We retrospectively analyzed the effectiveness of ruxolitinib in patients with myelofibrosis (MF) in real clinical practice in Russia. The study included 42 patients with MF with an average age of 56 years from 10 inpatient and outpatient clinics. The majority of patients (55 %) belonged to the intermediate risk group 1 on the DIPSS scale, 86 % had massive splenomegaly and 88 % had constitutional symptoms. The average initial dose of ruxolitinib was 15 mg twice a day. At the time of analysis, 74 % of patients continued taking ruxolitinib with an average duration of therapy of 20 months. A decrease in the palpable size of the spleen by at least 50 % was recorded in 36 % and 46 % of patients assessed after 3 and 6 months of treatment, respectively. A correlation was found between OV, initial splenomegaly and splenic response after 3 months of therapy. It should be noted that no deaths were recorded in patients with a decrease in the size of the spleen by ≥ 50 %. There were no cases of discontinuation of treatment due to side effects. In general, in our retrospective study, ruxolitinib effectively controlled constitutional symptoms and reduced the size of the spleen in patients with MF. An early splenic response after 3 months of therapy is apparently a prognostic factor for OS, and a decrease in the size of the spleen by less than 25 % should be considered as treatment ineffectiveness in patients with MF taking ruxolitinib.

Background. In accordance with the World health organization clinical guidelines, the analysis of somatic mutations in the CALR gene, as well as mutations in the JAK2 and MPL genes, are included in the list of criteria for the Ph-myeloproliferative neoplasms diagnosis.More than 50 different mutation variants have been found in the CALR gene, among which the most frequent are a 52 bp deletion (c.1092_1143del), also called type 1, and a 5 bp insertion (c.1154_1155insTTGTC), also called type 2 (88 %).The remaining 12 % are other type less frequent indels or combinations thereof.It is most convenient to use sequencing methods to identify all possible variants of CALR mutations. It is also important to develop inexpensive screening test that can detect any mutations in the analyzed DNA fragment of CALR gene. This method can be heteroduplex analysis followed by electrophoresis on polyacrylamide gel (PAGE).The objective: to develop and demonstrate the feasibility of using heteroduplex analysis with separation of the PCR product by electrophoresis on non-denaturing PAGE for the CALR exon 9 mutations detection as the screening test. Materials and methods. DNA samples of 13 CALR-positive patients with different phenotypic variants of Ph-myeloproliferative neoplasms were screened by heteroduplex analysis. For the most common variants of CALR mutations (c.1092_1143del and c.1154_1155insTTGTC), a threshold determination of the mutant allele presence was analyzed.Nucleotide sequence of exon 9 fragment was determined using Sanger sequencing. Also, all 13 samples were analyzed using the pyrosequencing method to assess the allelic burden level.Results. Heteroduplex analysis revealed mutations in exon 9 of the CALR gene in all 13 patients. The threshold determinations of the method in the case of the c.1154_1155insTTGTC and c.1092_1143del analysis are 6.25 % and 3.13 % of the mutant allele presence in the patient sample, respectively.Conclusion. The proposed variant of the heteroduplex analysis with separation of the PCR product by electrophoresis on non-denaturing PAGE can be recommended for use as the preliminary screening test which is carried out before the confirming sequencing methods for the different indels (or combinations thereof) CALR mutations determine.The presence of heteroduplexes indicates the presence of a mutation, even if the mutant product is not visualized (in case of small mutations).

Somatic mutations associated with oncological diseases, including Ph-myeloproliferative neoplasms (Ph-MPN), are very diverse, occur with different frequencies and different allelic burden levels. Therefore, at the initial stage of performing molecular-genetic diagnostic procedures, it is desirable to be able to conduct screening tests in the laboratory. This is especially important when analyzing rare and diverse mutations. Analysis of high resolution melting curves (HRM analysis), which has high sensitivity and is suitable for screening all types of mutations, in a number of studies is proposed for the analysis of Ph-MPN associated mutations in the JAK2 and CALR genes. For analysis of somatic mutations in the majority of literature sources that we reviewed, the authors use the LightCycler (Roche) thermocycler and much rarely the CFX96 (Bio-Rad), which is often presented in Russian scientific and practical and medical organizations. The aim of the study was to screen the somatic JAK2 and CALR mutations by HRM analysis using the CFX96 thermocycler and the Precision Melt Analysis software (Bio-Rad, USA) for patients with Ph-MPN. In the present research, HRM analysis was conducted on the DNA samples from patients with mutations in the JAK2 or in the CALR gene. The Precision Melt Analysis software identified all variants of the analyzed mutations, both a single nucleotide substitution in the JAK2 gene (with allelic burden level in the range of 5-40%), and various indel mutations in the CALR gene (with allelic burden level in the range of 40-50%) Therefore, the HRM analysis that was conducted on the CFX96 allows screening of highly specific mutation for the diagnosis of Ph-MPN in the exon 14 of the JAK2 gene and in the exon 9 of the CALR gene. The inclusion of this screening research in the laboratory testing algorithm improves the efficiency and accessibility of molecular genetic technologies in the diagnosis of Ph-MPN.

