pre-B Cell ALL Research Articles

Effect of GATA3 rs3824662 gene polymorphism in Han Chinese children with pre-B-cell acute lymphoblastic leukemia with 10 years follow-up.

To evaluate the influence of GATA3 rs3824662 on pre-B-cell acute lymphoblastic leukemia (pre-B-cell ALL) susceptibility and long-term prognosis in Han Chinese children with pre-B-cell ALL treated with the CCLG-2008 protocol at the Children's Hospital of Soochow University. A total of 256 patients with childhood pre-B-cell ALL under the CCLG-2008 protocol were enrolled in this study, and 174 healthy children were used as case controls. GATA3 rs3824662 genotyping was performed using a polymerase chain reaction, followed by Sanger sequencing. The association of genotype with clinical characteristics, treatment response, adverse events, and outcomes were analyzed. The A allele frequency of GATA3 rs3824662 in patients with pre-B cell ALL was significantly higher than that in healthy children (OR = 1.41, 95% CI = 1.042-1.908; P = 0.026). Among patients with pre-B-cell ALL, the GATA3 rs3824662 AA genotype was associated with poor prednisolone response and high blast cell burden on day 15 of the induction therapy (P = 0.011 and 0.007, respectively). Patients with the rs3824662 AA variant suffered more episodes of sepsis than those with the CC or CA variants (P = 0.021). The GATA3 rs3824662 AA genotype was significantly associated with sepsis [hazard ratio (HR) = 3.375; P = 0.01]. No significant differences were found in the cumulative incidence of relapse, overall survival, and event-free survival among all genotypes. GATA3 rs3824662 was associated with susceptibility in Han Chinese children with pre-B-cell ALL and could be a possible risk factor for poor early treatment response and treatment-related sepsis.

Clinical Features and Survival According to Minimal Residual Disease in a Colombian Population Diagnosed with B-Cell Acute Lymphoblastic Leukemia

IntroductionAcute lymphoblastic leukemia (ALL) is a clonal hematopoietic disorder that originates from B or T lymphoid progenitors and has well validated prognostic and predictive factors that influence outcomes. One of the strongest prognostic factors is the detection of minimal residual disease (MRD) which measures residual cell population after treatment when a morphologic complete response has been achieved. MRD positivity is associated with a higher risk of relapse and poor response to chemo or radiotherapyObjectivesThe aim of the study was to assess the prognostic impact of post-induction MRD status in a cohort of ALL Colombian patients in terms of relapse-free survival (RFS) and overall survival (OS).MethodsThis is a retrospective observational study conducted at a Colombian university hospital and included a cohort of ALL patients diagnosed between 2013 and 2020 treatment according to protocol PETHEMA (Spanish Program for Hematology Treatments). MRD was measured with 8-color flow cytometry evaluate on bone marrow. MRD status was classified as negative MRD (NMRD) or positive MRD (PMRD) based on a sensitivity threshold of <0,01% or ≥0,01% leukemic cells, respectively, following to international guides. Demographic and clinical characteristics were analyzed using descriptive statistics. Kaplan-Meier method was used to assess overall RFS and OS.ResultsA total 128 patients were included. The median age at diagnosis was 34 years (range 0-89 years), 54% were men, 26% were overweight, 22% obese, 6.2% had type 2 DM (T2DM), and most had a ECOG PS of £2 (94%). Most patients (80.5%) had high risk according PETHEMA, B-ALL and were classified as ALL-2 (58%) according to FAB classification with Pre-B cell ALL being the most common phenotype (54.7%). Ph+ ALL was diagnosed in 12% of patients. Most used treatments protocols were PETHEMA-AR and PETHEMA-RI in 43.8% and 11.6% of patients, respectively. Post-induction MRD measurement was available in 98 patients, 36 (36.7%) had NMRD and 62 (63.3%) PMRD. From the 36 patients with NMRD, eight patients (22.2%) received Allogeneic Hematopoietic Stem Cell Transplantation (alloHSCT): two of them, were transplanted in first complete remission, one because of high risk and the other one for BCR-ABL positivity. The other six patients received alloHSCT in second remission and all of them relapsed after late consolidations. Finally, alloHSCT was done in 28 patients with PMRD (45.2%).The 12-month OS for patients with NMRD was 68.7% (95%CI 50.5-81.2) compared to 63.7% (95%CI 50.3-74.4) in the ones with PMRD, p=0.375. 12-month RFS was 83.3% (95%CI 61.5-93.4) in patients with NMRD and 90.0% (95%CI 72.1-96.7) in patients with PMRD, p=0.436. (Figure 1). OS was significantly higher for the PMRD patients who underwent AlloHSCT 96.4% (95%CI 77.2-99.4) versus not underwent 36.8% (95%CI 20.6-53.2), HR: 0.39 (95%CI 0.005-0.29) p=0.002 (Figure 2).ConclusionMRD assessment is a strong prognostic in ALL, however it was not associated with significant differences in RFS or OS in this single institution cohort of Colombian patients. Patients with PMRD taken to AlloHSCT had superior OS compared to NMRD that underwent transplant. A small sample size and short follow-up could explain our results. Larger cohorts with extended follow up and with different MRD methods are needed to better understand the role of MRD assessment in minority ALL populations, such as Colombian patients. [Display omitted] DisclosuresPeña: Amgen: Research Funding. Salazar: Amgen: Research Funding. Sandoval-Sus: BMS: Other: Advisory Board, Speakers Bureau; SeaGen, Janssen, MassiveBio, TG: Other: Advisory Board. Sossa: Amgen: Research Funding.

