Monika Lejman

Role of 657del5 NBN mutation and 7p12.2 (IKZF1), 9p21 (CDKN2A), 10q21.2 (ARID5B) and 14q11.2 (CEBPE) variation and risk of childhood ALL in the Polish population.

Recent studies have shown that SNPs mapping to 7p12.2 (IKZF1), 9p21 (CDKN2A), 10q21.2 (ARID5B), and 14q11.2 (CEBPE) and carrier status for recessively inherited Nijmegen Breakage syndrome (NBS) influence childhood acute lymphoblastic leukemia (ALL) risk. To examine these relationship, we analysed 398 ALL cases and 731 controls from Poland. Statistically significant association between genotype at 7p12.2 (IKZF1), 10q21.2 (ARID5B) and the NBS associated locus, 8q21.3 (NBN) and ALL risk was found; odds ratios (ORs), 1.34 (P=0.002), 1.33 (P=0.003), and 1325.21 (P=0.0028), respectively. These data provide further insights into the biological basis of ALL highlighting the existence of both common and rare disease susceptibility variants.

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

Abstract 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.

Asparagine synthetase (ASNS) gene polymorphism is associated with the outcome of childhood acute lymphoblastic leukemia by affecting early response to treatment

The polymorphism of 14-bp tandem repeat sequence located in the ASNS gene probably acts as a transcriptional enhancer element and leads to higher expression of the gene in carriers of more than 2 repeats (>R2). We searched for an association with disease outcome in 264 children with ALL. A multivariate proportional hazard regression model adjusted for age at diagnosis (HR (95%CI)=1.05 (1.04-1.09)) and high-risk group (HR(95%CI)=3.47 (1.74-6.88)) revealed that R3 carriers with a poor response at day 15 had an increased risk of events, HR (95%CI)=2.72 (1.06-6.96). These results suggest a conditional interaction between the ASNS polymorphism and an early response to chemotherapy among pediatric patients with ALL.

Fluorescence in situ hybridization BCR/ABL fusion signal rate in interphase nuclei of healthy volunteer donors: a test study for establishing false positive rate

Fluorescence in situ hybridization (FISH) using chromosome-specific DNA probes is rapidly becoming a part of clinical laboratory practice. However, as a relatively new clinical test, it is not yet standardized and for practical reasons each laboratory must establish its own criteria. For this purpose we have evaluated the specificity of a dual-color BCR/ABL translocation probe by establishing the range of BCR/ABL fusion-positive scores in a healthy donor group. The false positive rate (FPR), determined by the percent of FISH BCR/ABL fusion-positive cells found in the specimens of healthy donors, was estimated at 2.3% (mean = 1%-4%). Thus the cut-off value for false positive nuclei was set at 5%.

Polymorphism in IKZF1 gene affects age at onset of childhood acute lymphoblastic leukemia

Abstract Acute lymphoblastic leukemia (ALL) is the most common childhood cancer, characterized by a peak of incidence between 2 and 5 years. Since recently conducted genome-wide association (GWA) studies revealed that the common low-penetrance susceptibility allele at 7p12.2 (IKZF1 gene) confers an increased risk of pediatric ALL, we investigated whether the risk allele at rs4132601 also coexists with well-established prognostic factors, among 508 Polish pediatric patients with newly diagnosed ALL. Additionally, to verify whether the risk allele is favored by somatic tumor evolution, we examined the incidence of IKZF1 deletions in leukemic clones derived from 153 previously genotyped cases of pediatric ALL. Results of the analysis provide statistically significant support for an association between the rs4132601 polymorphic site and age at diagnosis of childhood ALL (p = 0.04). No association between allele variant and occurrence of IKZF1 deletions was found. These data provide further evidence of a biological role of gene variants in the development of ALL.

Structural and numerical abnormalities resolved in one-step analysis: the most common chromosomal rearrangements detected by comparative genomic hybridization in childhood acute lymphoblastic leukemia

Comparative genomic hybridization (CGH) is a technique that permits detection of chromosomal imbalances. This method allows the detection of gains and losses of genetic material at a resolution lower than 5 Mb. The limitations of conventional cytogenetic studies, such as morphologically insufficient quality of metaphases or the mitotic index, can be eliminated by use of CGH. It is particularly important in the diagnosis of leukemias, and CGH could be a useful tool enabling more precise cytogenetic analysis of leukemic cells. A group of 89 children with acute lymphoblastic leukemia was studied by means of CGH using bone marrow obtained from all consecutive pediatric patients. CGH experiments were performed according to the manufacturers instruction with minor modifications. In addition, each sample was examined with standard GTG technique and fluorescence in situ hybridization. The conventional cytogenetics failed in 12 patients (13.5%), and 22 patients (24.7%) had a normal karyotype. Structural and numerical changes were found in 55 cases (61.8%) displaying a different abnormalities including deletions, trisomies, tetrasomies, isochromosomes, and markers with unknown origin. However, all samples were successfully analyzed by CGH. We have shown that high-resolution comparative genomic hybridization analysis is a reliable and relatively quick one-step method to identify main aberrations that would not be detected by either conventional G banding or by conventional fluorescence in situ hybridization. It should be considered to establish CGH as a routine analysis for screening patients with acute lymphoblastic leukemia.

