Cell Precursor ALL Research Articles

Regulation of Homeostatic and Malignant B Cell Development By the Tetraspanin CD53

Acute lymphoblastic leukemia (ALL) is the most frequent pediatric malignancy, most commonly originating from the transformation of progenitor cells of the B cell lineage (B cell precursor-ALL; BCP-ALL). Treatment of patients with high-risk or relapsed disease is difficult and prognosis remains poor in pediatric patients, with an even worse survival rate for adult BCP-ALL. Previous studies have shown an association of enhanced CD53 expression with many B cell malignancies, suggesting upregulation of CD53 may be implicated in carcinogenesis or maintenance of malignant cells. CD53 is a member of the tetraspanin family of transmembrane proteins, classically involved in cell adhesion, proliferation, and survival, and expressed exclusively on hematopoietic cells. While several studies have implicated a role for CD53 in regulating mature B cell proliferation, its role in early B cell development is not yet known. To elucidate the contribution of CD53 to normal and malignant B cell development, we have generated a CD53 knockout mouse.In our CD53-/- mouse, we observe no differences in total white blood cell counts, yet the fraction of peripheral blood B cells is significantly reduced by 31% compared to wild-type (WT) controls (28.3% vs. 19.5%; p<0.005). During homeostatic B lymphopoiesis, CD53 increases through development, beginning at the pre-pro-B cell stage and reaching highest expression on mature B cells. Further investigation into the loss of B cells revealed that immature pre-B cells in the bone marrow and mature B cells in the spleen and lymph nodes are significantly diminished upon loss of CD53, resulting from increased apoptosis in CD53-/- mice. B cell differentiation of CD53-/- hematopoietic stem cells (HSCs) in vitro corroborates the dependence on CD53 for normal differentiation, as CD53-/- cultures have 26% fewer B cells than controls (p=0.033). Investigation into the signaling differences between WT and CD53-/- B cell progenitors by mass cytometry (CyTOF) suggests that decreased PI3K/Akt and MAPK signaling could be driving this loss.With the observed loss of both B cell progenitors and mature B cells in CD53-deficient mice, CD53-/- mice were recently crossed to Eμ-Myc transgenic mice, a model of B-lineage leukemia/lymphoma, to generate WT, CD53-/-, Eμ-Myc+;CD53+/+, and Eμ-Myc+;CD53-/- groups to assess whether loss of CD53 alters the pathology or survival of these mice. As observed in human patients, moribund Eμ-Myc+ mice significantly upregulate CD53 on malignant cells, suggesting a potential role for CD53 during pathogenesis. Ongoing experiments are aimed at elucidating the mechanism by which CD53 promotes homeostatic B cell development and determining the potential of CD53 as a therapeutic target for B lineage malignancies. DisclosuresNo relevant conflicts of interest to declare.

Engineered Bcor Mutations Lead to Acute Lymphoblastic Leukemia of Progenitor B-1 Lymphocyte Origin in a Sensitized Background

Chromosomal translocations resulting in NUP98 fusion genes have been associated with a wide spectrum of hematologic malignancies, including MDS, AML, T-ALL, and B cell precursor (BCP) ALL. Based on gene expression profiles and murine transplantation experiments, it is thought that NUP98 fusions can confer aberrant self-renewal potential to hematopoietic cells. Approximately 90% of mice that express a NUP98-PHF23 (NP23) fusion in the hematopoietic compartment, under the control of Vav1 regulatory elements develop AML and/or T-ALL. However, approximately 10% of NP23 mice develop an aggressive acute lymphoblastic leukemia of B1-lymphocyte progenitor origin (pro B-1 ALL). Whole exome sequencing demonstrated that all NP23 pro-B1-ALL had acquired somatic frameshift mutations of the BCL6 co-repressor (Bcor) gene, and most had acquired mutations in the Jak/Stat pathway.To determine whether experimentally engineered Bcor mutations would lead to pro B-1 ALL, we used CRISPR-Cas9 to introduce Bcor indel mutations into NP23 hematopoietic stem and progenitor cells through the use of Bcor single guide RNAs (Bcor sgRNA). Recipient mice transplanted with NP23 bone marrow (BM) or fetal liver (FL) cells that had been transduced with a Bcor sgRNA developed pro B-1 ALL, characterized by a B-1 progenitor immunophenotype, clonal Igh gene rearrangement, and Bcor indel mutation, whereas control recipients did not. In addition, similar to some human BCP ALL, the Bcor sgRNA/NP23 murine pro B-1 ALL had acquired somatic mutations in Jak kinase genes.A distinct subset of pediatric BCP ALL are characterized by rearrangement and overexpression of the CRLF2 gene (designated CRLF2r); the CRLF2 gene is the receptor for thymic stromal lymphopoietin (TSLP), a cytokine that plays a role in normal progenitor B1 cell development. The NP23 pro-B1 ALL are similar to CRLF2r BCP ALL in terms of a preferential V heavy chain (VH) usage, gene expression profile, and propensity for acquired JAK/STAT pathways mutations. JAK inhibitors (ruxolitinib and tofacitinib) induced apoptosis and inhibited the growth of pro B-1 ALL cell lines established from Bcor sgRNA/NP23 recipients, at clinically achievable concentrations (10-100 nM). Taken together, these findings demonstrate that a CRISPR-induced Bcor frameshift collaborates with an NP23 transgene to predispose B-1 progenitors to leukemic transformation. These two events are unlikely to be sufficient for leukemic transformation, as we detected spontaneous Jak pathway mutations that were required for continued growth of the leukemic cells. This constellation of mutations (NP23 expression leading to increased stem cell self-renewal, Bcor frameshift leading to impaired B cell differentiation, and Jak pathway mutations leading to dysregulated proliferation) is similar to that seen in human BCP ALL patients, and suggests that the NP23/Bcor mutant mice and cell lines will be a useful model for human pro-B1 ALL. DisclosuresAplan:NIH Office of Technolgy Transfer: Employment, Patents & Royalties: NUP98-HOXD13 mice.

Acute Lymphocytic Leukemia in the Latin American Population

Introduction: An interesting epidemiological aspect of ALL is the variation between ethnic groups. Among all subtypes of leukemia Latinos have lower incidence rates. However, in remarkable contrast, ALL has a higher incidence rate in the Latino population and also carries a more dismal prognosis (Barrington-Trimis J, et al Blood 2015).The majority of reported data among Latinos either exclusively looks at B cell precursor (BCP) ALL or does not distinguish between subtypes. In this study we used the National Cancer Institute Surveillance, Epidemiology, and End Results (SEER) database to delineate the differences in incidence rates of B-ALL and T-ALL across ethnic groups in the United States.Epidemiologically, highest rates of ALL worldwide have been reported in both adult and pediatric Latino patients. However, determining who is to be considered Latino is not always straightforward. Many studies utilize self reported ethnicity and databases such as the Surveillance, Epidemiology, and End Results (SEER) Program of the National Cancer Institute (NCI) employ complex algorithms to determine ethnicity. Of note, the North American Association of Central Cancer Registries (NAACCR) Hispanic Identification Algorithm (NHIA) used by SEER includes European Spaniards but excludes South American Brazilians. This is a point of special interest due to the fact that although the overall incidence of ALL in the Brazilian population is low, the regional distribution is strikingly heterogeneous, with higher rates in regions with higher indigenous ancestry.Methods: Data from the SEER-18 database was used compare incidence rates of T-ALL and B-ALL. Patients in which ethnicity was reported were included. Due to the utilization of cytogenetics and subsequent changes in ICD coding over the years we chose to look at the most recent data reported in 2014. We compared rates in non-Hispanic Whites, Latinos (Hispanic), Blacks and Asian-Pacific Islanders (API). Age-adjusted incidence rates per 100,000 person-years were calculated.Results: AAIR of B-ALL in the Hispanic population is consistently higher than that of Non-Hispanic Whites throughout the years, ranging from 1.0-2.4 per 100,000. Blacks had the lowest incidence overall, with rates approximately one third of those found in Latinos.The distribution of ALL throughout the Americas can perhaps shed some epidemiological light on the genetic origins of ALL in Latinos. The ancestry across Latin America is mixed but historical idiosyncrasies can perhaps explain the distribution of ALL in the Americas. We reviewed the available literature and found that throughout the Americas, higher incidence rates are seen in areas of higher Native American ancestry (Fig 1).Discussion:Our finding shows that the increase of ALL among Latinos is accounted for by the high rate of B cell origin. The increased incidence of ALL in the Latino population has been linked to polymorphisms in genes such as the transcription factor GATA3 (Perez-Andreu V, Nat Genet 2013). The underlying mechanism of disease has yet to be elucidated and is likely related to BCP- ALL and, more specifically, Ph-Like ALL. This variation appears to be related to Native American ancestry. Understanding the molecular mechanism and epidemiological origins of this subset of patients with aggressive features will lead to improved treatment and overall survival in this minority healthcare disparity. DisclosuresDouer:Shire: Membership on an entity's Board of Directors or advisory committees; Gilled Sciences, Inc: Consultancy; Spectrum: Consultancy; Jazz Pharmaceuticals: Consultancy; Amgen: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria.

