Topic: 4. Acute myeloid leukemia - Clinical Background: Currently, a fundamental role in the prognosis of acute myeloid leukemia (AML) is associated with chromosomal aberrations and genetic abnormalities, which allow not only correctly stratifying patients into risk groups, thereby optimizing the intensity of cytostatic chemotherapy (CT) and timely planning of allogeneic hematopoietic stem cell transplantation (allo-HSCT), but also allow to use the entire arsenal of target drugs. The most common mutations among AML patients are FLT3 (ITD and TKD), which are considered to be potential predictors of poor disease prognosis. Aims: To assess the effect of the mutational status of the FLT3 gene and the impact of the FLT3-ITD allele burden (AB) value on the course of AML. Methods: A retrospective analysis of the FLT3 gene mutation status effect on the clinical features and course of the disease was carried out with involvement of 199 AML patients. The dynamics of the FLT3-ITD gene mutation AB value against the background of standard chemotherapy and allo-HSCT programs, as well as the impact of this dynamics on survival rates, were analyzed in 91 AML patients. Results: In the study group of the AML patients, the frequency of FLT3 gene mutations accounted for 30.5% of all considered cases (FLT3-ITD - 24% (48/199), FLT3-TKD - 5.5% (11/199), a co-occurrence of FLT3-ITD and FLT3-TKD - 1.0% (2/199)), the rest - 69.5% (138/199) of patients had no mutations in the FLT3 gene. Two groups were identified in accordance with the FLT3-ITD gene mutation AB value: low AB (<0.5) - n=50 (55%) group and high AB (≥0.5) - n=41 (45%) group. The presence of mutations in the FLT3 gene was characterized by a higher level of peripheral blood leukocytes (p<0.001). At the same time, patients with high AB in the FLT3-ITD gene, compared with patients with low AB, demonstrated high leukocytosis (p<0.001) and bone marrow blastosis (p=0.04). The percentage decrease in the FLT3-ITD AB associated with administration of chemotherapy significantly correlated with relapse-free survival and OS (r=0.45; p<0.01 and r=0.46; p<0.01, respectively). A significant decrease in AB after CT compared with pre-treatment data was found only in patients with complete remission of the disease (p<0.01). Mutations in the NPM1 gene were detected significantly more often in the FLT3-ITD group of patients – 37 (32.4%) out of 114 patients (p=0.0001). However, the presence of mutation in the NPM1 gene did not improve OS in patients with high FLT3-ITD AB. The results of overall survival depending on the mutational status are presented in Figures 1 and 2. The use of allo-HSCT as a therapeutic option significantly improves OS: in the group with high FLT3-ITD after allo-HSCT+ (n=15) treatment the follow-up median was not reached, while in the group that did not get allo-HSCT- (n=10) it was 2.5 months (p=0.00008). Summary/Conclusion: AML patients with mutations in the FLT3 gene remain one of the complex categories of patients requiring timely prescription of targeted drugs and allo-HSCT, which allows achieving improved survival rates.Keywords: FLT3, Acute myeloid leukemia

The DBSCAN clustering algorithm, an in-house method of unsupervised machine learning, was used to explore specific histotopographical features of the megakaryocytic lineage in the bone marrow biopsies of patients with JAK2- or CALR-mutated essential thrombocythemia and prefibrotic primary myelofibrosis. Ninety-five bone marrow biopsies of patients with essential thrombocythemia and primary myelofibrosis were investigated to assess the histotopography of megakaryocytes, specifically, the mean number of megakaryocytes in one cluster, as well as the mean number of clusters and megakaryocytes per 1 mm2 of section area. The logistic regression model was statistically significant: χ2 = 14.703, p = 0.023, Nagelkerke R2 = 19.6%. Analysis of the histotopographical features of megakaryocytes allowed correct differentiation between essential thrombocythemia and primary myelofibrosis in 71.6% cases. The differences in the histotopographical features of megakaryocytes in the bone marrow of with JAK2- or CALR-mutated essential thrombocythemia and primary myelofibrosis revealed by the DBSCAN clustering algorithm indicate that a relationship exists between the disease and the pattern of megakaryocytic lineage development that can be used to create a logistic regression model for differentiating these diseases.

