Childhood Acute Lymphoblastic Leukemia Patients Research Articles

Minimal Residual Disease Monitoring In Acute Lymphoblastic Leukemia Patients Using Immune Receptor Sequencing

BackgroundThe assessment of residual tumor cells persisting after therapy, or minimal residual disease (MRD), is a central component of accurate disease prognosis in acute lymphoblastic leukemia (ALL) (Pui et al, JCO 2011). MRD assessment has recently been shown to be useful for monitoring disease before and after stem cell transplantation and during salvage therapy for early detection of an imminent relapse (Bruggemann et al., SemOncol 2012). Allele-specific oligonucleotide (ASO)-PCR can be used to assess MRD; however, this technique requires preparation of clonotype-specific primers for each individual which is laborious and time-consuming. We recently demonstrated the utility of sequencing-based MRD assessment in lymphoid malignancies (Faham et al., Blood 2012). This quantitative approach, termed the LymphoSIGHT™ platform, relies on amplification and sequencing of immunoglobulin and T-cell receptor gene segments using consensus primers and can address some of the limitations associated with traditional MRD detection techniques. This sequencing platform has a sensitivity to detect one cancer cell per million leukocytes in peripheral blood and bone marrow samples. In this retrospective study, we evaluated the ability of the sequencing and ASO-PCR methods to detect MRD prior to clinical relapse in 17 patients with childhood ALL. MethodsUsing the sequencing assay, we analyzed bone marrow and/or peripheral blood samples collected at the diagnostic and relapse time points from 17 childhood ALL patients. Diagnostic and relapse samples were assessed for clonal rearrangements of immunoglobulin (IGH-VDJ, IGH-DJ, IGK) and T cell receptor (TRB, TRD, TRG) genes. Following identification of leukemia specific clonotype(s), we measured the corresponding MRD levels in 66 follow-up samples that were collected prior to clinical relapse. We analyzed the time from MRD positivity to clinical relapse for each patient using the sequencing and ASO-PCR methods. ResultsSequencing detected the presence of a high-frequency clonal rearrangement of at least one receptor (“calibrating receptor”) in all the 17 childhood ALL patients; all patients had at least 2 calibrating receptors at diagnosis and/or relapse, 15 patients had at least 3 calibrating receptors, and 6 patients had 4 or more. The IGH-VDJ and TRG assays were the most frequent gene rearrangement: at least one IGH-VDJ and/or TRG clonal rearrangement was detected in 13 ALL patients. TRD was the third most informative receptor, with clonal rearrangements being detected in 11 patients. In the majority of patients, most leukemic clones identified at diagnosis were also present at high levels in the relapse sample. We analyzed the time from MRD positivity to clinical relapse in the patient cohort. In 2 patients (Figure 1, Patients G and P), we observed MRD positivity by sequencing 16 and 6.5 months prior to relapse, respectively. ASO-PCR also detected MRD positivity in Patient P at the 6.5 month time point. In 6 patients there was a sample collected within 1-3 months prior to relapse, and sequencing detected MRD in 4 of these patients (67%, Figure 1 Patients B, A, R, I), while ASO-PCR detected MRD in 2 of the 6 patients (33%, Figure 1 Patients B, R). ConclusionsThe clinical value of monitoring MRD by ASO-PCR for assessment of treatment response and outcome has been established in multiple lymphoid malignancies. This preliminary study provides further support for the sequencing-based MRD monitoring in ALL patients. This new approach offers improvements over ASO-PCR in the ability to monitor multiple clonal sequences and in the sensitivity to detect the leukemic cell. Thus, the sequencing method represents a new potential approach for MRD monitoring. However a larger number of patients must be analyzed to support its clinical application. [Display omitted] Disclosures:Faham:Sequenta, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Pepin:Sequenta, Inc.: Employment, Equity Ownership. Carlton:Sequenta, Inc.: Employment, Equity Ownership.

