Childhood B-precursor Acute Lymphoblastic Leukemia Research Articles

The NSD2 p.E1099K Mutation Is Enriched at Relapse and Confers Drug Resistance in a Cell Context–Dependent Manner in Pediatric Acute Lymphoblastic Leukemia

The NSD2 p.E1099K (EK) mutation is observed in 10% of acute lymphoblastic leukemia (ALL) samples with enrichment at relapse indicating a role in clonal evolution and drug resistance. To discover mechanisms that mediate clonal expansion, we engineered B-precursor ALL (B-ALL) cell lines (Reh, 697) to overexpress wildtype (WT) and EK NSD2, but observed no differences in proliferation, clonal growth, or chemosensitivity. To address whether NSD2 EK acts collaboratively with other pathways, we used short hairpin RNAs to knockdown expression of NSD2 in B-ALL cell lines heterozygous for NSD2 EK (RS4;11, RCH-ACV, SEM). Knockdown resulted in decreased proliferation in all lines, decreased clonal growth in RCH-ACV, and increased sensitivity to cytotoxic chemotherapeutic agents, although the pattern of drug sensitivity varied among cell lines implying that the oncogenic properties of NSD2 mutations are likely cell context specific and rely on cooperative pathways. Knockdown of both Type II and REIIBP EK isoforms had a greater impact than knockdown of Type II alone, suggesting that both SET containing EK isoforms contribute to phenotypic changes driving relapse. Furthermore, in vivo models using both cell lines and patient samples revealed dramatically enhanced proliferation of NSD2 EK compared with WT and reduced sensitivity to 6-mercaptopurine in the relapse sample relative to diagnosis. Finally, EK-mediated changes in chromatin state and transcriptional output differed dramatically among cell lines further supporting a cell context-specific role of NSD2 EK. These results demonstrate a unique role of NSD2 EK in mediating clonal fitness through pleiotropic mechanisms dependent on the genetic and epigenetic landscape. IMPLICATIONS: NSD2 EK mutation leads to drug resistance and a clonal advantage in childhood B-ALL.

ETV6/RUNX1-positive childhood acute lymphoblastic leukemia in China: excellent prognosis with improved BFM protocol

BackgroundIn childhood B-precursor acute lymphoblastic leukemia (B-ALL), the ETV6/RUNX1 fusion transcript is considered to have an excellent outcome. However, few studies of children with ETV6/RUNX1-positive ALL from China have been conducted. It is largely unknown whether clinical outcomes for patients with this genotype and important factors that influence such outcomes are similar to those reported in other countries. Therefore, it is important to analyze the outcomes of children with ETV6/RUNX1-positive ALL treated at our institution with the aim of identifying significant prognostic variables in a Chinese population.MethodsWe studied the clinical characteristics and treatment outcomes for 77 pediatric patients diagnosed with ETV6/RUNX1-positive ALL between 2005 and 2015 at our institution.ResultsThe 5-year event-free survival (EFS) and the disease-free survival (DFS) were reported to be 90% ± 3% and 96% ± 3% respectively. Two patients had a relapse at a median of 42 months from diagnosis and the 5-year cumulative incidence of relapse was 2.1%. Despite intensive chemotherapy or allogeneic hematopoietic cell transplantation, the 2 relapsed patients succumbed to the disease progression and the 5-year overall survival (OS) was 97% ± 2%. Multivariate analysis for EFS revealed that the minimal residual disease (MRD) ≥10− 3 on Day + 33 negatively affected the outcome.ConclusionsIn conclusion, patients with ETV6/RUNX1 fusion transcript can achieve a high rate of complete remission and the long-term curative effect was excellent under risk-stratified treatment. In case of relapse, the MRD level at the end of induction therapy should be taken into consideration while deciding the appropriate chemotherapy dosage.

Induction of Apoptosis in Cancer Cells of pre-B ALL Patients after Exposure to Platelets, Platelet-Derived Microparticles and Soluble CD40 Ligand.

Objective The in vitro treatment of tumor cells with platelet (Plt) causes inhibition of tumor cell growth, although mechanism of this effect is not clear yet. Induction of apoptosis has been proposed as a mechanism of Plt effects on tumor cells. The purpose of this study was to clarify the role of Plts and Plt-derived components in the induction of apoptosis in the blood mononuclear cells of patients with leukemia. Materials and Methods In this experimental study, peripheral blood mononuclear cells (PBMCs) were isolated from whole blood of five patients with childhood B-precursor acute lymphoblastic leukemia (pre-B ALL) and encountered with Plts, Plt-derived microparticles (Plt-MPs) as well as purified soluble CD40L (sCD40L). After 48 hours of co-culture, the anti-cancer activity of the aforementioned factors was surveyed using examination of apoptosis markers of the cells including active caspase-3 and CD95 using ELISA and flow cytometer techniques, respectively. Additionally, staining of the cells with 7-Aminoactinomycin D (7-AAD) was evaluated by flow cytometer technique. Trypan blue exclusion test and WST-1 method were also used to compare the death/survival status of the cells. Results Levels of CD95 and caspase-3 were significantly increased in the all treated groups (P<0.05). On the other hand, trypan blue, 7-AAD and WST-1 methods showed significantly lower number of the live cells in the treated groups (P<0.05). ConclusionThis study can show the ability of Plts, Plt-MPs and sCD40L for the induction of apoptosis in PBMCs of pre-B-ALL patients. Further studies are necessary to elucidate the different effects of platelets on cancer cells in vitro and in vivo.

