Childhood B-cell Acute Lymphoblastic Leukemia Research Articles

Functional Study of Novel Gene Fusions and Patterns of Epigenetic Regulator Mutations in Adult and Childhood B-Cell Acute Lymphoblastic Leukemia

Background.In previous study of adult and childhood B-cell acute lymphoblastic leukemia, through whole exome and transcriptome sequencing we identified three new categories of fusion genes with prognostic significance and much more mutations of epigenetic regulators in adult versus pediatric B-ALL cases. To address how these gene alterations contribute to leukemogenesis and affect prognosis, we performed detailed data analysis and functional study of the three new categories of B-ALL. In addition, the changes of epigenetic regulator were scrutinized in a systematic way.Methods.Differentially expressed genes analysis and gene set enrichment analysis (GSEA) were performed. The regulation of target genes by distinct fusions was further verified through lentivirus mediated overexpression, shRNA induced knockdown, quantitative real-time PCR as well as transcriptional reporter assays. The modification of histone acetylation was evaluated by Western bloting. To observe the effect of fusion genes in vivo, murine retroviral bone marrow transplantation models were established.Results.Through in-depth data analysis, differentially expressed genes and signaling pathways related to fusion genes were identified. Alternative splicing of transcription factor ERG was seen in most of the cases with DUX4 fusions. In some of the cases, both ERG deletion leading to frameshift of the open reading frame and an abnormal short transcript of ERG were also observed. This group can also be characterized by some outlier genes (AGAP1, STAP1, PTPRM1, PCDH17 and PLEKHA6). Overexpression of DUX4-IGH in human B-ALL cell line induced the appearance of the short form of ERG and upregulation of these outlier genes. In mouse models, both DUX4-IGH and the short ERG inhibited the development of B-cell lineage. NT5E, also known as CD73, which has been reported to promote anthracycline resistance, was significantly downregulated in this group, which could explain the favorable prognosis of this type of B-ALL. Pre-BCR signaling pathway maintaining the survival of leukemic cells at the pre-B stage was activated in patients with MEF2D fusions. Genes related to protein amimo acid phosphorylation and cell morphogenesis involved in differentiation were significantly upregulated in this group. Notably, this group exhibited rather poor survival rate, and the expression of CD73 in this group was as high as that in BCR-ABL1 and BCR-ABL1 like group. When juxtaposed to the upstream of ZNF384, EP300 and CREBBP lost the histone acetylation domain, which could reduce the histone acetylation level and be involved in the leukemogenesis. Indeed, EP300-ZNF384 induced the rapid emergence of acute leukemia in mouse with a median survival of 100 days.In addition, we checked the sequence variations in our whole exome/genome dataset containing 203 cases. When mutations of different gene categories were scrutinized, abnormalities of epigenetic regulators (ER) drew our attention. Using the ER database that St. Jude Children's Research Hospital established, we found a total of 140 somatic mutations in 80 ER classified into eight classes. Forty two percent of the 203 patients (56.5% of adult and 30.6% of children) harbored at least one gene mutation among the eight classes of ER. The fact that abnormalities of MLL2 and SETD2, the two ER genes with highest mutation rates, were mutually exclusive in most affected cases suggests that they should bear somehow functional resemblance.Conclusions.Detailed study of MEF2D, ZNF384 and DUX4-IGH gene fusions expands the extant knowledge about molecular subtypes of B-ALL, and provides more potential targets as well as promotes further personalized treatment in both childhood and adult B-ALL. The overview of ER alterations in adult and childhood B-ALL proves the importance of these genes in the pathogenesis of B-ALL, especially in adult cases. DisclosuresNo relevant conflicts of interest to declare.

Inhibition of MEK and DDR Pathways Induces Synergistic Killing of Novel Mll-Af4 B-ALL Model Harboring Activated Ras Mutations