Identification Of BCL6 As a Therapeutic Target In MLL-Rearranged ALL

BackgroundBCL6 has been identified in diffuse large B cell lymphoma (DLBCL), where it is frequently translocated to immunoglobulin loci and acts as a protoncogene and transcriptional repressor. We found in Ph+ acute lymphoblastic leukemia (ALL) that BCL6 is involved in a novel form of drug resistance to tyrosine kinase inhibitors (TKI) by protecting cells from p53-mediated apoptosis (Duy et al., Nature 2011). Our current study is focusing on the function of BCL6 in Non-Ph+ALL based on the finding that high BCL6 levels represent a predictor of poor clinical outcome. ResultsWe analyzed the gene expression data from 207 children with high-risk B cell precursor ALL enrolled in the pediatric clinical trial (COG P9906) and found that high expression levels of BCL6 at the time of diagnosis correlated with a poor overall and relapse-free survival (OS p=0.007; RFS p=0.02). Furthermore, multivariate analyses showed that patients with high BCL6 expression levels and established predictors of poor clinical outcome such as high white blood counts (WBC), a positive minimal residual disease (MRD) or mutations in the tumor suppressor IKZF1 had a significantly poor OS and RFS. Matched sample pairs from 49 patients at diagnosis and relapse showed that BCL6 levels were increased at relapse compared to diagnosis (p=0.003). We next studied BCL6 protein levels in Non-Ph+ cell lines and childhood ALL patient samples and found that BCL6 levels are particularly high in MLL-AF4 ALL (n=19). Surprisingly, the patients from the clinical trial that had high BCL6 levels and MLLR rearrangements were those with the worst clinical outcome (OS p= 0.0009 and RFS p= 0.000208). We next tested if the MLL-AF4 oncogene drives aberrant BCL6 expression. First, we performed a ChIP-analysis with the oncoprotein MLL-AF4 and found that the BCL6 promoter is directly bound by MLL-AF4, suggesting that MLL-AF4 may indeed drive BCL6 expression. We then performed a BCL6 Western Blot of inducible MLL-AF4-transgenic pro-B cells, demonstrating 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 further elucidate the BCL6 signaling pathway, we performed a ChIP-analysis with human MLL-AF4 leukemia cells and found that tumor suppressor genes (e.g. CDKN1B and BACH2) and B cell linage specific genes like PAX5 and CD19 are repressed by BCL6, potentially explaining the mixed lineage phenotype of MLLR driven B-ALL. We used a genetic mouse model of childhood ALL based on bone marrow precursor cells from BCL6-/- mice to decipher the function of BCL6. Since mutations in the RAS pathway are found in about 30% of childhood ALL cases, we transduced B cell progenitor cells from BCL6-/- and BCL6+/+ mice with Nras using an inducible oncogenic TET-NRASG12D system. Similar to MLL-AF4 driven cells, activation of NRASG12D results in higher BCL6 protein expression. Strikingly, BCL6-deficiency results in a failure of NRASG12DALL cells to initiate leukemia. Clinical relevanceTo verify if the high BCL6 expression levels in MLL-AF4 patients are important for the disease progression, we transduced primary human childhood 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 children with MLL-AF4 leukemia, we treated human MLL-AF4 driven primary human xenograft cells with a recently developed retro-inverso BCL6 peptide inhibitor (RI-BPI). 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. ConclusionsThese findings identify BCL6 as an important factor in leukemia initiation and survival and its pharmacological inhibition as a novel strategy to treat childhood ALL. 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 currently used chemotherapeutics as potential approach to reduce the risk of ALL relapse and improve overall outcome. Disclosures:No relevant conflicts of interest to declare.

Abstract B68: Small GTPase RANBP16 as a novel target in acute lymphoblastic leukemia chemoresistance.

