and rituximab for 8 cycles, and he achieved complete remission (CR). Maintenance therapy included 26 months of 6-mercaptopurine, vincristine, methotrexate, and prednisone. He relapsed 5 months after completion of therapy and was found to have a P2RY8-CRLF2 fusion. IgH gene rearrangement analysis by next generation sequencing (LymphoTrack assay) demonstrated clonal rearrangement. Re-induction therapy included vincristine, pegaspargase, and prednisone. He completed 2 additional courses of chemotherapy but continued to have MRD both by flow cytometry and LymphoTrack. Given persistent MRD with CD19+ lymphoblasts, he was treated with blinatumomab at the FDA recommended dosing; he had no signs of toxicity. He achieved negative MRD by flow cytometry and LymphoTrack after 1 cycle. He then proceeded to a T-cell depleted peripheral blood SCT from a 10/10 matched sibling after conditioning with total body irradiation, thiotepa, and cyclophosphamide. Post-SCT course was uncomplicated and he is now 1 year post-SCT with no evidence of leukemia and 100% donor chimerism. Conclusion Blinatumomab is clinically effective and safe for patients with relapsed/refractory pre-B ALL, yet there is limited information on its use in DS-ALL patients. These patients have been largely excluded from blinatumomab trials given their altered immune system and unknown level of innate T cell dysfunction, potentially altering the efficacy of blinatumomab. We present here the case of a 22 year old male with relapsed DS-ALL who remained MRD positive after several cycles of re-induction chemotherapy, but achieved a CR following 1 cycle of blinatumomab with no toxicities, and safely proceeded to SCT. Although this is a single case, it demonstrates clinical efficacy of blinatumomab in relapsed DS-ALL, suggesting that these patients' T cells can be engaged to recognized CD19-positive leukemic blasts. Furthermore, blinatumomab represents a particularly attractive bridging therapy to SCT for DS-ALL patients who experience high rates of TRM with traditional agents.

Cytogenetic and molecular assessment of childhood acute lymphoblastic leukemia patients from 2014 to 2017 in Ahvaz

Background: Acute lymphoblastic leukemia (ALL) is the most common hematological malignancy in children that is caused by gene mutations and chromosomal rearrangements in lymphoid cells. Aim: In this study, for the first time, the prevalence of cytogenetic and molecular genetic abnormalities was discussed in children with ALL from 2014 to 2017 in Ahvaz. Materials and Methods: A total of 72 children were diagnosed as ALL patients by morphology, clinical examinations, and flow cytometry assays. Cytogenetic and molecular genetic analysis was done on bone marrow (BM) samples by BM culture and reverse transcription-polymerase chain reaction technique, respectively. Descriptive data analysis was done using SPSS software. Chi-square and independent samples t-test was used to assess the correlation between variables. Results: Sixty-five cases (90.3%) were preB lineage and 7 cases (9.7%) were T-lineage out of 72 ALL patients, t(9,22) BCR-ABL (p190) is the most frequent cytogenetic and molecular genetic abnormality in preB ALL (7%) and T-ALL patients (28.6%), respectively. t(4,3) inv (16) and t(2,8) were introduced as novel cytogenetic abnormalities in preB ALL cells. No significant correlation was found between gender, molecular genetic abnormalities, and white blood cell count in patients. Conclusion: For the first time in this study, the highest percentage of cytogenetic and molecular genetic abnormalities was related to t(9,22) BCR-ABL in both ALL subtypes in children. The evaluation of cytogenetic and molecular genetic abnormalities in children with ALL is essential in estimating the prognosis in both preB and T-ALL subtypes, which will be a great contribution to achieve a better diagnosis and develop appropriate therapeutic approaches.