Childhood B-cell acute lymphoblastic leukemia (B-ALL) that harbor a translocation of the MLL1 and AF4 genes are considered high-risk with poor prognosis (event-free survival (EFS) of 35%-50%), especially when compared to non-MLL-rearranged (MLL-R) childhood ALL (EFS >85%). An important obstacle to developing new therapeutic approaches for this patient population is the lack of models that faithfully recapitulate the short latency and aggressiveness of this disease. Recently, whole genome sequencing of patient childhood MLL-R leukemias revealed that activating mutations of the proto-oncogenes involved in signaling, most prominently, N or K-RAS were found in nearly 50% of patients. Patients with these co-occurring mutations have an even poorer overall survival rate, indicating that a model harboring both mutations is of extreme interest.Here, we report the generation of a highly aggressive, serially transplantable B-ALL by the retroviral overexpression of activating N-RasG12D mutant in bone marrow of an inducible knock-in Mll-Af4 murine model that we have previously published. Recipient mice injected with Mll-Af4/N-RasG12Dpre-leukemic bone marrow cells developed an acute B-ALL (B220+CD43+IgM-) with short latency to development of disease (median 35 days). Furthermore, the resultant primary B-ALL was serially transplantable into sub-lethally irradiated recipients with accelerated latency to secondary and tertiary disease developing at a median of 20 and 12 days, respectively.As our model includes an activating mutation in N-Ras, we wanted to see if the cells would be sensitive to small molecule inhibitors of downstream effectors of Ras. Pre-leukemic Mll-Af4/N-RasG12D cells were sensitive to two different MEK inhibitors, Trametinib or PD901, in vitro. Furthermore, in vivo treatment of tertiary B-ALL mice with Trametinib showed significant reduction in leukemia burden after 7 days of treatment, as well as increase in survival, compared to vehicle controls. However, prolonged in vivo treatment with Trametinib eventually led to loss of sensitivity and development of B-ALL in our mouse model, suggesting that Trametinib alone is insufficient to prevent leukemia progression.As single agent MEK inhibition was insufficient to generate long-term durable responses, we conducted RNA-Sequencing of primary Mll-Af4/N-RasG12D leukemias to discover pathways amenable for therapeutic intervention. Gene set enrichment analysis suggested that targeting the DNA damage response (DDR) pathway as an attractive therapeutic opportunity. We were able to demonstrate an increased basal level of replicative stress in our Mll-Af4/NrasG12D pre-leukemic cells and sensitivity to small molecule inhibition of ATR, a master regulator of the G2 to M transition of cell cycle progression, with AZ20, a selective ATR inhibitor. In vitro and in vivo treatment with AZ20 led to increased leukemia cytotoxicity. However, similar to Trametinib treatment, tertiary B-ALL mice eventually succumbed to disease with prolonged AZ20 treatment in vivo.Since neither single agent MEK nor ATR inhibition could prevent leukemic progression in vivo, we tested the combination and found increased cytotoxicity and cell cycle arrest in vitro at concentrations well below the IC50, as compared to single agent treatment. In vivocombination treatment also demonstrated decreased leukemia burden and significant prolonged survival compared to either AZ20 or Trametinib alone. Lastly, we tested out the efficacy of combination therapy in human B-ALL patient derived xenograft harboring both MLL-AF4 and activating N-RASmutations. 250,000 human primary leukemic blasts were transplanted into non-irradiated immune-compromised mice and treated with vehicle, single agent, or the combination for 14 days. Similar to the results seen in our mouse model, combination treatment with Trametinib and AZ20 led to significant reductions in leukemic burden.In summary, our model of B-ALL faithfully recapitulates the short latency and aggressiveness of this disease and was predictive of response in human patient samples harboring MLL-AF4 and activating N-RAS mutations to small molecule inhibitor therapy to MEK and DDR pathways. In the future, this model can be used as a platform to not only better understand the molecular events governing and sustaining leukemogenesis, but also as a discovery platform for novel therapeutic combinations. DisclosuresArmstrong:Epizyme, Inc: Consultancy; Vitae Pharmaceuticals: Consultancy; Imago Biosciences: Consultancy; Janssen Pharmaceutical: Consultancy.

RANK-RANKL Mediated Bone Destruction in B-Cell Acute Lymphoblastic Leukemia

Survival rates for pediatric B-Cell Acute Lymphoblastic Leukemia (B-ALL) have improved dramatically over the past 40 years approaching a current long-term survival rate of 85%. However childhood B-ALL patients continue to confront co-morbidities and their long-term consequences. For example, osteopenia and osteoporosis associated fractures are a common complication of pediatric leukemia at diagnosis, during treatment and in long-term B-ALL survivors. The STeroid-associated Osteoporosis in the Pediatric Population (STOPP) study reported that at ALL diagnosis, 16% of children and adolescents present with bone pain, vertebral compression and low vertebral Bone Mineral Density (BMD) scores, with the greatest incidence of vertebral fractures (VF) seen in the first year following diagnosis (J Clin Endocrinol Metab. 2015, 100:3408-17). Glucocorticoid treatment further elevated fracture risk in this population. These data underscore the need to identify molecular mechanism by which leukemic cells contribute to bone loss, and provide targeted therapies to limit these effects.Our laboratory previously showed that Rag2-/- p53-/- Prkdcscid/scid triple mutant (TM) and p53-/- Prkdcscid/scid double mutant (DM) mice develop spontaneous B-ALL, but only TM animals exhibit dissemination of leukemic blasts to the leptomeninges of the CNS, a poor prognosis feature observed in pediatric and adult ALL patients. We observed that TM leukemic mice also displayed fragile vertebral bones. Using comparative transcriptome analysis, we found that RANKL (Receptor Activator of the Nuclear factor-kB Ligand), a Tumor Necrosis Factor (TNF) superfamily member ligand and a key regulator of B cell and osteoclast differentiation, was expressed at greater levels in TM compared to the DM leukemia cells. RANKL binds to its receptor RANK, which is expressed in osteoclast precursor cells. RANK-RANKL interaction induces signaling in the osteoclast precursors and drives their differentiation into mature bone resorbing osteoclasts (Proc. Natl. Acad. Sci. 1999, 96:3540-3545). Upon adoptive leukemia cell transfer into immune deficient mice, RANKL+ TM but not DM cells caused decreased vertebral trabecular bone density in the recipients. Treatment with the recombinant RANKL antagonist protein Osteoprotegerin (OPG-Fc) inhibited the growth and dissemination of RANKL+TM leukemic cells and attenuated bone destruction in the recipient mice. These data suggested that TM mouse leukemia cells cause bone loss in the absence of glucocorticoid or other chemotherapy agents.We then examined the potential role of RANKL in osteoporosis associated with human B-ALL. RANKL mRNA was expressed by a majority of primary human adult and pediatric B-ALL. To determine whether primary patient B-ALL can cause bone loss, we transplanted RANKL+ human B-ALL samples of multiple cytogenetic high-risk subgroups (Complex, hypo-diploid and Mixed Lineage Leukemia (MLL) rearranged) into NOD.SCID.gC-/-(NSG) recipient mice. Micro-CT imaging and bone density measures in the xenotransplant recipients revealed extensive vertebral trabecular bone destruction. Immuno-histological analysis of the human B-ALL recipient mice demonstrated extensive osteoporotic damage of the long bones and marked RANKL protein expression in the long bones of mice harboring extensive human B-ALL cell burden compared to NSG control mice. To determine whether RANKL-RANK interaction was required for the B-ALL mediated bone destruction, cohorts of NSG mice engrafted with human B-ALL were treated with recombinant OPG-Fc compared to a matched Fc control protein. OPG-Fc treatment did not attenuate leukemia cell expansion and bone marrow burden, but despite bulky disease, the treatment conferred robust protection from bone destruction suggesting that RANKL was a critical mediator of this clinical complication.Our data demonstrate a central role of the RANK-RANKL axis in B-ALL-mediated bone disease and identify an actionable therapeutic target to reduce acute and long-term morbidity. Denosumab, an anti-RANKL antibody has been approved for the treatment of bone metastasis by solid tumors and for post-menopausal osteoporosis. Our pre-clinical studies suggest that Denosumab and other agents that inhibit the RANK-RANKL pathway may be efficacious in patients with B-ALL associated bone degeneration. DisclosuresNo relevant conflicts of interest to declare.

