Current Genetic Models for Prediction of Primary Myelofibrosis

LB Polushkina1, VA Shuvaev1, MS Fominykh1, YuA Krivolapov2, EA Belyakova2, ZP Asaulenko2, EV Motyko1, LS Martynenko1, MP Bakai1, NYu Tsybakova1, SV Voloshin1,3, SS Bessmeltsev1, AV Chechetkin1, IS Martynkevich1

1 Russian Research Institute of Hematology and Transfusiology, 16 2-ya Sovetskaya str., Saint Petersburg, Russian Federation, 191024

2 II Mechnikov North-Western State Medical University, 41 Kirochnaya str., Saint Petersburg, Russian Federation, 191015

3 SM Kirov Military Medical Academy, 6 Akademika Lebedeva str., Saint Petersburg, Russian Federation, 194044

For correspondence: Lyubov Borisovna Polushkina, PhD in Biology, 16 2-ya Sovetskaya str., Saint Petersburg, Russian Federation, 191024; e-mail: polushkina.lb@gmail.com

For citation: Polushkina LB, Shuvaev VA, Fominykh MS, et al. Current Genetic Models for Prediction of Primary Myelofibrosis. Clinical oncohematology. 2019;12(4):391–7 (In Russ).

DOI: 10.21320/2500-2139-2019-12-4-391-397


ABSTRACT

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; = 0.021). TN was associated with a high IPSS risk (= 0.011), low hemoglobin level (median 101 g/L; = 0.006), continuing drop in platelet count (median 266 × 109/L; = 0.041), increased lymphocyte count (median 26.9 × 109/L; = 0.001). The detection of terminating mutations in ASXL1 correlated with palpable enlarged spleen (= 0.050), reduced platelet count (median 184 × 109/L; = 0.016), leukocyte count > 25 × 109/L (= 0.046), and blast count ≥ 1 % (< 0.001). Univariate regression analysis showed that terminating mutations in ASXL1 (hazard ratio [HR] 2.9; = 0.018), unfavorable karyotype (HR 8.2; < 0.001), and TN (ОР 8.1; < 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.

Keywords: primary myelofibrosis, mutations, karyotype, prediction.

Received: April 8, 2019

Accepted: September 1, 2019

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REFERENCES

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Targeted Therapy of Myelofibrosis

OYu Vinogradova1,3,4, VA Shuvaev2, IS Martynkevich2, MM Pankrashkina1,3, MS Fominykh2, EV Efremova2, KYu Krutikova2, LB Polushkina2, NN Sharkunov1, SV Voloshin2, AV Chechetkin2

1SP Botkin Municipal Clinical Hospital, 5 2-i Botkinskii pr-d, Moscow, Russian Federation, 125284

2Russian Research Institute of Hematology and Transfusiology, 16 2-ya Sovetskaya str., Saint Petersburg, Russian Federation, 191024

3Dmitrii Rogachev National Medical Pediatric Hematology, Oncology and Immunology Research Center, 1 Samory Mashela str., Moscow, Russian Federation, 117198

4NI Pirogov Russian National Research Medical University, 1 Ostrovityanova str., Moscow, Russian Federation, 117997

For correspondence: Ol’ga Yur’evna Vinogradova, MD, PhD, 5 2-i Botkinskii pr-d, Moscow, Russian Federation, 125284; Tel.: 8(495)945-97-61; e-mail: olgavinz@mail.ru.

For citation: Vinogradova OYu, Shuvaev VA, Martynkevich IS, et al. Targeted Therapy of Myelofibrosis. Clinical oncohematology. 2017;10(4):471–8 (In Russ).

DOI: 10.21320/2500-2139-2017-10-4-471-478


ABSTRACT

Background. Myelofibrosis (primary myelofibrosis, post-essential trombocythemia myelofibrosis, post-polycythemia myelofibrosis) is the most complex and pressing problem among all Ph-negative myeloproliferative diseases. The present article summarizes the author’s experience of using new Janus kinase inhibitors in routine clinical practice, and compares the data with the results of other clinical research.

Aim. To evaluate the use of ruxolitinib in patients with myelofibrosis.

Materials & Methods. Our analysis includes 48 patients (21 men and 27 women) with histologically verified myelofibrosis (primary myelofibrosis in 36 cases, post-essential trombocythemia myelofibrosis in 10 cases, and post-polycythemia myelofibrosis in 2 cases) in a chronic stage. All patients received ruxolitinib. Median age at the start of therapy was 60 years (range from 35 to 79). Massive splenomegaly (≥ 10 cm below the costal margin) was found in 34 (71 %) of 48 patients. The initial dose of ruxolitinib was determined by the platelet level. The efficacy of the therapy was evaluated in accordance with ELN 2013 criteria.