Clinical significance of minimal residual disease in peripheral blood on day 8 induction in childhood B-precursor acute lymphoblastic leukemia: Report from Children’s Cancer Hospital in Egypt.

e21008 Background: Minimal residual disease (MRD) in childhood acute lymphoblastic leukemia (ALL) during the early stages of therapy is statistically significant indicator of outcome. Aim: To study...

Obesity is associated with residual leukemia following induction therapy for childhood B-precursor acute lymphoblastic leukemia

AbstractObesity is associated with poorer event-free survival (EFS) in pediatric acute lymphoblastic leukemia (ALL). Persistent minimal residual disease (MRD) in the bone marrow as measured by multidimensional flow cytometry (MDF) is a key early prognostic indicator and is strongly associated with EFS. We therefore hypothesized that obesity during induction would be associated with positive end-of-induction MRD (≥0.01%). We analyzed MDF of end-induction bone marrow samples from a historical cohort of 198 children newly diagnosed with B-precursor ALL (BP-ALL) and treated with Children’s Oncology Group induction regimens. We assessed the influence of body mass index on risk for positive end-induction MRD in the bone marrow. In our cohort of BP-ALL, 30 children (15.2%) were overweight and 41 (20.7%) were obese at diagnosis. Independent of established predictors of treatment response, obesity during induction was associated with significantly greater risk for persistent MRD (odds ratio, 2.57; 95% confidence interval, 1.19 to 5.54; P = .016). Obesity and overweight were associated with poorer EFS irrespective of end-induction MRD (P = .012). Obese children with newly diagnosed BP-ALL are at increased risk for positive end-induction MRD and poorer EFS.

Bioinformatics analysis of gene expression profiles in childhood B-precursor acute lymphoblastic leukemia

ObjectivesTo explore the underlying molecular mechanisms of childhood B-precursor acute lymphoblastic leukemia (ALL) by bioinformatics analysis and find potential targets for childhood ALL diagnosis and treatment.MethodsGene expression profile GSE28460 was downloaded from the Gene Expression Omnibus, including 49 diagnostic and relapse bone marrow samples with childhood B-precursor ALL. The differentially expressed genes (DEGs) were identified by paired t-test. Pathway enrichment analysis of DEGs and transcription factors (TFs) enrichment analysis were performed, followed by construction of co-expressed, DEGs, and susceptibility gene protein–protein interaction (PPI) network. Based on these three networks, relevant regulatory network modules and the important DEGs in the modules were identified.ResultsTotal of 947 DEGs were identified. Up-regulated DEGs enriched 20 pathways including cell cycle, and down-regulated DEGs significantly enriched Jak-STAT signaling pathways. CDK1 and BRCA1 were found to have more hubs in both co-expressed network and PPI network. Besides, total of five modules in INTS10, MCM, BRCA1, GYPA, and VCAN1 families were identified and a pathway of INTS10-INTS6-POLR2A-MAGI2 was selected.ConclusionCell cycle and Jak-STAT signaling pathway were closely associated with relapse of childhood B-precursor ALL. The DEGs, such as PTTG1, PIK3CA, CDK1, and BRCA1 may be the potential targets for childhood ALL diagnosis and treatment.

Focused screening of a panel of cancer‐related genetic polymorphisms reveals new susceptibility loci for pediatric acute lymphoblastic leukemia

A genetic predisposition to acute lymphoblastic leukemia (ALL) in childhood is well established. Currently known risk loci, however, explain only one third of the estimated total risk related to common genetic variations. We genotyped 1,421 polymorphisms in 407 candidate genes from the SNP500Cancer database (National Cancer Institute) using the Illumina Cancer SNP Panel. We investigated 78 cases (aged 0-19 years at diagnosis, and mixed ethnic background) of childhood B-precursor ALL and compared genotype data with those of 1,417 HapMap controls. To account for the ethnic diversity of the study population, structured association by genetically matching cases and controls using identity-by-state similarity was used. Case-control association analyses were performed using Cochran-Mantel-Haenszel tests, adjusted for the population substructure. Common variations rs6966 (3' UTR of PPP1R13L, chr 19q13.32, P = 4.55 × 10(-9)) and rs414580 (intron 2 of MSR1, chr 8p22, P = 6.09 × 10(-8)) were significantly associated with ALL. These SNPs remained significant after adjustment for multiple testing. The SNP rs6966 tags a haplotype block which includes SNPs in PPP1R13L and ERCC2 genes, which are related to DNA repair and cell survival. rs6966 and rs414580 conferred allelic odds ratios of 3.74 (95% confidence interval [CI] 2.31-6.04) and 3.93 (95% CI 2.31-6.69), respectively. These findings reveal two independent novel susceptibility loci for childhood ALL.

Anti-CRLF2 Antibody-Armored Biodegradable Nanoparticles for Childhood B-ALL.