Abstract 1409: Identification of microRNA upregulated in pediatric precursor-B cell acute lymphoblastic leukemia patients with post-induction MRD positivity

Abstract Background and goal: MicroRNAs (miRNA) are small non-coding RNA that regulate post-transcriptional gene expression, influencing cell proliferation, differentiation and death. Extensive research has demonstrated that deregulation of miRNA-associated pathways may participate in the development of cancers. Precursor-B cell acute lymphoblastic leukemia (pre-B ALL) is the most common pediatric malignancy. Over decades, medical advances have helped the risk stratification and treatment modalities for these patients resulting in better favorable prognosis. Unfortunately, approximately 5-10% of standard risk patients and 15-20% of high or very-high risk patients will experience disease relapse. One of the factors involved in risk stratification is the minimal residual disease (MRD) status from bone marrow testing at the end of the initial induction phase of chemotherapy. MRD positivity escalates the risk and may signify some degree of therapy resistance. The aim of this study is to evaluate expression levels of miRNAs in MRD positive samples as compared to MRD negative. Methods: Bone marrow samples of 14 pre-B cell ALL pediatric patients with MRD positivity (n=5) and MRD negative (n=9) at the end of induction chemotherapy were analyzed. Next-generation sequencing technology was utilized to study the miRNAs profiles of these samples (smallRNA-seq) and to identify potential mRNA targets of selected miRNAs overexpressed in NALM6 B-ALL cell lines for 72 hours (RNA-seq). Analysis was done in Partek Flow and with the web-based tool InteractiVenn. Results: Twelve miRNAs were upregulated in patients with MRD positivity compared to those who were MRD negative (p values &amp;lt; 0.05). To investigate the potential role of these mRNAs in pre-B ALL cells, we selected 3 of those miRNAs (hsa-miR-34a-5p, -33b-5p, and -210-3p) to transfect the pre-B-ALL cell line NALM6. We identified 145 potential direct target genes for hsa-miR-33b-5p, 79 potential targets for hsa-miR-34a5-p and 142 potential targets for hsa-miR-210-3p. Thirty-five genes were common among all treatments including IL31A, PDZK1 and INSL3. Individual (per miRNA) pathways associated with these genes included immune response, metabolism and cell signaling. One common pathway associated with the differential expression profile of these overexpressed miRNAs was alcohol intake, known to have deleterious consequences in the fetus and inducing, among other things, transgenerational epigenetic alterations that may be associated with malignancy. Conclusions: Our work identifies potential biomarkers of MRD positivity after induction chemotherapy and identifies pathways associated with this phenomenon. Future plans include the manipulations of several of these pathways to determine their role in the proliferation and aggressiveness of pre-B ALL leukemia cell with the idea of devising new treatment options for those patients with post-induction MRD positivity. Citation Format: Andrea Bauchat, Lauren Raney, Jone Garai, Li Li, Randall Craver, Jovanny Zabaleta. Identification of microRNA upregulated in pediatric precursor-B cell acute lymphoblastic leukemia patients with post-induction MRD positivity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1409.

Hematopoietic Stem Cell Transplantation with Human Placenta-Derived Stem Cells Combined with Unrelated Cord Blood for Malignant and Non-Malignant Disorders (IND 14949)

Background Myeloablative (MAC) or reduced toxicity conditioning (RTC) followed by UCBT in children with malignant and non-malignant diseases is safe and effective (Geyer/Cairo, BJH 2011). However, concentration of CD34+ HPCs in UCB is low, leading to delayed hematopoietic reconstitution and high incidence of engraftment failure (Satwani/Cairo, BBMT 2013). HPDSCs are rich in HPCs, low in HLA Class I/II expression and T-cells, and have regenerative, anti-inflammatory, and immunosuppressive properties (Liao/Cairo, Stem Cells Translational Medicine 2018). Objective To determine the safety and efficacy of HPDSC with UCBT in children with malignant and non-malignant diseases. Design/Method 4-6/6 HLA matched UCB with TNC ≥ 5 × 107/kg (4/6 HLA match) or ≥ 3.5 × 107/kg (5-6/6 HLA match) were included. Patients received MAC or RTC followed by UCB plus HPDSC infusion. GVHD prophylaxis consisted of tacrolimus and mycophenolate mofetil as previously described (Bhatia/Cairo, BBMT 2009). Results To date, 28 patients ≤18 years were enrolled, 15 males and 13 females. Mean age in years ± SD was 6.8±5.5. There were 12 patients with non-malignant disease including ALD (2), CAT (1), CGD (1), SCID (2), dyskeratosis congenita (1), congenital neutropenia (1), SAA (2), LCH (1), and SDS (1) and 16 patients with malignant disease including pre B-cell ALL- CR1 (4), pre B-cell ALL - CR2 (4), T-cell ALL – CR1 (1), AML-CR1 (4), JMML-CR1 (1), TLL - CR1 (1), and BL - CR2 (1). Mean UCB TNC±SD and CD34±SD infused were 7.7 × 107±5.0/kg and 0.48 × 106±0.59/kg, respectively. There were no SAEs associated with HPDSC infusions. Four patients were removed prior to day 180 due to progressive disease (1) and engraftment failure necessitating alternative treatment (3). Probability of neutrophil engraftment was 89.2%, median day 22 (13-53). Of neutrophil engrafted evaluable patients (n=22), the probability of platelet engraftment by day 100 was 90.9%, median day 42 (20-98). Of neutrophil engrafted, evaluable patients at day 30, 60, 100 and 180, mean whole blood UCB chimerism was 92, 99, 96 and 99%, respectively. Average whole blood HPDSC chimerism was Conclusions These results suggest that UCBT with HPDSC is safe and well tolerated, has a lower than expected probability of Grade 2-4 aGVHD, and results in robust immune reconstitution. A larger cohort and longer follow-up is required to determine the clinical significance of these findings.