Combining Biology and Chemistry for a New Take on Chemotherapy: Antibody-Drug Conjugates in Hematologic Malignancies.

This review is about the antibody-drug conjugate (ADC), a form of drug delivery consisting of a monoclonal antibody, linker, and cytotoxic payload. We summarize the history of ADC development, highlighting the three FDA-approved ADCs currently available. Gemtuzumab ozogamicin is a CD33-targeted ADC linked to calicheamicin. It is approved for CD33+ AML in the first line or the relapsed or refractory (R/R) setting. Brentuximab vedotin is a CD30-targeted ADC bound to MMAE. It is approved for the treatment of certain R/R CD30+ lymphomas. Recently, it has been approved for first line therapy with chemotherapy in advanced HL. Inotuzumab ozogamicin is a CD22-directed ADC attached to calicheamicin indicated for the treatment of adults with R/R B cell precursor ALL. Three ADCs have been approved for the treatment of various hematologic malignancies. We discuss the pertinent human trials that led to FDA approval. We include our perspectives about drug resistance, toxicities, and future development.

Recent Advances in the Biology and Treatment of T Cell Acute Lymphoblastic Leukemia.

This article provides an overview of the current knowledge regarding the biology and treatment of T cell acute lymphoblastic leukemia (T-ALL) and highlights the most recent findings in this field over the past 5years. Remarkable progress has been made in the genomic landscape of T-ALL over the past few years. The discovery of activating mutations of NOTCH1 and FBXW7 in a majority of patients has been a seminal observation, with several early phase clinical trials currently exploring these as potential therapeutic targets. Characterization of early T cell precursor ALL, incorporation of minimal residual disease assessment into therapeutic protocols, and use of pediatric-intensive regimens along with judicious use of allogeneic HCT have significantly improved risk stratification and treatment outcomes. Improved risk stratification and the use of novel targeted therapies based on recent genomic discoveries are expected to change the therapeutic landscape of T-ALL and hopefully improve the outcomes of this historically poor prognosis disease.

CD20 expression sub-stratifies standard-risk patients with B cell precursor acute lymphoblastic leukemia.

Patients with standard-risk adult acute lymphoblastic leukemia (ALL) treated with chemotherapy do not have satisfactory outcomes. To more precisely classify ALL patients and optimize treatment, we re-evaluated the risk stratification system by examining CD20 expression and other classic risk factors at diagnosis. We retrospectively analyzed response to induction chemotherapy of 217 consecutive patients with newly diagnosed Philadelphia-negative B cell precursor-ALL. Survival analyses were conducted for the 136 patients who were intended to be treated with chemotherapy alone. Among the 217 patients, 69 (31.8%) were considered standard risk based on age <35 years, white blood cell count <30 × 109/L, absence of central nervous system involvement, and high-risk cytogenetic abnormalities. Seventy-four patients (34.1%) expressed CD20 on ≥20% of leukemia blasts and were considered CD20 positive. We found that fewer CD20-positive than CD20-negative patients achieved durable first complete responses (CR1 ≥3 months) (81.1% vs. 94.9%, P=0.002). Within the standard-risk group, more CD20-negative than CD20-positive patients achieved CR (100% vs. 83.3%, P=0.003) and durable CR1 (100% vs. 82.4%, P=0.014). For patients in the CD20-negative standard-risk, CD20-positive standard-risk, CD20-negative high-risk, and CD20-positive high-risk groups, the 3-year cumulative incidence of relapse was 42.6%, 70.0%, 59.3%, and 69.5%, respectively (P=0.118); the 3-year disease-free survival rates were 52.1%, 0%, 20.7%, and 13.7%, respectively (P=0.006); and the 3-year overall survival rates were 55.8%, 13.8%, 23.6%, and 16.9%, respectively (P=0.006). Our results suggest that patients with CD20-negative standard-risk B cell precursor-ALL have favorable prognosis compared with CD20-positive standard-risk or CD20-negative or -positive high-risk patients. CD20-positive standard-risk ALL patients may need other therapeutic modalities bridging to allogeneic hematopoietic stem cell transplantation.

Philadelphia-Like Acute Lymphoblastic Leukemia in Adults.

This study aims to provide an overview of the classification, incidence, genomic alterations, and clinical implications of Philadelphia-like Acute Lymphoblastic Leukemia (Ph-like ALL) in adults. Ph-like ALL is a high-risk subtype of B cell precursor ALL with characteristic genomic alterations in children and adults. A standard approach for diagnosis is missing and currently mainly based on gene expression analysis. The incidence is age depended and highest in adolescents and younger adults (age 16-39) where 19-28% of patients belong to this subtype. Ph-like ALL is associated with persistence of minimal residual disease (MRD) and inferior prognosis. Some genomic alterations respond to specific treatment approaches and provide hope for tailored therapies. Ph-like ALL in adults is an aggressive and high-risk subtype of B cell precursor ALL. Without consensus definition, diagnosis is difficult and current publications highlight the importance of stringent MRD monitoring to guide risk-adapted treatment strategies.

Oncogenic Feedback Activation Between BCL6 and MLL Promotes Malignant Transformation in MLL-RearrangedAcute Lymphoblastic Leukemia