B-precursor acute lymphoblastic leukemia (B-ALL) lymphoblast (blast) internalization of anti-cytokine receptor-like factor 2 (CRLF2) antibody-armored biodegradable nanoparticles (AbBNPs) are investigated. First, AbBNPsaere synthesized by adsorbing anti-CRLF2 antibodies to poly(D,L-lactide- co -glycolide) (PLGA) nanoparticles of various sizes and antibody surface density (Ab/BNP) ratios. Second, AbBNPs are incubated with CRLF2-overexpressing (CRLF2+) or control blasts. Third, internalization of AbBNPs by blasts is evaluated by multicolor flow cytometry as a function of receptor expression, AbBNP size, and Ab/BNP ratio. Results from these experiments are con-firmed by electron microscopy, fluorescence microscopy, and Western blotting. The optimal size and Ab/BNP for internalization of AbBNPs by CRLF2+ blasts is 50 nm with 10 Ab/BNP and 100 nm with 25 Ab/BNP. These studies show that internalization of AbBNPs in childhood B-ALL blasts is AbBNP size-and Ab/BNP ratio-dependent. All AbBNP combinations are non-cytotoxic. It is also shown that CD47 is very slightly up-regulated by blasts exposed to AbBNPs. CD47 is "the marker of self" overexpressed by blasts to escape phagocytosis, or "cellular devouring", by beneficial macrophages. The results indicate that precise engineering of AbBNPs by size and Ab/BNP ratio may improve the internalization and selectivity of future biodegradable nanoparticles for the treatment of leukemia patients, including drug-resistant minority children and Down's syndrome patients with CRLF2+B-ALL.

Key pathways are frequently mutated in high-risk childhood acute lymphoblastic leukemia: a report from the Children's Oncology Group

AbstractWe sequenced 120 candidate genes in 187 high-risk childhood B-precursor acute lymphoblastic leukemias, the largest pediatric cancer genome sequencing effort reported to date. Integrated analysis of 179 validated somatic sequence mutations with genome-wide copy number alterations and gene expression profiles revealed a high frequency of recurrent somatic alterations in key signaling pathways, including B-cell development/differentiation (68% of cases), the TP53/RB tumor suppressor pathway (54%), Ras signaling (50%), and Janus kinases (11%). Recurrent mutations were also found in ETV6 (6 cases), TBL1XR1 (3), CREBBP (3), MUC4 (2), ASMTL (2), and ADARB2 (2). The frequency of mutations within the 4 major pathways varied markedly across genetic subtypes. Among 23 leukemias expressing a BCR-ABL1-like gene expression profile, 96% had somatic alterations in B-cell development/differentiation, 57% in JAK, and 52% in both pathways, whereas only 9% had Ras pathway mutations. In contrast, 21 cases defined by a distinct gene expression profile coupled with focal ERG deletion rarely had B-cell development/differentiation or JAK kinase alterations but had a high frequency (62%) of Ras signaling pathway mutations. These data extend the range of genes that are recurrently mutated in high-risk childhood B-precursor acute lymphoblastic leukemia and highlight important new therapeutic targets for selected patient subsets.

Enhanced Elimination of 6-Mercaptopurine or Methotrexate-Treated Acute Lymphoblastic Leukemia Cells by Natural Killer Lymphocytes In Vitro: A Potential Mechanism of Action of Maintenance Chemotherapy

AbstractAbstract 2128 Background:Maintenance chemotherapy (MC) is an important component of childhood B-precursor acute lymphoblastic leukemia (ALL) therapy; however, it is not necessary in the treatment of mature B cell neoplasms. The operational mechanisms of MC are not understood. Improvement in immunologic function including near normal levels of natural killer (NK) lymphocytes was reported during ALL MC. We hypothesize that in addition to their direct cytotoxicity, MC drugs alter surviving lymphoblasts, rendering them susceptible to innate immune response, likely through cell mediated cytotoxicity via stress proteins such as NKG2D ligands, co-stimulatory or adhesion molecules. Objective:The effect of 6-mercaptopurine (6MP) or methotrexate (MTX) treatment of B-precursor and mature B leukemia/lymphoma cells in their elimination by NK lymphocytes was investigated in this study. Design and Methods:Allogeneic NK cell-mediated elimination of REH (TEL/AML-positive B-precursor ALL) and Raji (mature B cell lymphoma) cells treated with standard MC drugs was studied. High dose cytarabine (Ara-C) and MTX are used during the consolidation chemotherapy; therefore, Ara-C and MTX-resistant REH and Raji cell sub-lines were established by exposing wild type cells to increasing concentrations of drugs over several months. Natural killer cells from 17 healthy volunteers were separated using the MACS NK cell isolation kit. After purity evaluation, NK cells were incubated with interleukin-15 overnight. Leukemia cells were incubated in minimally toxic (20% cytotoxicity) concentrations of 6MP and MTX. The leukemia/lymphoma cells were then co-incubated with NK cells at different ratios. The NK cell-mediated leukemia/lymphoma cell cytotoxicity was measured by flow cytometric cell-mediated cytotoxicity assay, marking effector cells with lineage-specific monoclonal antibodies and staining target cells with propidium iodide and annexin-V and using microspheres for quantification of viable and apoptotic cells. The level of resistance of the respective cell sub-lines was evaluated using MTT assay. We also investigated whether NK cell exposure to same concentrations of MC drugs before co-incubation alters cytotoxicity. Surface expression of NKG2D ligands, ULBP 1, 2 and 3, MICA and MICB was studied by flow cytometry. Results:6-mercaptopurine treatment of REH cells and MTX treatment of Raji cells resulted in enhanced NK cell-mediated elimination when compared to untreated cells by 25% and 20%, respectively. The results were similar when NK cells were exposed to the same concentrations of MC drugs before co-incubation, indicating lack of negative effect of the drug exposure in NK cells' ability to kill. Similar experiments were conducted on resistant cells, in order to make the target cells more comparable to the residual lymphoblasts during MC. Most interestingly, the REH cells, but not the Raji cells, resistant to Ara-C and MTX showed about 14% and 4% enhancement of NK cell-mediated killing, respectively, after being exposed to the minimally toxic concentrations of MC drugs. This indicates that resistant B precursor ALL cells can be eliminated by NK cells upon MC drug exposure, but not mature B lymphoblasts, in this experimental setting. No increase in the expression of NKG2D ligands on drug treated ALL cells was observed. Conclusion:These findings suggest that enhanced susceptibility of drug-exposed leukemia cells to innate immune response may be an operational mechanism of MC. This mechanism may involve pathways other than NKG2D. Disclosures:No relevant conflicts of interest to declare.