Prognostic significance of P2RY8-CRLF2 and CRLF2 overexpression may vary across risk subgroups of childhood B-cell acute lymphoblastic leukemia.

The cytokine receptor-like factor 2 (CRLF2) gene plays an important role in early B-cell development. Aberrations in CRLF2 activate the JAK-STAT signaling pathway that contributes to B-cell acute lymphoblastic leukemia (B-ALL). The prognostic significance of CRLF2 overexpression and P2RY8-CRLF2 fusion in various B-ALL risk subgroups has not been well established. Two hundred seventy-one patients with newly diagnosed childhood B-ALL were enrolled from a Chinese population. The prevalence of CRLF2 overexpression, CRLF2-P2RY8 fusion, CRLF2 F232C mutation, and JAK2 and IL7R mutational status were analyzed, and the prognostic impact of CRLF2 overexpression and P2RY8-CRLF2 on B-ALL was evaluated by assessing their influence on overall survival and event-free survival. CRLF2 overexpression and P2RY8-CRLF2 were found in 19% and 10%, respectively, in the whole cohort. No correlation between CRLF2 overexpression and P2RY8-CRLF2 was observed. CRLF2 F322C and IL7R mutations were not detected in B-ALL cases overexpressing CRLF2, and no JAK2 mutations were found in the whole cohort either. The results showed that CRLF2 overexpression and P2RY8-CRLF2 were associated with a poor outcome in unselected B-ALL. Moreover, in an intermediate risk B-ALL subgroup P2RY8-CRLF2 was correlated with worse survival, whereas in high- and low-risk subgroups, CRLF2 overexpression predicted a poor outcome. Our findings suggest that P2RY8-CRLF2 is an independent prognostic indicator in intermediate risk B-ALL, while CRLF2 overexpression is correlated with an inferior outcome in high- or low-risk B-ALL. Our study demonstrates that the impact of P2RY8-CRLF2 and CRLF2 overexpression on B-ALL survival may differ across risk subgroups. © 2016 Wiley Periodicals, Inc.

MiR-pharmacogenetics of methotrexate in childhood B-cell acute lymphoblastic leukemia.

Methotrexate (MTX), the key drug in childhood B-cell acute lymphoblastic leukemia (B-ALL) therapy, often causes toxicity. An association between genetic variants in MTX transport genes and toxicity has been found. It is known that these transporters are regulated by microRNAs (miRNAs), and miRNA single nucleotide polymorphisms (SNPs) interfere with miRNA levels or function. With regard to B-cell ALL, we have previously found rs56103835 in miR-323b that targets ABCC4 associated with MTX plasma levels. Despite these evidences and that nowadays a large amount of new miRNAs have been annotated, studies of miRNA polymorphisms and MTX toxicity are almost absent. Therefore, the aim of this study was to determine whether there are other variants in miRNAs associated with MTX levels. Blood samples of 167 Spanish patients with pediatric B-cell ALL treated with the LAL-SHOP protocol were analyzed. We selected all the SNPs described in pre-miRNAs with a minor allele frequency more than 1% (213 SNPs in 206 miRNAs) that could regulate MTX transporters because the miRNAs that target MTX transporter genes are not completely defined. Genotyping was performed with VeraCode GoldenGate platform. Among the most significant results, we found rs56292801 in miR-5189, rs4909237 in miR-595, and rs78790512 in miR-6083 to be associated with MTX plasma levels. These miRNAs were predicted, in silico, to regulate genes involved in MTX uptake: SLC46A1, SLC19A1, and SLCO1A2. In this study, we detected three SNPs in miR-5189, miR-595, and miR-6083 that might affect SLC46A1, SLC19A1, and SLCO1A2 MTX transport gene regulation and could affect MTX levels in patients with pediatric B-cell ALL.

