The kinetics of blast clearance are associated with copy number alterations in childhood B-cell acute lymphoblastic leukemia.

We analyzed the pattern of whole-genome copy number alterations (CNAs) and their association with the kinetics of blast clearance during the induction treatment among 195 pediatric patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) who displayed intermediate or high levels of minimal residual disease (MRD). Using unsupervised hierarchical clustering of CNAs > 5 Mbp, we dissected three clusters of leukemic samples with distinct kinetics of blast clearance [A - early slow responders (n=105), B - patients with persistent leukemia (n=24), C - fast responders with the low but detectable disease at the end of induction (n=66)] that corresponded with the patients' clinical features, the microdeletion profile,the presence of gene fusions and patients survival. Low incidence of large CNAs and chromosomal numerical aberrations occurred in cluster A which included ALL samples showing recurrent microdeletions within the genes encoding transcription factors (i.e., IKZF1, PAX5, ETV6, and ERG), DNA repair genes (XRCC3 and TOX), or harboring chromothriptic pattern of CNAs. Low hyperdiploid karyotype with trisomy 8 or hypodiploidy was predominantly observed in cluster B. Whereas cluster C included almost exclusively high-hyperdiploid ALL samples with concomitant mutations in RAS pathway genes. The pattern of CNAs influences the kinetics of leukemic cell clearance and selected aberrations affecting DNA repair genes may contribute to BCP-ALL chemoresistance.

Prognostic significance of IKZF1 deletions and IKZF1plus profile in children with B-cell precursor acute lymphoblastic leukemia treated according to the ALL-IC BFM 2009 protocol.

The strongest predictors of outcome in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) are minimal residual disease (MRD) and specific molecular abnormalities. One unfavorable prognostic factor is the presence of IKZF1 gene aberrations, particularly when co-occurring with high MRD level at the end of induction treatment. The present study determines the predictive value of a recently-defined IKZF1-plus (IKZF1plus ) microdeletion profile in 373 children with BCP-ALL treated according to the ALL-intercontinental Berlin-Frankfurt-Munster protocol 2009 protocol. IKZF1-wild type (IKZF1wt ) patients demonstrated lower leukemic burden parameters than those carrying IKZF1 deletion (IKZF1del [n = 26, 7.0%]) or IKZF1plus pattern (n = 34, 9.1%): (i) median blast percentage at diagnosis (78.0% vs. 86.9% vs. 86.0%; p = 0.021); (ii) median MRD level at day 15 of induction protocol (0.3% vs. 2.1% vs. 0.8%; p = 0.011); (iii) poor steroid response (7.6% vs. 26.5% vs. 12.5%; p = 0.010). Minimal residual disease level at day 33 (MRD33) exceeding 10-4 was more frequently observed in both the IKZF1del and IKZF1plus subgroups than in IKZF1wt patients (n = 9 [36.0%] vs. n = 13 [41.9%] vs. n = 70 [24.0%], p = 0.051). IKZF1plus individuals showed a tendency for a lower MRD reduction between day 15 and 33 compared to IKZF1del patients (p = 0.124). IKZF1del and IKZF1plus patients showed decreased relapse-free survival (HR [95%CI] for IKZF1wt as reference = 2.72 [1.21-6.11] and 2.00 [0.87-4.49], respectively, p = 0.023). Both genetic markers including IKZF1del and IKZF1plus microdeletion profile provide additional predictive value of treatment outcome in childhood BCP-ALL and may contribute to more efficient patient stratification; the same is true in MRD guided protocols, which are based on flow cytometric measurements on day 15 of induction protocol.