T-Cell Receptor Gene Deletions Are Associated with High Risk Features and Worse Outcome In Childhood Precursor B-Cell Acute Lymphoblastic Leukemia (ALL)

AbstractAbstract 275T-cell receptor (TCR) gene rearrangements occur in 50–80% of childhood precursor B-cell ALL and have been used for minimal residual disease (MRD) detection. However, TCR rearrangement has never been examined for its contribution to disease classification and outcome. We used Molecular Inversion Probe technology (Affymetrix) to analyze precursor B-cell ALL samples (n=48) from the University of Utah for genome-wide copy number changes, and evaluated the relationship between focal TCR deletions (indicating rearrangement) and clinical features. Copy number deletions were detected by Nexus Copy Number (BioDiscovery, Inc.) and were based on 5 consecutive probes having a value of <1.5 (diploid copy number=2). We then validated our findings in the diagnostic B-precursor ALL samples (n=221) from the Children's Oncology Group (COG) High Risk (HR)-ALL TARGET (Therapeutically Applicable Research to Generate Effective Targets) Project which includes 500K SNP array (Affymetrix) data from patients enrolled on COG P9906. In the Utah cohort, we observed the following TCR deletions: TCR-α (14q11.2)=23/48 (48%), TCR-β (7q34)=18/48 (38%), TCR-γ (7p14.1)=29/48 (60%), TCR-δ (14q11.2)=23/48 (48%), or any TCR deletion (TCR-'any')=36/48 (75%). Utah samples contained 37.5% NCI-Rome designated “High Risk” patients, and 15/18 (83%) of these patients had TCR-'any' deletions. Fisher's Exact Test (two-tailed) was used to look for associations in the Utah cohort between TCR-α,-β,-γ,-δ, or -‘any’ deletions and high risk clinical features. TCR-α deletion was significantly associated with Day 28 MRD Positivity (p=0.0486); there was significant association between TCR-β deletion and Day 7 M3 Marrow (>25% blasts; p=0.019). TCR-δ deletion was also significantly associated with Day 28 MRD Positivity (p=0.0206). Due to the small sample size and insufficient follow-up, outcome analyses were not done in this cohort. The COG HR-ALL TARGET cohort had the following TCR deletions: TCR-α=53/221 (24%), TCR-β=24/221 (11%), TCR-γ=55/221 (25%), TCR-δ=53/221 (24%), or TCR-'any'=96/221 (43%). Fisher's Exact test (two-tailed) was used to look for associations between TCR deletions and clinical features; outcome analyses were conducted using the Kaplan-Meier method and the log-rank test. In the TARGET cohort, we found TCR-α, TCR-γ, and TCR-δ deletions were significantly associated with occurrence of relapse and/or death (p=0.0209, p=0.0143, and p=0.0209, respectively). TCR-β deletion was associated with age at diagnosis ≥ 10 yo (p=0.0225), a high risk feature, and Day 29 MRD Positivity (p=0.0084). In the high-risk TARGET validation cohort, 43 of the 78 children (55%) who experienced a relapse or died contained TCR deletions vs. 53 of the 143 children (37%) who were event-free with TCR deletions (p=0.0109). There was a significant difference in event-free survival (EFS) between patients with TCR-'any' deletions vs. those without the deletions (p=0.013), with the largest difference in survival occurring beyond two years of diagnosis (Figure 1). TCR deletions were uncommon in Hyperdiploid (0/4, 0%), E2A-PBX1 (n=1/25, 4%), and MLL (n=4/19, 21%) subtypes in the TARGET cohort, but were more frequent in TEL-AML1 (n=2/3, 67%) and Other (n=89/170, 52%) subtypes. In summary, both the Utah discovery cohort and the larger COG HR-ALL TARGET validation cohort showed focal TCR gene deletions that were linked to high risk clinical features, as well as associated with poor prognostic attributes such as end-of-induction MRD Positivity. Additionally, the larger TARGET cohort demonstrated that deletion of any TCR region was significantly associated with worse outcome. TCR gene deletions may indicate a more immature state of developmental arrest and could imply initial abortive T-cell lineage development prior to B-cell commitment and transformation. This represents the first study to reveal an association between TCR gene deletion status and clinical outcome in childhood B-precursor ALL, and these findings will need validation in additional pediatric and adult cohorts. Ultimately, focal TCR gene deletions may prove to be a valuable addition to the molecular risk classification schema currently applied to B-precursor ALL. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Excellent Outcome for ETV6/RUNX1-Positive Childhood Acute Lymphoblastic Leukemia (ALL) with Contemporary Therapy