Abstract 3: Amino acid profiles indicate dependence on different metabolic pathways between leukemia subtypes

Abstract Despite the growing body of evidence that the tumor microenvironment protects leukemia cells from chemotherapeutic stresses (1-3), the effect of many extracellular metabolites remains largely unknown. To explore the influence of extracellular metabolites on different leukemia subtypes, cells were treated for 24 hours in vitro with either a supplemental amino acid or amino acid derivative. From this initial screening, a subset of metabolites were chosen for metabolomics analysis. Mass spectrometry (UPLC-MS/MS) was performed on intracellular fractions to identify metabolic differences that resulted from supplementation. Metabolite profiles were also compared between leukemia cell types, namely AML, pre-B cell ALL, and T cell ALL. Of the metabolites tested, lysine and 4-hydroxyphenylpyruvate, an intermediate of tyrosine and phenylalanine metabolism, had the greatest impact on global amino acid profiles. In AML and T cell ALL cell lines, intracellular glutamate, glutamine, proline, and aspartate were increased relative to their respective controls. These amino acids can enter the tricarboxylic acid (TCA) cycle as either α-ketoglutarate or oxaloacetate, suggesting a central role of the TCA cycle in both AML and T cell ALL metabolism. Interestingly, these metabolites were not significantly increased in pre-B cell ALL, signifying the inverse it true for pre-B cells. This observation provides metabolomics evidence that is consistent with a previous study that reported downregulated expression of TCA cycle related genes in pre-B cell ALL (4). Our findings indicate that uptake and metabolism of amino acids and their derivatives is distinct for different leukemia types. Moreover, supplementation with a single metabolite can result in global changes in intracellular metabolite profiles, suggesting an influence not only as an energy substrate, but on overall metabolic pathway activity. Specifically, we conclude that the TCA cycle is more active in AML and T cell ALL and can be modulated by changing the extracellular environment, while pre-B cells are less sensitive to amino acid modulation. (1) Meads MB, et al. Clin Cancer Res 2008;14(9):2519-2526. (2) Ayala F, et al. Leukemia 2009;23:2233-2241. (3) Konopleva M, et al. Drug Resist Updat 2009;12:103-113. (4) Boag JM, et al. Leukemia 2006;20:1731-1737. Citation Format: Shannon R. Sweeney, Enrique Sentandreu, Stefano Tiziani. Amino acid profiles indicate dependence on different metabolic pathways between leukemia subtypes. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3.

ROR1 Protein Expression Is Regulated By UHRF1 in t(1;19) Pre B-Cell ALL

Introduction: Acute lymphoblastic leukemia is the most common form of pediatric cancer, and a translocation between chromosomes 1 and 19 represents one of the most frequent cytogenetic abnormalities, accounting for ~5% of ALL cases. The 1;19 rearrangement results in a fusion between the E2A and PBX1 genes, and pre B-ALL cells carrying the E2A-PBX1 fusion are consistently arrested at an intermediate stage of B cell lineage development in which the cells express a functional pre B-cell receptor (pre-BCR). We have previously shown that receptor orphan tyrosine kinase receptor 1 (ROR1) is highly expressed on t(1;19) pre B-ALL blasts and required for their viability, even though ROR1 is not directly regulated by either the E2A-PBX1 fusion or the pre-BCR. Moreover, ROR1 exhibits compensatory signaling cross-talk with the pre-BCR such that maximal killing of t(1;19) pre B-ALL cells is achieved by combining silencing of ROR1 with siRNA and inhibition of the pre-BCR with kinase inhibitors such as dasatinib. However, clinical employment of this strategy is hampered as ROR1 is a pseudokinase whose biology is poorly understood and no ROR1 small-molecule inhibitors currently exist. Therefore, it is critical to advance our understanding of the upstream regulatory mechanisms responsible for ROR1 expression to uncover new therapeutic strategies for t(1;19) pre B-cell ALL.Methods: To identify potential regulators of ROR1 expression, we designed two siRNA panels targeting transcription factors predicted to bind the ROR1 locus or an array of chromatin remodeling factors and epigenetic regulators. Pre B-ALL cell lines that are positive or negative for the 1;19 rearrangement were screened with these siRNA panels and any gene targets required for the viability of t(1;19) pre B-cell ALL cells but not control cells lines were considered candidate regulators of ROR1 expression. Candidates were validated for the capacity to regulate ROR1 expression by qRT-PCR and immunoblot.Results: Screening of the t(1;19) pre B-cell ALL cell line, RCH-ACV, with our siRNA panels targeting transcription factors and chromatin remodeling/epigenetic regulators identified four potential regulators of ROR1 that were required for viability of RCH-ACV cells but not our negative control cell line, REH (t(12;21)-positive). Among these four candidates was UHRF1, which interacts with histone and DNA methyltransferases. Interestingly, silencing of UHRF1 by siRNA led to reduced ROR1 protein levels, but had no significant impact on ROR1 mRNA expression. Since one primary function of UHRF1 is to regulate DNA methylation, we hypothesized that UHRF1 regulates ROR1 protein expression through an intermediate gene whose transcription is impacted by UHRF1-mediated DNA methylation. To test this hypothesis, RCH-ACV cells were treated with the RNA synthesis inhibitor actinomycin D 48 hours after silencing UHRF1. This resulted in a transient rescue of ROR1 protein levels, indicating that UHRF1 regulation of ROR1 protein depends on the transcription of an intermediate gene whose gene product subsequently regulates ROR1 protein levels.Conclusions: Data from siRNA screens and subsequent validation experiments suggest that UHRF1 functions to regulate ROR1 protein levels, but not mRNA expression. This is intriguing due to the primary role of UHRF1 as a modulator of DNA and histone methylation. The observed rescue of ROR1 protein with actinomycin D suggests that UHRF1 regulation of ROR1 protein occurs through an undefined intermediary whose locus is methylated in a UHRF1-dependent manner and whose gene product modulates ROR1 protein levels. Inhibitors of additional stages of gene transcription, translation, and stabilization are currently being tested to continue characterizing UHRF1-dependent regulation of ROR1. Cumulatively, this work has identified a novel regulatory mechanism of ROR1 protein expression by UHRF1 in t(1;19) pre B-cell ALL. In addition to advancing our understanding of ROR1 biology, this regulatory mechanism suggests a new therapeutic strategy to target ROR1-expressing cancers. DisclosuresTyner:Incyte: Research Funding; Aptose Biosciences: Research Funding; Janssen Pharmaceuticals: Research Funding; Constellation Pharmaceuticals: Research Funding; Array Biopharma: Research Funding.