Background:BCL6 is known as a protooncogene and transcriptional repressor in diffuse large B cell lymphoma, where it is frequently involved in chromosomal rearrangements. We recently identified BCL6 as a novel mediator of drug resistance to tyrosine kinase inhibitors (TKI) in Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) and chronic myeloid leukemia (Duy et al., Nature 2011; Hurtz et al., J Exp Med 2011). In addition,BCL6 directly competes with the tumor suppressor BACH2 for p53 promotor binding to protecting cells from p53-mediated apoptosis in multiple ALL subgroups (Swaminathan et al., Nature Medicine 2013). Based on this, our current study is focusing on the function of BCL6 in different subtypes of human ALL.Results: Analysis of gene expression data from 207 children with high-risk B cell precursor ALL (COG P9906) showed that high expression levels of BCL6 at the time of diagnosis correlated with a poor overall and relapse-free survival (p=0.007). Furthermore, 49 matched sample pairs from patients at diagnosis and relapse showed increased BCL6 levels at relapse compared to diagnosis (p=0.003). To test whether or not there are specific subtypes of leukemia with high BCL6 expression levels, we studied BCL6 expression via western blot and immunohistochemistry staining in Non-Ph+ cell lines and ALL patient samples (n=76). Interestingly, BCL6 levels are particularly elevated in MLL-rearranged (MLLr) ALL cases. In addition, patients from the clinical trial that had high BCL6 levels and had MLL rearrangements had the worst clinical outcome (p=0.0009). We next tested if MLLr oncogenes drive aberrant BCL6 expression. First, we performed a ChIP-analysis using antibodies against MLL, AF4, and ENL, which provided evidence for direct binding to the BCL6 promoter. We then performed a BCL6 Western blot analysis of inducible MLL-AF4-transgenic and retrovirally transduced MLL-ENL pre-B cells, demonstrating that both oncogenes are sufficient to induce ~10-fold upregulation of BCL6 protein levels. Additionally, we used a newly developed conditional BCL6 knock out/reporter mouse model to decipher the function of BCL6. We transduced B cell progenitor cells from BCL6fl/fl mice with MLL-ENL and either with a control or Cre-expression vector. Using the BCL6 reporter capability of the mouse we found that BCL6 is significantly higher expressed in MLL-ENL transduced cells. To test if MLL is required for BCL6 upregulation, we used a conditional MLL knock out mouse and found that after Cre-mediated deletion of MLL, pro-B, mature-B, and MLL-AF4 transduced ALL cells almost lost the ability to upregulate BCL6. Interestingly, using inducible BCL6 transgenic and knockout as well as retrovirally transduced pre-B and ALL cells showed that overexpression of BCL6 leads to higher expression levels of MLL and deletion of BCL6 results in lower expression levels of MLL. Therefore, we conclude that MLL and BCL6 both cooperate and activate each other's expression in an activating feedback loop. Strikingly, Cre-mediated BCL6- deficiency results in apoptosis of MLL-ENL transduced cells.Clinical relevance: To verify if the high BCL6 expression levels in MLL-AF4 patients are important for the disease progression, we transduced primary human ALL xenografts with a dominant-negative BCL6-mutant (BCL6-DN). Expression of BCL6-DN rapidly induced cell cycle arrest and cell death. To test if pharmacological inhibition of BCL6 is of potential use for patients with MLLr leukemia, we treated multiple MLL-AF4 rearranged human xenograft cases with a RI-BPI a BCL6 peptide inhibitor. Strikingly, treatment with RI-BPI not only compromised colony formation in methylcellulose it also prevents leukemia-initiation in transplant recipient mice. RI-BPI also had a strong synergistic effect when combined with the chemotherapy drug Vincristine, which represents the backbone for most high risk regimen in pediatric ALL.Conclusions: These findings identify BCL6 as a central factor in leukemia initiation and survival and its pharmacological inhibition as a novel strategy to treat MLL-rearranged ALL. Aberrant expression of BCL6 in MLLr ALL is the direct consequence of the MLLr oncogenic activity in these cells. Based on these findings, we propose combinations of BCL6 inhibitors with currently used chemotherapeutics as potential approach to reduce the risk of ALL relapse and improve overall outcome. DisclosuresArmstrong:Epizyme, Inc: Consultancy. Ernst:Amgen: Other: stocks. Melnick:Janssen: Research Funding.

Non-Functional ("haploinsufficient"), but Not Dominant Negative Clonal IKZF1 Deletions Confer an Adverse Prognosis in Adult BCR-ABL-Negative Acute Lymphoblastic Leukemia

Background: IKAROS (IKZF1) is a key transcription factor for B-cell development. The IKZF1 protein exerts its functions as a dimer (homodimer or heterodimer with other IKAROS family members). Various IKZF1 intragenic deletions have been identified as recurrent aberrations in acute lymphoblastic leukemia: those resulting in the loss of transcription of one allele or at least loss of the dimerization domain ("haploinsufficient", "non-functional") and those involving only the DNA-binding domain but retaining the dimerization domain of the transcription factor, leading to so-called "dominant negative" isoforms.Several studies in pediatric ALL have confirmed the negative prognostic impact of IKZF1 alterations although there is still no consensus if they are independent risk factors in a multivariate analysis. The situation in adult ALL is less clear and few larger studies have addressed this issue, in particular the prognostic impact of different types of IKZF1 alterations.Objectives: We investigated archived patient samples obtained in the context of the German Multicenter ALL Study Group (GMALL) therapy studies for different IKZF1 alterations to assess their prognostic implications and their molecular pattern.Methods: We used RT-PCR to determine the presence of aberrant IKZF1 transcript variants and additionally investigated all patients by DNA-based PCRs for the IKZF1 aberrations D4-7, D2-7, D4-8, D2-8. Rare transcript variants, such as D2-3, D2, D5-7 were molecularly characterized on the DNA level by specific PCRs. IKZF1 aberrations were quantified using gel densitometry and/or quantitative real time PCR such that clonal and subclonal aberrations could be distinguished. All chromosomal breakpoints were molecularly characterized.Results: In total, 482 B cell precursor ALL patients aged between 15 and 65 years were included in the analysis. Samples were obtained at primary diagnosis and tested BCR-ABL -negative. Other molecular aberrations were tested according to the GMALL standards: 39 MLL-AF4-positive, 4 MLL-ENL, 1 MLL-AF9, 30 TCF3-PBX1, 3 ETV6-RUNX1. The immunophenotypes were the following: 424 pre B/common, 58 pro B.One hundred and twenty-eight out of 482 (27%) patients carried at least one IKZF1 deletion, with 37 patients carrying more than one deletion. Altogether there were 175 IKZF1 deletions, 71 cases of D4-7, 47 with D2-7, 26 with D4-8, 19 with D2-3, 10 with D2-8, and 1 with D5-7 and D2 each. Taken together 56 patients (12%) carried only non-functional mutations while 50 (10%) had dominant-negative mutations only. There was a group of 22 patients with both types of mutations (5%).Evaluating the prognostic impact, we considered the effect of non-functional and dominant-negative mutations separately. Patients with a non-functional IKZF1 mutation had a reduced overall survival (OS at 5 ys 37% vs 59% in IKZF1-unmutated patients, N=78/404, p=0.001) while dominant-negative mutations had no effect on OS (OS at 5 ys 54% compared to 56%, N=72/410, p=0.95). However, solely present subclonal non-functional mutations did not affect survival (OS at 5 ys 59% compared to 59%, N=23/404). In the dominant-negative group, clonality had no influence on OS (p=0.56, N=45/73/351). Taken together, patients with clonal non-functional IKZF1 had a significantly worse OS compared to those without (0.28 vs 0.59, N=53/427, p<.0001). This was true for patients with standard risk as defined by GMALL risk stratification (0.37 vs 0.68, N=24/243, p.0002). High risk patients also tended to do worse, which however did not reach significance (0.26 vs 0.46, N=30/184, p=.06).With regard to molecular analysis 193 chromosomal fusion sites were characterized. In the great majority (>95%) putative cryptic recombination sites were identified in the near vicinity thus underlining the causal role of the VDJ recombination machinery in the generation of these aberrations.Conclusions: We conducted a detailed and large-scale molecular analysis of intragenic IKZF1 deletions and identified clonal non-functional IKZF1 deletions as adverse prognostic factor in adult and adolescent B cell precursor ALL patients treated according to the GMALL protocols. Subclonal, or dominant negative IKZF1 mutations had no prognostic effect. It remains open for further analysis whether clonal non-functional IKZF1 deletions represent an independent prognostic factor in MRD-based risk stratification. DisclosuresWäsch:German Cancer Aid: Research Funding; Comprehensiv Cancer Center Freiburg: Research Funding; Janssen-Cilag: Research Funding; MSD: Research Funding.

