Chronic Myeloproliferative Neoplasia Research Articles

Analysis of the contribution of polymorphisms of hemostasis system genes and folate cycle genes to the genetic presposition to the development of thrombosis in patients with Ph-negative myeloproliferative neoplasia in the Republic of Uzbekistan

Thrombotic complications often complicate the course of the underlying disease in patients with chronic myeloproliferative neoplasia. However, a thromboembolic condition is not observed in all patients.The aim of the study was to assess the carriage frequency of polymorphic genes of the blood coagulation system and folate metabolism genes and the contribution of genetic variants to the development of thrombosis in patients with chronic Ph-negative myeloproliferative neoplasia (CMPN).Materials and methods. Molecular genetic testing for the presence of genetic variants with an assessment of their frequency of occurrence and allelic load was carried out in 142 patients with CMPN. We studied polymorphisms of the folate cycle genes – A2756G (Asp919Gly) of the MTR gene, C677T (Ala22Val) of the MTHFR gene, A66G (ILe22Met) of the MTRR gene (rs1801394), as well as mutations of the genes of blood coagulation factors – G(455)A of the FGB gene, G20210A of the F2 gene, G10976A (Arg353Gln) of the F7 gene, G1691A (Arg506Gln) of the F5 gene. The study was carried out using real-time polymerase chain reaction (PCR); The biological material for the test was whole blood.Results and discussion. The study showed that only 6 out of 50 (12%) patients with thrombotic complications had no changes in the genes studied, while 88% of patients had one or another genotypic variant, which may indicate a high probability of involvement of a hereditary genetic factor in the development of hypercoagulability and thrombotic complications in patients with chronic MPN.Conclusions. A comprehensive study of the role, interaction and operating conditions of genes that control blood coagulation processes in patients with Ph-negative chronic MPN will make it possible to understand the causes of hemostatic system disorders and develop effective measures to prevent thrombotic complications in this category of patients.

A potentially new thromboembolic event scoring system in polycythaemia vera patients: An audit of the Hungarian Philadelphia negative chronic myeloproliferative neoplasia register.

The Hungarian National Registry for Philadelphia chromosome negative myeloproliferative neoplasms was used to analyse the thromboembolic events (TE) of Hungarian patients with polycythemia vera (PV). Data from 351 JAK2 V617F-positive patients diagnosed with PV were collected online from 15 haematology centres reporting clinical characteristics, therapeutic interventions and thromboembolic events. TE events were evaluated before and after diagnosis based upon the Landolfi and Tefferi risk assessment scales. TE were reported on 102 patients before diagnosis and 100 during the follow-up period. Comparing to the frequency of major arterial events before PV diagnosis, we can notice a decreasing tendency after diagnosis: from 12.3% to 2.6% (p < .00003). There was no significant change in the rate of major venous events (from 5.1% to 8.5%; p = .1134) or minor arterial events (from 11.7% to 17.4%; p = .073). Bleeding events were recorded in 5.7% of patients. Despite treatment with HU + ASA, 44 patients (43.1%) with prior TE had recurrent thromboembolic complications. The particular analysis of our data revealed a new TE scoring system based on: age, gender, previous TE and iron deficiency at the time of diagnosis. Our registry enables characterisation of patients with PV. The high level of recurrent TE events highlights the need for more effective and risk-adapted therapy.

Assessment of Dysgranulopoiesis By Optical Cytology Compared to Distribution of Monocyte Subsets By Flow Cytometry in Peripheral Blood for Predicting the Diagnosis of CMML