AbstractAbstract 495The t(12;21)(p13;q22) translocation causes the fusion of the ETV6 and RUNX1 genes and occurs in approximately 25% of cases of childhood B-precursor ALL. The presence of ETV6/RUNX1 has been associated with a lower rate of relapse in multiple studies. However, it is not clear whether the ETV6/RUNX1 fusion retains independent prognostic significance in the context of current risk-adapted therapy and whether the outcome of these children can be further improved.We studied clinical features, treatment response and adverse events of 168 children with newly diagnosed ETV6/RUNX1-positive B-precursor ALL enrolled in St Jude Total Therapy studies XIIIA (1991-1994; N=36), XIIIB (1994-1998; N=38) and XV (2000-2007; N=94). Results were compared to those of 495 ETV6/RUNX1-negative patients with B-precursor ALL. Total XV study used levels of minimal residual disease (MRD) during treatment for risk assignment.Presence of ETV6/RUNX1 was significantly related with age 1–9 years old (p<0.001), lower prevalence of central nervous system (CNS) disease at diagnosis (p<0.001) and classification as low-risk ALL (p<0.001). ETV6/RUNX1 patients had significantly lower rates of MRD positivity on day 19 (p<0.001) and day 46 of remission induction therapy (p=0.01) than non ETV6/RUNX1 patients. Event-free survival (EFS) did not differ between patients with or without ETV6/RUNX1 in Total XIIIA (p=0.14; 5-year EFS 86.1 ± 5.7% versus 75.6 ± 4.8%) or XIIIB (p=0.76; 5-year EFS 84.1 ± 6.8% versus 81.2 ± 3.8%). Likewise, overall survival (OS) did not differ among patients treated in either Total XIIIA (p=0.41; 5-year OS 88.9 ± 5.2% versus 83.3 ± 4.2%) or Total XIIIB (p=0.67; 86.5 ± 5.6% versus 85.0 ± 3.5). By contrast, patients with ETV6/RUNX1 ALL had significantly better EFS (p=0.008) and marginally better OS (p=0.057) than those without ETV6/RUNX1 in Total XV; 5-year EFS estimate was 97.6± 2.8% versus 86.0 ± 3.3% for patients without the fusion and 5-year OS was 98.9 ± 1.8% versus 93.3 ± 2.4%. Overall, among 168 patients with ETV6/RUNX1 ALL, there were 17 treatment failures, including 2 deaths in complete remission, 6 secondary malignancies (4 in Total XIIIA and 2 in Total XIIIB) and 9 relapses (6 hematologic, 1 combined hematologic and CNS and 2 isolated CNS relapses). Three relapses occurred during or within 6 months of completion of therapy while 6 occurred later. The median time to relapse was 38 months (range 6–139 months) as compared to 36.5 months (range 3–119 months) in ETV6/RUNX1-negative patients (p=0.28). In the most recent Total XV study, the only causes of treatment failure were 2 hematologic relapses. Within the ETV6/RUNX1 group, age, gender, race, presenting leukocyte count, CNS status, risk group and even MRD at day 19 and day 46 were not associated with 5-year EFS or OS. Only treatment protocol, i.e. Total XV (versus Total XIIIA or XIIIB) was significantly associated with improved EFS (p=0.03) and OS (p=0.04) (Figure 1). With the Total XV treatment strategy, conventional risk factors such as age and leukocyte count did not have prognostic significance among patients with ETV6/RUNX1-positive ALL. In this protocol, there were 5 ETV6/RUNX1 patients who were ≥10 years old and 17 who were 1–9 years old and had a leukocyte count ≥50 × 109/L at presentation with a good MRD decline (<1% on Day 19 and <0.01% on day 46). These patients received lower-risk therapy and none of them have relapsed to date. The Total XV regimen intensified asparaginase, dexamethasone (post-remission) and vincristine; drugs which are known to be especially cytotoxic to ETV6/RUNX1 lymphoblasts in vitro. Patients on Total XV might have also benefited from the higher doses of methotrexate administered during the consolidation phase, as ETV6/RUNX1 lymphoblasts accumulate lower amounts of methotrexate polyglutamates. In addition, measures such as avoiding cranial radiotherapy and limiting the use of epipodophyllotoxins and alkylating agents led to decreased adverse events like secondary malignancies for all patients.In conclusion, stringent risk-directed therapy featuring intensive asparaginase, dexamethasone and high-dose methotrexate resulted in excellent outcome for children with ETV6/RUNX1 ALL.Kaplan-Meier Estimates of EFS and OS in ETV6/RUNX1-positive Patients by Study. [Display omitted] Disclosure:Bhojwani:MedImmune: Research Funding. Sandlund:Seattle Genetics: Research Funding. Jeha:Genzyme: Honoraria, Research Funding. Relling:St. Jude Children's Research Hospital: Employment, Patents & Royalties; Enzon Pharmaceuticals: Research Funding. Pui:EUSA Pharma: Honoraria; Enzon: Honoraria; Sanofi-Aventis: Honoraria.

