SPECTRUM OF AFFECTED GENES IN UKRAINIAN PATIENTS WITH PRIMARY MYELOFIBROSIS

In this study we aimed to identify the spectrum of affected genes in Ukrainian patients diagnosed with primary myelofibrosis (PMF). DNA samples were obtained from peripheral blood leukocytes of 30 Ukrainian PMF patients. Using Whole Exome Sequencing, we detected previously reported and unreported sequence variants considered as pathogenic or potentially pathogenic. Canonical mutations of usual MPN-driver genes were detected in 70% of PMF patients, including JAK2V617F in 43.3%, MPLW515 in 10% and CALRmutations (type 1-like and 2-like) in 16.7% of patients. Also, a non-canonical MPLP222S variant was detected in one patient negative for these mutations. However, in cell culture assay, MPLP222S expression in Ba/F3 cells did not demonstrate differences in phosphorylation of JAK/STAT signaling proteins in response to TPO stimulation compared to MPLWT expression. Overall, more pathogenic and potentially pathogenic sequence variants were found among PMF patients negative for canonical mutations in three driver genes (JAK2, MPL andCALR), compared to patients, positive for one of these mutations. The mean numbers of variants were 5 (range: 4 7) versus 3.3 (range: 1 9), respectively (p = 0.03). The most frequently affected among Ukrainian PMF patients were genes ASXL1, PEG3, EZH2, ATM, U2AF1, andCDH23in addition to JAK2, MPL andCALR. Pathogenic or potentially pathogenic sequence variants of ASXL1and EZH2genes were identified in 23.3% and 16.7% of cases, respectively. Genes JARID2, RBBP8, RTEL1, SUZ12, BRCA2, LAMB4, NF1, RBM12B, RBM43, and RBP3 were recurrently affected in PMF patients who were also positive for usual mutations in three driver genes. Among PMF patients negative for usual mutations in three driver genes recurrently affected genes were DNMT3Aand TET2in addition to mentioned earlier, and some genetic variants were identified in RTEL1, SUZ12, CBL, CUX1, FLT3LGand UMODL1genes in single cases. Also, we identified several genes affected in single cases (KIT, SF3B1, GNAS) in PMF patients negative for canonical mutations in three driver genes which may be potential MPN drivers

13q Deletions detected by fluorescence in situ hybridization for diagnosis and prognosis of chronic lymphoproliferative neoplasms

Aim. Determination of deletion of the long arm of chromosome 13 in the patients with chronic lymphocytic leukemia, diffuse large B-cell lymphoma and multiple myeloma to provide prognostic assessments of Chronic Lymphoproliferative Neoplasms (CLPN) sub-variants progression, and early detection of therapy resistant cases and relapses of CLPN. Methods. Preparations of bone marrow cells from all patients (n = 115) with CLPN were studied. Fluorescence in situ hybridization was performed using commercial test Vysis LSI D13S319 (13q14.3) Spectrum Orange/ Vysis LSI 13q34 Spectrum Green FISH probe kit (Abbott Molecular, USA). Results. The molecular cytogenetic investigations have revealed deletions of 13q in 38 % of the patients with CLPN. We also present a clinical case where the deletion of 13q is detected along with other cytogenetic aberrations that significantly impair a disease prognosis. Conclusion. The analysis of deletions of the long arm of chromosome 13 is an important diagnostic and prognostic criterion, which assists to optimizes the treatment of the patients with CLPN

Features of hematological parameters in CALR-positive patients with myeloproliferative neoplasms

Визначена частота мутацій 1-го та 2-го типів гена CALR у 144 хворих на ЕТ та ПМФ. Мутації гена CALR були виявлені у 26 (18,1%) випадків, із них у 12 (28,6%) хворих на радіаційно-асоційовані МПН. Показано, що мутації гена CALR виявляються частіше серед хворих на радіаційно-асоційовані МПН (р = 0,05). Хворі на ЕТ та ПМФ характеризуються підвищеною кількістю тромбоцитів (р = 0,0004). Кількість еритроцитів (р = 0,04) та рівень гемоглобіну (р = 0,05) є нижчими серед CALR-позитивних хворих, ніж у хворих із JAK2 V617F- позитивним мутаційним статусом; Определена частота мутаций 1-го и 2-го типов гена CALR у 14больных ЭТ и ПМФ. Мутации гена CALR были обнаружены в 26 (18,1%) случаях, из них у 12 (28,6%) больных радиационно-ассоциированные МПН. Показано, что мутации гена CALR оказываются чаще среди больных радиационно-ассоциированными МПН (р = 0,05). Больные ЭТ и ПМФ характеризуются повышенным количеством тромбоцитов (р = 0,0004). Количество эритроцитов (р = 0,04) и уровень гемоглобина (р = 0,05) является ниже среди CALR- положительных больных, чем у больных с JAK2 V617F-положительным мутационным статусом; Classic chronic Ph-negative myeloproliferative neoplasms (MPNs) are an unique group of hematologic diseases encompassing polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). Our understanding of the mechanisms of MPNs development was dramatically changed in 2005, when recurrent mutation of tyrosine kinase JAK2 V617F was described in most patients with PV and in 50-60% patients with ET and PMF. Later, other MPNs-causing mutations were reported in MPL, CALR genes as well as in JAK2 gene involving other localizations. In most cases mutations in these genes arise in mutually exclusive way and cause establishment of myeloproliferative phenotype predominantly through JAK-STAT3/5 signalling pathway. Therefore, MPNs have common molecular genetic features and can mimic each other. However, due to impact of the factors such as specific disease-causing mutations in one of three mentioned genes, experiencing of low doses of ionizing radiation, they can present with different clinical characteristics. Aim of our research was to study the frequency of the most common CALR gene mutations and their association with clinical hematological characteristics in Ukrainian patients with ET and PMF with accent on radiation-associated MPNs. 144 patients with ET and PMF (including 42 patients who were previously exposed to low doses of ionizing radiation) were tested for presence of JAK2 V617F mutation using allele-specific PCR, 1st type CALR (52 bp deletion) and 2nd type CALR (5 bp insertion) gene mutations using RT-PCR. Hematological parameters in 144 MPN patiens were analysed. CALR gene mutations were detected in 26 (18,1%) cases, including 12 (28,6%) patient with MPNs previously exposed to ionizing radiation. In our study we showed that CALR gene mutations are more common among MPN patients who were previously exposed to ionazing radiation (p = 0,05). Platelets count is higher (p = 0,0004), but red blood cells count (p = 0,04) and level of hemoglobin (p = 0,05) are lower in CALR-positive patients, than in JAK2-positive patients