Introduction The diagnosis of chronic myelomonocytic leukemia (CMML) according to the WHO 2017 requires the presence of absolute (≥ 1x109/L) and relative (≥ 10%) monocytosis. Optical cytology represents the basis for its diagnosis and is essential to evaluate dysmyelopoiesis and to identify promonocytes and blasts. Although dysplasia is not a specific finding, its presence along with a persistent monocytosis is highly suggestive of CMML. In this sense, dysgranulopoiesis is an almost constant finding in CMML. The study of monocyte subsets distribution in peripheral blood (PB) by multiparametric flow cytometry (MFC) has proven to be a very useful tool, since the presence of >94% of classical phenotype monocytes (MO1) allows to predict the diagnosis of CMML with a sensitivity and specificity greater than 90% (Selimoglu-Buet et al. Blood 2015). The aim of this study was to evaluate the predictive capacity of morphological evaluation of PB smears for the diagnosis of CMML in a series of patients with absolute and relative monocytosis. Additionally, these results were compared with the analysis of monocyte subsets distribution in PB by MFC. Methods 162 patients with absolute and relative monocytosis were studied. 114 PB smears were available for morphological assessment and 160 MFC analysis of monocyte subsets distribution. The final diagnosis for these patients was: CMML (n=76), Phi negative chronic myeloproliferative neoplasia (MPN) (n=9) and reactive monocytosis (n=77). Genomic information (cytogenetics and NGS) was available in 66 CMML and all of MPN. After anonymizing PB smears, a cytological assessment was performed without additional clinical or analytical data (eg.: age, blood counts, biochemistry). This way we tried to compare the predictive capacity for the diagnosis of CMML of both techniques without the influence of additional data other that the known absolute and relative monocytosis. The cytological evaluation of PB smears consisted fundamentally of a qualitative evaluation of dysgranulopoiesis: nuclear hyposegmentation, chromatin hypercondensation and/or cytoplasmic hypo-degranulation. The sensitivity, specificity, Youden index, positive predictive value (PPV) and negative predictive value (NPV) of both techniques were analyzed. The Youden index allows to evaluate the balance between sensitivity and specificity ([sensitivity + specificity] -100). The capacity for diagnostic prediction of CMML for both techniques was assessed using the C-index. Finally, Cohen's Kappa index was used to evaluate the concordance between both techniques. Results Table 1 shows the results of sensitivity, specificity, Youden index, PPV, NPV and C-index of both techniques. The Kappa index showed a very good degree of agreement (K=0.715; p<0.001) between both techniques. Conclusions The presence of >94% MO1 had a better balance between sensitivity and specificity than the assessment of dysgranulopoiesis in PB. It also had a high sensitivity, demonstrating that it can be very useful screening tool. Despite this, the specificity and PPV of the assessment of dysgranulopoiesis were higher than those of the MFC, demonstrating that the identification of morphological dysplasia would allow the diagnosis of CMML to be predicted with a higher degree of certainty in the initial workup of absolute and relative monocytosis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A case of &lt;i&gt;CALR&lt;/i&gt; mutation in &lt;i&gt;JAK2&lt;/i&gt;-negative patient with polycythemia

JAK2 mutations can be associated with any phenotypic form of chronic myeloproliferative neoplasia, while MPL and CALR mutations occur, as a rule, in cases of essential thrombocythemia and primary myelofibrosis and they are not observed in polycythemia vera. In this article we describe a clinical case of CALR mutation (c.1154_1155insGTGTC; p.E386fs*46) presence in a JAK2 -negative polycythemia vera patient at age 36. In January 2018 changes in his hemogramm were recorded for the first time. In June 2018, based on a diagnostic study of bone marrow trepanobiopsy, a diagnosis of polycythemia vera was made. Molecular genetic study of the patient’s DNA didn’t reveal mutations in the JAK2 (12 and 14 exons) and the MPL genes. CALR mutation was revealed during the screening by heteroduplex analysis with the electrophoresis in polyacrylamide gel. Then the mutation was identified by Sanger’s DNA sequencing as с.1154_1155insGTGTC; p.E386fs*46. The allelic burden level as determined by pyrosequencing was 20 % (June 2018). In conclusion we can suppose that the revealed CALR mutation с.1154_1155insGTGTC; p.E386fs*46 plays its role in our patient’s polycythemia phenotype.

Anti–IL-6 cytokine treatment has no impact on elevated hematocrit or splenomegaly in a polycythemia vera mouse model

Somatic mutations in JAK2, MPL and Calreticulin and inflammation play a key role in pathophysiology of chronic myeloproliferative neoplasia (CMN). One of the most prominent cytokines elevated in serum of Polycythemia vera patients is interleukin-6 (IL-6). Currently, it is being discussed whether suppression of inflammation by anti-cytokine approaches as anti-IL-6 treatment may be therapeutically useful in CMN. We here sought to investigate the efficacy of anti-IL-6 treatment on inflammatory cytokines, hematocrit and splenomegaly in CMN like disease. JAK2-V617F knock-in mice (JAK2+/V617F) were treated for three weeks with anti-IL-6 antibody (Ab) or IgG-control. Upon anti-IL-6 Ab treatment, serum levels of CXCL2 and CXCL10 were significantly reduced. In addition, CXCL1, CCL11, M-CSF, G-CSF, IL-17, IL-12p40 and CCL2 were reduced by a factor of 0.3 -- 0.8. Partly, this was also achieved by applying high-dose IgG. Hematocrit, erythrocyte and leukocyte counts were elevated in JAK2+/V617F mice but were not reduced by anti-IL6 Ab treatment. In addition, there was no apparent amelioration of splenomegaly and spleen histopathology. In conclusion, anti-IL-6 Ab treatment did not result in improvement of hematological disease parameters but was shown to modulate the serum cytokine signature.