Results. Median duration of treatment was 18 months (range from 1 to 50 months). Symptoms of intoxication were relieved in 33 of 37 patients (89 %). The spleen size decreased in 64 % of patients. In 33 % of cases spleen size did not change, whereas an increase was observed in 3 % of patients. In the majority of patients hemoglobin level remained stable through the course of treatment. Three of 14 transfusion dependent patients did not require blood transfusions after 3 months of therapy. In patients with high thrombocyte levels prior to ruxolitinib therapy the mean level was approaching normal by the end of the 1st month of treatment. The median JAK2V617F mutant allele burden at the beginning treatment was 56.5 % (n = 20; 22.5–126.1 %). After 6 moths of treatment it accounted for 62.3 % (n = 11; 25.4–79.7 %) and in 12 months accounted for 47.4 % (n = 12; 14.2–102.2 %). By the time of the analysis 42 of 48 patients continued the ruxolitinib treatment (88 %). Death occurred in 4 patients. Overall 1-year (92 %) and 2-year (87 %) survival corresponds to the data of COMFORT-I, COMFORT-II and JUMP clinical trials.

Conclusion. Ruxolitinib showed to be an effective treatment for myelofibrosis. The most pronounced and rapid effect ruxolitinib had on the spleen size and the symptoms of intoxication. The tolerability of ruxolitinib was satisfactory in the majority of patients. According to the author’s data, ruxolitinib had a small impact on the JAK2V617F mutant allele burden. The overall survival rate in patients with myelofibrosis, receiving ruxolitinib in the clinical setting was similar to that of in the clinical trials.

Keywords: primary myelofibrosis, post-essential trombocythemia myelofibrosis, post-polycythemia myelofibrosis, JAK2V617F, ruxolitinib, clinical practice, targeted therapy.

Received: February 11, 2017

Accepted: May 22, 2017

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REFERENCES

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  15. Verstovsek S, Mesa RA, Gotlib J, et al. Long-term treatment with ruxolitinib for patients with myelofibrosis: 5-year update from the randomized, double-blind, placebo-controlled, phase 3 COMFORT-I trial. J Hematol Oncol. 2017;10(1):55. doi: 10.1186/s13045-017-0417-z.
  16. Al-Ali HK, Griesshammer M, le Coutre P, et al. Safety and efficacy of ruxolitinib in an open-label, multicenter, single-arm phase 3b expanded-access study in patients with myelofibrosis: a snapshot of 1144 patients in the JUMP trial. Haematologica. 2016;101(9):1065–73. doi: 10.3324/haematol.2016.143677.

Myelofibrosis Models: Literature Review and Own Data

AA Silyutina, II Gin, NM Matyukhina, EN Balayan, PA Butylin

VA Almazov Federal North-West Medical Research Center, 2 Akkuratova str., Saint Petersburg, Russian Federation, 197341

For correspondence: Pavel Andreevich Butylin, PhD, 2 Akkuratova str., Saint Petersburg, Russian Federation, 197341; e-mail: butylinp@gmail.com

For citation: Silyutina AA, Gin II, Matyukhina NM, et al. Myelofibrosis Models: Literature Review and Own Data. Clinical oncohematology. 2017;10(1):75–84 (In Russ).

DOI: 10.21320/2500-2139-2017-10-1-75-84


ABSTRACT

Background & Aims. Chronic myeloproliferative disorders typically develop during a long latent period, and it complicates the study of the mechanism of its pathogenesis. Observations from the clinical practice should be confirmed by experiments. The mechanisms of oncological transformation related to mutations associated with chronic myeloproliferative diseases were confirmed in transgene animal models. Biological models permitted to determine a complex nature of myelofibrosis. However, studies of the cellular mechanisms of myelofibrosis require new models. This paper presents a review of published models of myeloproliferative disorders, mainly, primary myelofibrosis, and results of studies of a new cell line with expression of JAK2 V617F. The aim of this study is to create a new cell line with expression of transforming JAK2 V617F mutation in acute monocytic leukemia THP-1 cells.

Methods. Transgenic cell lines were created on the basis of monocytic leukemia THP-1 cell line that can differentiate into macrophages. Direct mutagenesis was used to cause V617F mutation. Two cell lines were created: one with JAK2 expression with V617F mutation, the other with wild type JAK2.