Association of germline genetic variants in RFC, IL15 and VDR genes with minimal residual disease in pediatric B-cell precursor ALL

Minimal residual disease (MRD) enables reliable assessment of risk in acute lymphoblastic leukemia (ALL). However, little is known on association between MRD status and germline genetic variation. We examined 159 Caucasian (Slavic) patients with pediatric ALL, treated according to ALL-IC-BFM 2002/2009 protocols, in search for association between 23 germline polymorphisms and MRD status at day 15, day 33 and week 12, with adjustment for MRD-associated clinical covariates. Three variants were significantly associated with MRD: rs1544410 in VDR (MRD-day15); rs1051266 in RFC (MRD-day33, MRD-week12), independently and in an additive effect with rs10519613 in IL15 (MRD-day33). The risk alleles for MRD-positivity were: A allele of VDR (OR = 2.37, 95%CI = 1.07–5.21, P = 0.03, MRD-day15); A of RFC (OR = 1.93, 95%CI = 1.05–3.52, P = 0.03, MRD-day33 and MRD-week12, P < 0.01); A of IL15 (OR = 2.30, 95%CI = 1.02–5.18, P = 0.04, MRD-day33). The risk for MRD-day33-positive status was higher in patients with risk alleles in both RFC and IL15 loci than in patients with risk alleles in one locus or no risk alleles: 2 vs. 1 (OR = 3.94, 95% CI = 1.28–12.11, P = 0.024), 2 vs. 0 (OR = 6.75, 95% CI = 1.61–28.39, P = 0.012). Germline variation in genes related to pharmacokinetics/pharmacodynamics of anti-leukemic drugs and to anti-tumor immunity of the host is associated with MRD status and might help improve risk assessment in 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.

Heterozygous carriers of germline c.657_661del5 founder mutation in NBN gene are at risk of central nervous system relapse of B-cell precursor acute lymphoblastic leukemia

Isolated central nervous system (CNS) relapse is observed in 3-8% of patients with acute lymphoblastic leukemia (ALL) and accounts for 30-40% of all disease recurrence. Although introduction of intensive CNSdirected therapy significantly improved 5-year overall survival of patients diagnosed with leukemia CNS relapse, such aggressive treatment results in long-term side effects. Therefore, more accurate risk stratification of CNS relapse in newly-diagnosed ALL pediatric patients is needed. In contrast to somatic gene defects, only a small number of studies have investigated the host genetic factors and their association with leukemia relapse. Since germline NBN c.657_661del5 mutation increases the risk of developing pediatric ALL and other types of cancers in Slavic populations, and there is a high frequency in the Polish population (1:190 individuals), we investigated its possible effect on leukemia development and progression among heterozygous carriers. NBN germline c.657_661del5 mutation leads to an impaired function of the nibrin protein, which acts as an element of the Mre11-Rad50-Nbs1 (MRN) complex in DNA double-strand breaks (DSB) repair processes. Patients with a homozygous deletion in the NBN gene, diagnosed with Nijmegen-breakage syndrome (NBS, OMIM #251260), are known to often present CNS involvement at diagnosis of pediatric ALL. In contrast to patients with NBS, who mainly develop lymphoma or Tcell ALL, heterozygous carriers of the c.657_661del5 deletion in the NBN gene are mainly diagnosed with B-cell precursor ALL (BCP-ALL). Therefore, in this unique group of patients, it is possible to investigate whether germline c.657_661del5 heterozygous mutation in the NBN gene has an impact on the biology and course of childhood BCPALL. We retrospectively evaluated 578 pediatric patients diagnosed with BCP-ALL (median age 4.51 years, range 0.1317.99 years; details are available in the Online Supplementary Methods). In total, 17 (2.94%) patients harbored the c.657_661del5 heterozygous mutation in the NBN gene. There was no significant difference in baseline clinical and biological features of leukemia and risk group

Paternally Inherited GABRB3 Intragenic Deletion in a Boy with Autistic Featuresand Angelman Syndrome Phenotype Case Report and Literature Review

We report on a 4 year old patient with a unique paternally inherited single-exon GABRB3 gene deletion and clinical findings of severe speech delay, intellectual disability, autistic features, unusual behavior, tremor, and history of seizures and gait abnormalities. Similarities and significant differences with other cases involving rearrangements of 15q11-q13 are discussed. Further on, we provide literature review of the clinical picture of GABRB3 mutations.