Epigenetic analysis of childhood acute lymphoblastic leukemia

We used a chromosome 3 wide NotI microarray for identification of epigenetically inactivated genes in childhood acute lymphoblastic leukemia (ALL). Three novel genes demonstrated frequent methylation in childhood ALL. PPP2R3A (protein phosphatase 2, regulatory subunit B'', alpha) was frequently methylated in T (69%) and B (82%)-ALL. Whilst FBLN2 (fibulin 2) and THRB (thyroid hormone receptor, beta) showed frequent methylation in B-ALL (58%; 56% respectively), but were less frequently methylated in T-ALL (17% for both genes). Recently it was demonstrated that BNC1 (Basonuclin 1) and MXS1 (msh homeobox 1) were frequently methylated across common epithelial cancers. In our series of childhood ALL BNC1 was frequently methylated in both T (77%) and B-ALL (79%), whilst MSX1 showed T-ALL (25%) specific methylation. The methylation of the above 5 genes was cancer specific and expression of the genes could be restored in methylated leukemia cell lines treated with 5-aza-2’-deoxycytidine. This is the first report demonstrating frequent epigenetic inactivation of PPP2R3A, FBLN2, THRB, BNC1 and MSX1 in leukemia. The identification of frequently methylated genes showing cancer specific methylation will be useful in developing early cancer detection screens and for targeted epigenetic therapies.     

Quantitative expression of TEL in childhood acute lymphoblastic leukemia

Loss of heterozygosity of chromosome 12p in human precursor B-cell ALL invariably results in loss of TEL coding sequences. Accompanied by a 12;21 translocation, such loss of heterozygosity ensures complete loss of the wild-type TEL. No inactivating mutations of the retained TEL allele have been reported in leukemias with hemizygous deletion. However, only minimal data reported the expression of the wild-type TEL in ALL. We now demonstrate that quantitative real-time RT-PCR from leukemic RNA samples could be indicative of compromised TEL expression in childhood ALL and therefore loss of TEL function.     

Molecular diagnostics in the treatment of childhood acute lymphoblastic leukemia

Somatically acquired genetic alterations play an important role in the pathogenesis of acute lymphoblastic leukemia. The molecular analysis of these alterations has increased our understanding of the mechanisms of leukemogenesis. In addition, this information has led to improvements in our abilities to predict treatment response and to deliver the optimal intensity of treatment to individual patients. For example, the prognosis for patients with acute lymphoblastic leukemia whose leukemic cells express the TEL-AML1 fusion is favorable when they are treated on modem chemotherapy protocols, whereas patients whose leukemic lymphoblasts contain the MLL-AF4 or the BCR-ABL fusion sometimes require allogeneic hematopoietic stem cell transplantation for cure. Molecular techniques are also used to detect minimal residual disease and genetic polymorphisms that are important in optimizing drug therapy.     

Hungarian experience in the treatment of childhood acute lymphoblastic leukemia

The Hungarian Pediatric Oncology Study Group treated 362 acute lymphoblastic leukemia patients between 1990 and 1995 using the the ALL-BFM 90 protocol. The modified protocol, ALL-BFM 95, was used later to treat 257 patients. The two protocols were similarly successful to treat low and medium risk cases. However, the ALL-BFM 95 protocoll was more efficient to treat high risk patients and resulted in 10% increase in survival. The Western-European results are superior by 10-15% compared to the Hungarian data mainly due to the relatively high proportion of the early death in Hungary. Improvement of these data can only be expected from the development of the diagnostic potentials and from further improvement of treatment of the high isk patients.     

Lymphoblast cell size and prognosis in acute lymphoblastic leukemia in childhood.

The significance of cell size as a prognostic indicator in acute lymphoblastic leukemia (ALL) is controversial. Accuracy in measurement of cell size can be improved by determination of cell areas instead of single cell diameters. In the present study cell areas of 200 cells were determined in pretreatment bone marrows of 35 children with ALL. For better comparison with other studies which had used cell diameters only, the measured area was expressed as circle area from which the circle diameter was calculated. Cells with a diameter of greater than 12 micron were defined as macrolymphoblasts (MLB). Several clinical characteristics considered to be risk factors in ALL were ascertained for each patient. The duration of first complete remission was used to assess the prognostic significance of cell size and of number of risk factors. In contrast to previous reports patients with more than 25% MLB had longer remissions. However, nearly all patients of this group had no or one risk factor only. When patients with more than one risk factor were excluded from statistical analysis, the group with more than 25% MLB had no longer a better prognosis compared to the group with 25% MLB or less. Thus, in this study the percentage of MLB was not an independent prognostic indicator for risk of relapse in ALL.     