The publication contains materials of the reports presented at the II Conference “Current Issues of Diagnosis and Treatment of Ph-Negative and Ph-Positive Myeloproliferative Neoplasms” held from 15 to 16 March 2019 at the National Research Center for Hematology (Moscow). The conference was organized to enable professional communication of the clinicians specializing in the treatment of myeloproliferative neoplasms (MPN), and the researchers in the related fields as well as to allow the exchange of views on the implementation of current diagnosis and treatment methods in Ph-negative and Ph-positive MPNs. Reports covered a wide range of rare and non-standard settings. Of particular importance was the opportunity to debate them in detail at panel discussions and interactive sessions. This format of the conference allowed to provide expert opinions in the present publication. It emphasizes the importance of complex diagnosis in MPN using morphological examination of bone marrow core biopsy samples and molecular genetic testing. Accordingly, the second day of the conference was devoted to a thorough analysis of the morphological characteristics of the cases presented and based on bone marrow core biopsy samples.

Введение: Тромбозы являются одним из основных осложнений Ph-негативных миелопролиферативных новообразований (МПН), таких как истинная полицитемия (ИП), эссенциальная тромбоцитемия (ЭТ) и первичный миелофиброз (ПМФ). Имеются многочисленные свидетельства активации тромбоцитов, лейкоцитов, эндотелия и коагуляции при МПН; однако результаты определения показателей гемостаза, применимых в клинической практике для выявления протромботических состояний при МПН, неоднозначны. Цель исследования: оценить состояние плазменного звена гемостаза у пациентов с Ph-негативными МПН и выявить показатели, отражающие протромботическую направленность изменений в системе гемостаза при данных патологиях. Материалы и методы: В исследование было включено 116 пациентов с МПН (43 с ИП, 35 с ЭТ, 38 с ПМФ). Пациенты находились на антиагрегантной, циторедуктивной и таргетной терапии ингибиторами янус-киназ. Контрольная группа состояла из 43 практически здоровых лиц. В плазме крови всех обследованных определяли индекс активированного парциального тромбопластинового времени (АПТВ) — отношение АПТВ исследуемого образца к АПТВ нормальной плазмы, протромбиновый тест по Квику (ПТ), содержание фибриногена, активности фактора VIII (FVIII) и естественных антикоагулянтов антитромбина (АТ) и протеина С (PC), уровень свободного протеина S (PS), а также показатели теста генерации тромбина (ТГТ), в том числе с добавлением тромбомодулина (ТМ). Результаты: У пациентов с МПН по сравнению с контрольной группой наблюдали сдвиг АПТВ и ПТ в сторону гипокоагуляции, было удлинено время инициации генерации тромбина, снижена скорость и количественные показатели ТГТ без добавления ТМ. При проведении ТГТ с добавлением ТМ у больных МПН выявлено снижение чувствительности к ТМ, которое коррелировало с падением активности PC и уровня свободного PS. Содержание фибриногена и активность FVIII у обследованных пациентов были повышены. Заключение: Для пациентов с МПН характерны разнонаправленные изменения показателей плазменного звена гемостаза. Важной детерминантой развития протромботических состояний при МПН выступает недостаточная эффективность естественных антикоагулянтов системы протеина С, приводящая к выраженному дисбалансу в системе гемостаза. Background: Thromboses are one of the main complications of Ph-negative myeloproliferative neoplasms (MPN), such as polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). There are abundant evidences of platelet, leukocyte, endothelial, and coagulation activation in MPN; however, studies of hemostatic parameters applicable in clinical practice for revealing of prothrombotic states in MPN are not identical. Objectives: to assess plasma hemostasis in patients with Ph-negative MPN and to identify parameters reflecting the prothrombotic tendency of hemostatic changes in these pathologies. Patients/Methods: The study included 116 patients with MPN (43 with PV, 35 with ET, 38 with PMF). Patients were treated with antiplatelet, cytoreductive and targeted therapy with Janus kinases inhibitors. The control group consisted of 43 healthy individuals. In blood plasma of all examined persons we determined the index of activated partial thromboplastin time (APTT) — the ratio of APTT of the test sample to APTT of normal plasma, Quick prothrombin test (PT), fi brinogen content, activities of factor VIII (FVIII) and natural anticoagulants — antithrombin (AT) and protein C (PC), free protein S (PS) level, as well as thrombin generation assay (TGA) parameters, including thrombomodulin (TM) addition. Results: As compared with control group, in patients with MPN a shift in APTT and PV to hypocoagulation was observed, the initiation time of thrombin generation was prolonged, the rate and quantitative parameters of TGA without TM addition were decreased. In patients with MPN TGA with TM addition showed reduced sensitivity to TM that correlated with decreased PC activity and reduction of free PS level. Fibrinogen content and FVIII activity in the examined patients were increased. Conclusions: Patients with MPN are characterized by multidirectional changes of plasma hemostasis parameters. The important determinant of prothrombotic states development in MPN is insufficient effectiveness of the natural anticoagulants of protein C system that leads to expressed hemostasis imbalance.