Abstract Background: Despite the improvements made on acute lymphoblastic leukemia (ALL) treatment around 20% of the patients still present treatment failure thus showing how the molecular mechanisms responsible for chemotherapy resistance still need to be clarified. The present study was undertaken to address the possible involvement of the small GTPase RANBP16 (a nuclear export receptor also known as Exportin 7) to acute lymphoblastic leukemia chemotherapy response. Experimental procedures: global gene expression analysis was carried out among B-ALL patients classified as prednisone poor responders (PPR) versus prednisone good responders (PGR) using Agilent platform. Subsequent validation was performed by quantitative RT-PCR in childhood ALL patients under the Brazilian Group for Treatment of Childhood Leukemia - GBTLI 99. To address the role of RANBP16 in chemoresistance, the cell lines ReH (B-ALL) and Jurkat (T-ALL) were transduced with lentivirus containing either shRNA for the target gene or empty vector. Puromycin selection was performed to generate stable transformed lineages. Gene knockdown was confirmed by qRT-PCR and Western blot. Drug cytotoxic assay was performed in a 4-day XTT cell proliferation assay and IC50 value was calculated with Calcusyn software. Annexin V staining and Colony-forming assay (CFA) was tested in triplicate and t-test was used to analyze the data (alpha value was set at 5%). Results: Initially, in a gene expression analysis by microarray we observed that RANBP16 was highly expressed in B-ALL samples from prednisone poor responders compared to good responder patients. Validation of this initial finding by quantitative RT-PCR in 87 B-ALL patients showed that RANBP16 up-regulation is indeed related to lower EFS (event free survival) rates, suggesting a role of this molecule in B-ALL poor outcome. RANBP16 knockdown induced a significantly reduction of IC50 values to prednisolone (0.5-150ug/mL) and daunorrubicin (10-150 nM) in both cell lines and a 7.0-fold increase in apoptosis rates in prednisolone resistant Jurkat cell line (as compared to the empty vector). Colony forming assays showed that RANBP16 knockdown significantly reduces the self-renewal capability after prednisolone treatment. Response to vincristine (1.0-20nM) was not modified by the knockdown condition. Conclusions: This data suggests that the RANBP16 gene can play an important role in leukemic cells sensibility to the glucocorticoid prednisolone and could be considered as a target for further investigations on new therapeutic interventions for ALL treatment. Further experiments, are still need to clearly demonstrate the role of this specific RAN-GTPase in glucocorticoid-mediated response. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B68. Citation Format: Vanessa S. Silveira, Veridiana K. Suazo, Kleiton S. Borges, Augusto F. Andrade, Gustavo AV Cruzeiro, Luiz Gonzaga Tone, Carlos Alberto Scrideli. Small GTPase RANBP16 as a novel target in acute lymphoblastic leukemia chemoresistance. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B68.

DNA repair gene XRCC1 polymorphisms and susceptibility to childhood acute lymphoblastic leukemia: a meta-analysis.

To estimate the relationship between genetic polymorphisms of X-ray repair cross-complementing group 1 (XRCC1) and the susceptibility to childhood acute lymphoblastic leukemia (ALL). Relevant case-control studies were enrolled in the meta-analysis. We applied Rev Man 4.2 software to pool raw data and test studies' heterogeneity and to calculate the incorporated odds ratio (OR) and 95% confidence interval (95% CI). Our data showed that the OR for the Gln allele of the Arg399Gln polymorphism, compared with the Arg allele, was 1.35 (95% CI, 1.16-1.57; P<0.0001) for childhood ALL patients. Similarly, the homozygous genotype Gln/Gln and heterozygous genotype Arg/Gln both significantly increased the risk of childhood ALL compared with the wild genotype Arg/Arg (OR =1.58; 95% CI, 1.13-2.21; P=0.008; OR =1.51; 95% CI, 1.21-1.87; P=0.0002). The dominant model of Arg399Gln was associated with childhood ALL risk (OR =1.54; 95% CI, 1.25-1.89; P<0.0001). The ethnic subgroup analysis demonstrated that the Gln allele in all five ethnic groups was prone to be a risk factor for childhood ALL just with different degrees of correlation while Arg194Trp SNP showed a protective or risk factor or irrelevant thing in different races. XRCC1 399 polymorphism may increase the risk of childhood ALL. Different ethnic groups with some gene polymorphism have different disease risks.

Unrelated cord blood transplantation for central nervous system relapse in high-risk childhood acute lymphoblastic leukemia

Few clinical studies have investigated the role of unrelated cord blood transplantation (CBT) for central nervous system (CNS) relapse of childhood acute lymphoblastic leukemia (ALL) patients with high-risk factors. The aim of this report is to identify the potential benefits of unrelated CBT in high-risk childhood ALL with CNS relapse who has been treated on CNS-directed treatment strategies. Eleven childhood ALL patients with CNS relapse who underwent unrelated CBT enrolled in our study between 2001 and 2011, and all of the patients had features associated with poor outcomes, such as high white blood cells at diagnosis, ph + chromosome, or a history of bone marrow relapse. All transplants were performed with myeloablative-conditioning therapy (BU/cyclophosphamide (CY2) or total body irradiation/CY) plus highly CNS-active agents (carmustine or high-dose cytarabine). All patients achieved neutrophil engraftment and platelet engraftment. A total of nine patients (81.8 %) developed pre-engraftment syndrome at a median of 7 days, and three patients developed acute graft-vs-host disease at a median of 21 days. The median follow-up after CBT was 28.5 months. The probability of overall survival at 9 years was 63.6 %, and no patient experienced a CNS relapse. Our experience suggests that unrelated CBT appears to be an effective treatment option for CNS relapse of childhood ALL patients associated with poor outcome features.

Thiopurine methyltransferase genotyping in Palestinian childhood acute lymphoblastic leukemia patients.

