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Ivanova M.I.,Medical University-Sofia | Shivarov V.S.,Medical University-Sofia | Hadjiev E.A.,Laboratory of Hematopathology and Immunology | Naumova E.J.,Medical University-Sofia
Leukemia Research | Year: 2011

MPL exon 10 mutations were the second class of mutations shown to be associated with the pathogenesis of some Philadelphia chromosome - negative myeloproliferative neoplasms (MPNs). Recently, their identification gained wide recognition in the diagnostic work-up for suspected cases of JAK2 V617F negative MPNs. Various molecular approaches have been applied, yet universally accepted method is still lacking. We aimed at development and validation of a novel bead-based liquid assay using Locked nucleic acids (LNA)-modified oligonucleotide probes for multiplexed detection of the following MPL mutations: W515L/K/A/R. Testing on both artificial plasmid constructs and on clinical samples revealed that the method was comparable in terms of specificity to direct sequencing and had a much higher sensitivity of 1% mutant alleles. This method could be successfully implemented in the diagnostic work-up for MPNs. Furthermore, this system allows further multiplexing for single-tube identification of different mutations associated with MPNs. © 2011 Elsevier Ltd.

Shivarov V.,Laboratory of Hematopathology and Immunology | Stoimenov A.,Laboratory of Cytogenetics and Molecular Biology | Spassov B.,National Hematology Hospital | Angelova S.,Laboratory of Cytogenetics and Molecular Biology | And 2 more authors.
Hematology | Year: 2014

MicroRNAs are a class of small noncoding RNAs playing a crucial role in physiological and pathological conditions, including acute myeloid leukemia. We aimed at examining whether in clinical settings the identification of patient-specific leukemia-associated profiles (LAMPs) of overexpressed microRNAs could be used for minimal residual disease (MRD) detection. Patients with identified LAMPs at diagnosis were further tested for the presence of LAMP after induction therapy. Those with identifiable MRD defined by LAMP expression after induction showed a trend toward a shorter overall survival. Larger studies are needed to confirm the utility of patient-specific LAMPs for MRD follow-up. © W. S. Maney & Son Ltd 2014.

Shivarov V.,Laboratory of Hematopathology and Immunology | Ivanova M.,Medical University-Sofia | Naumova E.,Medical University-Sofia
PLoS ONE | Year: 2014

Mutations in the human DNA methyl transferase 3A (DNMT3A) gene are recurrently identified in several hematologic malignancies such as Philadelphia chromosome-negative myeloproliferative neoplasms (MPN), myelodysplastic syndromes (MDS), MPN/MDS overlap syndromes and acute myeloid leukemia (AML). They have been shown to confer worse prognosis in some of these entities. Notably, about 2/3 of these mutations are missense mutations in codon R882 of the gene. We aimed at the development and validation of a novel easily applicable in routine practice method for quantitative detection of the DNMT3A p.R882C/H/R/S mutations bead-based suspension assay. Initial testing on plasmid constructs showed excellent performance of BNA(NC)-modified probes with an optimal hybridization temperature of 66°C. The method appeared to be quantitative and showed sensitivity of 2.5% for different mutant alleles, making it significantly superior to direct sequencing. The assay was further validated on plasmid standards at different ratios between wild type and mutant alleles and on clinical samples from 120 patients with known or suspected myeloid malignancies. This is the first report on the quantitative detection of DNMT3A R882 mutations using bead-based suspension assay with BNA(NC)-modified probes. Our data showed that it could be successfully implemented in the diagnostic work-up for patients with myeloid malignancies, as it is rapid, easy and reliable in terms of specificity and sensitivity. © 2014 Shivarov et al.

Shivarov V.,Laboratory of Hematopathology and Immunology | Gueorguieva R.,Yale University | Stoimenov A.,Laboratory of Cytogenetics and Molecular Biology | Tiu R.,Cleveland Clinic
Leukemia Research | Year: 2013

Somatic DNA methyl transferase 3A (DNMT3A) mutations have been recognized recently as recurrent molecular aberrations in acute myeloid leukemia (AML). The precise role of these mutations in leukemogenesis remains elusive but a number of studies have already been conducted to study their potential prognostic value in AML patients with variable results. We performed a meta-analysis on published data from over 4500 AML patients to provide robust evidence supporting DNMT3A mutation testing in clinical setting for AML patients. Our meta-analysis showed that DNMT3A mutations were associated with M4 and M5 AML subtypes. Those mutations conferred significantly worse prognosis with both shorter OS (p= 0.0004) and shorter RFS (p= 0.002). Notably, DNMT3A mutations appeared to be an independent adverse prognostic factor also in younger patients with normal cytogenetics AML (OS (p= 0.01) and RFS (p= 0.0005)) and also in the subgroup of patients with high risk genotypes defined according to the criteria of the European Leukemia Net (ELN) (OS (p= 0.002)). Therefore, DNMT3A mutational status can improve the risk stratification of AML patients in the setting of integrated mutational profiling. © 2013 Elsevier Ltd.

Mitova V.,Bulgarian Academy of Science | Slavcheva S.,Bulgarian Academy of Science | Shestakova P.,Bulgarian Academy of Science | Momekova D.,Medical University-Sofia | And 4 more authors.
European Journal of Medicinal Chemistry | Year: 2014

Macromolecular conjugates of a dinuclear platinum complex with a spermidine bridge were synthesized using poly(oxyethylene H-phosphonate)s as precursor polymer. The complex species were attached to the polymer chain via a phosphoramide bond resulting from the reaction between the H-phosphonate groups and the middle amino group of the spermidine moiety. 1H and 31P{H} DOSY NMR spectral data were used to prove the conjugation reaction and to characterize the new species. The conjugates exhibited profound cytotoxicity in a panel of five chemosensitive human tumor cell lines and one cisplatin-resistant model (HL-60/CDDP), and were found to induce apoptotic cell death. A flow cytometric analysis encountered a cisplatin-dissimilar modulation of the cell cycle progression in KG-1 leukemic cells, following exposure to the dinuclear agents. Moreover, the novel compounds displayed less pronounced inhibitory activity against cultured murine renal epithelial cells, as compared to cisplatin. © 2013 Elsevier Masson SAS. All rights reserved.

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