Co-Opting the Genetic Redundancy of the Pax Family to Treat PAX5 Mutated Pre-B ALL

Pre-B cell ALL is the most common pediatric malignancy. More than a third of cases acquire loss-of-function mutations of PAX5, a regulatory transcription factor which is required for the differentiation of B lymphocytes. Additionally, germline loss of function PAX5 mutation is a cause of familial pre-B cell ALL. These observations indicate that loss of PAX5 activity drives leukemogenesis. We have seen that PAX5 re-expression in PAX5 -mutated pre-B cell ALL restores differentiation capacity and promotes apoptosis. However, because the gene is deleted or otherwise mutated it may not be possible to easily repair or restore PAX5 expression in leukemic cells. Nevertheless, PAX5 possesses two closely related paralogs, PAX2 and PAX8, which are structurally and functionally similar to PAX5, yet are neither expressed in lymphocytes nor mutated in ALL. Toward the goal of exploiting PAX family genomic redundancy for therapeutic purposes, we have tested the ability of PAX2 and PAX8 to replace PAX5 in pre-B ALL cell lines. Preliminary results indicate that both PAX2 and PAX8 rescue B cell differentiation and function similarly to PAX5 in this context. These results include modulation of B cell developmental markers, CD10, CD19 and CD22, as well as induction of PAX5 target genes, CD79a and BACH2. Cells also display a reduction in size which may be indicative of the large to small B cell transition and importantly show increased levels of cell death following PAX2 and PAX8 expression. We have further investigated the epigenetic status of PAX2 and PAX8 promoters in B cells and find that both loci are hypermethylated, suggesting that demethylation with agents such as methotrexate, may represent a therapeutic entry point for activating expression of PAX5 paralogs. Finally, based on observations that electrical polarization during early embryogenesis regulates developmental transcription factor expression, including for PAX genes, we have tested the ability of approved drugs targeting ion channels for their ability to induce PAX factor expression and thereby complement for loss of PAX5 in pre-B cell ALL. The anti-parasitic drug ivermectin, which activates chloride channels, is one of several compounds that appear promising in early studies. DisclosuresNo relevant conflicts of interest to declare.

The Pre-BCR to the Rescue: Therapeutic Targeting of Pre-B Cell ALL

Pre B-ALL is an aggressive cancer of the blood for which treatment of patients with relapsed and refractory disease remains a challenge. In this issue of Cancer Cell, Geng and colleagues surveyed the activation status of the pre-B cell receptor and comprehensively investigated downstream signaling mechanisms currently targetable with small molecule inhibitors.

Mature B Cell Acute Lymphoblastic Leukemia Presenting with Hypercalcemia

Hypercalcemia is rarely observed in acute lymphoblastic leukemia. So far, all cases presenting with hypercalcemia are pre-B cell ALL. In this case, a mature B-cell ALL patient presenting with hypercalcemia is discussed. A threeyear-old boy had a history of fever, weakness, swelling and pain in both kness. In the patient’s blood smear, 93% L3 type blasts were seen and 90% L3 type large blasts with vacuoles were seen in the bone marrow aspiration smear. The flow cytometry results were as follows: CD10: 87%; CD19: 85%; KAPPA: 66%; and Lambda: 35% was compliant with mature B cell ALL. His calcium level: 15 mg/dl. Although previous cases imply that hypercalcemia is usually treated with pamidronate, calcium levels gradually decreased to normal levels within five days with intravenous fluid therapy, furosemide, and steroids in our case. Conclusion: Hypercalcemia is rare in leukemia. So far, all cases presenting with hypercalcemia are pre-B cell ALL. It is important to know that hypercalcemia could be seen in the mature B-cell ALL.

Management of Metachronous Bilateral Testis Cancer in a Patient with Pre-B Cell ALL.

We present a patient with a metachronous, second testicular cancer after being diagnosed with pre-B cell ALL and receiving induction chemotherapy for a bone marrow transplant. We discuss the management of bilateral testis masses in a young patient with a hematologic malignancy as well as the role of immunosuppressive chemotherapy in developing a second cancer. This case illustrates the importance of recognizing bilateral testicular cancer early, as well as the importance of follow-up care in oncology patients including routine measurements of tumor markers. A multidisciplinary approach between medical oncology and urology, including close monitoring of the contralateral testis, remains paramount to patient care.

Obinutuzumab (GA101) Significantly Improves Survival In CD20 Positive Pre-B Cell Lymphoblastic Leukemia (pre-B-ALL) Xenograft Models Compared To Rituximab (RTX): Potential Targeted Therapy In Patients With High Risk Pre-B-ALL

