Killer-cell immunoglobulin-like receptor genotype and haplotype combinations in children treated for acute lymphoblastic leukemia.

INTRODUCTION: Acute lymphoblastic leukemia (ALL) is the most common malignancy diagnosed in children. The factors predisposing to ALL remain mostly unknown. Natural killer (NK) cells are a component of innate immunity. Their role is to eliminate cells that were infected with viruses or underwent a neoplastic transformation. The activity of NK cells is regulated by their activating and inhibitory receptors, inter alia killer-cell immunoglobulin-like receptors (KIRs). The available data about a link between the incidence of ALL and KIR genotype are highly inconclusive, and further research is needed to explain whether such a relationship truly exists. The aim of this study was to analyze KIR genotype and haplotype combinations in children treated for ALL. MATERIAL AND METHODS: The study included 49 children diagnosed with ALL at 1.2-19.8 years of age. The control group was composed of 43 healthy subjects aged between 1.2 and 21.9 years. DNA was isolated using QIAamp DNA Mini kits. KIR genotypes were identified by a polymerase chain reaction (PCR) with sequence-specific primers (SSPs). The analysis also included KIR haplotype combinations: AA, AB and BB. RESULTS: Patients with ALL and controls did not differ significantly in the frequencies of individual KIR genes and haplotypes. However, the overall frequency of all 6 activating KIR genes in patients with ALL was significantly higher than in the controls (24.5% vs. 4.7%, p = 0.019). CONCLUSIONS: The findings presented here imply that individual KIR genes do not play a significant role in the pathogenesis of ALL. Nevertheless, a higher number of activating KIR genes may constitute a risk factor for this malignancy.

IDH2 mutations in patients with normal karyotype AML predict favorable responses to daunorubicin, cytarabine and cladribine regimen.

Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) genes occur in about 20% patients with acute myeloid leukemia (AML), leading to DNA hypermethylation and epigenetic deregulation. We assessed the prognostic significance of IDH1/2 mutations (IDH1/2+) in 398 AML patients with normal karyotype (NK-AML), treated with daunorubicine + cytarabine (DA), DA + cladribine (DAC), or DA + fludarabine. IDH2 mutation was an independent favorable prognostic factor for 4-year overall survival (OS) in total NK-AML population (p = 0.03, censoring at allotransplant). We next evaluated the effect of addition of cladribine to induction regimen on the patients' outcome according to IDH1/2 mutation status. In DAC group, 4-year OS was increased in IDH2+ patients, compared to IDH-wild type group (54% vs 33%; p = 0.0087, censoring at allotransplant), while no difference was observed for DA-treated subjects. In multivariate analysis, DAC independently improved the survival of IDH2+ patients (HR = 0.6 [0.37-0.93]; p = 0.024; censored at transplant), indicating that this group specifically benefits from cladribine-containing therapy. In AML cells with R140Q or R172K IDH2 mutations, cladribine restrained mutations-related DNA hypermethylation. Altogether, DAC regimen produces better outcomes in IDH2+ NK-AML patients than DA, and this likely results from the hypomethylating activity of cladribine. Our observations warrant further investigations of induction protocols combining cladribine with IDH1/2 inhibitors in IDH2-mutant.

International external quality assurance of JAK2 V617F quantification.

External quality assurance (EQA) programs are vital to ensure high quality and standardized results in molecular diagnostics. It is important that EQA for quantitative analysis takes into account the variation in methodology. Results cannot be expected to be more accurate than limits of the technology used, and it is essential to recognize factors causing substantial outlier results. The present study aimed to identify parameters of specific importance for JAK2 V617F quantification by quantitative PCR, using different starting materials, assays, and technical platforms. Sixteen samples were issued to participating laboratories in two EQA rounds. In the first round, 19 laboratories from 11 European countries analyzing JAK2 V617F as part of their routine diagnostics returned results from in-house assays. In the second round, 25 laboratories from 17 countries participated. Despite variations in starting material, assay set-up and instrumentation the laboratories were generally well aligned in the EQA program. However, EQA based on a single technology appears to be a valuable tool to achieve standardization of the quantification of JAK2 V617F allelic burden.

Selected Parameters of Angiogenesis and the JAK2, CALR, and MPL Mutations in Patients With Essential Thrombocythemia.

The aim of the study was to evaluate selected angiogenic factors in patients with essential thrombocythemia (ET) depending on JAK2V617F, calreticulin gene (CALR) and myeloproliferative leukemia virus oncogene (MPL) mutations. Sixty ET patients and 20 healthy volunteers were enrolled in the study. The following tests were performed: vascular endothelial growth factor- A (VEGF-A), soluble vascular endothelial growth factor receptor-1 (sVEGFR-1),soluble vascular endothelial growth factor receptor-2 (sVEGFR-2), platelet-derived growth factor( PDGF-BB), and stromal-derived factor-1α (SDF-1α). We observed an increased PDGF-BB level in patients with ET compared to the controls. Patients with CALR mutation had significantly higher concentration of PDGF-BB and lower concentration of SDF-1α than patients with JAK2V617F mutation. High concentration of PDGF-BB and low concentration of SDF-1α in patients with CALR(+) ET may indicate a contribution of these chemokines in disturbed Ca2+ metabolism in platelets.