Prognostic value of morphometry in acute lymphoblastic leukemia of childhood.

The prognostic value of morphometric features was studied in a group of 33 children with acute lymphoblastic leukemia (ALL) and compared with clinical and hematologic parameters. Air dried, May-Grünwald-Giemsa-stained specimens were prepared from iliac crest biopsies, and for each patient, 150 blasts and their nuclei were selected according to a stratified selection method and measured on a graphic tablet system. Univariate overall survival analysis showed the French-American-British (FAB) classification to be the strongest clinical parameter (P less than .0001). However, the significance was mainly due to the fact that both L3 cases died; the results for L1 and L2 were less satisfactory, with a survival rate (at 10 years) of 69% for the 26 L1 cases and 80% for the five L2 cases. The nuclear/cytoplasmic (N/C) ratio was the best morphometric feature (P less than .0001) and provided more satisfactory classification results than did FAB: only 2 of the 21 (10%) cases with N/C ratios greater than 0.90 died (7 and 9.5 years after the diagnosis, respectively), and 9 of the 12 (75%) cases with N/C ratios greater than or equal to 0.90 died. For recurrence-free survival analysis, essentially the same results were obtained. The N/C ratio retained its significant prognostic value after recurrence: 11 of the 15 patients with eventual recurrences died; 9 of them had an (original) N/C ratio less than or equal to 0.90. Three of the four recurring cases that survived after recurrence had an N/C ratio greater than 0.90 (P less than .03).(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of fragile sites in childhood acute lymphoblastic leukemia patients and normal controls

Summary A high concordance has been reported between fragile sites and breakpoints involved in chromosomal rearrangements in cancer. A prospective study on the role of fragile sites in the etiology of childhood acute lymphocytic leukemia (ALL), with appropriate comparisons to results obtained from normal controls, analyzed fluorodeoxyuridine-, aphidicolin-, and caffeine-induced fragile sites in the peripheral blood of seven ALL patients (three with cytogenetically normal karyotype and four with pseudodiploid karyotype) and eight normal controls. While extensive variations in the number and distribution of fragile sites was observed within each group, there was no significant difference in the mean total fragile sites and mean fragile sites per cell between the two groups (P>0.05) in all three treatments. Similarly, within the ALL patients, the two karyotypic groups did not exhibit any significant difference in fragility (P>0.05).     

Intrinsic and chemo-sensitizing activity of SMAC-mimetics on high-risk childhood acute lymphoblastic leukemia