Genomic Profiling Reveals Gain of Mutations in Histone Methylation Regulators in Relapsed Adult B Cell Precursor ALL

Introduction:B cell precursor acute lymphoblastic leukemia (BCP-ALL) represents the largest immunophenotypically defined subgroup of adult ALL. Yet, targeted therapies are assigned only to the minority of patients, whereas druggable lesions for the majority of adult BCP-ALL are still unknown. Thus, it remains necessary to explore their genomic landscape to unravel molecular alterations suitable for targeted therapies. We analyzed the mutational pattern of 206 candidate genes in adult BCP-ALL patients at diagnosis and matched relapse samples, focusing on therapeutically targetable alterations and their clonal evolution.Patients and methods:We performed targeted resequencing on diagnostic samples of patients with BCR-ABL negative BCP-ALL (n=89) as well as matched relapse samples (n=53). The mean age at first diagnosis was 44 years (range 17 - 79). Relapses were categorized as early (> 18 months from diagnosis, n=33) or late (< 18 months, n=20). All patients were enrolled on trials of the German Acute Lymphoblastic Leukemia Multicenter Study Group (GMALL). The targeted region comprised 206 genes known to be frequently mutated in leukemia and relevant in normal hematopoiesis. Customized biotinylated RNA oligo pools (SureSelect, Agilent) were used to select the targeted regions. We performed 100-bp paired-end sequencing on an Illumina Genome HiSeq1500 sequencing system. For a variant call, we required at least a read depth of 30 and a variant allele frequency (VAF) of 10%. We obtained an average coverage of 825 reads for the target region with over 98 percent of the targeted region been covered with a minimum of 30 reads. After exclusion of polymorphism annotated in dbSNP135, 625 protein changing single nucleotide variations and small indels were identified. 139 of the 206 target genes were mutated at least once.Results:On average three (median 3.0, range 1-12) genes were mutated in the 89 diagnostic BCP-ALL samples. The most frequent mutations included alterations in NRAS (18%), PAX5 (16%), TP53 (9%), JAK1/2 (8%) and IKZF1 (6%). Categorizing mutated genes according to their functional annotation revealed that 44 (49%) of patients harbored at least one mutation affecting epigenetic regulation, followed by adhesion/matrix proteins (48%), transcription factors (46%), RAS pathway genes (29%), kinase signaling (28%), and p53/cell cycle regulation (26%). Four (KMT2D, SETD2, KDM6A, KDM6B) of the twelve most frequently mutated genes, affecting 30 patients (34%), were epigenetic regulators specifically involved in histone methylation. Mutations in these genes were nearly mutually exclusive, suggesting their functional redundancy in this context.For 53 of the 89 patients matched relapse samples were available, showing a median frequency of 4.0 (range 1-36) mutated genes per sample. The frequency of mutations across gene families was similar compared to diagnostic samples, except for those involved in epigenetic regulation that showed a higher mutation rate at relapse (55%).In 68% of matched relapse samples, mutational gains and losses indicated mechanisms of clonal evolution. Among 28 patients (53%), which gained at least one mutation, the Lysine-Specific Methyltransferase 2D (KMT2D) was the most frequently affected gene with 7 patients acquiring KMT2D alterations at relapse. Other methyl-transferases (SETD2, KMT2C) as well as demethylases (KDM5A, KDM6A, KDM6B) acquired novel mutations at relapse, contributing to a total of 11 patients (21%) that gained alterations in histone methylation regulators. Notably, mutations in methylation regulators were only gained, but not lost at relapse. Nine of these 11 patients suffered an early relapse, implicating a selection advantage for clones harboring mutations in methylation regulating genes and their involvement in a more aggressive course of the disease.Conclusion:We describe for the first time a highly heterogeneous genomic mutational spectrum in adult BCP-ALL. A high mutation rate at diagnosis and further increase at relapse identified regulators of histone methylation as a most prominent target of recurrent alterations. Functional studies are needed to determine the direct biological consequence of these alterations, facilitating targeted therapeutic interventions with epigenetically active compounds. DisclosuresBaldus:Novartis: Research Funding.

Synergistic Activity of ABT-199 with Conventional Chemotherapy and Dinaciclib in B-Cell Precursor Acute Lymphoblastic Leukemia

Over the past decades, the use of optimized multi-agent chemotherapy protocols and improved risk stratification have led to superior patient outcome. However, avoiding therapy-related toxicity, decreasing the relapse rate of currently almost 20 % and improving the outcome of relapsed patients remain challenging problems. Aberrant activity of pathways involved in the regulation of survival and cell death contribute to development of leukemia and therapy failure. Anti-apoptotic BCL-2 family proteins are key regulators of apoptosis providing a promising target for novel directed therapies. The BH3 mimetic ABT-199 binds to BCL-2, counteracts its anti-death function and leads to apoptosis induction by direct release of pro-death BCL-2 family proteins such as BAX. BH3 mimetics are currently used pre-clinically and in first clinical trials. However the intrinsic or acquired resistance indicates the need for predictive markers and for effective combination treatment strategies.In this study, we addressed the activity of ABT-199 in B cell precursor (BCP) ALL. The effects of ABT-199 as single agent and in combination with chemotherapeutic drugs used in remission-induction therapy of pediatric ALL were analyzed using BCP-ALL cell lines (n=5) and a set of patient-derived primograft samples (n=9) established by transplantation of primary leukemia cells obtained from pediatric BCP-ALL patients at diagnosis onto immunodeficient NOD/SCID mice. Since ABT-199 does not bind to all antiapoptotic BCL-2 family members, sparing MCL-1, we included the cyclin-dependent kinase (CDK) inhibitor dinaciclib, which also exerts MCL-1 inhibitory effects. Cell viability was determined by flowcytometry according to fw/sc criteria and half maximal inhibitory concentrations (IC50) were estimated upon exposure to single agents and drug combinations. Combination indices (CI) indicating synergistic or additive effects were estimated according to Chou-Talalay. BCL-2 and MCL-1 protein levels were investigated by western blot analysis.Despite sensitivity in most of the BCP-ALL cell lines with IC50 values in the nanomolar range (mean IC50, 212 nM), one cell line, Nalm-6, showed ABT-199 resistance with an IC50 >10 µM. Interestingly, low protein levels of BCL-2 and increased MCL-1 expression were identified in this insensitive line, in contrast to high level BCL-2 and low MCL-1 expression in ABT-199 sensitive leukemias, indicating a potential marker to identify sensitive and resistant ALL. A pronounced synergism of ABT-199 in combination with chemotherapeutic agents was found with vincristine (CI 0.31 and 0.40, cell lines RS4;11 and Nalm-6) and even strong synergism with asparaginase and dexamethasone in ABT-199 monotherapy resistant Nalm-6 cells (CI 0.002, 0.14). Strikingly, the combination of the MCL-1 inhibitor dinaciclib with ABT-199 was highly effective and revealed a strong synergism for both compounds (CI 0.03).When we addressed the effects of ABT-199 on primograft ALL samples isolated from mice with full-blown leukemia, a pattern of high sensitivity (IC50 from 16 to 156 nM) similar to the cell lines was observed. In contrast, peripheral blood (PB) samples from healthy control donors were resistant to ex vivo exposure with ABT-199 (n=3, IC50 values > 1 µM). However, one primograft sample derived from a high risk patient showed ABT-199 insensitivity with an IC50 value of > 1 µM similar to healthy PBMCs. This primograft sample was also characterized by low BCL-2/high MCL-1 protein expression similar to Nalm-6. Most importantly, this resistant leukemia was also rendered ABT-199 sensitive by co-treatment with dinaciclib (CI 0.04).Taken together, we found efficacy of ABT-199 in the majority BCP-ALL cell lines and patient-derived primograft ALL samples and ABT-199 synergized with conventional chemotherapeutic agents. ABT-199 resistant leukemias characterized by low BCL-2/high MCL-1 expression were resensitized by addition of dinaciclib leading to strong synergism. These data indicate effective cell death sensitization by ABT-199 and potential strategies to overcome ABT-199 resistance in BCP-ALL. DisclosuresNo relevant conflicts of interest to declare.