Results. Both transgenic lines were characterized by increased JAK2 expression as compared to non-modified cells. In routine cultivation, transgenic THP-1 cells retained the morphology of monocytes. After treatment with phorbol ester, THP-1 differentiated into macrophages and become adherent to culture plastic. Adherent cells demonstrated the variety of shapes: some of them were spherical, the other ones had pseudopodia. No significant differences in viability of cells were observed. However, macrophages expressing mutant JAK2 and overexpressing the wild type JAK2 demonstrated a tendency to decreased amount unlivable cells during cultivation.

Conclusion. The obtained cell model can be used for estimating the influence of JAK2 V617F mutation on pro- and antifibrotic potential of macrophages that can help to investigate the pathogenetic role of macrophages in myelofibrosis development. In addition, this model can help to develop novel methods of therapy and diagnostics of primary and secondary myelofibrosis.

Keywords: Ph-negative chronic myeloproliferative disorders, primary myelofibrosis, JAK2 V617F, transgenic animals.

Received: September 15, 2016

Accepted: December 13, 2016

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Biology of Myeloproliferative Malignancies

AL Melikyan, IN Subortseva

Hematology Research Center, 4а Novyi Zykovskii pr-d, Moscow, Russian Federation, 125167

For correspondence: Irina Nikolaevna Subortseva, PhD, 4а Novyi Zykovskii pr-d, Moscow, Russian Federation, 125167; Tel.: +7(495)612-44-71; e-mail: soubortseva@yandex.ru

For citation: Melikyan AL, Subortseva IN. Biology of Myeloproliferative Malignancies. Clinical oncohematology. 2016;9(3):326-35 (In Russ).

DOI: 10.21320/2500-2139-2016-9-3-314-325


ABSTRACT

Chronic myeloproliferative diseases (WHO, 2001), or myeloproliferative neoplasms/malignancies (MPN) (WHO, 2008), are clonal diseases characterized by proliferation of one or more myelopoietic cell line in the bone marrow with signs of unimpaired terminal differentiation and is normally associated with changes in peripheral blood characteristics. The group of classical Ph-negative MPNs consists of polycythemia vera, essential thrombocythemia, primary myelofibrosis and unclassified MPNs. Acquired somatic mutations contributing to the pathogenesis of Ph-negative MPNs include JAK2 (V617F, exon 12), MPL, CALR gene mutations found in about 90 % of patients. However, these molecular events are not unique in the pathogenesis of the diseases. Mutations of other genes (ТЕТ2, ASXL1, CBL, IDH1/IDH2, IKZF1, DNMT3A, SOCS, EZH2, TP53, RUNX1, and HMGA2) are involved in formation of the disease phenotype. This review describes current concepts concerning the molecular biology of MPNs.


Keywords: chronic myeloproliferative diseases, myeloproliferative neoplasms, polycythemia vera, essential thrombocythemia, primary myelofibrosis, genes JAK2, CALR, and MPL.

Received: April 11, 2016

Accepted: April 11, 2016

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New Сytogenetic Approaches in Patients with Primary Myelofibrosis

TL Gindina, NN Mamaev, VV Baikov, MV Barabanshchikova, EN Nikolaeva, IA Petrova, IS Moiseev, OV Pirogova, YuYu Vlasova, MO Ivanova, EV Morozova, SN Bondarenko, BV Afanasev

R.M. Gorbacheva Scientific Research Institute of Pediatric Hematology and Transplantation; Academician I.P. Pavlov First St. Petersburg State Medical University, 6/8 L’va Tolstogo str., Saint Petersburg, Russian Federation, 197022

For correspondence: Tat’yana Leonidovna Gindina, PhD, 6/8 L’va Tolstogo str., Saint Petersburg, Russian Federation, 197022; Tel.: + 7(812)233-12-43; e-mail: cytogenetics.bmt.lab@gmail.com

For citation: Gindina TL, Mamaev NN, Baikov VV, et al. New Сytogenetic Approaches in Patients with Primary Myelofibrosis. Clinical oncohematology. 2016;9(1):61–9 (In Russ).

DOI: 10.21320/2500-2139-2016-9-1-61-69


ABSTRACT

Aim. To evaluate the potential of a new cytogenetic technique in patients with primary myelofibrosis (PMF).

Materials and methods. 48-hour blood cell cultures (according to Singh et al., 2013) were used for cytogenetic study in 11 PMF patients (5 female, 6 men, aged 32–60 years; median 48.6 years). GTG-banding and different types of fluorescence in situ hybridization (FISH) techniques were used for identification of chromosomal aberrations.