BackgroundThe genetic polymorphism of thiopurine methyltransferase (TPMT) is well characterized in most populations. Four common polymorphic alleles are associated with impaired activity of the enzyme. These are TPMT*2 (238G>C), TPMT*3B (c.460G>A), TPMT*3A (c.460G>A and c.719A>G) and TPMT*3C (c.719A>G). The aim of the present study was to determine the frequency of TPMT polymorphisms and their association with the occurrence of adverse events, during 6-mercaptopurine therapy in pediatric acute lymphoblastic leukemic (ALL) patients in Gaza Strip.MethodsA total of 56 DNA samples from all pediatric ALL patients admitted to the pediatric hematology departments of Gaza strip hospitals were analyzed. Genomic DNA from peripheral blood leukocytes was isolated and the TPMT*2, TPMT*3B TPMT*3A and TPMT*3C allelic polymorphism was determined by PCR-RFLP and allele specific PCR technique.ResultsNo TPMT*2, *3B or *3C alleles were detected. Only one, out of 56 patients, was found heterozygous for the TPMT*3A allele. Thus, the frequency of TPMT*3A allele was calculated to be 0.89%. Fourteen patients of ALL were suffering from myelotoxicity during 6-MP therapy. From our results, no significant association could be established between clinical and laboratory data and/or the presence of the mutation in TPMT gene.ConclusionTPMT*3A was the only deficiency allele detected in our population with an allelic frequency of 0.89%. Other polymorphic alleles in TPMT gene, or factors other than TPMT polymorphisms may be responsible for the development of myelosuppression in cases that don’t carry the investigated TPMT alleles (*2, *3A, *3B and *3C). Therefore, more studies are recommended to study such factors.

FLT3 mutations in patients with childhood acute lymphoblastic leukemia (ALL)

The type III receptor tyrosine kinase FLT3 is of importance in hematopoiesis. FLT3 is a frequently mutated gene in acute myeloid leukemia (AML) as well as prognostic marker. The most common mutation that occurs in patients includes internal tandem duplication (ITD) in the juxtamembrane domain. Point mutations in the kinase domain are also identified in many patients. These mutations result in constitutive activation of receptor as well as uncontrolled activation of survival pathways. Although 30 % of AML patients carry a mutation in FLT3 [2], its expression is not limited to only those patients. It has also been found to be expressed in acute lymphoblastic leukemia (ALL) and several studies identified FLT3 mutations in a portion of ALL patients. This study will identify possible correlation in between FLT3 and childhood ALL patients. With the search criteria depicted in Fig. 1a, we retrieved 12 references that included 1,213 childhood ALL patients with 76 patients (6.3 %) carrying FLT3 mutations (Table S1). All 12 studies described FLT3 mutations. Further review of those references identified 89 patients with MLL-rearrangement and 196 patients with hyperdiploid ALL. Although 6.3 % childhood ALL patients carried FLT3 mutations, 15.7 % of MLL-rearrangement and 14.4 % of hyperdiploid childhood ALL patients carried FLT3 mutations (data not shown). Only 1 % patients carried FLT3-ITD mutations (12/1,213). Childhood ALL patients with MLL-rearrangement displayed an overall odds ratio of 4.17 (95 % Cl = 2.60–9.88, p 0.0001) (Fig. 1b) and patients with hyperdiploid ALL showed an overall odds ratio of 4.03 (95 % Cl = 2.18–7.95, p 0.0001) (Fig. 1c). Studies with FLT3-ITD mutation (Fig. 1d) and FLT3-TKD mutations (Fig. 1e) showed that TKD mutations happened more frequently than ITD mutations. An increased rate of FLT3 mutations was observed in American population (odds ratio = 1.4, 95 % CI = 0.85–2.29), while in Japanese and European population, comparatively lower FLT3 mutations were identified (Fig. 1f). The receptor tyrosine kinase FLT3 is frequently mutated in AML and activating FLT3 mutations cause poor prognosis in this disease [2]. Several FLT3 inhibitors display promising results in clinical trials. Although several studies described presence of FLT3 mutations in childhood ALL, still we have limited knowledge in defining roles of FLT3 mutations. In this study, we carried out a meta-analysis to define the role of FLT3 mutations in childhood ALL. All 12 studies analyzed in this study were mainly from three different populations that cover 1,213 childhood ALL patients. The identification of 76 mutations in these populations suggests that FLT3 mutations in childhood ALL is less frequent than that of in AML. Although about 30 % of FLT3-ITD mutations were reported in AML [2], childhood ALL patients carry only 1 % FLT3-ITD mutations suggesting that targeting FLT3-ITD will not be beneficial in pediatric ALL. It is likely that mutations other than FLT3ITD mutations are more frequent, e. g. FLT3-TKD mutations. However, it is quite difficult to suggest a correlation between FLT3 mutations and ALL prognosis due to the Electronic supplementary material The online version of this article (doi:10.1007/s12032-013-0462-6) contains supplementary material, which is available to authorized users.