TNF superfamily proteins in the serum of patients with B-ALL--preliminary study.

TNF superfamily ligands such as B cell-activating factor (BAFF), a proliferation inducing ligand (APRIL) and TNF-related apoptosis inducing ligand (TRAIL), can play an important role in the pathogenesis and development of various B cell malignancies involving B-ALL. The study group consisted of 25 children suffering from newly diagnosed B cell precursor ALL (B- ALL), involving immunophenotype pre-B and common-cALL. Peripheral blood samples were collected at the time of diagnosis, before any treatment, and after treatment. Soluble APRIL, soluble BAFF, and soluble TRAIL concentrations were assessed using ELISA kits. The concentrations of sAPRIL and sBAFF in the sera of the patient group were higher compared to the values of the control specimens. Among all molecules examined, the highest concentrations were found for the sAPRIL molecule. Furthermore, sAPRIL and sBAFF concentrations were higher in patients with ALL-pre B than in patients with cALL. After the treatment, the sera of patients with ALL-pre B contained lower concentrations of sAPRIL, sBAFF, and sTRAIL than those of the same patients before therapy. Contrary to earlier indications of the key function of BAFF in B-ALL, the quantitative dominance of APRIL in the serum suggests that this molecule can play an equivalent or even more significant role in this patient group than BAFF.

First Description Of a t(10;11)(q22;q23)/MLL-TET1 Reciprocal Translocation In a T Lymphoblastic Lymphoma With Subsequent Lineage Switch To Acute Myelomonocytic Myeloid Leukemia

TET1 genomic breakpoints and clinical features of MLL-TET1 rearrangements have been described in 13 acute leukemia cases, 11 in AML, 2 in B cell-precursor ALL. The incidence of this rare translocation was evaluated to 0.3% of MLL rearranged AML cases (5 out of 1590 MLL [Meyer C. Leukemia 2013]). Although those cases are very uncommon, their study can improve our current understanding of leukemogenesis. We report here the first t(10;11) MLL-TET1 positive case of lymphoblastic T lymphoma occurring in a 31 years old male patient, with a subsequent transformation to AML.The patient was referred for a large mediastinal mass and right pleural effusion. Mediastinal and bronchus biopsies led to the diagnosis of a precursor-T cell lymphoblastic lymphoma, expressing CD3, CD5, CD4, CD8, CD10 antigens, without any expression of CD34 or CD79.Molecular analyses of the malignant T-cells showed a clonal TCR gamma-chain gene rearrangement together with HOXA10 overexpression. FISH analysis showed a MLL breakage. The partner gene, TET1, was identified using RP11-9E13 and RP11-314J18 BACs, corresponding to the recurrent translocation t(10;11)(q22;q23). MLL-TET1 fusion transcript was detected (intron 8 of TET1 fused to exon 8 of MLL), as well as its reciprocal transcript.The patient was treated following the (GELA) LL03 protocol, and was considered in complete remission after induction and consolidation phases. Fourteen months after the diagnosis, a bone marrow examination was performed for thrombopenia (6 G/L) which revealed a myelomonocytic acute leukemia with trilineage dysplasia.At this time, The MLL-TET1 fusion transcript as well as HOXA10 overexpression was still present but the TCR rearrangement was not detected. A non-familial donor allogeneic bone marrow transplant was performed in CR after intensive chemotherapy, that was complicated by a grade IV acute graft-versus-host disease. The patient died 54 days after the transplant of bacterial sepsis leading to multi-organ failure.MLL-TET1 fusions have been described in 13 cases in the literature, mainly in AMLs (11/13 cases, mostly AML M4 or M5 cases) and in B-ALLs (2/13 cases). The case reported here presents two interesting features. Firstly, this patient harbors the first MLL-TET1 fusion reported to date in T-ALL, and secondly this case presented a lymphoid to myeloid phenotype switch during the time course of the disease.This strongly suggests that the translocation occurred very early during hematopoietic differentiation, prior to the lymphoid/myeloid commitment. As described for the Ph1 chromosome rearrangement, the t(10;11) translocation may arise in hematopoietic stem cell rather than in committed progenitors. These features are also described in 8p11 stem cell syndrome that involves FGFR1. In both cases, genetic rearrangements arise in myeloid or lymphoid neoplasms with possible subsequent transformation.TET family enzymes convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and play a key role in active DNA demethylation. TET1 and TET2 are also the key enzymes responsible for the presence of 5hmC in mouse embryonic stem cells (ESCs) (Koh KP., Cell Stem Cell 2011), and TET1 functions to regulate the lineage differentiation potential of ESCs. In addition to its role in DNA demethylation, TET1 interacts physically with NANOG and NANOG/TET1 co-occupy genomic loci of genes associated with both maintenance of pluripotency and lineage commitment in embryonic stem cells (Costa Y., Nature 2013). Taken together, these observations enlighten possible mechanism of the lineage switch observed in this case, and may be a rationale for using demethylating agents in MLL-TET1 neoplasms. Disclosures:No relevant conflicts of interest to declare.

A Novel Model of Immune-Mediated Disease Equilibrium in Acute Lymphoblastic Leukemia

AbstractAbstract 3540Acute lymphoblastic leukemia (ALL) is a challenging target for immunotherapy due to its poor antigen presentation, lack of costimulatory molecule expression, and resistance to cytotoxic killing by immune effector cells. In order to develop new strategies to generate autologous immune-mediated protection against ALL progression, we sought to understand more about the immune requirements that must be met in order to achieve disease control. To do this, we have transfected B cell precursor ALL cell lines derived from leukemic Eμ-ret transgenic BALB/c mice with a lentiviral construct encoding green fluorescent protein (GFP) and firefly luciferase (luc) in tandem. The luciferase protein serves a dual purpose as a potent neoantigen capable of stimulating leukemia-specific immune responses and as a bioluminescent reporter for highly sensitive detection of minimal residual disease (MRD).Intravenous injection of 106 non-transfected Eμ-ret-derived leukemia cells is consistently lethal in wild type BALB/c mice within 3–5 weeks. However, adoptive transfer of 106 GFP/luc-expressing leukemia cells into wild type BALB/c mice does not lead to morbidity or mortality (p=0.025, logrank for survival). These mice show detectable disease in bone marrow by bioluminescent imaging below the limit of detection by peripheral blood flow cytometry that increases until day 7–10, followed by a spontaneous decrease in leukemia burden to undetectable levels by imaging. BALB/c mice lacking B and T cells (RAG1−/−) or B, T and NK cells (gamma common−/−) injected with GFP/luc ALL cells develop leukemia that is lethal by day 25 (p=0.037, logrank). Early T cell-depletion in wild type BALB/c mice on days 0, 3, 6, and 9 with CD4- and/or CD8-depleting antibodies also results in complete breakthrough of disease, while NK cell-depletion with asialo-GM1 on the same days does not significantly alter the timeframe of leukemia clearance (p<0.0001, 2-way ANOVA; Figure 1). These results demonstrate that the presence of a neoantigen is sufficiently immunogenic to generate effective T cell-mediated control of the disease.We have followed immunocompetent mice that received GFP/luc ALL for up to 12 months. While most mice have long-term remissions both by imaging and peripheral blood sampling, a subset have evidence of persistent MRD by bioluminescent imaging that waxes and wanes in luminosity but may not break through to a fulminant relapse. Relapses in these mice have occurred up to 11 months from injection. Late T cell-depletion with CD4 and CD8 antibodies (weekly x12, beginning at day 28) leads to an increased rate of leukemia breakthroughs and occurs more frequently in mice who had cleared their leukemia in the setting of early NK cell depletion (p<0.001, 2-way ANOVA), suggesting early crosstalk between NK and T cells to develop a sustained immune response. In addition, many mice relapse with GFP/luc-expressing leukemia, while a proportion of mice have late relapses with GFP/luc-negative leukemia, suggesting antigen loss is one of several potential mechanisms of immune evasion in ALL.Finally, BALB/c mice transgenic for GFP and luciferase do not generate T cell protection against outgrowth of injected GFP/luc ALL cells and succumb to disease by day 20 (p = 0.027 versus wild type mice, logrank). This result suggests that tolerance mechanisms, rather than antigen presentation deficiencies, may undermine host immune responses against ALL.We present a novel model of immune equilibrium in ALL, made evaluable by exquisitely sensitive bioluminescent imaging. Importantly, despite its well-documented lack of immunogenicity, we have shown ALL can be a sensitive target for long-term leukemia-specific T cell-mediated immune control. We are currently investigating additional cellular mechanisms of immune protection, the role of antigen spreading in control of ALL, and the mechanisms allowing leukemia to escape from immune control. Our approach will enable the development of strategies to break immune tolerance towards leukemia antigens and generate control of MRD in ALL. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