Heterogeneity of human WT1 gene.

The WT1 gene, characterized by an extremely complex structure, is located on chromosome 11. It is involved in cell growth and differentiation, and has a strong impact on consecutive stages of the functioning of the body. The WT1 gene may undergo many different mutations, as well as may be overexpressed without a mutation. The molecular basis of diseases such as Wilms tumor, WAGR, Denys-Drash or Frasier syndromes are congenital WT1 mutations, while somatic mutations of this gene occur in acute and chronic myeloid leukemia, myelodysplastic syndrome and also in some other blood neoplasms, as acute lymphoblood leukemia. Increased expression of this gene without its mutation is observed in leukemias and solid tumors. The WT1 may function both as a tumor suppressor gene and as an oncogene. The diversity of WT1 changes causes many controversies, therefore investigations are still carried out to determine the function of this gene, its interaction with other molecules and its prognostic significance in various diseases.

CEBPA copy number variations in normal karyotype acute myeloid leukemia: Possible role of breakpoint-associated microhomology and chromatin status in CEBPA mutagenesis.

Copy number variations (CNV) in CEBPA locus represent heterogeneous group of mutations accompanying acute myeloid leukemia (AML). The aim of this study was to characterize different CEBPA mutation categories in regard to biological data like age, cytology, CD7, and molecular markers, and identify possible factors affecting their etiology. We report here the incidence of 12.6% of CEBPA mutants in the population of 262 normal karyotype AML (NK-AML) patients. We confirmed that double mutant AMLs presented uniform biological features when compared to single CEBPA mutations and accompanied mostly younger patients. We hypothesized that pathogenesis of distinct CEBPA mutation categories might be influenced by different factors. The detailed sequence analysis revealed frequent breakpoint-associated microhomologies of 2 to 12bp. The analysis of distribution of microhomology motifs along CEBPA gene showed that longer stretches of microhomology at the mutational junctions were relatively rare by chance which suggests their functional role in the CEBPA mutagenesis. Additionally, accurate quantification of CEBPA transcript levels showed that double CEBPA mutations correlated with high-level CEBPA expression, whereas single N-terminal CEBPA mutations were associated with low-level CEBPA expression. This might suggest that high-level CEBPA expression and/or accessibility of CEBPA locus contribute to B-ZIP in-frame duplications.

[The hematological malignancies related to primary hypereosinophilia and their diagnostics]. / Nowotwory ukladu krwiotwórczego przebiegajace z pierwotna hipereozynofilia i ich diagnostyka.

Published in 2008, by experts of the World Health Organization, the new classification of hematological malignancies forced a change of look at chromosomal aberrations and gene mutations, which are important in establishing the diagnosis and prognosis for patients with these malignancies. The new classification includes a new category of neoplasms - hematological malignancies with hypereosinophilia. Due to the high diversity of causes of hypereosinophilia and underlying genetic changes, their differential diagnosis is based on classical cytogenetics, fluorescence in situ hybridization (FISH) and genetic molecular techniques. Cytogenetic analysis of bone marrow cells showed that the majority of hypereosinophilia cases can be characterized by the presence of normal karyotype. Therefore, routine cytogenetic diagnostics should be complemented by FISH with break-apart probes for potentially rearranged genes (e.g., CBFB, ETV6) and unique probes for fusion genes (e.g., FIP1L1-PDGFRA), specific for hypereosinophilia-associated diseases. In differential diagnosis of hypereosinophilia, the analysis of characteristic gene mutations (e.g., cKIT) and gene fusions (e.g., ETV6-PDGFRB) is also applied, using molecular genetic methods.

[Standardization of quantitative detection of BCR-ABL gene expression by RQ-PCR in patients with chronic myeloid leukemia in cooperation with European Leukemia Net]. / Standaryzacja ilosciowej oceny ekspresji genu BCR-ABL metoda RQ-PCR u chorych na przewlekla bialaczke szpikowa we wspólpracy z European Leukemia Net.

Monitoring of chronic myeloid leukemia treatment efficacy requires very sensitive methods of BCR-ABL gene detection based on polymerase chain reaction (PCR). The lack of comparability of BCR-ABL mRNA quantification results generated by various methodologies in different laboratories was the cause of an international multicenter trial initiation with the participation of 133 laboratories in 24 European countries cooperating within the "EUTOS for CML" project. Pracownia Diagnostyki Molekularnej Kliniki Hematologii is taking part in standardisation rounds organised since 2005. The compatibility of methodology used in Pracownia with European Leukemia Net (ELN) standards was confirmed, and correction factor for the expression of RQ-PCR results in an international scale was calculated. Pracownia was charge by ELN with a task of conducting the standardisation in polish molecular biology laboratories. Test probes were prepared and sent to eight cooperating laboratories. The results obtained in six laboratories were concordant with results from laboratory in Krakow after conversion to international scale, therefore it was possible to calculate individual correction factors. The participation of polish laboratories in international standardization process created the opportunity for unification of BCR-ABL quantification methodologies with recommendations of international experts, and showed that the quality of analyses performed in majority of them was satisfactory enough to calculate correction factor and to express the RQ-PCR results in widely accepted international scale.