Expression of CD13 in Philadelphia chromosome-negative B-cell acute lymphoblastic leukemia and its clinical significance

Objective To analyze the immunophenotypes of B-cell acute lymphoblastic leukemia(B-ALL) and correlation between the expression of CD13 and clinical, hematologic and biological parameters in patients with Philadelphia chromosome-negative(Ph-) B-ALL. Methods Multiparameter flow cytometry was used to analyze the immunophenotypes of the bone marrow blasts in 111 cases of adult and 32 cases of childhood B-ALL. Results The positive rates of CD13 and CD33 in childhood B-ALL patients were no differences compared with those in adult patients [31.3%(10/32) vs 19.8%(22/111), 18.8%(6/32) vs 29.7%(33/111), both P>0.05]. In de novo Ph- B-ALL, the frequency of CD13 in childhood patients was higher than that in adult patients [32.3% (10/31) vs 9.4% (6/64), P 0.05). Difference on time of relapse was found between CD13+ and CD13- groups in childhood Ph- B-ALL patients[(111±16) d vs (683±57) d, P<0.000 1]. The time of relapse between childhood and adult CD13+ Ph- B-ALL cases had statistical difference [(111±16) d vs (319±16) d,P<0.000 1]. Conclusions The positive rate of CD13 is higher in childhood Ph- B-ALL patients than that in adult Ph- B-ALL patients. CD13+ Ph- B-ALL patients, in particular, childhood Ph- CD13+ patients are more likely to relapse. Key words: Leukemia, B lymphoblastic, acute; Multiparameter flow cytometry; Immunophenotyping; CD13

Family-based exome-wide assessment of maternal genetic effects on susceptibility to childhood B-cell acute lymphoblastic leukemia in hispanics.

Children of Hispanic ancestry have a higher incidence of acute lymphoblastic leukemia (ALL) compared with other ethnic groups, but to the authors' knowledge, the genetic basis for these racial disparities remain incompletely understood. Genome-wide association studies of childhood ALL to date have focused on inherited genetic effects; however, maternal genetic effects (the role of the maternal genotype on phenotype development in the offspring) also may play a role in ALL susceptibility. The authors conducted a family-based exome-wide association study of maternal genetic effects among Hispanics with childhood B-cell ALL using the Illumina Infinium HumanExome BeadChip. A discovery cohort of 312 Guatemalan and Hispanic American families and an independent replication cohort of 152 Hispanic American families were used. Three maternal single-nucleotide polymorphisms (SNPs) approached the study threshold for significance after correction for multiple testing (P<1.0 × 10-6 ): MTL5 rs12365708 (testis expressed metallothionein-like protein [tesmin]) (relative risk [RR], 2.62; 95% confidence interval [95% CI], 1.61-4.27 [P = 1.8 × 10-5 ]); ALKBH1 rs6494 (AlkB homolog 1, histone H2A dioxygenase) (RR, 3.77; 95% CI, 1.84-7.74 [P = 3.7 × 10-5 ]); and NEUROG3 rs4536103 (neurogenin 3) (RR, 1.75; 95% CI, 1.30-2.37 [P = 1.2 × 10-4 ]). Although effect sizes were similar, these SNPs were not nominally significant in the replication cohort in the current study. In a meta-analysis comprised of the discovery cohort and the replication cohort, these SNPs were still not found to be statistically significant after correction for multiple comparisons (rs12365708: pooled RR, 2.27 [95% CI, 1.48-3.50], P = 1.99 × 10-4 ; rs6494: pooled RR, 2.31 [95% CI, 1.38-3.85], P = .001; and rs4536103: pooled RR, 1.67 [95% CI, 1.29-2.16] P = 9.23 × 10-5 ). In what to the authors' knowledge is the first family-based based exome-wide association study to investigate maternal genotype effects associated with childhood ALL, the results did not implicate a strong role of maternal genotype on disease risk among Hispanics; however, 3 maternal SNPs were identified that may play a modest role in susceptibility. Cancer 2016;122:3697-704. © 2016 American Cancer Society.

Application of Combined Detection of Fusion Gene and BIOMED-2 Standardized Ig Gene Rearrangement System in Childhood B-cell Acute Lymphoblastic Leukemia

To explore the application of combined detection of fusion gene and BIOMED-2 standardized immunoglobulin (Ig) gene rearrangement system in diagnosis and treatment of children with acute lymphoblastic leukemia (ALL). Multiplex-PCR amplifications and RQ-PCR of RNA/DNA were performed using ALL fusion gene detection kit and BIOMED-2 primer. The Ig gene rearrangements were analyzed by using PCR fragment analysis system. Out of 251 children with B-ALL, 77 cases were TEL-AML1(+) , 28 cases were E2A-PBX1(+) , 10 cases were MLL-AF4(+) , 11 cases were BCR-ABL(+) , the total positive rate was 50.2%, 82.5% showed IgH VH-JH rearrangement, 53.4% showed IgK rearrangement. The positive rate of combined detection of fusion gene and gene rearrangement was 99%. E2A-PBX1(+) and MLL-AF4(+) with IgK(+) gene rearrangement group was compared with negative control group, the difference was statistically significant (P < 0.001 or P = 0.005); 105 ALL fusion gene positive cases had been detected by fluorescence in situ hybridization (FISH) simultaneously, the accordance rate of fusion gene and FISH was more than 94%. The combined detection of ALL fusion gene and BIOMED-2 standardized clonality analysis system can improve the positive detected rate of B-ALL dramatically, and make the grouping of disease prognosis more accurately; this combined detection is a more faster and sensitive method than FISH.