Advantages and Limitations of SNP Array in the Molecular Characterization of Pediatric T-Cell Acute Lymphoblastic Leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is a highly heterogeneous disease, and numerous genetic aberrations in the leukemic genome are responsible for the biological and clinical differences among particular ALL subtypes. However, there is limited knowledge regarding the association of whole-genome copy number abnormalities (CNAs) in childhood T-ALL with the course of leukemia and its outcome. The aim of this study was to identify the pattern of whole-genome CNAs in 86 newly diagnosed childhood T-ALL cases using a high-density single-nucleotide polymorphism array. We analyzed the presence of whole-genome CNAs with respect to immunophenotype, clinical features, and treatment outcomes. A total of 769 CNAs, including trisomies, duplications, deletions, and segmental loss of heterozygosity, were detected in 86 analyzed samples. Gain or loss of chromosomal regions exceeding 10 Mb occurred in 46 cases (53%), including six cases (7%) with complex chromosomal alterations. We observed that microdeletions in selected genes (e.g., FIP1L1 and PDGFRB) were related to the clinical features. Interestingly, 13% of samples have a duplication of the two loci (MYB and AIH1-6q23.3), which never occurred alone. Single-nucleotide polymorphism array significantly improved the molecular characterization of pediatric T-ALL. Further studies with larger cohorts of patients may contribute to the selection of prognostic CNAs in this group of patients.

MLPA as a complementary tool for diagnosis of chromosome 21 aberrations in childhood BCP-ALL.

Chromosome 21 abnormalities are the most frequent genetic findings in childhood B cell precursor acute lymphoblastic leukemia (BCP-ALL) cases. Majority of patients are effectively diagnosed with fluorescence in situ hybridization (FISH) and karyotyping; however, some cases may require additional tools to be used. Bone marrow samples of 373 childhood BCP-ALL patients were tested for chromosome 21 copy number variations (CNVs) with Multiplex Ligation-dependent Probe Amplification (MLPA) P327 array. Results from MLPA and cytogenetics were compared between groups according to the type of abnormality found on chromosome 21. Out the group of 235 patients, chromosome 21 multiplication was found by FISH assay in 56 cases (23.81%), ETV6-RUNX1 fusion in 34 (14.47%) and iAMP21 in 3 (1.28%) children, remaining 142 (60.43%) patients had no known chromosome 21 aberration. Median peak ratios of all tested probes in MLPA in aforementioned groups were 1.47 (IQR 1.28-1.77) vs. 1.00 (IQR 1.00-1.09) vs. 2.79 (IQR 1.97-2.83) vs. 1.00 (1.00-1.11), respectively. Aforementioned peak ratio of ETV6-RUNX1 fusion group was similar with patients of no known chromosome 21 aberration (p = 0.71). Interestingly, both groups differed from patients with chromosome 21 multiplication (p < 10-5) and with iAMP21 (p < 10-5). All cases of iAMP21 were correctly recognized by MLPA. MLPA seems to be good additional tool in the diagnostic process of chromosome 21 CNVs, especially in cases with iAMP21.

Sensitive detection of FGFR1 N546K mosaic mutation in patient with encephalocraniocutaneous lipomatosis and pilocytic astrocytoma.

Encephalocraniocutaneous lipomatosis (ECCL) is a rare neurocutaneous disorder, with only about 100 cases reported worldwide. It is characterized by congenital lesions of the eye, skin, and central nervous system. Only recently, potential causative FGFR1 point mutations have been identified in brain tumors and cultured skin biopsies from patients with this condition. Here, we analyzed the molecular status of a patient with ECCL and a coexisting pilocytic astrocytoma with detected FGFR1 N546K mutation. The presence of the alteration in both affected and unaffected tissues has been evaluated using Sanger sequencing and droplet digital polymerase chain reaction (ddPCR) technique. The ddPCR analysis showed differential distribution of the alteration in all specimens, including unaffected and untreated samples. Therefore, we confirm that FGFR1 N546K is a plausible causative mutation of ECCL patients and could be associated with a risk of brain tumor development. We also show the usefulness of sensitive ddPCR method for detection of low levels of autosomal mosaic mutation in blood or swabs. We suggest that utilization of this method may improve the diagnostic process, especially when targeted therapies are considered.

Microarray testing as an efficient tool to redefine hyperdiploid paediatric B-cell precursor acute lymphoblastic leukaemia patients.