Allergy and Risk of Childhood Acute Lymphoblastic Leukemia: A Population-based and Record-based Study

A deficit of normal immune stimulation in early childhood is a suspected risk factor for both childhood acute lymphoblastic leukemia (ALL) and allergies. The present study utilized a population-based case-control design using medical claims data from the National Health Insurance Research Database of Taiwan to evaluate the association between allergy and childhood leukemia. Eight hundred forty-six childhood ALL patients who were newly diagnosed during 2000 to 2008 and were older than 1 but less than 10 years of age were individually matched with 3,374 controls based on sex, birth date, and time of diagnosis (reference date for the controls). Conditional logistic regression was performed to assess the association between childhood ALL and allergies. An increased risk of ALL was observed with having an allergy less than 1 year before the case's ALL diagnosis (odds ratio (OR) = 1.7, 95% confidence interval (CI): 1.5, 2.0), more than 1 year before the case's diagnosis (OR = 1.3, 95% CI: 1.1, 1.5), and before the age of 1 year (OR = 1.4, 95% CI: 1.1, 1.7). These results suggest that the pathogenesis of childhood ALL and allergy share a common biologic mechanism.

Second Neoplasms After Treatment of Childhood Acute Lymphoblastic Leukemia

AbstractAbstract ▪661▪This icon denotes a clinically relevant abstract Purpose:The 10-year overall survival (OS) for childhood ALL is now 80% or higher with many contemporary treatment programs. Although, second neoplasms (SMN) after diagnosis of childhood acute lymphoblastic leukemia (ALL) are rare events, toxic death in remission and death due to a SMN may constitute up to one third of all deaths among children with ALL. Previously reported cumulative incidences of SMN have varied between treatment protocols from less than 1% to 10% or more due to differences in the antileukemic therapy and duration and structure of follow-up. Method:18 collaborative groups from Europe, Asia and the US analyzed risk factors and outcome of 642 SMNs occurring as first event after childhood ALL entered onto ALL trials between 1980 and 2007. Result:186 patients had acute myeloid leukemias (AML), 138 brain tumors, 78 carcinomas, 81 lymphomas, 69 myelodysplasias (MDS), and 90 others malignancies. The median time interval to SMN was 4.8 years, being shortest for hematological malignancies (2.9 years) and longest for patients with carcinomas (10.1 years), meningiomas (16.2 years) and melanomas (10.0 years). AML/MDS (t-MN) with monosomy 7 or 5q- was associated with high-hyperdiploid ALL karyotypes, whereas MLL-rearranged AML/MDS was associated with ALL translocations (p=0.03). The pattern of SMN was significantly influenced by the preceding ALL therapy. The vast majority of CNS tumors had received cranial irradiation. The 12 CNS tumor patients who had not received CNS-irradiation were diagnosed at significantly shorter intervals after ALL than the 97 CNS tumors that occurred after CNS irradiation (median, 6.6 versus 9.1 years, P=0.01). 38 of 50 (76.0%) cases of t-MN with an aberrant karyotype and previous exposure to epipodophyllotoxins had 11q23/MLL rearrangements, whereas only four (8.0%) had monosomy 7 and none had 5q-. Patients developing myeloid malignancies had received higher maintenance therapy 6-mercaptopurine starting doses than patients with other SMNs (p< 0.001). Transplantation during first remission of ALL had been performed in 29 of the 510 ALL patients (5.7%) with available information. One of 74 (1.4%) patients with CNS tumors and seven of 193 (3.6%) with t-MN had been transplanted as compared to 9 of 32 (28.1%) with carcinomas and 8 of 52 (15.4%) with other SMNs (P<0.001). The 5 year OS for all SMNs was 40.4±2.1. Acute myeloid leukemia, myelodysplastic syndrome, and non-meningioma brain tumors (n=116) had the poorest outcome (5-year survival rate, 18.1±2.9%, 31.1±6.2%, and 18.3±3.8%, respectively). Allogeneic stem-cell transplantation failed to improve outcome of secondary myeloid malignancies after adjusting for waiting time to transplantation. The OS was 30.3±4.4% for 119 transplanted AML/MDS patients vs 11.4±4.0% for 66 non-transplanted patients (p< 0.001), but this difference in survival was eliminated after adjustment for waiting time to transplant (4.1 months). Five year OS rates were above 90% for patients with meningiomas, Hodgkin disease, thyroid carcinoma, basal cell carcinomas, and glandula parotis tumors, and 68.5±6.4% for NHL. Conclusion:Disease- and treatment-related factors influence risk of SMN among childhood ALL patients, being most pronounced for central nervous system irradiation. Except for brain tumors, AML and MDS, survival after SMN was very similar to their primary counterparts. Disclosures:No relevant conflicts of interest to declare.