Results. The incidence of abnormal karyotypes in blood cultures was significantly higher than that in standard bone marrow cultures (82 vs 27 %; < 0.01). The polyploid clones were found in blood cultures of 45 % of patients. Structural chromosomal aberrations were found in chromosomes 6, 1, 3, as well as 16 and 17 (in 2 and 1 patients with each aberration, respectively). In all but one patients these abnormalities in diploid and polyploid metaphases were identical. Partial 1q trisomy resulted from adding of additional (1q21–1q44) material translocated to the short arm of chromosome 5 to the material of 2 normal homologue of chromosome 1. It seems that 1q+, i(17q) and some others chromosomal abnormalities were secondary, whereas 6p21 locus involvement may be a primary defect in PMF. The t(3;6)(q25;p21) translocation described for the first time and confirmed by FISH should be considered a variant of well-known translocation t(1;6). Allo-HSCT in 2 patients with 1q+ was successful, whereas there were problems with engraftment in a female patient with prognostically unfavorable t(3;3)(q21;q26) translocation associated with the EVI1 gene overexpression.

Conclusion. Cytogenetic examinations in blood cultures provide important additional information about PMF patients.


Keywords: primary myelofibrosis, 48-hour peripheral blood cell culture, cytogenetics.

Received: July 14, 2015

Accepted: November 1, 2015

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In vitro model of myelofibrosis using human platelet lysate

Ye.N. Bulycheva, N.T. Siordiya, E.G. Lomaia, A.Yu. Zaritskiy, and P.A. Butylin

V.A. Almazov Federal Heart, Blood and Endocrinology Center, Saint Petersburg, Russian Federation


ABSTRACT

The development and studies of the myelofibrosis (MF) in vitro model is an important issue, since such model can lead to understanding of pathogenesis and identifying the new targets for therapy.

Objectives. Here, we studied the properties of mesenchymal stromal cells (MSCs) cultured in the medium containing the human platelet lysate (HPL).

Design and methods. Bone marrow MSCs from healthy donors and a patient with primary myelofibrosis (PMF) were cultured in the media containing various HPL concentrations. We measured the proliferative activity, the collagen type I and III expression, and capability to differentiate into the osteogenic or adipogenic lineages. The concentrations of the vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), transforming growth factor beta (TGF-b), and hepatocyte growth factor (HGF) were measured in HPL from 17 patients with primary myelofibrosis or post-polycythemia vera myelofibrosis (post-PV MF) using the specific ELISA kits.

Results. The highest MSC proliferative rate was found in the cultures with high HPL concentrations (10–20 %). The ratio of collagen type III/collagen type I expression was the highest in the cultures containing 10 % HPL. The use of HPL for MSCs culturing caused no changes in their osteogenic differentiation capability, but the increase in the HPL concentration resulted in the decreased capability to differentiate into the adipogenic lineage. Further, we observed the significantly increased VEGF and bFGF concentrations in HPL from MF patients, compared to the age-matched healthy controls (2.5- and 2.4-fold, respectively, < 0.01), while the TGF-b and HGF concentrations showed the trend towards an increase, but with no significant difference from the controls. MSCs cultured with HPL from MF patients showed a higher proliferative rate compared to HPL from healthy donors. MSCs from the PMF patient tended to proliferate more actively compared to the cells from healthy donors.

Conclusion. MSCs culturing using varying HPL concentrations can be used as an adequate MF in vitro model, since it leads to pro-fibrotic changes in the bone marrow stromal cells.


Keywords: primary myelofibrosis, platelet lysate, mesenchymal stromal cells, myelofibrosis in vitro model.

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Selection of patients for iron-chelating therapy

S.V. Gritsaev, B. Davaasambuu, N.A. Romanenko, and K.M. Abdulkadyrov

Russian Research Institute of Hematology and Transfusiology, FMBA, Saint Petersburg, Russian Federation


ABSTRACT

Emergence of iron aggressive forms in patients with hematological or oncohematological diseases is potentially life-threatening. This can be caused by several situations: multiple allogeneic RBC transfusions, increased intestinal absorption of iron, or damaging effects of chemotherapeutic agents. The objective of the study was to identify candidates for iron-chelating therapy. The data on 727 patients with screening blood tests were analyzed. The highest serum ferritin level was revealed in patients with thalassemia, PMF, or MDS. They received 80, 37, or 35 RBC transfusions, respectively. The lowest serum ferritin levels were found in patients with CLL or hemolytic anemia that received lesser number of RBC transfusions, namely, 18.5 and 16.5, respectively. The correlation between serum ferritin level and the total number of RBC transfusions was revealed: r = 0.462; p = 0.000. We concluded that iron-chelating therapy is primarily indicated to the patients with MDS or PMF in whom the high serum ferritin level is due to excessive post-transfusion iron.


Keywords: myelodysplastic syndromes, primary myelofibrosis, thalassemia, acute leukemia, multiple myeloma, serum ferritin, allogeneic RBC transfusions.

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