The aim of our study was to characterize genetic alterations in a cohort of paediatric patients with B-cell progenitors (BCP-ALL) and a hyperdiploid karyotype. In our study, we analysed 55 childhood hyperdiploid BCP-ALL patients using single nucleotide polymorphism (SNP) microarray testing. The group consisted mostly of patients with the modal number of chromosomes between 54 and 58 (34 cases). Within this group, Trisomy 4 and Trisomy 10 (30 cases) were the most frequent cases. Additionally, a total of 93 structural abnormalities mainly affecting chromosomes 1, 6, 9, 12, and 17 as well as 68 copy number alterations (CNAs) were identified. The microarray testing revealed a loss of ETV6, IKZF1, CDKN2A/CDKN2B, PAX5, and RB1. Moreover, chromosomal abnormalities resulting in the loss of heterozygosity (LOH) were also observed. Currently, patients with hyperdiploidy constitute a genetically heterogeneous group, and therefore, it is insufficient to rely only on banding cytogenetic analysis for the identification of hyperdiploid karyotype. Microarray testing has been proven an effective and satisfactory tool for the analysis of molecular karyotypes and to redefine the prognostic criteria in hyperdiploid patients.

GATA3 germline variant is associated with CRLF2 expression and predicts outcome in pediatric B-cell precursor acute lymphoblastic leukemia.

The germline variant at rs3824662 in GATA3 is a risk locus for Philadelphia-like acute lymphoblastic leukemia (Ph-like ALL), the biological subtype of B-cell precursor (BCP)-ALL defined by a distinct gene expression profile and the presence of specific somatic aberrations including rearrangements of CRLF2. In this study, we investigated whether rs3824662 in GATA3 associates with CRLF2 expression in leukemic cells and predicts prognosis in pediatric BCP-ALL patients treated according to the ALL Intercontinental Berlin-Frankfurt-Münster (IC BFM) 2009 (n = 645) and the ALL IC BFM 2002 (n = 216) protocols. High expression of CRLF2 was observed at both protein and mRNA levels (fourfold higher in AA than in CA + CC) among GATA3 AA variant carriers, independent of the presence of P2RY8-CRLF2 fusion. Additionally, the AA variant at rs3824662 was a significant factor affecting minimal residual disease level at the end of induction phase and overall survival regardless of the risk group and the protocol. The germline variant at rs3824662 in GATA3 is a prognostic factor which associates with CRLF2 expression in leukemic cells supporting the hypothesis that GATA3 may have a regulatory effect on the CRLF2 pathway in pediatric BCP-ALL.

Surface expression of Cytokine Receptor-Like Factor 2 increases risk of relapse in pediatric acute lymphoblastic leukemia patients harboring IKZF1 deletions.

We prospectively examined whether surface expression of Cytokine Receptor-Like Factor 2 (CRLF2) on leukemic blasts is associated with survival and induction treatment response in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) patients. Flow cytometric analysis of bone marrow-derived leukemia cells revealed that 7.51% (29/286) of 386 pediatric BCP-ALL patients were CRLF2-positive (CRLF2pos) at diagnosis. The median minimal residual disease (MRD) was lower in CRLF2pos than CRLF2-negative (CRLF2neg) patients on day 15 (MRD15) after induction therapy [0.01% (0.001-0.42%) vs. 0.45% (0.05-3.50%); p=0.001]. By contrast, the MRD15 was higher in Ikaros family Zinc Finger Protein 1 (IKZF1)-deleted BCP-ALL patients than in BCP-ALL patients without IKZF1 deletions [1.18% (0.06-12.0%) vs 0.33% (0.03-2.6%); p=0.003]. Subgroup analysis showed that MRD15 levels were lower in IKZF1Δ/CRLF2pos patients than in IKZF1Δ/CRLF2neg patients [0.1% (0.02-5.06%) vs. 2.9% (0.25-12%); p=0.005]. Furthermore, MRD15 levels were higher in IKZF1WT/CRLF2neg patients than in IKZF1WT/CRLF2pos patients [0.40% (0.04-2.7%) vs. 0.001% (0.001-0.01%)]. Despite the low MRD15 levels, IKZF1Δ/CRLF2pos patients showed poorer relapse-free survival (RFS) than other patient groups (p=0.003). These findings demonstrate that surface CRLF2 expression is associated with increased risk of relapse in pediatric BCP-ALL patients harboring IKZF1 deletions.

Biallelic loss of CDKN2A is associated with poor response to treatment in pediatric acute lymphoblastic leukemia.