A specific linkage between the incidence of TP53 mutations and type of chromosomal translocations in B‐precursor acute lymphoblastic leukemia cell lines

Chromosomal translocation plays an essential role in leukemogenesis of childhood B-precursor acute lymphoblastic leukemia (ALL) in combination with specific gene mutations. Although several reports suggest correlation of TP53 mutation with the type of chromosomal translocation in childhood B-precursor ALL, it has not been directly confirmed in a single cohort study due to the limited incidence of TP53 mutation in primary samples at diagnosis. Leukemia cell lines generally show higher incidence of gene mutations compared with primary samples. Thus, to clarify possible differences in cooperation of the p53 pathway for leukemogenesis with the type of chromosomal translocation, TP53 gene mutation was analyzed in 32 B-precursor ALL cell lines. TP53 mutation was observed in 40.6% (13 cell lines), and missense mutation of R248Q was the most commonly observed (5 cell lines). Of note, TP53 mutation was frequently observed among MLL-rearranged and t(1;19)-positive ALL cell lines, but was very rare among Philadelphia chromosome-positive and t(17;19)-positive ALL cell lines. Although the number of samples analyzed is limited, this is the first direct observation of the correlation between the incidence of TP53 mutation and types of translocation in B-precursor ALLs, suggesting a functionally different fusion gene product in the p53 pathway for leukemogenesis.

Conditioning with Treosulfan and Fludarabine followed by Allogeneic Hematopoietic Cell Transplantation for High-Risk Hematologic Malignancies

In this prospective study 60 patients of median age 46 (range: 5-60 years), with acute myelogenous leukemia (AML; n= 44), acute lymphoblastic leukemia (ALL; n = 3), or myelodysplastic syndrome (MDS; n = 13) were conditioned for allogeneic hematopoietic cell transplantation with a treosulfan/fludarabine (Flu) combination. Most patients were considered at high risk for relapse or nonrelapse mortality (NRM). Patients received intravenous treosulfan, 12 g/m(2)/day (n = 5) or 14 g/m(2)/day (n = 55) on days -6 to -4, and Flu (30 mg/m(2)/day) on days -6 to -2, followed by infusion of marrow (n = 7) or peripheral blood stem cells (n = 53) from HLA-identical siblings (n = 30) or unrelated donors (n = 30). All patients engrafted. NRM was 5% at day 100, and 8% at 2 years. With a median follow-up of 22 months, the 2-year relapse-free survival (RFS) for all patients was 58% and 88% for patients without high-risk cytogenetics. The 2-year cumulative incidence of relapse was 33% (15% for patients with MDS, 34% for AML in first remission, 50% for AML or ALL beyond first remission and 63% for AML in refractory relapse). Thus, a treosulfan/Flu regimen was well tolerated and yielded encouraging survival and disease control with minimal NRM. Further trials are warranted to compare treosulfan/Flu to other widely used regimens, and to study the impact of using this regimen in more narrowly defined groups of patients.

Evolution of Gene Expression Signatures in Relapsed Childhood Acute Lymphoblastic Leukemia Differs Based on Timing of Relapse.

The outcome for childhood acute lymphoblastic leukemia (ALL) following marrow relapse remains bleak in spite of numerous approaches to further intensify therapy. Understanding the biological basis of relapse and chemoresistance, as well as identifying and validating potential new targets, are the goals of our study. Previously we determined global gene expression patterns of matched diagnosis and relapse leukemic blasts in 32 patients (64 samples) with childhood B-precursor ALL using Affymetrix U133A arrays (Blood 2006; 108(2):711–7). We now have extended this analysis to 60 patients (120 samples). Thirty-six patients relapsed early (within 36 months of initial diagnosis), while 24 patients relapsed late. Within the TEL/AML1 subset (n=12 patients), time to relapse was inversely proportional to the correlation co-efficient of expression profiles of the diagnosis and relapse matched pair samples, suggesting that the later the relapse, the more distinct the relapse clone is from the diagnostic clone. A supervised pairwise analysis in all 60 patients identified 292 probesets that were differentially expressed between diagnosis and relapse (FDR < 10%). In a relative enrichment analysis, multiple genes mediating cell death were down-regulated at relapse (p=0.00003), suggesting that the leukemia cells had evolved mechanisms to enhance survival. These included p21, TNFPAI3, RIPK2, BCLAF1, STK17B. In concert, DNA replication genes were up-regulated at relapse (p=0.00002). Differences in pathways leading to early vs. late relapse were evident. Early relapse was characterized by an over-expression of cell cycle genes reflecting a proliferative state. At the time of relapse, a marked over-representation of genes involved in the progression through the M phase of the cell cycle was observed in early relapse compared to late relapse (p=1.3E-08). Late relapse was characterized by the over-expression of genes involved in nucleoside biosynthesis, particularly targets of antifolates (DHFR, MTHFD1, TYMS). A small number of gene expression patterns were common to both early and late relapse, including up-regulation at relapse of BIRC5 (survivin): an attractive target for therapeutic intervention. In conclusion, analysis of an expanded cohort of matched diagnosis/relapse pairs has validated and extended our previous findings that early relapse is associated with a proliferative gene expression signature. In addition we have now identified pathways operative in late relapse. Targeting these individual genes and pathways may lead to innovative strategies to treat or prevent relapsed ALL.