Bcl-G acquitted of murder!

Bcl-G acquitted of murder!

Effectiveness of pamidronate as treatment of symptomatic osteonecrosis occurring in children treated for acute lymphoblastic leukemia

Osteonecrosis (ON) is a severe complication of acute lymphoblastic leukemia (ALL) treatments. Recent studies suggest that bisphosphonates might reduce pain and loss of motor function in patients with ON. We assessed the effects of pamidronate compared to standard care in patients with symptomatic ON (sON) and studied whether steroids might be continued after diagnosis of ON in some patients. We evaluated 17 patients with sON as complication of primary ALL treatment between 2000 and 2008. Fourteen patients were treated with pamidronate. Mobility and pain control were monitored in all patients. Affected joints were classified by magnetic resonance imaging (MRI) at ON diagnosis and after 6-72 months. Out of 220 patients with ALL, 17 (7.7%) patients developed sON. The median age at ALL diagnosis was 11 years (range: 2.7-16.6 years) and sON occurred a median of 13.4 months (range: 2.5-34 months) after ALL diagnosis. Affected joints were hip, knee and ankle. MRI scans showed 7 severe, 4 moderate, and 6 mild ON lesions. Fourteen patients showed improvement in pain (77% of patients) and motor function (59% of patients), even though corticoids were reintroduced in 4 patients. MRI demonstrated improvement, stability or worsening in 6, 3, and 5 cases, respectively. Pamidronate seems to be effective in the management of pain and motor function recovery in sON. Further studies are needed to provide evidence as to whether bisphosphonates can be recommended for the treatment or the prevention of ON in childhood ALL patients.

No evidence of increased asparagine levels in the bone marrow of patients with acute lymphoblastic leukemia during asparaginase therapy

Mesenchymal cells (MSCs) in bone marrow (BM) may produce asparagine and form protective niches for leukemic cells. In vitro, this led to high levels of asparagine and conferred asparaginase resistance to acute lymphoblastic leukemia (ALL) cells. The aim of this study was to investigate whether MSCs or other cells in BM indeed produce such significant amounts of asparagine in vivo as to result in clinical asparaginase resistance. Twenty-six patients with newly diagnosed ALL were enrolled. All children received induction chemotherapy according to the Dutch Childhood Oncology Group (DCOG) ALL-10 protocol. Asparaginase was administered from days 12-33. Asparaginase, asparagine, aspartic acid, glutamine, and glutamic acid levels were measured in BM and blood at diagnosis, days 15, 33, and 79. Median asparaginase trough levels were not significantly different at days 15 and 33. Only at diagnosis, asparagine level was significantly higher in BM than in blood (P = 0.001). Asparagine levels were all below the lower limit of quantification in BM and blood at days 15 and 33. However, aspartic acid level in BM was significantly higher than in blood (P < 0.001) at diagnosis, and also at days 15, 33, and 79. We demonstrate higher aspartic acid levels in BM compared to blood; however, no increased asparagine levels were seen during induction therapy containing asparaginase in BM when compared to blood. Therefore, increased asparagine synthesis by MSCs is of relevance for resistance to asparaginase of leukemic cells in vitro, but it is questionable whether this leads to asparaginase resistance in childhood ALL patients.