BackgroundObinutuzumab (GA101) is a type II, glycoengineered, humanized anti-CD20 monoclonal antibody (Ab). Obinutuzumab binds with high affinity and selectivity to a type II binding region of the CD20 protein present on all B cells except stem or plasma cells. Unlike type I anti-CD20 monoclonal Abs, type II anti-CD20 Abs promote direct cell death induction without the need for a cross-linking Ab. Patients who relapse with CD20+ B-NHL and early relapsed pre-B-ALL have a dismal prognosis, often associated with chemotherapy resistance (Cairo et al. JCO, 2012, Barth/Cairo et al. BJH, 2013) and require alternative therapeutic strategies. RTX in combination with FAB/LMB 96 chemotherapy is a safe, well-tolerated treatment that is associated with > 95% EFS in children with newly diagnosed and advanced mature B-Cell NHL (Goldman/Cairo et al. Leukemia, 2013). Resistance to RTX, however, may predispose patients with CD20+ NHL/Pre-B-ALL to an increased risk of relapse and or disease progression (Barth/Cairo et al. BJH, 2013). ObjectiveTo evaluate the anti-tumor activity of obinutuzumab vs RTX against pre-B-LL in-vivo in severe combined immune-deficient IL2 receptor gamma chain knockout (NSG) and NOD/SCID mice. MethodsU698-M (Pre-B cell ALL, CD20+, DSMZ, Germany), Loucy cells (T-ALL, CD20- ATCC, Manasass, VA) were maintained in RPMI with 10% FBS at 370C, 5% CO2. The mammalian expression construct ffLUCZeo-pcDNA (kindly provided by L.Cooper, MD,PhD) was transfected into U698M and Loucy (Luc+) tumor targets for later evaluation by bioluminescent imaging (BLI). Six to 8 week old female NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ, no functional T, B and NK cells) and NOD/SCID (absence of functional T and B cells) mice were bred in-house and treated in pathogen free conditions at the animal facility. The in vivo cytotoxic activity of obinutuzumab and RTX was determined by dividing NSG and NOD/SCID mice into 5 groups: PBS only (control), isotype control (IgG), obinutuzumab (10 mg/kg) (kindly provided by C. Klein, Roche Glycart AG), obinutuzumab (30 mg/kg), and rituximab (30 mg/kg). Mice received intravenous injections of Luc+ U698M and Loucy cells at 5x106 tumor cells/mouse. 6-8 days after tumor cell injection, mice were injected every 7 days with the respective therapy for 8 weeks. Mice were closely monitored for tumor burden progression/regression and survival for up to 12 weeks (approx. 80 days) via BLI using the IVIS Spectrum System. ResultsWe observed that obinutuzumab was significantly more effective than RTX when administered at the same doses in established pre-B-ALL xenografts. In the NSG mouse model, at 30 days tumor volume in mice receiving 30 mg/kg of obinutuzumab was significantly decreased when compared to mice receiving 10 mg/kg obinutuzumab or 30 mg/kg of RTX (p<0.0001, p<0.002 respectively). At 11 weeks, mice receiving 30 mg/kg of obinutuzumab showed no increase in tumor volume compared to week 4. The overall survival following obinutuzumab at 30 mg/kg was 80 days compared to 48 days by obinutuzumab at 10mg/kg (p=0.001) and 30 days following RTX treatment at 30mg/kg)(p=0.002) (Figure-1A). Relative tumor luminescence activity on day 8 was significantly decreased in mice receiving 30 mg/kg of obinutuzumab (8x105 RLU) compared to 10 mg/kg of obinutuzumab (7.7x105 RLU) or 30 mg/kg of RTX (9.9x105 RLU) compared to IgG isotype (2.0x106 RLU, p=0.02,p=0.07,p=0.1, respectively).Similarly, in the NOD/SCID mouse model, at day 80, we observed a significant decrease in tumor luminescence when mice were treated with 30 mg/kg obinutuzumab compared to 10 mg/kg obinutuzumab or 30 mg/kg rituximab (p=0.001). Furthermore, at day 80 there was 100% survival in animals with 30 mg/kg obinutuzumab compared to 60% following rituximab at 30 mg/kg ( p=0.001) or 40% obinutuzumab (10mg/kg; p=0.001) (Figure1B). [Display omitted] ConclusionThese preliminary studies in two groups of pre-B-ALL xenograft mice (NSG and NOD/SCID), demonstrated that the antitumor effect of obinutuzumab at 30 mg/kg significantly increases survival and decreases the tumor burden in Pre-B-ALL xenografts compared to equal dose of RTX. Further studies are underway to determine the mechanism(s) responsible for the significantly increased efficacy of obinutuzumab vs. RTX in pre-B-ALL xenografts. Disclosures:Cairo:Roche/Genentech: advisory board Other.

Chromothripsis-Mediated Structural Variations and Clonal Evolution In Recurrent Childhood High Hyperdiploid Acute Lymphoblastic Leukemia