SOX4 enables Oncogenic Survival Signals in Acute Lymphoblastic Leukemia

Abstract 863 Background:The SOX4 (SRY-related HMG-box) transcription factor is expressed in early B- and T- cell development. In the absence of SOX4, B cell development is arrested at the pro-B to pre-B cell transition. In Sox4−/− mice, pro-B cells fail to proliferate in response to IL7 and to expand and differentiate past the pre-B cell receptor checkpoint. Interestingly, SOX4 functions a transcription factor yet closely interacts with membrane-proximal cytokine receptor signaling. The PDZ domain-containing adaptor protein syntenin (SDCBP) recruits the Sox4 protein directly to the cytoplasmic tail of the cytokine receptors. With regard to its receptor-proximal activation, Sox4 resembles the SMAD and STAT transcription factors. A role for Sox4 in acute myeloid leukemia was proposed based on the identification of viral insertions, and Sox4-overexpression causes myeloid leukemia. In this study, we found SOX4 was strongly upregulated upon tyrosine kinase inhibitor (TKI) treatment in Ph+ ALL and high expression of Sox4 correlates with poor clinical outcome of patients with ALL. Results:To elucidate the potential contribution of Sox4 to BCR-ABL1-driven B cell lineage leukemia, we studied a conditional Sox4 knockout mouse model. To this end, we transformed bone marrow B cell precursors from Sox4fl/fl mice with BCR-ABL1 to test its function in a mouse-model for Ph+ ALL. Tamoxifen (4-OHT)-inducible activation of Cre and subsequent deletion of loxP-flanked Sox4 alleles in B cell precursor ALL resulted in reduced mRNA levels of multiple genes that play a known role in survival signaling in Ph+ ALL cells including BCLXL (BCL2l1), Mapk8, Map2k6, PI3K/AKT (Pik3r2, Pik3r3, Rps6ka2, Mtor). Overexpression of Sox4 had the opposite effect and resulted in increased mRNA levels of BCLXL (Bcl2l1), Mapk8, PI3K/AKT (Pik3r2, Pik3r3). Single-locus quantitative ChIP confirmed binding of SOX4 to BCL2l1, PIK3R3 and MAPK8 promoters in human Ph+ ALL cells via its putative DNA binding motif. Transcriptional regulation of components within the PI3K/AKT pathway is indeed functionally relevant since Cre-mediated deletion of Sox4 caused multiple de-phosphorylation events including AKTS473, SRCY416 and RPS6S235/6. In addition, inducible deletion of Sox4 resulted in strong upregulation of both Arf and p53 protein, suggesting that Sox4 mediates survival signaling both by activation of BCLXL (Bcl2l1) and repression of Arf/p53. Consistent with Sox4-mediated negative regulation of Arf/p53, deletion of Sox4 resulted in G0/G1 arrest of BCR-ABL1 ALL cells and loss of self-renewal capacity, failure to form colonies in methylcellulose replating assays and delayed the onset of disease and substantially prolonged overall survival of recipient mice. In agreement with reduced expression of BCLXL (Bcl2l1) and increased levels of Arf/p53, the overall outcome of Sox4 deletion is rapid loss of viability and apoptosis in BCR-ABL1-transformed ALL but not normal pre-B cells. Loss of viability upon Sox4 deletion in the leukemia cells was rescued by transduction with constitutively active (myristoylated; CA) mutants of AKTCA and the p110 catalytic subunit of PI3K (p110αCA) and as well with BCLXL. Clinical relevance:These findings are also relevant to human disease since promoter CpG methylation analysis (HELP assay) revealed lower levels SOX4 promoter methylation in human Ph+ALL cells compared to their normal pre-B cell counterparts. In addition, samples from patients with high risk ALL (n=31) show a trend towards higher SOX4 mRNA levels compared to patients with intermediate risk ALL (n=23, p=0.07; ALL REZ BFM 2002). Analyzing data from a larger study based on 207 patients with ALL (COG P9906) showed a statistically significant association between high mRNA levels of SOX4 and poor clinical outcome (shorter overall survival). Conclusion:Collectively, these findings identify SOX4 as a critical upstream regulator of survival signaling in Ph+ ALL. Pathways affected by SOX4 include PI3K/AKT signaling downstream of BCR-ABL1, activation of BCLXL (BCL2L1) and negative regulation of Arf and p53. Disclosures:No relevant conflicts of interest to declare.