Genome-wide copy number profiling reveals molecular evolution from diagnosis to relapse in childhood acute lymphoblastic leukemia

AbstractThe underlying pathways that lead to relapse in childhood acute lymphoblastic leukemia (ALL) are unknown. To comprehensively characterize the molecular evolution of relapsed childhood B-precursor ALL, we used human 500K single-nucleotide polymorphism arrays to identify somatic copy number alterations (CNAs) in 20 diagnosis/relapse pairs relative to germ line. We identified 758 CNAs, 66.4% of which were less than 1 Mb, and deletions outnumbered amplifications by approximately 2.5:1. Although CNAs persisting from diagnosis to relapse were observed in all 20 cases, 17 patients exhibited differential CNA patterns from diagnosis to relapse. Of the 396 CNAs observed in 20 relapse samples, only 69 (17.4%) were novel (absent in the matched diagnosis samples). EBF1 and IKZF1 deletions were particularly frequent in this relapsed ALL cohort (25.0% and 35.0%, respectively), suggesting their role in disease recurrence. In addition, we noted concordance in global gene expression and DNA copy number changes (P = 2.2 × 10−16). Finally, relapse-specific focal deletion of MSH6 and, consequently, reduced gene expression were found in 2 of 20 cases. In an independent cohort of children with ALL, reduced expression of MSH6 was associated with resistance to mercaptopurine and prednisone, thereby providing a plausible mechanism by which this acquired deletion contributes to drug resistance at relapse.

Prenatal origin of childhood acute lymphoblastic leukemia, association with birth weight and hyperdiploidy

Recent studies with very small numbers of patients showed that in some cases of childhood acute lymphoblastic leukemia (ALL), preleukemic cells are detectable on Guthrie cards that were used for newborn screening. We present here the largest series of ALL patients (n=32) in whom Guthrie cards were analyzed for the presence of preleukemic cells. Rearranged immunoglobulin heavy-chain genes were used as a marker for leukemic clones. We combined our set of patients with 17 previously published cases. Preleukemic cells were detected in 31 of all 49 patients (63%). Positive screening cards were not associated with patient's age at diagnosis but were almost always found in patients with hyperdiploidy (10/11; 91%; P=0.04). High birth weight is an established risk factor for childhood ALL. Positive screening cards were strongly associated with low birth weight (P=0.01). In conclusion, the majority of childhood B-precursor ALL arise prior to birth. In the search for causes of childhood leukemia we should concentrate on prenatal factors as well as postnatal factors. Our results suggest that autologous cord bloods could be a poor choice as the source of stem cells for transplantation in leukemia, which may contain preleukemic cells. Pending the development of suitable methods, childhood leukemia is a potentially screenable disease.

SIMULTANEOUS OCCURRENCE OF POSTERIOR REVERSIBLE LEUKOENCEPHALOPATHY SYNDROME IN TWO CASES OF CHILDHOOD ACUTE LYMPHOBLASTIC LEUKEMIA INDUCTION CHEMOTHERAPY

We describe two cases Posterior Reversible Encephalopathy Syndrome (PRES) occurring in childhood B-precursor acute lymphoblastic leukemia within a week of each other during induction chemotherapy in our ward. We review the literature related to the possible etiology of PRES in these cases.

Pre-Clinical Evaluation of the Anti-CD22 Immunotoxin CAT-8015 in Combination with Chemotherapy Agents for Childhood B-Precursor Acute Lymphoblastic Leukemia (Pre-B ALL).