Background. There are problems related to both quantitative assessment of an allele burden level of a mutant gene and interpretation of results in DNA samples with the burden level of the mutant allele less than 15–20 %, when using Sanger sequencing for analyzing somatic mutations. Applied Biosystems (USA) has developed new software Minor Variant Finder, which allows determining mutations with the allele burden level from 5 %.The objective: to determine the allele burden level and identification of minor variants of somatic mutations in the ASXL1, JAK2 genes and BCR-ABL oncogene using Minor Variant Finder software in patients with myeloproliferative neoplasms.Materials and methods. The level of mutant allele burden for 15 patients with myeloproliferative neoplasms was determined by the identified mutations using the Minor Variant Finder software, after analysis of point somatic mutations in the ASXL1, JAK2 genes and BCR-ABL oncogene by Sanger sequencing.Results. The allele burden level in all 5 ASXL1-positive samples and BCR-ABL-positive sample was determined as higher than 20 % using the Minor Variant Finder software. The allele burden level in 2 cases was higher than 20 % and in 7 cases lower than 20 %, when we analyzed 9 JAK2-positive samples.Conclusion. Minor Variant Finder software can be used to estimate the allele burden level and to identify minor variants of somatic mutations in the ASXL, JAK2 and BCR-ABL genes.

Detection of FLT3 gene mutations in acute myeloid leukemia is now recognized as an unfavorable factor that affects the disease course, emerging the risk of relapses and overall survival shortening and disease-free survival of patients. The aim of the study was to determine the frequency of mutations of the gene FLT3 and to assess their impact on clinical indicators, overall survival and disease-free survival in patients with acute myeloid leukemia. We compared complete blood count parameters, karyotype, duration of overall survival and disease-free survival in 199 patients with acute myeloid leukemia depending on the presence or absence of mutations of the FLT3 gene. Significant differences across these groups were discovered only in WBC and blasts between the group of patients with acute myeloid leukemia (FLT3+) and without mutations in the FLT3 gene (FLT3-). The differences between two groups were also identified in patients chromosomal aberrations. Significant differences (p=0,00024) in the duration of overall survival between groups of patients with acute myeloid leukemia with mutations of FLT3-ITD+, FLT3-TKD+ and FLT3- were demonstrated. Median overall survival was: 1 6 months for patients with mutation FLT3-ITD+ and 17 months for FLT3-TKD+ patients and not achieved for FLT3- patients. The use of modern molecular genetic methods of research in acute myeloid leukemia allows to improve the diagnosis of the disease, as well as to carry out risk stratification and individualize therapy. The use of targeted therapy for FLT3-positive patients who are not candidates for hematopoietic stem cell transplantation will increase the effectiveness of the treatment and improve the performance of overall survival and disease-free survival.