Targeting BCL6-Mediated Drug-Resistance in High-Risk Childhood ALL

Abstract 776 Background:BCL6 is known as a protooncogene and transcriptional repressor in Diffuse Large B cell lymphoma (DLBCL), where BCL6 is frequently involved in chromosomal rearrangements. We recently found that BCL6 mediates a novel form of drug resistance to tyrosine kinase inhibitors (TKI) in Ph+ ALL and CML (Duy et al., Nature 2011; Hurtz et al., J Exp Med 2011). This was based on the finding that Ph+ ALL and CML cells respond to TKI-mediated inhibition of BCR-ABL1 kinase activity by upregulation of BCL6, which protects from p53-mediated apoptosis. Our current study was prompted by the finding that high expression levels of BCL6 represent a predictor of poor clinical outcome in children with Non-Ph ALL. We therefore performed detailed studies of expression and function of BCL6 in various subtypes of Non-Ph childhood ALL. Results:In a gene expression analysis of 207 children with high-risk B cell precursor ALL (COG P9906 trial), we found that high expression levels of BCL6 at the time of diagnosis represents a strong predictor of poor overall (OS; p=0.007) and relapse-free (RFS; p=0.02) survival. Also, high expression levels of BCL6 are predictive of positive minimal residual disease (MRD+) status on day 29 after the onset of chemotherapy. For 49 patients in this clinical trial, matched sample pairs at diagnosis and relapse were available. In these patients, BCL6 expression was significantly higher at relapse compared to diagnosis (p=0.003). We next studied BCL6 mRNA (n=122) and protein (n=21) levels in a larger cohort of primary childhood ALL patient samples and found that BCL6 expression levels are particularly high in MLL-AF4 infant ALL. This finding was unexpected and we tested if the MLL-AF4 oncogene drives aberrant BCL6 expression in these cells. First, ChIP-analysis revealed that the chimeric oncoprotein MLL-AF4 directly binds to the BCL6 promoter, suggesting that MLL-AF4 may indeed drive BCL6 expression. In support of this hypothesis, BCL6 Western blot analyses of inducible MLL-AF4-transgenic pro-B cells demonstrated that activation of the MLL-AF4 transgene is sufficient to induce ∼10-fold upregulation of BCL6 protein levels. We conclude that aberrant expression of BCL6 in childhood ALL can be the direct consequence of MLL-AF4 activity. To investigate the potential function of BCL6, we used a genetic mouse model of childhood ALL based on bone marrow precursor cells from BCL6−/− mice. Since mutations in the RAS pathway are found in about 30% of childhood ALL cases, we transformed B cell progenitor cells from BCL6−/− and wildtype mice with oncogenic NRASG12D, which represents the most frequent RAS lesion in B cell lineage leukemia. Surprisingly, BCL6-deficiency results in a failure of NRASG12D ALL cells to initiate leukemia, while NOD/SCID mice that were transplanted with BCL6 wildtype NRASG12DALL succumbed to the disease. Clinical relevance:To verify if these observations are relevant to human disease, we transduced primary human childhood ALL xenografts with MLL-AF4 gene rearrangement with a dominant-negative BCL6-mutant (BCL6-DN). Expression of BCL6-DN rapidly induced cell cycle arrest and cell death. To test if BCL6 inhibition is of potential use for children with MLL-AF4 leukemia, we used a recently developed retro-inverso BCL6 peptide inhibitor (RI-BPI, Cerchietti et al., Blood 2009). RI-BPI is able to inhibit BCL6 function and is currently under clinical development for the treatment of BCL6-dependent DLBCL. Treatment of the primary human MLL-AF4 ALL xenograft cells with RI-BPI compromised colony formation in methylcellulose and leukemia-initiation in transplant recipient mice and had a strong synergistic effect when combined with Vincristine, which represents the backbone for most high risk regimen in pediatric ALL. Conclusions:These findings identify BCL6 as a therapeutic target in high-risk childhood leukemia and its pharmacological inhibition as a novel strategy as therapeutic adjuvant. Interestingly, MLL-AF4 ALL cells exhibit constitutively high expression levels of BCL6. Aberrant expression of BCL6 in MLL-AF4 ALL is the direct consequence of MLL-AF4 activity in these cells. Based on these findings, we propose combinations of BCL6 inhibitors (e.g. RI-BPI) with standard chemotherapy as potential approach to reduce the risk of ALL relapse and improve overall outcome. Disclosures:No relevant conflicts of interest to declare.

IKZF1 Deletion Is Strongly Associated with Risk of Relapse in Pediatric B Cell Precursor ALL with TCF3-PBX1

AbstractAbstract 1454 IntroductionRecent progress in the treatment of pediatric B cell precursor (BCP)-ALL has improved the outcome of TCF3-PBX1 fusion positive BCP-ALL. However, approximately 10% of those cases still experience relapse and reliable genetic prognostic marker has not been identified in this sub-group. Recently, IKZF1 deletion was reported to be associated with poor outcomes in pediatric BCP-ALL. Therefore, we sought to determine whether IKZF1 deletion plays a role in determining poor outcomes in pediatric BCP-ALL patients with TCF3-PBX1 fusion. Materials and MethodsDiagnostic bone marrow or peripheral blood samples of 53 pediatric TCF3-PBX1 fusion positive BCP-ALL patients treated according to Japan Association of Leukemia Study (JACLS) ALL02 protocol from April, 2002 to May, 2008 were examined in this study. The patients with Down syndrome were excluded in this genetic analysis. The diagnosis of BCP-ALL was based on morphological findings on bone marrow aspirates and immuno-phenotype analyses of leukemic cells by flow cytometry. Conventional cytogenetic analyses were done as part of the routine work-up. Molecular studies using quantitative RT-PCR for the detection of TCF3-PBX1 were performed as part of the routine work-up. The deletion of IKZF1, CDKN2A, CDKN2B, PAX5, ETV6, RB1, BTG1, EBF1 and the PAR1 region, which includes CRLF2, CSF2RA, and IL3RA was determined using multiplex ligation-dependent probe amplification (MLPA) in 53 patients whose diagnostic DNA samples were available. JAK2 mutations were also determined by direct sequencing of the exon 16, 20 and 21 in the patients with IKZF1 deletion. ResultsThe median age at diagnosis was 5 years (range 1 –14 years). The median leukocyte count at diagnosis was 21,800 × 106 /L (range 2,700 – 183,300). The patients consisted of 28 male and 25 female patients. Twenty seven patients were categorized in NCI-SR and 26 patients in NCI-HR. Forty seven patients were classified as prednisolone good responder, six patients as prednisolone poor responder. The deletion of IKZF1 gene was present in 2 of 53 (3.8 %) patients, which was less frequent in BCP-ALL without TCF3-PBX1 fusion. JAK2 mutation was not determined in those two patients with IKZF1 deletion. The deletion of PAX5 was also less frequent (TCF3-PBX1 positive vs. negative: 17.9 % vs. 32.5 %). The deletion of CDKN2A and CDKN2B was identified in 12.8 % and 10.3 % of these patients, respectively, which was also less frequent than in BCP-ALL patients without TCF3-PBX1 (del. CDKN2A: 46.6 %, del. CDKN2B: 39.9 %). However, the deletion of RB1 was identified in 17.9 % of 53 patients, which was more than in those without TCF3-PBX1 fusion (3.0 %). The deletion of ETV6, EBF1 or BTG1 was not identified in 53 patients. Age at onset, initial WBC count and NCI risk were not associated with the relapse rate. Interestingly, patients with IKZF1 gene deletion had significantly higher relapse rate than those without IKZF1 deletion (2/2 vs. 5/51, p=0.015). However, the deletion of CDKN2A, CDKN2B, PAX5 and RB1 was not associated with the relapse rate. DiscussionsThis study determined that the presence of IKZF1 deletion was strongly correlated with risk of relapse in TCF3-PBX1 positive BCP-ALL, although the frequency was less than in those without TCF3-PBX1 negative. Thus, we expect that IKZF1 deletion might be an independent predictor of treatment outcome and represents a candidate of prognostic marker in pediatric BCP-ALL with TCF3-PBX1. Further molecular studies are required to determine genetic alterations in relapsed patients of TCF3-PBX1 positive BCP-ALL without IKZF1 deletion. Disclosures:No relevant conflicts of interest to declare.

Abstract 458: B cell precursor ALL cells alter their immunophenotype, cell cycle, and chemosensitivity through contact with mesenchymal stromal cells

Abstract Background: The outcome of B cell precursor ALL (BCP-ALL) in childhood has steadily improved, but 10 to 20 % of the patients still have recurrence of the disease. We speculate that BCP-ALL cells may obtain a characteristic of chemoresistance and similar phenotype of leukemic stem cell through the interaction with mesenchymal stromal cells (MSCs). Materials and methods: Five BCP-ALL cell lines established from pediatric patients at author's institution were cultured in serum free medium. Primary BCP-ALL samples and specimens were obtained from patients with informed consents. Human bone marrow mesenchymal cells transfected with telomerase reverse transcriptase (hTERT) gene were passaged in cell culture medium with 10% fetal bovine serum and moved into serum free medium when incubating with leukemic cells. The BCP-ALL cells were analyzed in doubling time with time-lapsing images, and examined for expression of stem cell markers, hematopoietic stem/progenitor cell markers, and multidrug-resistance transporters with flowcytometry or confocal microscopy. Chemosensitivity of each cells to different anti-cancer drug were estimated by cell counting kit-8 (Doujindo) for suspending cells, and Vybrant cell adhesion assay kit (Invitrogen) for adherent cells. Results: The doubling time of BCP-ALL adherent with MSCs were prolonged, compared with that of suspending cells. The suspending BCP-ALL cells incubated with or without MSCs showed positive to CD38 and negative to CD34, CD133, P-glycoprotein (Pgp), and BCRP/ABCG2, whereas the cells adherent with MSCs altered the expressions into CD34+, CD38-, CD133+, Pgp+, and BCRP/ABCG2+. The stem cell markers, Oct3/4, Nanog, SSEA-1 and Sox-2 were all negative in any condition. The alterations in phenotype were universally observed in all 5 cells examined. The primary bone marrow sample biopsied from a BCP-ALL patient was positive to CD34, CD133, Pgp and BCRP/ABCG2+ although that aspirated was all negative to those antibodies. BCP-ALL cells adherent with MSCs demonstrated remarkably decreased chemosensitivity against methotrexate, predonisolone, 6-mercaptoprine, vincristine, and cytrabine, compared with suspending cells. Discussions: Although leukemic stem cells in BCP-ALL have not been proved, our data showed that BCP-ALL cells become positive to hematopoietic stem/progeniter cell surface markers such as CD34 and CD133, and obtain chemoresistance through contact with MSCs. The chemoresistance in BCP-ALL cells adherent with MSCs were suspected to result from expression of multidrug-resistant proteins. BCP-ALL cells may escape from cytotoxic effect of chemotherapeutic agents through interaction with MSCs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 458. doi:10.1158/1538-7445.AM2011-458