Aim. To assess the efficacy of targeted therapy with ruxolitinib in patients with myelofibrosis in real clinical practice in Russia. To determine the prognostic value of spleen reduction in the early stages of ruxolitinib treatment and its effect on overall survival. Materials & Methods. The present retrospective study was based on the data of 10 centers of Russia. It included 56 myelofibrosis (primary or post-polycythemic and post-throm-bocythemic) patients who received ruxolitinib. The median age of patients was 56 years (range 26-76 years). Most of them (59 %) were considered intermediate-1 risk according Results. By the start of data collection most of patients (79 %) had been treated with ruxolitinib. In no case therapy was withdrawn for the reason of drug toxicity. On ruxolitinib constitutional symptoms were reversed in 70 %, 87 %, and 98 % of patients by months 1, 3 and 6, respectively. In 36 % and 46 % of patients by months 3 and 6, respectively, > 50 % decrease in spleen size was observed. Overall, in 31 % and 27 % of cases the size of the spleen decreased by less than 25 % by months 3 and 6, respectively. The factors affecting the changes in spleen size have not been identified. The probability of overall survival by years 2 and 5 of follow-up was 97 % and almost 70 %, respectively. This parameter was significantly affected by the extent of spleen size reduction by month 3 of follow-up as well as by its initial size. Conclusion. Ruxolitinib shows high efficacy for both decrease of general myelofibrosis symptoms and reduction in spleen size. The extent of spleen reduction is an important prognostic factor. It seems, that in patients with insufficient spleen reduction an increase in drug dose is advisable. If it is not possible, alternative methods of treatment should be sought.

Цель. Оценить эффективность таргетной терапии руксолитинибом у пациентов с миелофиброзом в реальной клинической практике в России. Определить прогностическое значение динамики уменьшения размеров селезенки в ранние сроки лечения руксолитинибом и его влияние на общую выживаемость. Материалы и методы. Настоящий ретроспективный анализ проведен по данным 10 центров России. В исследование включено 56 пациентов с миелофиброзом (первичным или постполицитемическим и посттромбоцитемическим), получавших руксолитиниб. Медиана возраста пациентов составила 56 лет (диапазон 26–76 лет). Большинство из них (59 %) были с промежуточной-1 группой риска по шкале DIPSS, имели массивную спленомегалию (80 %) и конституциональные симптомы (86 %). Исходная доза препарата составляла 30 мг в сутки в 64 % случаев. При этом уровень тромбоцитов ≥ 200 × 109/л наблюдался у 61 % пациентов. Размеры селезенки оценивались пальпаторно. Результаты. К началу сбора данных большинство пациентов (79 %) продолжали лечение руксолитинибом. Ни в одном случае причиной прекращения терапии не была токсичность препарата. На фоне приема руксолитиниба конституциональные симптомы были купированы у 70, 87 и 98 % пациентов к 1, 3 и 6 мес. терапии соответственно. Уменьшение размеров селезенки на ≥ 50 % отмечено у 36 и 46 % пациентов к 3 и 6 мес. лечения соответственно. Всего в 31 и 27 % случаев размеры селезенки сократились на менее 25 % к 3 и 6 мес. терапии соответственно. Не удалось выявить факторы, влияющие на динамику изменения размеров селезенки. Вероятность общей выживаемости к 2 и 5 годам наблюдения составила 97 и почти 70 % соответственно. На этот показатель статистически значимо влияла степень уменьшения размеров селезенки к 3 мес. наблюдения, а также ее исходные размеры. Заключение. Руксолитиниб демонстрирует высокую эффективность в отношении как уменьшения общих симптомов миелофиброза, так и сокращения размеров селезенки. Степень редукции размеров селезенки является важным прогностическим фактором. У пациентов с недостаточным сокращением размеров селезенки целесообразно увеличение дозы препарата. При невозможности этого необходим поиск альтернативных методов лечения.

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.

MiR-155 is involved in various physiological processes in the cell, including hematopoiesis, immunity, inflammation and differentiation. Increased expression of miR-155 is observed in many malignant diseases, including lymphomas, acute myeloid leukemia and CLL. However, a comparative study of the miR-155 expression in the blood leukocytes in patients with chronic myeloid and lymphoproliferative diseases has not yet been carried out. To investigate the expression of miR-155 in the blood cells of patients with lympho- and ph-negative myeloproliferative neoplasms. MiR-155 expression were studied in the blood leukocytes of 28 patients with B-CLL, 52 patients with MPN and 51 donors by "real time" PCR method. The study revealed an increase in miR-155 in blood leukocytes in both patients with CLL and patients with MPN compared with the control group. In accordance with the results of the ROC analysis, the sensitivity and specificity of blood leukocytes testing on miR-155 expression level was 81.8% and 78.4%, respectively, for CLL and 55.1% and 82.4%, respectively, for MPN. At the same time, in patients with CLL who received therapy, the level of miR-155 was significantly lower compared with those who did not receive therapy. Thus, the involvement of miR-155 in the pathogenesis of chronic myeloid and lymphoproliferative diseases was demonstrated.

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.