Aim. To study the relationship of karyotype, JAK2, CALR, and MPL driver mutations and ASXL1 mutation status with the progression and prediction of primary myelofibrosis (PMF). Materials & Methods. The trial included 110 PMF patients (38 men and 72 women), median age was 59 years (range 18-82) with median follow-up after diagnosis of 2.6 years (range 0.1-23). The patients were examined for JAK2, CALR, MPL, and ASXL1 mutations. Restriction fragment length polymorphism technique was used for the analysis of V617F substitution in JAK2 and 515 codon mutation in MPL. CALR (exon 9) and ASXL1 (exon 12) mutation tests were performed using Sanger direct sequencing. In 48 (44 %) out of 110 patients bone marrow cell karyotype was determined. Clinical and hematological parameters and median overall survival (OS) of patients were analyzed with regard to detected genetic aberrations and combinations of them. Results. JAK2, CALR, MPL mutations were detected in 55 (50 %), 28 (25.5 %), and 7 (6.4 %) out of 110 patients, respectively. Triple negative (TN) status was identified in 20 (18.2 %) out of 110 examined patients. ASXL1 mutations were detected in 22 (20 %) out of 110 patients. Out of 48 patients in 32 (66.7 %) normal karyotype, in 3 (6.3 %) favorable karyotype, in 4 (8.3 %) intermediate-prognosis karyotype, and in 9 (18.7 %) unfavorable karyotype were detected. The comparison of clinical and hematological parameters showed a number of significant differences. JAK2-positive patients had a higher hemoglobin level (median 129 g/L; p = 0.021). TN was associated with a high IPSS risk (p = 0.011), low hemoglobin level (median 101 g/L; p = 0.006), drop in platelet count (median 266 x 109/L; p = 0.041), increased lymphocyte count (median 26.9 х 109/L; р = 0.001). The detection of terminating mutations in ASXL1 correlated with palpable enlarged spleen (р = 0.050), reduced platelet count (median 184 х 109/L; р = 0.016), leukocyte count > 25 х 109/L (р = 0.046), and blast count > 1 % (р < 0.001). Univariate regression analysis showed that terminating mutations in ASXL1 (hazard ratio [HR] 2.9; р = 0.018), unfavorable karyotype (HR 8.2; р < 0.001), and TN (ОР 8.1; р < 0.001) had prognostic value for OS. ASXL1 mutation was associated with significantly worse OS in TN patients. Median OS of ASXL1-negative patients without high-risk chromosomal aberrations was significantly longer than in patients with high-risk karyotype and/ or ASXL1 mutation. Conclusion. Several genetic defects in tumor cells are associated with phenotypic manifestations of PMF. Based on the results of cytogenetic analysis and mutation determination of JAK2, CALR, MPL, and ASXL1, patients can be classified in different “genetic” risk groups when PMF is diagnosed.

Aim. To analyze the effect on prognosis of mutations that are typical of acute myeloid leukemia (AML) patients. Materials & Methods. The study included 620 AML patients surveyed at Hematology Clinics of Saint Petersburg (Russia) and Charite Clinic (Berlin, Germany). G-banding of chromosomes was employed for cytogenetic testing. Aberration screening in DNMT3A, IDH1/2 genes was based on real-time polymerase chain reaction (PCR) with subsequent analysis of melting and sequencing profiles. Mutations in FLT3, NPM1 genes were revealed by PCR. Results. Mutations were identified in 343 (55.3 %) out of 620 patients. Significantly more often mutations were discovered in patients with normal karyotype (NK) (p = 0.001). FLT3-ITD mutation was associated with reduced medians of overall survival (OS) and disease-free (DFS) survival: 11.3 vs. 15.8 months with FLT3-ITD- (p = 0.005) and 10.0 vs. 13.3 months with FLT3-ITD+ (p = 0.009), respectively. The relation of FLT3-ITD allele burden to OS duration was also assessed. In the ITDlow/ITD- group the OS median was considerably longer than in the ITDhigh group (p = 0.028). In the group of patients with 1 mutation in NPM1 gene OS and DFS were much better in comparison with other patients (medians of 27.4 and 13.9 months, respectively, p = 0.040; 19.3 and 12.0 months, p = 0.049). Negative impact of mutations in DNM-T3A gene was noticed while assessing OS median: 12 (DN-MT3A+) and 15 months (DNMT3A-), respectively (p = 0.112). Mutations in IDH1 gene correlated with a better OS than in the group without mutations (p = 0.092). The rs11554137 polymorphism in IDH1 gene was associated with worse OS in the group of patients with NK (p = 0.186). In 144 patients various mutation combinations (from 2 to 5) were identified. It was demonstrated that mutations in FLT3 (FLT3-ITD), NPM1, DNMT3A, and IDH2 were identified significantly more often in combinations with other mutations (p = 0.001): NPM1+/ FLT3-ITD+ (20.8 %), NPM1+/FLT3-ITD+/DNMT3A+ (8.3 %), and FLT3-ITD+/DNMT3A+ (8.3 %). Patients with 1 mutation had a noticeably longer OS median compared with patients with 2 mutations (18.1 and 12.2 months; p = 0.003). In patients with NPM1+ according to their OS the most unfavorable additional mutation was FLT3-ITD (median 27.4 vs. 9.2 months; p = 0.019) and the combination of NPM1+/FLT3-ITD+/DNM-T3A+ (median 27.4 vs. 14.6 months; p = 0.141). OS of patients with DNMT3A+ showed a downward trend if FLT3-ITD additional mutation was identified (17.3 vs. 7.1 months; p = 0.074). Conclusion. Mutations in FLT3, DNMT3A, IDH1/2, NPM1 genes frequently occur in AML intermediate-risk patients, i.e. they determine the intermediate prognosis group in AML. The studied mutations considerably impact prognosis. It is important to take into consideration mutation type, its allele burden, and the presence of additional mutations. A patient with 2 mutations has a considerably worse OS compared with a patient with 1 mutation. The studied group of patients with the combination of NPM1+/FLT3-ITD+, NPM1+/ FLT3-ITD+/DNMT3A+, DNMT3A+/FLT3-ITD+ mutations has the poorest prognosis. Comprehensive analysis of genetic damages in AML patients allows to most accurately predict the course and prognosis of the disease and to plan targeted therapy.

Background & Aims. The presence of different molecular genetic markers of clonality (mutations in JAK2, MPL, CALR) or their absence (triple negative status, TN) in essential thrombocythemia (ET) indicates a biological heterogeneity of the disease and can determine its clinical forms. The aim was to evaluate the association of molecular genetic markers with the clinical form and the prognosis of ET. Materials & Methods. We analyzed the data of 240 patients with ET at the age of 20-91 years (median age 58.7 years), who were observed in the Russian Research Institute of Hematology and Transfusiology from 1999 to 2016 (median observation period 37.2 months). Results. The JAK2V617F (JAK2+) mutation was found in 182 (75.9 %) of 240 patients. CALR (CALR+) mutations were found in 30 (12.5 %): type 1 (CALP7+) mutations in 13/30 (43.3 %) and type 2 (CALR2+) in 17/30 (56.7 %). MPL (MPL+) mutations were found in only 2 (0.8 %) of 240 patients. None of the mutations were detected in 26 (10.8 %) of 240 patients (TN status). Significantly higher platelet counts were observed in CALP7+ and CALR2+ subgroups during the primary diagnosis of ET compared with JAK2+ and TN groups. The mean platelet counts were 1252 * 109/L for CALR2+ and 1079 * 109/L for CALP7+ vs 841 * 109/L (p < 0.001; p = 0.06) and 775 * 109/L (p < 0.001; p = 0.04) for JAK2+ and TN, respectively. Thrombosis was diagnosed in 50 (27.4 %) of 182 patients of the JAK2+ subgroup, in 8 (30.7 %) of the 26 patients of the TN subgroup, and in 2 (18.2 %) of 11 patients of the CALP7+ subgroup. No thrombosis was found in the CALR2+ and MPL+ subgroups (p < 0.001). In general, the CALP7+ status was characterized as the most favorable in terms of prognosis (5-year overall survival rate of 100 %), compared to the least favorable TN status (5-year overall survival rate of 85 %). Conclusion. Mutations in the CALR gene were characterized by a more favorable prognosis in comparison with JAK2+and TN, as well as a decrease in the risk and frequency of thrombosis, despite higher platelet counts. TN-status of ET was associated with unfavorable prognosis.