Impact of mTOR expression on clinical outcome in paediatric patients with B-cell acute lymphoblastic leukaemia - preliminary report.

Aim of the studyTo characterise expression of mTOR (mammalian target of rapamycin) in childhood B-cell acute lymphoblastic leukaemia (ALL), and to evaluate a possible link between mTOR and clinical characteristics.Material and methodsThe examined group consisted of 21 consecutive patients, aged 1–18 years, diagnosed with B-cell ALL in 2010, and 10 relapsed B-cell ALL patients diagnosed for the first time between 2009 and 2011, who developed relapse before 2014. All subjects were treated in the Department of Paediatric Haematology and Oncology of the Medical University of Warsaw according to the ALL-IC BFM 2002 Protocol. We evaluated mTOR and phospho-mTOR expression by immunohistochemistry using rabbit monoclonal antibodies.ResultsmTOR expression was found to be significantly associated with the risk of relapse and was more frequent in ALL recurrence. No significant relationship was detected between mTOR expression and other features of high-risk disease in paediatric ALL.ConclusionsmTOR activity could be considered a high-risk feature in paediatric B-cell ALL. Expression of mTOR kinase is observed remarkably more frequently in disease recurrence than at first diagnosis, indicating higher proliferative and survival potential of leukaemic cells in relapse. Routine analysis of mTOR activity could be performed to select patients that may potentially benefit from mTOR inhibitors (MTI) treatment.

Autophagy Collaborates with Ubiquitination to Down-Regulate Oncoprotein E2A/Pbx1 in B Cell Acute Lymphoblastic Leukemia

Background: B cell acute lymphoblastic leukemia (B-ALL) accounts for the most cancer incidences in children. The t(1;19) translocation leukemia accounts for a quarter of pre-B ALL and up to 5% of all ALL patients, in which the transcriptional activator E2A and homeobox pre-B-cell leukemia transcription factor 1 (PBX1) fuses, resulting in expression of the chimeric transcription factor E2A/PBX1. E2A/PBX1 has been proved to be an oncogene and could induce malignant transformation.Methods: (1) Childhood B-ALL patients were collected and the stem/progenitor cells (CD34+CD38-) and leukemia cells (CD19+) were sorted with BD FACS Aria III. The autophagy level in these cells was measured by real-time Q-PCR, including gene expression of Beclin1, Atg7, Atg5, LC3 and p62. Normal bone marrow cells from healthy donors were used as control. (2) E2A/PBX1 fusion gene positive pre-B ALL 697 cells were used to establish leukemia mouse model and the autophagy activity in the mice was enhanced by administration of rapamycin. Mice were sacrificed three weeks post treatment, leukemia phenotype was then identified and E2A/PBX1 oncoprotein of liver was detected by western blotting. (3) Autophagy and ubiquitination were manipulated with inhibitors or starvation in 697 cells and the degradation mechanism of E2A/PBX1 was explored. Co-localization of E2A/PBX1-LC3 and E2A/PBX1-Ub was observed by confocal microscopy and quantified by Amnis image flow cytometry.Results: (1) B-ALL primary cells from childhood patients show down-regulated level of autophagy. (2) The NOD-SCID mouse model study shows that activating autophagy of mice by rapamycin improved the survival of leukemia animals, prevented leukemiagenesis by inhibition on the transplanted leukemia cells (examined by blood cell counting, liver HE staining and expression of CD 45, 10, 19 from transplanted human 697 cells by flow cytometry), promoted the degradation of oncoprotein E2A/PBX1 (by Western blotting)), and more importantly, restored hematopoietic stem cells (LSKCD34- cell number detected by flow cytometry). (3) The ALL 697 cell line study shows that activation of autophagy by rapamycin and starvation could down-regulate E2A/PBX1 expression detected by flow cytometry and western blotting. The confocal microscopic results show co-localization of E2A/PBX1 with autophagy marker GFP-LC3 in both rapamycin and starvation treatment groups. To confirm the degradation mechanism, autophagy inhibitor (3-MA or Baf-A1) and ubiquitin-proteasome inhibitor (MG132) were used to treat 697 cells. The results show that inhibition of autophagy in the early stage by 3-MA fails to degrade E2A/PBX1 in 697 cells, but ubiquitination also contributes to the degradation of E2A/PBX1. Quantitative analysis shows increased co-localization percentage of E2A/PBX1-LC3 in rapamycin and starvation treatment groups and increased co-localization of E2A/PBX1 with Ubiquitin in starvation group; but MG132 treatment inhibited the co-localization of E2A/PBX1-Ub induced by starvation, indicating a collaborative role between autophagy and ubiquitination in the degradation of E2A/PBX1.Conclusions: B-ALL primary cells from patients show low autophagy activity; Autophagy activation fights against B-ALL by inhibition on transplanted leukemia cells, degradation of oncoprotein E2A/Pbx1 and restoration of hematopoietic stem cells in the NOD-SCID B-ALL mouse model; autophagy collaborates with ubiquitination in the degradation of E2A/PBX1 in the 697 cells, thereby proposing a novel strategy for targeted therapy on childhood B-ALL. DisclosuresNo relevant conflicts of interest to declare.

Identification and characterization of OSTL (RNF217) encoding a RING-IBR-RING protein adjacent to a translocation breakpoint involving ETV6 in childhood ALL.