High hyperdiploid acute lymphoblastic leukemia (HeH-ALL) is characterized by 51-67 chromosomes and nonrandom gains of specific chromosomes (X, 4, 6, 10, 14, 17, 18, and 21). It presents the most frequent numerical cytogenetic alteration in childhood pre B-cell ALL occurring in 25-30% of cases. Recurrent disease will affect 15-20%. Pre-leukemic HeH clones are generated in utero, but cooperating oncogenic lesions are necessary for overt leukemia and remain to be determined. Recently, a phenomenon termed chromothripsis has been described in which massive structural variations occur in a single aberrant mitosis. Whole or partial chromosomes are shattered and some fragments are lost in the process of rejoining. Thus, characteristically, chromosomal copy numbers oscillate between two copy number states. Chromothripsis has been suggested to be a tumor-driving alteration that may be present in 2-3% of all human cancers. Its role as a potential cooperating or initiating lesion in HeH-ALL has not been determined.We applied state-of-the-art whole-genome next-generation-sequencing to analyze structural variations in six pediatric patients with recurrent HeH-ALL. Matched sample sets taken at diagnosis, remission and/or relapse were compared. Paired end sequencing was carried out on a Genome Analyzer IIx or a HiSeq 2000 (Illumina), respectively. Reads were aligned against the human reference genome (GRCh37) using BWA. Translocations were detected by GASV. Copy number variations were analyzed by FREEC. Structural variations were validated by PCR/Sanger sequencing and FISH.Of the six patients analyzed, five harbored on average one interchromosomal translocation or intrachromosomal inversion, but one patient presented with massive genomic rearrangements (Figure). These affected chromosome 3, 11, 12 and 20. Ten copy number shifts on chromosome 3 oscillating between two copy number states (2 and 3) indicated that these rearrangements were caused by chromothripsis. Breakpoint sequencing revealed that one of the identified translocations (t(12;20)(p13.1;p12.3)), was indeed a three-loci-rearrangement composed of small fragments derived from chromosomes 3, 12 and 20. Characteristically for chromothripsis, the breakpoints clustered closely. Three breakpoints separated by 224 bp and 64 kb were located in the transducin (beta)-like X-linked receptor 1 (TBL1XR1) gene. Other genes repeatedly targeted included the MACRO domain-containing protein 2 (MACROD2) gene (a deacetylase involved in deacetylation of lysine residues in histones and other proteins), the KIAA1467 gene (a transmembrane protein of the integrin alpha FG-GAP repeat containing 3 (ITFG3) family), and a novel regulatory lincRNA (ENSG00000243276). MACROD2 was previously observed as a target of chromothripsis in a colorectal carcinoma. Thus, the characterized breakpoints may identify fragile genomic sites prone to chromothriptic rearrangement. DNA repair was effectuated by non-homologous-end-joining as typical addition of non-template nucleotides with microhomologies of two to four nucleotides at the breakpoints demonstrated. Copy number profiles of this patient showed that at least two distinct leukemic clones could be identified at diagnosis. One had acquired chromothriptic alterations and presented the dominant clone at relapse indicating chemotherapy resistance and tumor-driving potential. Prior whole-exome sequencing did not reveal mutations in known oncogenes or tumor suppressor genes. Therefore, loss of function or expression of genes affected by chromosomal rearrangements, such as TBL1XR1 that is recurrently mutated in childhood ALL with ETV6-RUNX1 translocation, may account for the tumor-driving effect. All leukemic cells at diagnosis showed conformity concerning number and pattern of whole chromosome gains demonstrating that chromothripsis was not an initiating oncogenic event, but occurred secondary to high hyperdiploidy. Further aberrations (t(4;7), loss of 4q) were gained by the chromothriptic clone and could be detected by FISH in minor subclones pointing at ongoing clonal evolution. Taken together, our study reveals chromothripsis as a novel assisting and tumor-driving lesion in HeH ALL.Chromothripsis in HeH-ALL. Copy number variations and translocations at diagnosis (left) and relapse (right). (magenta: chromothriptic translocations; green: other translocations) [Display omitted] Disclosures:No relevant conflicts of interest to declare.

A recurrent germline PAX5 mutation confers susceptibility to pre-B cell acute lymphoblastic leukemia

Somatic alterations of the lymphoid transcription factor gene PAX5 (also known as BSAP) are a hallmark of B cell precursor acute lymphoblastic leukemia (B-ALL), but inherited mutations of PAX5 have not previously been described. Here we report a new heterozygous germline variant, c.547G>A (p.Gly183Ser), affecting the octapeptide domain of PAX5 that was found to segregate with disease in two unrelated kindreds with autosomal dominant B-ALL. Leukemic cells from all affected individuals in both families exhibited 9p deletion, with loss of heterozygosity and retention of the mutant PAX5 allele at 9p13. Two additional sporadic ALL cases with 9p loss harbored somatic PAX5 substitutions affecting Gly183. Functional and gene expression analysis of the PAX5 mutation demonstrated that it had significantly reduced transcriptional activity. These data extend the role of PAX5 alterations in the pathogenesis of pre-B cell ALL and implicate PAX5 in a new syndrome of susceptibility to pre-B cell neoplasia.

A Bach2 Link between Pre-B Cell Receptor Checkpoint and Pre-B Cell ALL

Bach2 is a transcription factor required for affinity maturation of B cells. A recent study reveals, quite unexpectedly, that Bach2 also plays a key role in the pre-B cell receptor checkpoint and functions as a tumor suppressor in pre-B cell acute lymphocytic leukemia.

Mer receptor tyrosine kinase is a therapeutic target in pre–B-cell acute lymphoblastic leukemia

AbstractAcute lymphoblastic leukemia (ALL) is currently treated with an intense regimen of chemotherapy yielding cure rates near 85%. However, alterations to treatment strategies using available drugs are unlikely to provide significant improvement in survival or decrease therapy-associated toxicities. Here, we report ectopic expression of the Mer receptor tyrosine kinase in pre–B-cell ALL (B-ALL) cell lines and pediatric patient samples. Inhibition of Mer in B-ALL cell lines decreased activation of AKT and MAPKs and led to transcriptional changes, including decreased expression of antiapoptotic PRKCB gene and increase in proapoptotic BAX and BBC3 genes. Further, Mer inhibition promoted chemosensitization, decreased colony-forming potential in clonogenic assays, and delayed disease onset in a mouse xenograft model of leukemia. Our results identify Mer as a potential therapeutic target in B-ALL and suggest that inhibitors of Mer may potentiate lymphoblast killing when used in combination with chemotherapy. This strategy could reduce minimal residual disease and/or allow for chemotherapy dose reduction, thereby leading to improved event-free survival and reduced therapy-associated toxicity for patients with B-ALL. Additionally, Mer is aberrantly expressed in numerous other malignancies suggesting that this approach may have broad applications.