Evolutionary Trajectory of Hyperdiploid ALL: Insights From Monozygotic Twins

AbstractAbstract 28Identical twins have provided substantial insight into the natural history, developmental timing and sequence of genetic events in ALL. Much of these data are focussed on ALL with ETV6-RUNX1 fusions. More limited data exists for the most prevalent subset of childhood ALL with hyperdiploidy though retrospective screening from cord blood (Maia et al. Genes Chromosomes and Cancer, 2004) provide evidence that hyperdiploidy itself can be an early, possibly initiating and pre-natal event.We have now explored the genetic complexity of hyperdiploid ALL using four pairs of monozygotic twins, three concordant and one discordant for ALL. In parallel with our prior studies on ETV6-RUNX1 ALL (Bateman et al. Blood, 2010), we hypothesized that hyperdiploidy itself would be a consistent pre-natal event and all other potential ‘driver’ mutations would be secondary and probably post-natal. If correct, this twin pair should share identical hyperdiploid karyotypes but have different copy number alterations (CNA) or other secondary mutations (e.g. in FLT3 or RAS).We used Affymetrix SNP 6.0 mapping arrays to identify CNA using the Genome Orientated Laboratory File (GOLF) v3.2 software package and dCHIP. The SNP array was performed using leukaemic DNA compared to matched remission DNA for all cases. The mutation screen included the activating D835 FLT3, NRAS exon 2 (codons 12 and 13) exon 3 (codon 61), KRAS exon 2 (codons 12 and 13) and exon 3 (codon 61), PTPN11 (exon 3 and exon 13). In the discordant twin pair, Fluorescence In-Situ Hybridisation (FISH) was used to interrogate the pre-leukaemic cells of the unaffected twin for the CNA found in the corresponding affected twin.In the identical twin pairs concordant for hyperdiploid ALL, the whole chromosome gains were identical within each twin pair apart from an additional but sub-clonal gain of chromosome 10 in one twin of twin pair set 2. The median number of whole chromosome gains was 56 (Range 55 to 59). The total number of CNA found was 6 (Range 0 to 4 per case) (excluding T cell receptor and immunoglobulin rearrangements). These included genes previously found to be recurrently altered in hyperdiploid ALL such as PAX5, CDKN2A/2B and ADD3. All the CNA abnormalities detected were different within each twin pair. The mutation screen revealed an NRAS exon 2 codon 13, GGT to GAT (glycine to aspartic acid) mutation in one twin of twin pair set 1, which was absent in leukaemic DNA of the co-twin.In the twin set discordant for ALL, the CNA at 19p13.3 (incl TCF3 gene) found in the leukaemic DNA of twin 1 were absent from the hyperdiploid pre-leukaemic cells of the unaffected twin 2. These pre-leukaemic cells were found at a consistent frequency between 0.0012% and 0.002% (over a period of 13 months).These data accord with the view that hyperdiploidy is a pre-natal and probably initiating event in childhood B cell precursor ALL and that the set of additional ‘driver’ genetic changes including CNA and sequence based mutations are all secondary. As all of the latter are single twin restricted, it is likely, though not formally proven, that they occur post-natally and perhaps more proximal to disease diagnosis. These data extend our understanding of the history of B cell precursor ALL. Whole genome sequencing is required to complete the picture of essential genetic events and their timing. Disclosures:No relevant conflicts of interest to declare.

A Novel B Cell Precursor ALL Cell Line (018Z) with Prominent Neurotropism and Isolated CNS Leukemia in a NOD/SCID/huALL Xenotransplantation Model.

Abstract 1630Poster Board I-656Acute lymphoblastic leukemia (ALL) is the most frequent malignant disease in childhood. Despite efforts achieved in treatment resulting in improved long term survival about 20% of the patients encounter relapse of their disease. Involvement of the central nervous system (CNS) is a common feature at diagnosis but also at relapse. About 10% to 20% of relapse patients present with isolated CNS leukemia as first event mainly occurring early within 24 months after diagnosis and associated with a survival of about 50%. However, achievement of effective CNS control is essential for successful treatment. Model systems to study the mechanisms of CNS leukemia but also new treatment strategies are required.Here we present and characterize a new pediatric B cell precursor (BCP) ALL cell line (018Z) which was established by passage in a NOD/SCID/huALL xenotransplantation model and exhibits a distinct CNS phenotype if re-transplanted onto NOD/SCID mice.Leukemia cells isolated from a male, adolescent patient at diagnosis were transplanted onto unconditioned NOD/SCID mice by intravenous injection. The patient had no CNS disease at diagnosis, however he died in remission after induction therapy. Upon onset of leukemia in the recipients the animals were killed and the engrafted leukemia cells were isolated and further transplanted onto secondary and tertiary recipients. Cells isolated from subsequent recipients were transferred into cell culture flasks and kept under standard culture conditions in RPMI medium supplemented with 20% fetal calf serum. Autonomous proliferation of the cells was observed for now more than three years.The cell line remained immunophenotypically stable as regularly assessed by flowcytometry (every 20 passages, from cell line passage 20 up to 160) showing the same BCP-ALL subtype. Infection of the cells with Epstein Barr Virus or Human Herpes Virus 8 was ruled out.Cytogenetic analysis of the cell line revealed presence of 47, XY, +8, del(9)(p13). The same karyotype was found in two samples isolated from previous xenograft passages (P4 and P6) and in leukemia cells obtained at diagnosis. The same karyotype was also detected if the 018Z cell line cells were re-transplanted and leukemia cells isolated from the recipients. Interestingly, at diagnosis two additional karyotypes were observed but could not be detected in the xenografts or cell line indicating selection and subsequent proliferation of one clone engrafted [47, XY, +8, del(9)(p13)]. Interestingly, chromosome arm 9p abnormalities have been reported to be associated with adverse events in pediatric BCP ALL, including onset of CNS leukemia (Heerema et al., Blood, 1996, 94(5): 1537-44).The cell line was further characterized and re-transplanted in the NOD/SCIDhuALL model. Unlike leukemia manifestation observed after transplantation of other samples, recipients transplanted with the 018Z cell line presented with a rapid onset of symptoms like seizures and palsies of the limbs pointing to CNS involvement. Infiltration of BCP ALL cells in the CNS of the recipients was detected by immunohistochemistry of the brain and by flowcytometry in cell suspensions prepared from the mice' meninges while only below 5% of human leukemia cells were detected in peripheral blood, spleen or bone marrow.Taken together, we established a novel BCP ALL cell line showing manifestation of CNS leukemia with rapid onset of about 6 weeks after re-transplantation onto NOD/SCID mice and providing a model system to study CNS disease in ALL. DisclosuresNo relevant conflicts of interest to declare.