SMAC-mimetics represent a targeted therapy approach to overcome apoptosis resistance in many tumors. Here, we investigated the efficacy of the SMAC-mimetic BV6 in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). In ALL cell lines, intrinsic apoptosis sensitivity was associated with rapid cIAP degradation, NF-κB activation, TNF-α secretion and induction of an autocrine TNF-α-dependent cell death loop. This pattern of responsiveness was also observed upon ex vivo analysis of 40 primograft BCP-ALL samples. Treatment with BV6 induced cell death in the majority of ALL primografts including leukemias with high-risk and poor-prognosis features. Inhibition of cell death by the TNF receptor fusion protein etanercept demonstrated that BV6 activity is dependent on TNF-α. In a preclinical NOD/SCID/huALL model of high-risk ALL, marked anti-leukemia effectivity and significantly prolonged survival were observed upon BV6 treatment. Interestingly, also in vivo, intrinsic SMAC-mimetic activity was mediated by TNF-α. Importantly, BV6 increased the effectivity of conventional induction therapy including vincristine, dexamethasone and asparaginase leading to prolonged remission induction. These data suggest SMAC-mimetics as an important addendum to efficient therapy of pediatric BCP-ALL.In childhood acute lymphoblastic leukemia (ALL), major improvements in therapy and supportive care have led to increased survival rates.¹ In current treatment protocols, patients are assigned to multi-agent chemotherapy regimens of different intensities based on the patient''s individual risk profile and probability to encounter relapse.² However, 20% of patients relapse or do not respond well to therapy.^{3, 4} Therefore, new treatment strategies are required to overcome resistance mechanisms and enhance outcome. Defects in cell death mechanisms contribute to resistance and treatment failure in many cancers, including leukemia.^{5, 6} We previously described, that deficient apoptosis signaling in primary, patient-derived leukemia cells is indicative for early patient relapse and associated with rapid engraftment in NOD/SCID mice.^{7, 8, 9, 10}Two major apoptosis signaling pathways are known, the death receptor (extrinsic) and mitochondrial (intrinsic) pathway.¹¹ Within the extrinsic pathway, activation of death receptors of the tumor necrosis factor (TNF) superfamily activate caspase-8 via a death-inducing signaling complex (DISC) leading to activation of effector caspases.¹² Activation of intrinsic signaling triggers mitochondrial release of cytochrome c and SMAC (second mitochondria-derived activator of caspase). Released cytochrome c forms a complex together with caspase-9, the apoptosome, which cleaves and activates caspase-3. SMAC antagonizes inhibitor of apoptosis proteins (IAPs), thus enabling apoptosis signaling.¹³IAPs constitute a family of structurally related proteins, defined by the presence of at least one baculoviral IAP repeat (BIR) domain, which can bind to active subunits of caspases, thereby inhibiting their function. Some IAPs (e.g., cIAP) also possess a RING domain conferring E3 ligase activity mediating ubiquitination reactions.¹⁴Since IAPs are often overexpressed and associated with inferior outcome in different malignancies including childhood acute leukemias, they may serve as an attractive target for therapeutic intervention.^{15, 16, 17, 18} Previously, IAP antagonists, binding to selected BIR domains of IAPs and leading to cell death sensitization were designed.^{16, 19, 20, 21} Recently, a new class of molecules mimicking the N-terminal domain of SMAC (SMAC-mimetics) was developed, which bivalently bind to both BIR2 and BIR3 domains, and possess additional intrinsic apoptogenic activity via a TNF-α feed forward loop.^{22, 23} Within this TNF-α loop, SMAC-mimetics stimulate the E3-ubiquitine ligase activity of cIAPs, leading to autoubiquitination and subsequent proteasomal degradation of cIAPs. Depletion of cIAPs enables accumulation of NF-κB-inducing kinase (NIK), resulting in non-canonical activation of NF-κB and NF-κB target gene expression, including TNFA, which stimulates TNFR1 in an autocrine loop.²³ In the absence of cIAP, TNFR1-stimulation triggers the assembly of the secondary RIP1-containing cytoplasmatic complex (complex II), leading to caspase-8 activation and cell death induction.²⁴ SMAC-mimetics may induce cell death dependently or independently of TNF-α,^{25, 26, 27} and we previously showed, that counteracting caspase inhibition by IAP antagonists sensitizes cancer cells for death receptor ligand- and chemotherapy-induced apoptosis.^{28, 29}Here, we analyze the therapeutic potential of the SMAC-mimetic BV6 in pediatric B-cell precursor (BCP)-ALL in vitro, ex vivo and in a preclinical model in vivo, and define the molecular requirements for intrinsic activity.     

HLA complex-linked heat shock protein genes and childhood acute lymphoblastic leukemia susceptibility