Genomic aberrations involving ETV6 on band 12p13 are amongst the most common chromosomal abnormalities in human leukemia. The translocation t(6;12)(q23;13) in a childhood B-cell acute lymphoblastic leukemia (ALL) cell line fuses ETV6 with the putative long non-coding RNA gene STL. Linking STL properties to leukemia has so far been difficult. Here, we describe a novel gene, OSTL (annotated as RNF217 in Genbank), which shares the first exon and a CpG island with STL but is transcribed in the opposite direction. Human RNF217 codes for a highly conserved RING finger protein and is mainly expressed in testis and skeletal muscle with different splice variants. RNF217 shows regulated splicing in B cell development, and is expressed in a number of human B cell leukemia cell lines, primary human chronic myeloid leukemia, acute myeloid leukemia with normal karyotype and acute T-ALL samples. Using a yeast two-hybrid screen, we identified the anti-apoptotic protein HAX1 to interact with RNF217. This interaction could be mapped to the C-terminal RING finger motif of RNF217. We propose that some of the recurring aberrations involving 6q might deregulate the expression of RNF217 and result in imbalanced apoptosis signalling via HAX1, promoting leukemia development.

Repetitive genomic elements and overall DNA methylation changes in acute myeloid and childhood B-cell lymphoblastic leukemia patients.

Aberrant epigenetic regulation is a hallmark of neoplastic cells. Increased DNA methylation of individual genes' promoter regions and decreases in overall DNA methylation level are both generally observed in cancer. In solid tumors, this global DNA hypomethylation is related to reduced methylation of repeated DNA elements (REs) and contributes to genome instability. The aim of the present study was to assess methylation level of LINE-1 and ALU REs and total 5-methylcytosine (5metC) content in adult acute myeloid leukemia (AML) (n = 58), childhood B-cell acute lymphoblastic leukemia (ALL) (n = 32), as the most frequent acute leukemias in two age categories and in normal adult bone marrow and children's blood samples. DNA pyrosequencing and ELISA assays were used, respectively. Global DNA hypomethylation was not observed in leukemia patients. Results revealed higher DNA methylation of LINE-1 in AML and ALL samples compared to corresponding normal controls. Elevated methylation of ALU and overall 5metC level were also observed in B-cell ALL patients. Differences of REs and global DNA methylation between AML cytogenetic-risk groups were observed, with the lowest methylation levels in intermediate-risk/cytogenetically normal patients. B-cell ALL is characterized by the highest DNA methylation level compared to AML and controls and overall DNA methylation is correlated with leukocyte count.

TET2 Promoter DNA Methylation and Expression Analysis in Pediatric B-Cell Acute Lymphoblastic Leukemia

TET2 is a novel tumor suppressor gene involved in several hematological malignancies of myeloid and lymphoid origin. Besides loss-of-function mutations and deletions, hypermethylation of the CpG island at the TET2 promoter was found in human cancer. Previous analysis revealed no TET2 mutations in acute lymphoblastic leukemia (ALL). Since the TET2 promoter methylation status in pediatric ALL has not been reported, the aim of the present study was to determine if promoter hypermethylation may be a mechanism of TET2 inactivation in a group of pediatric ALL cases. Methylation of TET2 promoter region in one (1/45) ALL B-common patient was detected by methylation specific polymerase chain reaction (PCR) and subsequently analyzed by bisulfite sequencing. We found no correlation between promoter methylation and gene expression, measured by quantitative reverse transcriptase-PCR, however the level of TET2 expression in ALL group was significantly decreased compared to children’s normal peripheral blood mononuclear cells and isolated B-cells. TET2 promoter hypermethylation seems to have limited clinical relevance in childhood B-cell ALL due to its low frequency.

Genome-Wide DNA Methylation Analysis Reveals Biological and Clinical Insights In Relapsed Childhood Acute Lymphoblastic Leukemia: A Report From The COG ALL Target Project