Despite advances in curative treatment, childhood ALL remains a leading cause of cancer-related mortality in pediatrics and survivors are at risk of multiple late effects. Targeted therapies that overcome chemotherapy resistance and decrease nonspecific toxicities are needed. The B-lineage differentiation antigen CD22 represents a relevant target for pre-B ALL. Monotherapy with the anti-CD22 immunotoxin RFB4(dsFv)-PE38 (CAT-3888 or BL22) has been well tolerated in a pediatric Phase I trial. To date however, only transient clinical activity has been observed with this agent in children with ALL. We conducted cytotoxicity studies to evaluate combination regimens of a higher affinity anti-CD22 immunotoxin (CAT-8015 or HA22) with standard ALL chemotherapeutic agents.Methods: CD22+ human malignant cell samples studied included cell lines of Burkitt lymphoma (CA46) and pre-B ALL (REH and KOPN-8), as well as primary lymphoblasts from 2 children with multiply relapsed pre-B ALL. Flow cytometry was used to determine CD22 expression and anti-CD22 antibody binding using the BD Quantibrite System. Asparaginase, cytarabine, doxorubicin, methotrexate, and vincristine were added to cells individually and in various combinations with CAT-8015. Total incubation time was 2 days for cell lines and 5 days for primary patient blasts. Cell viability was assessed using WST-8. The Chou-Talalay median-effect method was used for data analysis (Chou TC, Talalay P. Adv Enzyme Regul 1984; 22:27–55).Results: All samples were sensitive to CAT-8015 and to most chemotherapy agents tested. Simultaneous addition of a fixed concentration of CAT-8015 with varying concentrations of each chemotherapy drug led to a 2- to 4-fold reduction in IC50 in comparison to individual agents. Assays with simultaneous addition of equipotency ratios of CAT-8015 and chemotherapy agents had synergistic or additive interactions. These interactions were sequence dependent, and the addition of CAT-8015 followed by chemotherapy led to greater positive interactions than the reverse order. Further assays with simultaneous addition of CAT-8015 and doublet chemotherapy drugs showed enhanced synergistic and additive interactions compared to assays with only doublet agents.Conclusions: CAT-8015 combined with standard chemotherapy agents results in synergistic or additive cytotoxicity against CD22-positive Burkitt lymphoma and pre-B ALL cell lines and primary blasts from children with pre-B ALL. High affinity anti-CD22 recombinant immunotoxins have the potential to overcome chemotherapy resistance and to decrease nonspecific treatment-associated toxicities. In addition, CAT-8015 might synergistically enhance chemotherapy-induced cytotoxicity, thus improving efficacy and allowing the use of less toxic regimens in the future. Clinical trials of recombinant immunotoxins targeting CD22 in combination with standard ALL chemotherapy regimens are planned.

Targeting CD22 in Childhood B-Precursor Acute Lymphoblastic Leukemia (Pre-B ALL): Pre-Clinical Studies and Phase I Trial of the Anti-CD22 Immunotoxin CAT-3888 (BL22).

Although most children with pre-B ALL are cured, treatment is associated with multiple toxicities and outcome after relapse is poor. New therapies are needed to overcome drug resistance and reduce non-specific toxicities of chemotherapy. We evaluated the B-lineage differentiation antigen CD22 and the anti-CD22 immunotoxin RFB4(dsFv)-PE38 (CAT-3888 or BL22) in childhood ALL.Methods: Blasts were obtained from children with pre-B ALL. Flow cytometry was used to determine CD22 expression and anti-CD22 antibody binding using the BD Quantibrite System. 72 hour in vitro protein synthesis cytotoxicity assays were conducted. Murine xenografts (human ALL cell line EU-1 into CB17 SCID mice) were treated with varying doses of CAT-3888. A pediatric Phase I trial of CAT-3888 was initiated.Results: All cases (n=69) were CD22 positive. Median CD22 site density (studied in 28 cases) was 4,099 sites/cell (range, 451 – 9,380). In vitro cytotoxicity to CAT-3888 was assessed in 43 cases and the median IC50 was 10 ng/ml. Murine xenografts treated with CAT-3888 had significant prolongation of leukemia free survival in a dose responsive fashion (p<0.05). 21 subjects (18 ALL, 3 lymphoma; 3–22 years of age) were treated on a Phase I trial of CAT-3888 at doses of 10–40 mcg/kg QOD for 3–6 doses with cycles repeated every 21–28 days. Treatment was well tolerated and adverse events attributed to CAT-3888 were reversible. Pharmacokinetic parameters appeared to be influenced by disease burden consistent with rapid drug binding by CD22 positive blasts. All subjects were heavily pre-treated and had progressive high burden disease at the time of treatment. Transient clinical activity was observed in 17 of 21 subjects as evidenced by decreased peripheral blood blast count (8), decreased marrow infiltration (6), decreased extramedullary infiltration (3), increased platelet count (2), increased neutrophil count (1), increased reticulocyte count (1), improved PET scan (1), decreased tumor-associated pain (2), and/or peripheral blast count stabilization (5). A dose response was apparent with 6 of 12 (50%) achieving disease stabilization at or above 30 mcg/kg (vs. 0/9 at lower doses). Three subjects developed high-titer neutralizing antibodies, 1 of which had pre-existing low titer antibodies from prior pseudomonas infection.Conclusions: CD22 represents a relevant target for childhood pre-B ALL. All cases tested were CD22+ and most blasts were highly sensitive to CAT-3888 in vitro at concentrations below clinically achievable levels. CAT-3888 showed a dose response effect and prolonged leukemia free survival in murine ALL xenografts. CAT-3888 is well tolerated in children and activity has been seen in most subjects treated on the Phase I trial. A new higher affinity anti-CD22 immunotoxin (CAT-8015 or HA22) is in development.