Three heat shock protein 70 (HSP70) genes, HSPA1L, HSPA1A, and HSPA1B, are located within the human leukocyte antigen (HLA) class III region. HSPs act as stress signals and regulate natural killer cell response to cancer. HSP70 gene polymorphisms show disease associations partly due to their linkage disequilibrium with HLA alleles. To systematically evaluate their associations with childhood acute lymphoblastic leukemia (ALL), we examined the three functional single nucleotide polymorphisms (SNPs) rs2227956 (T493M) in HSPA1L, rs1043618 in HSPA1A 5′UTR, and rs1061581 (Q351Q) in HSPA1B by TaqMan assays or polymerase chain reaction–restriction fragment length polymorphism in 114 ALL cases and 414 controls from Wales (UK), in 100 Mexican Mestizo ALL cases and 253 controls belonging to the same ethnic group, and in a panel of 82 HLA-typed reference cell line samples. Homozygosity for HSPA1B rs1061581 minor allele G was associated with protection (odds ratio (OR) = 0.37, 95% confidence interval (CI) = 0.16–0.78; P = 0.007) with gene-dosage effect (additive model) reaching significance (P = 0.0001) in the Welsh case–control group. This association was replicated in the second case–control group from Mexico (OR (recessive model) = 0.49, 95% CI = 0.24–0.96; P = 0.03), and the pooled analysis yielded a strong association (Mantel–Haenszel OR = 0.43, 95% CI = 0.27–0.69, P = 0.0004). The association was stronger in males in each group and in the pooled analysis. A three-SNP haplotype including the major allele A of rs1061581 showed a highly significant increase in Welsh cases compared with respective controls (6.7% vs 1.8%; P = 0.0003) due to the difference between male cases and controls. The protective allele of rs1061581 occurred more frequently on the HLA-DRB3 haplotypes (especially DRB1*03) in the cell line panel, but the HSPA1B association was independent from the HLA-DRB4 association previously detected in the same case–control group from Wales (adjusted P = 0.001). Given the cancer promoting roles played by HSPs intracellularly as well as roles in immune surveillance when expressed on the cell surface and the known correlations between expression levels and the HSP polymorphisms, these results are likely to indicate a primary association and warrant detailed assessment in childhood ALL development.     

Childhood acute lymphoblastic leukemia

As cure rates in childhood acute lymphoblastic leukemia reach 80%, emphasis is increasingly placed on the accurate identification of drug-resistant cases, the elucidation of the mechanisms involved in drug resistance and the development of new therapeutic strategies targeted toward the pivotal molecular lesions. Pharmacodynamic and pharmacogenomic studies have provided rational criteria for individualizing therapy to enhance efficacy and reduce acute toxicity and late sequelae. Currently, assessment of the early response to treatment by measurement of minimal residual disease (MRD) is the most powerful independent prognostic indicator. MRD is affected by both the drug sensitivity of leukemic cells and the pharmacodynamic and pharmacogenetic properties of the host cells. Rapid advances in biotechnology and bioinformatics should ultimately facilitate the development of molecular diagnostic assays that can be used to optimize antileukemic therapy and elucidate the mechanisms of leukemogenesis. In the interim, prospective clinical trials have provided valuable clues that are further increasing the cure rate of childhood acute lymphoblastic leukemia.     

Identification of prognostic protein biomarkers in childhood acute lymphoblastic leukemia (ALL)

Early response to 7 days of prednisolone (PRED) treatment is one of the important prognostic factors in predicting eventual outcome in childhood acute lymphoblastic leukemia (ALL). Using proteomic tools and clinically important leukemia cell lines (REH, 697, Sup-B15, RS4; 11), we have identified potential prognostic protein biomarkers as well as discovered promising regulators of PRED-induced apoptosis. After treatment with PRED, the four cell lines can be separated into resistant (REH) and sensitive (697, Sup-B15, RS4;11). Two dimensional gel electrophoresis (2-DE) and MALDI-TOF/TOF MS identified 77 and 17 significantly differentially expressed protein spots (p<0.05) in PRED-sensitive and PRED-resistant cell lines respectively. Several of these were validated by Western blot including proliferating cell nuclear antigen (PCNA), cofilin 1, voltage-dependent anion-channel protein 1 (VDAC1) and proteasome activator subunit 2 (PA28β). PCNA is a promising protein because of its important roles both in cell cycle regulation and survival control. We subsequently validated PCNA in 43 paired bone marrow samples from children with newly diagnosed ALL (Day 0) and 7 days after PRED treatment (Day 8). ROC curve analysis confirmed that PCNA was highly predictive of PRED response in patients (AUC=0.81, p=0.007) and most interestingly, independent of the molecular subtype, providing a promising universal prognostic marker.     