▪Although survival of children with B-cell acute lymphoblastic leukemia (B-ALL) has improved substantially over time, 15% to 20% of patients will relapse, and most of those who experience a bone marrow relapse will die. A better understanding of genetic and epigenetic aberrations in relapsed ALL will facilitate new strategies for risk stratification and targeted therapy. In this collaborative study with the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) project, we performed high resolution genome-wide DNA methylation profiling using the HELP (HpaII tiny fragment Enrichment by Ligation-mediated PCR) array on a total of 178 (110 diagnosis, 68 relapse) leukemia samples from 111 patients with childhood B-ALL enrolled on the Children's Oncology Group (COG) clinical trials who experienced relapsed, and 12 normal preB samples isolated from the bone marrows of 12 healthy individuals. The HELP array covers 117,521 CpG sites, annotated to ∼22,000 gene promoters. For eight diagnosis/relapse pairs, base-pair resolution DNA methylation using the eRRBS (enhanced Reduced Representation Bisulfite Sequencing) method was also performed on Illumina HiSeq2000.The median relapse time for the 111 patients was 21.8 months (range 2.1 to 56.2). Unsupervised clustering analysis using the HELP data revealed seven clusters: one cluster contained only the 12 normal preB samples; four clusters were enriched with MLLr, ETV6/RUNX1, Trisomy 4+10, and TCF3/PBX1 samples, respectively. The sixth cluster was not enriched for specific cytogenetic cases, but interestingly, all cases in this cluster were NCI High Risk (age>10 years or WBC>=50,000; p<0.0001, Fisher's Exact test) while the seventh cluster has a mixture of other cases.Supervised analysis of HELP profiles between paired relapse/diagnosis samples (n=67) revealed a markedly aberrant DNA methylation signature (1011 probesets, 888 genes, FDR<0.01 and methylation difference dx >25%, paired t-test), with 70% of the genes hyper- and 30% hypo-methylated in relapse samples. Using a Bayesian predictor and leave-one-out cross validation, this methylation signature could predict a sample as diagnosis or relapse with 95.3% accuracy. When comparing early (<36 months; n=50) versus late relapses (>=36 months; n=18), we detected a profound hypermethylation signature in early relapse (96.6% of the 610 probesets, 544 genes, FDR<0.01, dx >25%).Finally, we identified 1800 probesets (1658 genes) as differentially methylated within all cytogenetic subtypes described above compared to the normal preB samples (Dunnett's test with normal preB as reference, FDR<0.01, dx>25%). Again the majority (70%) of those genes were hypermethylated in relapse as compared to diagnostic and normal preB.The base-pair resolution and more comprehensive eRRBS methylation analysis for the eight pairs of samples identified 39,679 CpG sites as differentially methylated (dx >25%, FDR<0.01), with 78.2% CpG sites hyper- and 21.2% hypo-methylated in relapse samples. Remarkably, the hypermethylated CpGs are primarily in promoter regions (50%, defined as +/-1kb to TSS), followed by intergenic (26%), then intragenic (14%), and exonic (10%) regions. In contrast, the hypomethylated CpGs are mainly in intragenic (48%), followed by intergenic (31%), exonic (14%) and promoter (7%) regions. The hypermethylated CpGs were mainly in CpG islands (86%) or CpG shores (10%), while hypomethylated CpGs were not (CpG islands: 8%, CpG shores: 27%). We further identified 3040 differentially methylated regions (DMRs) with a median size 426 bp. 78.4% of those DMRs were hyper- (1362 gene promoters) and 21.6% hypo-methylated (98 promoters) in relapse compared to diagnostic samples. Gene set enrichment and Ingenuity pathway analysis showed epigenetically disrupted pathways that are highly involved in cell signaling, and embryonic and organismal development.Taken together, our genome-wide high resolution DNA methylation analysis on a large cohort of relapsed childhood B-ALL from the COG trial identified unique methylation signatures that correlated with relapse and with specific genetic subsets. Those methylation signatures featured prevailing promoter hypermethylation and to a lesser extent, intrageneic hypomethylation. Epigenetically dysregulated gene networks in those relapse samples involved cell signaling, and embryonic and organismal development. Disclosures:No relevant conflicts of interest to declare.

Variation at 10p12.2 and 10p14 influences risk of childhood B-cell acute lymphoblastic leukemia and phenotype

AbstractAcute lymphoblastic leukemia (ALL) is the major pediatric cancer diagnosed in economically developed countries with B-cell precursor (BCP)-ALL, accounting for approximately 70% of ALL. Recent genome-wide association studies (GWAS) have provided the first unambiguous evidence for common inherited susceptibility to BCP-ALL, identifying susceptibility loci at 7p12.2, 9p21.3, 10q21.2, and 14q11.2. To identify additional BCP-ALL susceptibility loci, we conducted a GWAS and performed a meta-analysis with a published GWAS totaling 1658 cases and 4723 controls, with validation in 1449 cases and 1488 controls. Combined analysis identified novel loci mapping to 10p12.2 (rs10828317, odds ratio [OR] = 1.23; P = 2.30 × 10−9) and 10p14 marked by rs3824662 (OR = 1.31; P = 8.62 × 10−12). The single nucleotide polymorphism rs10828317 is responsible for the N215S polymorphism in exon 7 of PIP4K2A, and rs3824662 localizes to intron 3 of the transcription factor and putative tumor suppressor gene GATA3. The rs10828317 association was shown to be specifically associated with hyperdiploid ALL, whereas the rs3824662-associated risk was confined to nonhyperdiploid non–TEL-AML1 + ALL. The risk allele of rs3824662 was correlated with older age at diagnosis (P < .001) and significantly worse event-free survivorship (P < .0001). These findings provide further insights into the genetic and biological basis of inherited genetic susceptibility to BCP-ALL and the influence of constitutional genotype on disease development.Key Points

PAX5 alteration-associated gene-expression signatures in B-cell acute lymphoblastic leukemia

The paired box domain gene 5 (PAX5) is frequently altered in both childhood and adult B-cell acute lymphoblastic leukemia, and takes part in leukemogenesis. We analyzed data from the database of Gene Expression Omnibus (accession number: GSE11877) using bioinformatical and statistical methods. The results showed that cases of PAX5 alteration can cluster using unsupervised clustering algorithms, and one gene, zinc and ring finger 1 (ZNRF1), was characterized and validated by quantitative RT-PCR. ZNRF1 may be associated with leukemogenesis of ALL with PAX5 alteration.

Imbalances of Chromosome 21 in MDS/AML Include Cryptic Deletions but NOT RUNX1 Amplifications.