A Rare Finding in Acute Lymphocytic Leukemia: Philadelphia Chromosome

Patient: 48-year-old Caucasian male Chief Complaint: Gastrointestinal bleeding Medical History: Transient ischemic attack, knee surgery, daily tobacco use History of Present Illness: Patient diagnosed with pre B-cell ALL with Philadelphia chromosome. Patient underwent chemotherapy and entered remission. Patient came out of remission and did not undergo chemotherapy. Present Illness is pre-B-cell ALL with Philadelphia chromosome. Physical Exam Findings: Petechiae on lower extremities Principal Lab Findings: [Table 1][1] * Ph : Philadelphia ALL : acute lymphoblastic leukemia AML : acute myelogenous leukemia FISH : fluorescent in situ hybridization MPO : myeloperoxidase NSE : non-specific esterase TdT : terminal deoxynucleotidyl transferase PAS : periodic acid schiff CD : cluster of differentiation [1]: #T1

Abstract IA16: Engineered T cell therapies for cancer.

Abstract Chimeric antigen receptors (CARs) combine an antigen recognition domain of a specific antibody with a intracellular signaling domains into a single engineered protein. CD19 is an ideal target for CARs since expression is restricted to normal and malignant B cells. In preclinical studies, inclusion of the CD137 (4-1BB) signaling domain results in remarkable antitumor activity and in vivo persistence of anti-CD19 CARs. We have previously reported dramatic in-vivo expansion, persistence and anti-tumor activity of CAR modified autologous T cells targeted to CD19 (CART19 cells) in 3 patients with CLL with relatively short follow up (Porter, et al NEJM 2011; Kalos et al Sci Trans Med 2011). Currently, 11 patients have been treated with CART19 cells. There were 9 adults (median age 65 years (range 51-78)) treated for relapsed, refractory and high risk CLL and two pediatric cases treated for relapsed refractory pre-B cell ALL. CLL patients had received a median of 5 prior regimens excluding single agent rituxumab (range 2-10) and all had active disease at the time of cell infusion. The pediatric patients with ALL had chemotherapy refractory relapse, and one was resistant to allogeneic transplantation and CD19 directed blinotumomab. A median of 7.5 x 10^8 total cells (range 1.7-50) corresponding to 1.45 (range 0.14-5.9 x 108) genetically modified cells were infused. In the patients in complete remission, CAR modified CART19 cells are detectable in the peripheral blood by flow cytometry at the most recent follow up for at least 2 years. All patients in CR have B cell aplasia. No patient with CR has progressed. Citation Format: Carl H. June. Engineered T cell therapies for cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr IA16.

The Novel CXCR4 Antagonist POL5551 Decreases Surface CXCR4 (s-CXCR4) Expression, Inhibits Chemotaxis, and Enhances Chemosensitivity in Acute Lymphoblastic Leukemia (ALL)

Abstract 780 Background:The interaction between the cell surface receptor CXCR4 and the chemokine SDF-1 (CXCL12) is critical in signaling between leukemic blasts and the bone marrow microenvironment. We previously demonstrated that CXCR4 is an important mediator of chemotherapy resistance, as chemotherapy-induced upregulation of s-CXCR4 in acute myeloid leukemia (AML) cell lines and primary samples led to increased SDF-1-mediated chemotaxis and increased protection by normal human bone marrow stroma from chemotherapy-induced apoptosis. We also showed that stromal protection and chemotherapy resistance could be reversed by treatment with the FDA-approved CXCR4 inhibitor plerixafor, both in vitro in stromal co-cultures of pre-B cell ALL cell lines and in vivo in xenografts of primary samples of infant MLL-rearranged ALL. Therefore, disruption of the CXCR4/SDF-1 axis is a rational means to target extrinsic survival mechanisms in acute leukemia.The novel Protein Epitope Mimetic (PEM) POL5551 is a selective and potent antagonist of CXCR4. Treatment with POL5551 inhibits vascular accumulation of CXCR4+ smooth muscle cells but its effects on ALL have not been reported. We hypothesized that treatment of ALL cell lines with POL5551 would 1) decrease s-CXCR4 expression, 2) inhibit SDF-1-mediated chemotaxis, and 3) reverse stromal-mediated protection from chemotherapy-induced apoptosis. Methods/Results:Pre-B cell ALL (697, HB11;19, NALM-6, SEMK2) and T cell ALL cell lines (CCRF-CEM-1301, Jurkat, Molt-4) were treated with dose ranges of POL5551. Cells were harvested at multiple time points over 72 hours and s-CXCR4 was measured by FACS. S-CXCR4 was potently and markedly reduced in all cell lines, with IC50 levels of <5 nM at 1 hour and IC50 levels of <20 nM at 48 hours. In comparison, 3- to 30-fold higher doses of plerixafor were needed to achieve similar levels of reduction. Simultaneous measurement of cell proliferation using the WST-1 proliferation assay demonstrated that treatment with POL5551 neither increased nor decreased leukemia cell proliferation in a significant manner.To ascertain the functionality of s-CXCR4 inhibition, we performed chemotaxis assays. Leukemia cells were treated with 10 nM POL5551 or vehicle control and placed into hanging cell culture inserts. Migration through a permeable membrane toward an SDF-1 gradient was then measured after 24 hours. Compared to control-treated cells, POL5551-treated cells had significantly decreased SDF-1-induced chemotaxis (average 38% reduction in chemotaxis in pre-B cell lines, p<0.001; average 41% reduction in T cell lines, p=0.05).We also performed co-culture experiments with normal human bone marrow stroma in the presence and absence of POL5551 to further demonstrate the functional effects of s-CXCR4 inhibition. Specifically, we cultured leukemia cells off stroma (O), on stroma (S), or pretreated with POL5551 for 30 minutes prior to plating on stroma (P+S). Cells from each culture condition were then treated with dose ranges of chemotherapy. Following treatment, we measured apoptosis by staining with Annexin V/7-AAD. IC10 through IC90 values were obtained using Calcusyn. To quantify stromal protection, we calculated a Protective Index (PI), defined as the S IC values divided by the O IC values. Thus, PI >1 signified stromal protection, while PI ≤1 signified no stromal protection. To quantify the ability of POL5551 to reverse stromal protection, we calculated a Reversal Index (RI), defined as the P+S IC values divided by the O IC values. Therefore, PI > RI indicated a decrease in stromal protection, while RI ≤1 indicated a reversal of stromal protection. Overall, stroma protected leukemia cells from chemotherapy-induced apoptosis. Importantly, treatment with POL5551 abrogated stromal-mediated protection and restored chemosensitivity (eg, PI 1.182 vs. RI 0.956 for NALM-6 treated with daunorubicin +/− 20 nM POL5551, p<1×10e-9). Conclusions:The novel CXCR4 antagonist POL5551 is a potent inhibitor of CXCR4 in pre-B and T ALL cell lines with activity at nanomolar concentrations in decreasing s-CXCR4 expression, inhibiting SDF-1-induced chemotaxis, and reversing stromal-mediated protection from chemotherapy in vitro. Therefore, if our findings are confirmed in primary samples and in vivo, interruption of leukemia-microenvironment signaling with POL5551 may prove to be an effective strategy in the treatment of pre-B and T cell ALL. Disclosures:Chevalier:Polyphor Ltd: Employment. Dembowsky:Polyphor Ltd: Employment.