The inactivation of tumor suppressor genes located within 9p21 locus (CDKN2A, CDKN2B) occurs in up to 30% of children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL), but its independent prognostic significance remains controversial. In order to investigate the prognostic impact of deletions and promoter methylation within 9p21, 641 children with newly diagnosed BCP-ALL using methylation specific multiplex ligation-dependent probe amplification (MS-MLPA) were investigated. A total of 169 (26.4%) microdeletions in 9p21 were detected, of which 71 were homozygous. Patients with CDKN2A homozygous deletions were older at diagnosis (p < .001), more frequently steroid resistant (p = .049), had higher WBC count (p < .001), higher MRD at Day 15 (p = .013) and lower relapse-free survival [p = .028, hazard ratio: 2.28 (95% confidence interval: 1.09-4.76)] than patients without these alterations. CDKN2A homozygous deletions coexisted with IKZF1 and PAX5 deletions (p < .001). In conclusion, CDKN2A homozygous deletions, but not promoter methylation, are associated with poor response to treatment and increased relapse risk of pediatric BCP-ALL.

Functional characteristic of PC12 cells with reduced microsomal glutathione transferase 1.

Microsomal glutathione transferase 1 (MGST1) possesses glutathione transferase and peroxidase activities and is active in biotransformation of xenobiotics and in defense against oxidative stress. To assess MGST1 role in the development and functioning of PC12 cells, we constructed a cell line with reduced MGST1 (PC12_M). Real-time PCR and immunoblot assays showed MGST1 expression lowered to 60 % and immunocytochemical analyses demonstrated an altered concentration and distribution of the enzyme. PC12_M cells revealed a larger tendency to grow in clusters, weaker adhesion, irregular shape of bodies, short neurite outgrowth and higher percentage of necrotic cells (34 %). The total GSTs activity determined with non-specific substrate CDNB (1-chloro-2,4-dinitrobenzene) decreased by 15-20 %, whereas that with DCNB (2,4-dichloro-1-nitrobenzene), a substrate more specific for cytosolic GSTs, was similar to the one in control cells. This suggests that reduction of MGST1 cannot be compensated by other glutathione transferases. In PC12_M cells the total glutathione content was higher by 15-20 %, whereas the GSSG/GSH ratio was lower than in control cells. Moreover, the laminin-dependent migration rate was much faster in control cells than in PC12_M, suggesting some alterations in the metastatic potential of the line with suppressed MGST1. The amount of MAP kinases (p38, JNK, ERK1/2) was elevated in PC12_M cells but their phosphorylation level declined. Microarray analysis showed changed expression of several genes, which may be linked with differentiation and necrosis of PC12_M cells. Our data suggest that MGST1 could be an important regulator of PC12 cells development and might have significant effects on cell growth and proliferation, probably through altered expression of genes with different biological function.

Adaptation of microsomal glutathione transferase 1 in PC12 cells with modified PMCA isoforms composition.

Microsomal glutathione transferase 1 (MGST1) is an integral homo-trimeric membrane protein with transferase and peroxidase activities. With glutathione as a co-substrate, it scavenges toxic compounds and may exert anti-apoptotic effect. We examined the effect of suppression of plasma membrane Ca(2+)-ATPase isoforms--PMCA2 or PMCA3 on MGST1 in PC12 cells. GSH level was significantly higher in PMCA2-reduced line, but similar GSSG/GSH ratios in all cell lines suggested an efficient protection or absence of oxidative stress. The ATP concentration decreased in both modified lines, although in PMCA2-suppressed cells the decrease was higher. Total GSTs activity in postmitochondrial fraction increased by 30% in the cells with reduced PMCA3. After treatment with MGST1 activator N-ethylmaleimide (NEM), the activity increased in both transfected lines by 30-40%. Real-time PCR also showed a higher mRNA expression of MGST1 in these lines. Staining with antibody recognizing all cytosolic and membrane-bound GSTs revealed the difference in oligomeric forms of GSTs, and specific anti-MGST1 antibody showed the presence of MGST1 hexamers in the transfected cells. Formation of similar hexamers was detected in the control line after treatment with peroxynitrite. Modification of MGST1 under reduced PMCAs amount may represent an adaptive mechanism that offers protection against the cytotoxicity mediated by increased Ca2+.