Improvements in the survival of children and adolescents with acute lymphoblastic leukemia

Over the past 50 years, the treatment of young patients with acute lymphoblastic leukemia (ALL) has significantly improved, and is one of the success stories of cancer treatment. This success is measured by the improved survival of childhood ALL patients from less than 10% in the 1960s to more than

Association between HLA-DQB1 gene and patients with acute lymphoblastic leukemia (ALL)

Acute lymphoblastic leukemia (ALL) affects both children and adults. Survival in ALL has improved in recent decades due to recognition of its biological heterogeneity. Although children have higher remission and cure rates than adults, both populations have benefited from these improvements. Our aim in this study is to determine the association between HLA-DQB1 genes with childhood and adult ALL patients. To define this association, we compared HLA-DQB1 allele frequencies and allele carrier frequencies in a cohort of 135 adults and children with ALL with 150 controls, using polymerase chain reaction with sequence-specific primers. Allele carrier frequencies in childhood ALL show a deficiency in DQ2 (*0201) (P 0.049 and RR 0.75), but an increase in DQ5 (*0501-*0504) and DQ7 (*0301, *0304) compared to the control group (P 0.001 RR 1.89, P 0.003 RR 1.48, respectively). Allele carrier frequencies in adult ALL indicated an increase in DQ5 (*0501-*0504) (P0.045 RR 2.28). Allelic frequencies in childhood ALL revealed the same increase in DQ5 and DQ7, and a decrease in DQ2. In adult ALL it shows a decrease in DQ7. Therefore, our results in adult ALL were similar to childhood ALL addressing DQ5 allele carriers, which showed an increase in both age groups. We suggest that DQ5 could be more strongly considered as an ALL susceptibility allele, and that this allele may underlie a pathogenic phenotype with a major role in the immunologic process involved in both adults and children with ALL.

Genome-wide analysis of cytogenetic aberrations in ETV6/RUNX1-positive childhood acute lymphoblastic leukaemia

The chromosomal translocation t(12;21) resulting in the ETV6/RUNX1 fusion gene is the most frequent structural cytogenetic abnormality among patients with childhood acute lymphoblastic leukaemia (ALL). We investigated 62 ETV6/RUNX1-positive childhood ALL patients by single nucleotide polymorphism array to explore acquired copy number alterations (CNAs) at diagnosis. The mean number of CNAs was 2·82 (range 0-14). Concordance with available G-band karyotyping and comparative genomic hybridization was 93%. Based on three major protein-protein complexes disrupted by these CNAs, patients could be categorized into four distinct subgroups, defined by different underlying biological mechanisms relevant to the aetiology of childhood ALL. When recurrent CNAs were evaluated by an oncogenetic tree analysis classifying their sequential order, the most common genetic aberrations (deletions of 6q, 9p, 13q and X, and gains of 10 and 21) seemed independent of each other. Finally, we identified the most common regions with recurrent gains and losses, which comprise microRNA clusters with known oncogenic or tumour-suppressive roles. The present study sheds further light on the genetic diversity of ETV6/RUNX1-positive childhood ALL, which may be important for understanding poor responses among this otherwise highly curable subset of ALL and lead to novel targeted treatment strategies.

Chromosomal Aberrations in ETV6/RUNX1-positive Childhood Acute Lymphoblastic Leukemia using 244K Oligonucleotide Array Comparative Genomic Hybridization

BackgroundAcute lymphoblastic leukemia (ALL) is a heterogeneous form of hematological cancer consisting of various subtypes. We are interested to study the genetic aberration in precursor B-cell ALL with specific t(12;21) translocation in childhood ALL patients. A high resolution 244K array-based Comparative Genomic Hybridization (array-CGH) was used to study eleven ETV6/RUNX1-positive childhood acute lymphoblastic leukemia (ALL) patients.Result155 chromosomal aberrations (119 losses, 36 gains) were reported in the array findings, corresponding to 76.8% deletions and 23.2% amplifications. The ETV6 gene deletion occurred in 4 of the patients, corresponding to 45% of the sample. The most common alterations above 1 Mb were deletion 6q (13%), 12p (12%) and 9p (8%), and duplication 4q (6%) and Xq (4%). Other genes important in ALL were also identified in this study including RUNX1, CDKN2A, FHIT, and PAX5. The array-CGH technique was able to detect microdeletion as small as 400 bp.ConclusionThe results demonstrate the usefulness of high resolution array-CGH as a complementary tool in the investigation of ALL.

Gene Dose Effects of GSTM1, GSTT1 and GSTP1 Polymorphisms on Outcome in Childhood Acute Lymphoblastic Leukemia