PB2236 APPLICATION OF HETERODUPLEX ANALYSIS FOR CARL MUTATION DETECTION

Background:CARL exon 9 somatic mutations are highly specific to confirm the diagnosis of essential thrombocythemia (ET) and myelofibrosis (MF). Moreover, the cases of CALR mutations in the absence of JAK2 mutations when polycythemia vera (PV) or idiopathic erythrocytosis (IE) is diagnosed are reported in the literature. CALR mutations are concentrated in exon 9 and they involve various deletions and insertions as well as their combinations that lead to the frameshifting. Nowadays over 50 different CALR mutations associated with chronic myeloproliferative neoplasia (CMN) have been identified and classified. To identify all possible variants, it is necessary to use sequencing. However, due to the high cost of sequencing, developing a two‐stage algorithm for detect mutations in CARL exon 9 using inexpensive screening is of immediate practically necessity. We have previously proposed a two‐stage algorithm for detect mutations in JAK2 exon 12 using inexpensive screening test by heteroduplex analysis (Subbotina T et al, Haematologica 2017).Aims:The aim of this study was to demonstrate the feasibility of using heteroduplex analysis with separation of the PCR product by electrophoresis on non‐denaturing PAGE as the preliminary screening test for detection of CARL exon 9 somatic mutations.Methods:6 DNA samples of patients with different phenotypic form of CMN and different level of CARL mutation allele burden were included in this study. The informed consents from these patients were obtained. The presence of the CARL mutations and allele burden levels were identified by pyrosequencing method with PyroMark Q24 (Qiagen, Germany) and by Sanger's DNA sequencing with AB3500 (USA). The PCR with the additional stage of formation heteroduplexes was performed using the Real‐time PCR kit (Syntol, Russia) and CFX 96 Real Time System (Biorad, USA). PCR products were analyzed by electrophoresis in 8% PAGE.Results:The ET or MF diagnoses for five of six patients were confirmed by bone marrow trephine biopsies histological examination. It is important to note one out of six patients had a confirmed diagnosis of PV, which is a rare event. The results of electrophoresis on non‐denaturing PAGE are reported in Figure 1. All six patients have six different indel mutation variants in the 9 exon of the CARL: с. 1154_1155insGTGTC; с. 1128_1129insCTTTGCTT and c. 1131_1133delAGA; c.1100_1151del (52); c.1154_1155insTTGTC; c.1092_1143del (51); c.1154_1155insTTGTC, accordingly. The minimum number of inserted nucleotides were 5 and the maximum number of falling nucleotides were 52. Variants of CARL mutations for № 1, 4, 5 and 6 patients have been already described. Patients № 2 and № 3 carry new (yet included in the COSMIC website) mutations in CARL: с. 1128_1129insCTTTGCTT and c. 1131_1133delAGA; c.1100_1151del (52). The allele burden level ranged from 25% to 50%.Summary/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 pyrosequencing or sanger's sequencing. The two‐stage approach allows to optimize the algorithm of the CARL exon 9 mutation detection and to improve the efficiency of testing for patients suspected of having CMN.image

Long-Term Survival in the Concomitant Evolution of Two Cancers: The Idiopathic Myelofibrosis and the Clear Cell Renal Carcinoma; A Case Report

Introduction: The association of chronic myeloproliferative neoplasia with urogenital cancer is rare (1.6% - 1.9%), with the death risk of 1.21 - 2.28 times higher in patients with solid cancer and chronic myeloproliferative disease. Case Presentation: We present the case of a patient diagnosed with triple-negative idiopathic myelofibrosis treated and monitored at the Department of Hematology of the Clinical Emergency County Hospital, Brasov, Romania. After five years of idiopathic myelofibrosis, which was treated with hydroxyurea, a second solid neoplasia was identified as left renal cell carcinoma in Robson stage I. The complete remission of the renal carcinoma was achieved with the left nephrectomy sustained for a duration of approximately four years, overlapping with the partial remission of myelofibrosis under treatment with ruxolitinib. The recovery of the renal carcinoma significantly influenced myelofibrosis therapy and subsequently led to the patient’s development. The global survival, in this case, is 124 months for myelofibrosis and 64 months for the concomitant evolution of the two cancers. Conclusions: Despite a number of peculiarities (age at diagnosis and complications during the treatment of two cancers), the patient had a prolonged survival.