Atherogenic low density lipoprotein phenotype in long-term survivors of childhood acute lymphoblastic leukemia

Survivors of childhood acute lymphoblastic leukemia (ALL) have an increased risk of cardiovascular disease. Small density lipoproteins are atherogenic but have not been studied in this population. We conducted a cross-sectional analysis of 110 ALL survivors (mean age, 24.3 years) to determine prevalence of small dense LDL (pattern B) phenotype in ALL survivors and identify associated factors. Lipid subfractions were measured using Vertical Auto Profile-II. Participants with greater than 50% of LDL-cholesterol (LDL-c) in small dense LDL fractions (LDL₃₊₄) were classified as LDL pattern B. Visceral and subcutaneous adipose tissue (VAT, SAT) volumes were also measured by computed tomography. While the mean LDL-c level of ALL survivors was 108.7 ± 26.8 mg/dl, 36% (40/110) of survivors had atherogenic LDL pattern B. This pattern was more common in males (26/47; 55%) than in females (14/63; 22%, P = 0.001) and more common in survivors treated with cranial radiotherapy (15/33; 45%) than in those who were treated with chemotherapy alone (25/77; 33%; P = 0.04, adjusted for age, gender, history of hypertension, and smoking history). VAT was associated with atherogenic lipids: LDL pattern B and LDL₃₊₄ levels. This association was independent of other measures of body fat. We conclude that a substantial proportion of ALL survivors had an atherogenic LDL phenotype despite normal mean LDL-c levels. An atherogenic LDL phenotype may contribute to the increase in cardiovascular mortality and morbidity in this population.     

Identification of potential predictive markers of dexamethasone resistance in childhood acute lymphoblastic leukemia

Response to dexamethasone (DEXA), as a hallmark drug in the treatment of childhood acute lymphoblastic leukemia (ALL), is one of the pivotal prognostic factors in the prediction of outcome in ALL. Identification of predictive markers of chemoresistance is beneficial to selecting of the best therapeutic protocol with the lowest effect adverse. Hence, we aimed to find drug targets using the 2DE/MS proteomics study of a DEXA-resistant cell line (REH) as a model for poor DEXA responding patients before and after drug treatment. Using the proteomic methods, three differentially expressed proteins were detected, including voltage dependent anion channel 1 (VDAC1), sorting Nexin 3 (SNX3), and prefoldin subunit 6 (PFDN6). We observed low expression of three proteins after DEXA treatment in REH cells. We subsequently verified low expression of resulted proteins at the mRNA level using the quantitative PCR method. These proteins are promising proteins because of their important roles in drug resistance and regulation of apoptosis (VDAC1), protein trafficking (SNX3), and protein folding (PFDN6). Additionally, mRNA expression level of these proteins was assessed in 17 bone marrow samples from children with newly diagnosed ALL and 7 non-cancerous samples as controls. The results indicated that independent of the molecular subtypes of leukemia, mRNA expression of VDAC1, SNX3, and PFDN6 decreased in ALL samples compared with non-cancerous samples particularly in VDAC1 (p?<?0.001). Additionally, mRNA expression of three proteins was also declined in high-risk samples compared with standard risk cases. These results demonstrated diagnostic and prognostic value of these proteins in childhood ALL. Furthermore, investigation of protein-protein interaction using STRING database indicated that these proteins involved in the signaling pathway of NR3C1 as dexamethasone target. In conclusion, our proteomic study in DEXA resistant leukemic cells revealed VDAC1, SNX3, and PFDN6 are promising proteins that might serve as potential biomarkers of prognosis and chemotherapy in childhood ALL.     

New chromosomal translocations correlate with specific immunophenotypes of childhood acute lymphoblastic leukemia

Cytogenetic analysis of leukemic cells obtained at diagnosis from 122 patients with childhood acute lymphoblastic leukemia (ALL) disclosed chromosomal translocations in 36 cases. Two new nonrandom translocations were identified and found to be associated with specific immunophenotypes of the disease. The first, identified in 4 of 16 cases of T-cell ALL positive for sheep erythrocyte receptors (E+), involved the short arm (p) of chromosome 11 and the long arm (q) of chromosome 14 and was designated t(11;14) (p13;q13). The second, found in 7 of 23 cases with a pre-B-cell phenotype, involved the long arm of chromosome 1 and the short arm of chromosome 19; it was designated t(1;19) (q23;p13.3). A third abnormality involving a common breakpoint on chromosome 12 (band p 12) was also identified. These two new differentiation-specific translocations suggest a mechanism for aberrant expression of genes that influence lymphoid cell growth and development, as well as leukemogenesis.