Children with acute lymphoblastic leukemia (ALL) react very differently to chemotherapy. One explanation for this is inherited genetic variation. The glutathione S-transferase (GST) enzymes inactivate a number of chemotherapeutic drugs administered in childhood ALL therapy. Two multiplexing methods were applied for genotyping the GSTM1 and GSTT1 genes (distinguishing between 0, 1, or 2 gene copies) and the GSTP1 313 A>G polymorphism, simultaneously. A total of 263 childhood ALL patients were genotyped. No gene dose effect on outcome was demonstrated with either GST polymorphisms. Grouping of GSTM1 and GSTT1 into poor (0 or 1 gene copy)-and good metabolizers (at least 2 gene copies)-showed that the poor metabolizers had a trend toward a better outcome (event-free survival =91.8%) compared with the good metabolizers (event-free survival =83.2%). Similarly, in the adjusted analysis the good metabolizers demonstrated a 2.2-fold higher risk trend of experiencing an event (resistant disease or relapse) compared with the poor metabolizers (P=0.066; hazard ratio =2.248; 95% confidence interval, 0.948-5.327). In conclusion, our results suggest that the combined gene dose of GSTM1 and GSTT1 may influence outcome in childhood ALL.

Early Response to Dexamethasone as Prognostic Factor: Result from Indonesian Childhood WK-ALL Protocol in Yogyakarta

Early response to treatment has been shown to be an important prognostic factor of childhood acute lymphoblastic leukemia (ALL) patients in Western studies. We studied this factor in the setting of a low-income province in 165 patients treated on Indonesian WK-ALL-2000 protocol between 1999 and 2006. Poor early response, defined as a peripheral lymphoblasts count of ≥1000/μL after 7 days of oral dexamethasone plus one intrathecal methotrexate (MTX), occurred in 19.4% of the patients. Poor responders showed a higher probability of induction failures compared to good responders (53.1% versus 23.3%, P < 0.01), higher probability of resistant disease (15.6% versus 4.5%, P = 0.02), shorter disease-free survival (P = 0.034; 5-year DFS: 24.9% ± 12.1% versus 48.6% ± 5.7%), and shorter event-free survival (P = 0.002; 5-year EFS: 9.7% ± 5.3% versus 26.3% ± 3.8%). We observed that the percentage of poor responders in our setting was higher than reported for Western countries with prednisone or prednisolone as the steroids. The study did not demonstrate a significant additive prognostic value of early response over other known risk factors (age and white blood cell count) for DFS and only a moderately added value for EFS.

Up-regulation of β-arrestin1 in children with acute lymphoblastic leukemia and its clinical significance

Objective To explore the expression of β-arrestin1 in newly-diagnosed childhood with acute lymphoblastic leukemia(ALL) and its possible clinical significance.Methods A total of 155 newly-diagnosed childhood ALL patients were enrolled in this study.Fifty-one sex-and age-matched patients without malignant hematological diseases were used as controls.The bone marrow samples were collected and from which the mononuclear cells were isolated.The β-arrestin1 mRNA expression was detected by quantitative real-time RT-PCR,and β-arrestin1 protein expression was detected by Western blotting analysis and immunofluorescence assays.Spearman correlation coefficient was used to analyze the correlations of β-arrestin1 expression with the clinical and biological features ALL patients.Results The expression of β-arrestin1 mRNA and protein in ALL patients was significantly higher than that in the controls(P0.01).β-arrestin1 mRNA expression in ALL patients of high risk(HR) group and medium risk(MR) group was significantly higher than that of the standard risk(SR) group,and that in HR group was significantly higher than that in MR group(P0.05).Spearman correlation analysis showed that β-arrestin1 expression was positively correlated with WBC count(r=0.458,P=0.002) and risk classification(r=0.344,P=0.022) of ALL patients.Conclusion Bone marrow β-arrestin1 expression is closely correlated with the risk classfication of child ALL patients,which is of great significance for the diagnosis,therapy strategy and prognosis of ALL.

Methotrexate consolidation treatment according to pharmacogenetics of MTHFR ameliorates event-free survival in childhood acute lymphoblastic leukaemia

Recent advances in treatment for childhood acute lymphoblastic leukaemia (ALL) have significantly increased outcome. High-dose methotrexate (MTX) is the most commonly used regimen during the consolidation period, but the optimal dose remains to be defined. We investigated the usefulness of the MTHFR genotype to increase the MTX dosage in the consolidation phase in 141 childhood ALL patients enrolled in the ALL/SHOP-2005 protocol. We also investigated the pharmacogenetic role of polymorphisms in genes involved in MTX metabolism on therapy-related toxicity and survival. Patients with a favourable MTHFR genotype (normal enzymatic activity) treated with MTX doses of 5 g m⁻² had a significantly lower risk of suffering an event than patients with an unfavourable MTHFR genotype (reduced enzymatic activity) that were treated with the classical MTX dose of 3 g m⁻² (P=0.012). Our results indicate that analysis of the MTHFR genotype is a useful tool to optimise MTX therapy in childhood patients with ALL.