Chemical Biology Strategy Reveals Cell Lineage- and Cell Differentiation-Specific Small Molecule Inhibitors of NF-κB.

AbstractAbstract 2421Members of the nuclear factor-kB (NF-κB) family of transcription factors play important roles in cell-signaling involved in host-defense, immune responses, inflammation, and cancer. Multiple stimuli operating through different receptors are capable of activating NF-κB, including the Tumor Necrosis Factor (TNF) family cytokines, Toll-like receptors (TLRs), Nucleotide-binding Leucine Rich Repeat Proteins (NLRs), DNA-damaging agents, and others. Drugs that selectively block activation of NF-κB through one or more of these receptors could have activity against various types of cancer in which activation of NF-κB plays a role. We used a chemical biology strategy to identify novel chemical inhibitors of cell pathways responsible for NF-κB activation. Screening of a large chemical library (>330K compounds from NIH) was conducted using human 697 pre-B-cell leukemia cells with a stably integrated NF-κB-luciferase reporter gene and stimulating the Carma/Bcl-10/MALT pathway using protein kinase C (PKC) activators (PMA/Ionomycin). Confirmed hits were counter-screened against HEK293T-NF-κB-luciferase cells treated with TNF-a, with the goal to eliminate non-pathway specific inhibitors of NF-κB. Among the hit compounds, we identified oxadiazole- and oxazole-based chemical probes as potential cell lineage- and cell differentiation-specific inhibitors of NF-κB (“NLDSi” = NF-κB lineage, differentiation-specific inhibitors). Using a panel of cell lines with stably integrated NF-κB-driven luciferase reporter genes, NLDSi compounds suppressed PMA/Ionomycin-induced NF-κB activity in a concentration-dependent manner (IC50=0.1–1.0 μM) in the pre-B-cell leukemia cell-line 697, but not in the T-cell leukemia line JURKAT, the myeloma cell-line RPMI8226, or HEK293 epithelial cells. These compounds also suppressed NF-κB activity (measured by reporter gene) in 697 cells stimulated with BAFF or treated with doxorubicin. Examining NF-κB target gene expression by q-RT-PCR (mRNA level) and immunoblotting (protein level) showed that NLDSi compounds reduced PMA/Ionomycin-induced TRAF1 and A20 expression in 697 cells and in the pre-B-cell leukemia cell line REH, but not in several mature B-cell lines, including BJAB, Daudi, OCILy3, and OCILy10. NLDSi compounds also inhibited NF-κB target gene expression in pre-B-cell lines stimulated with BAFF or treated with lipopolysaccharide (LPS) or doxorubicin (Dox). Nevertheless we found these compounds could suppress of PMA/Ionomycin-induced expression of TRAF1 and A20 gene in the primary leukemia cells of patients with chronic lymphocytic leukemia (CLL). Taken together, the compounds described here appear to selectively suppress NF-κB activation in the B-cell lineage at specific stages of differentiation. As such, these small molecules could serve as chemical probes for uncovering novel targets that regulate NF-κB activity and potentially lead to development of therapies that selectively block certain types of NF-κB activation in pre-B cell ALL, CLL, or other hematologic malignancies. (Supported by NIH grants X01 MH077633-1, U54–005033, and P01-CA-81534). Disclosures:No relevant conflicts of interest to declare.

Normal Karyotype CALLA-Positive Adult Pre-B ALL: Dismal Outcome With Chemotherapy for Patients With Loss/Gain of ABL1 and/or BCR FISH Signals

Diagnostic karyotype and molecular studies represent the most powerful prognostic indicators in acute myeloid leukemia and provide the framework for risk stratification. Risk stratification in ALL has also a vital role in predicting outcome and identifying patients at higher risk of relapse with multiagent chemotherapy, but the role of diagnostic karyotype and molecular markers in adult ALL is limited to few well recognized cytogenetic abnormalities. We report a case series of 6 adult ALL patients with a characteristic molecular abnormality that have done poorly with chemotherapy. Between April 2004 and November 2009, 72 adult ALL patients (Pre-B-cell 61; T-cell 11) were referred to and treated at the Leukemia/BMT Program of BC in Vancouver, Canada. FISH for BCR-ABL fusion was positive in 12 of 61 Pre-B cell ALL patients. An additional 6 patients were negative for this typical fusion but had FISH abnormalities related to BCR and/or ABL1. In this report, we describe the clinical and hematopathologic characteristics of these 6 patients and their poor outcome. We review the literature where only 2 similar cases with normal karyotype Pre-B ALL and associated FISH BCR/ABL1 numerical abnormalities were found. We recommend screening all adult pre-B ALL patients with normal karyotype for this clonal abnormality and suggest classifying these ALL patients into the high-risk category.