AbstractAbstract 2561We present the results of a study demonstrating that the genome profile of RUNX1 in MDS/AML is characterised by hitherto unreported partial deletions and absence of amplifications. This is in stark contrast to reports of chromosome 21 amplifications in ALL. We speculate that the absence of RUNX1 deletions results from them being well below a size detectable by commercial FISH probes.Extra chromosome 21 is the second most common acquired trisomy after (+) 8 in adult myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). It is rarely observed as sole abnormality but seen as part of complex karyotype in some 3–7% of the AML (Atlas of Genetics and Cytogenetics in Oncology and Haematology, http://atlasgeneticsoncology.org). Although the gene(s) in trisomy 21 associated with leukemia are unknown, the 21q22 region appears to be critical since it houses the RUNX1 gene. Multiple amplified copies of the RUNX1 carried by marker chromosomes, such as iAML21, are described in both acute lymphoblastic leukemia (ALL) and AML. A common 5.1 Mb amplification containing the RUNX1, miR-802 and genes mapping to the Down syndrome critical region identified in 91 children with iAML21, was shown to be the likely initiating event in this rare form of childhood B-cell ALL (Rand et al., Blood, 2011). In contrast, recent studies of AML in a Down syndrome and a constitutionally normal individual showed lack of RUNX1, ETS2 and ERG involvement (Canzonetta et al., BJH, 2012). Here we present 16 MDS/AML cases with imbalances of chromosome 21 identified by genomic array screening from a cohort of 83 cases. Whole genome screening (aCGH) was performed on presentation samples of MDS /AML and de novo AML cases using an oligonucleotide array platform (Agilent) at 60K, 244K, 400K and 1M density. G banding and FISH analysis were also successfully performed.Gain of an extra copy (trisomy) of chromosome 21 (+21) was found in 9 patients, all but one with complex karyotypes. In 2 AMLs high level amplifications were detected at 21q22, which involved the ETS2 and ERG but not the RUNX1 sequences. While several commercially available RUNX1 FISH probes showed gene multiple signals, custom FISH probes covering the relevant regions confirmed that the amplifications excluded the RUNX1 but affected both EST2 and ERG thus rendering the commercial probes unfit to assess CNA in this genome area.In another two cases with trisomy 12, cryptic loss of 43Kb and 98Kb resp. within the RUNX1 sequences was detected and confirmed by FISH. Furthermore, similar deletions within the 21q22.12 were also found in another 7 cases all of which had diploid set of chromosome 21 but had multiple changes at G banding level and high TGA score. These RUNX1 deletions were variable in size, ranging from 98Kb to 2.7Mb. Although our observations excluded clinical correlations it is note worthy that most of the patients with RUNX1 loss have not achieved complete cytogenetic remission.These findings suggest role for the RUNX1 loss as indicator of progressive disease and provide a novel insight into pathogenesis of MDS/AML. Disclosures:No relevant conflicts of interest to declare.

DNA Methylation and miRNA Expression Profiling in Childhood B-Cell Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is the most common cancer in children in the modern world. Recent efforts in characterizing the genetic contribution to this disease through uncovering gene mutations, deletions and structural variation by genome-scale methods have only accounted for a modest proportion of children with ALL. This suggests that either further genetic contributions to ALL have yet to be characterized or other factors, such as epigenetic aberrations are involved. A number of DNA methylation and miRNA profiling studies have investigated the role of both in childhood ALL. Here, we review these profiling efforts, summarize their major findings and speculate as to what the future may hold.

Replication analysis confirms the association of ARID5B with childhood B-cell acute lymphoblastic leukemia

Although childhood acute lymphoblastic leukemia is the most common pediatric cancer, its etiology remains poorly understood. In an attempt to replicate the findings of 2 recent genome-wide association studies in a French-Canadian cohort, we confirmed the association of 5 SNPs [rs7073837 (P=4.2 x 10(-4)), rs10994982 (P=3.8 x 10(-4)), rs10740055 (P=1.6 x 10(-5)), rs10821936 (P=1.7 x 10(-7)) and rs7089424 (P=3.6 x 10(-7))] in the ARID5B gene with childhood acute lymphoblastic leukemia. We also confirmed a selective effect for B-cell acute lymphoblastic leukemia with hyperdiploidy and report a putative gender-specific effect of ARID5B SNPs on acute lymphoblastic leukemia risk in males. This study provides a strong rationale for more detailed analysis to identify the causal variants at this locus and to better understand the overall functional contribution of ARID5B to childhood acute lymphoblastic leukemia susceptibility.

Outcome of childhood B-cell non-Hodgkin lymphoma and B-cell acute lymphoblastic leukemia treated with the Tokyo Children's Cancer Study Group NHL B9604 protocol

From June 1996 to January 2001, 91 patients with B-cell non-Hodgkin lymphoma or B-cell acute lymphoblastic leukemia up to 18 years of age were enrolled in Tokyo Children's Cancer Study Group (TCCSG) NHL B9604 protocol study. Five-day intensive chemotherapy courses including high-dose methotrexate and high-dose cyclophosphamide were used for localized disease (Groups A and B). High-dose cytarabine was added for advanced disease (Groups C and D). Fifteen patients experienced an adverse event. There were three induction failures, eight relapses (three local, four bone marrow (BM), one BM + local), two toxic deaths and two second malignant neoplasm. Event-free survival at 6 years in Group D and in all patients was 82.4% ± 9.2% and 81.9% ± 4.4%, respectively. The TCCSG NHL B9604 protocol achieved an excellent treatment outcome especially in patients with the most advanced disease (Group D: high BM blast cell burden and/or central nervous system involvement).