Comprehensive clinical, molecular and histopathological analysis of bone marrow in chronic myeloproliferative neoplasia

ObjectivesTo analyze the possible correlation between molecular changes in the JAK2, MPL and CALR genes, the morphological pattern of bone marrow and the clinical-hematologic profile of patients. Patients and methodsWe conducted a retrospective study that included 140 patients diagnosed with Philadelphia-negative myeloproliferative neoplasia (Ph-MPN) in a single center. ResultsIn essential thrombocythemia (ET), the patients with the JAK2 V617F mutation presented more leucocytes and neutrophils than patients who presented the CALR mutation, who had more platelets and a greater need for cytoreductive therapy. These findings support the fact that the mutational state in ET appears to define subtypes of patients with substantially different clinical courses and prognoses. In myelofibrosis, the mutational state appears to influence the histopathological changes found in the bone marrow biopsy, which did not occur in polycythemia vera or ET.

In Vivo Blockade of Beta-1 and Beta-2 Integrin Activity Inhibits Splenomegaly in JAK2-V617F Positive Myeloproliferative Disease

Introduction:Recently, we showed that in chronic myeloproliferative neoplasia (CMN), β1 and β2 integrins are overactivated on leukocytes in patients and in a JAK2-V617F knock-in mouse model (Gupta et al., Leukemia, 2017; Edelmann-Stephan et al., JCI, In-Press Preview) resulting in increased adhesion of leukocytes to vascular cell adhesion molecule 1 (VCAM-1) and to intercellular cell adhesion molecule 1 (ICAM-1). VCAM-1 and ICAM-1 are abundantly expressed on endothelial cells and in spleen tissue. In JAK2-V617F induced CMN, current therapeutic strategies for symptomatic splenomegaly as hydroxyurea, JAK inhibitors or allogeneic stem cell transplantation (SCT) are effective, however, often limited in duration of efficacy or in eligibility for SCT. Therefore, there is a need to deeper understand the pathophysiology of splenomegaly in CMN in order to develop novel therapeutic approaches. We sought to investigate the effects of in vivo blockade of β1 and β2 integrin overactivation on spleen size, cellular composition of spleen and peripheral blood counts.Material and Methods:11 weeks old Vav1-Cre x JAK2+/V617F mice were treated with a combination of 200µg neutralizing anti-α4β1-integrin (VLA4) antibody (BioXCell, USA) plus 200µg neutralizing anti-β2 integrin antibody (BD Pharmingen, USA) (N=6) and with isotype controls (each 200µg) (N=3), respectively. Animals were injected i.p. on day 1 and day 8. Small animal MRI was used to serially assess the development of spleen volume on days 1, 8, and 15. Abdominal MRI-images employing a slice thickness of 1-2 mm were obtained. The volume of spleens were computed according to radiology standards. Peripheral blood was collected via retro-orbital bleeding and blood counts were measured on days 1, 8, and 15. Thereafter, mice were sacrificed by cervical dislocation and spleens were removed for further analysis by FACS.Results and Discussion:The applied antibody injections were well tolerated and all animals survived the planned observation period. At the start of the experiments (day 1), splenomegaly was confirmed by MRI. As expected, the spleen volumes were similar in the cohorts of isotype and β1/β2 integrin antibody treated JAK2+/V617F mice: 550±76 mm3 (mean±SD) and 518±96 mm3, respectively (Fig. 1). At day 8, upon a single injection of β1/β2 integrin antibodies, a significant decrease in mean spleen volume from baseline 518±96 mm3 to 414±32 mm3 (p=0.0302) was observed (Fig. 1). The correspondent mean±SD for the isotype antibody treated animals was 594±63 mm3. Thus, on day 8, a 30.3% reduction in spleen volume was demonstrated upon β1/β2 integrin antibody treatment in comparison to isotype treatment (p=0.0006). On day 15, the mean spleen volumes of isotype-treated and β1/β2 integrin antibody-treated mice were 665±187 mm3 and 460±69 mm3 (p=0.0410). These results demonstrate that in JAK2-V617F induced disease, blocking of overactivated β1/β2 integrins reduces splenomegaly within a short period of time (8 days). Longer application schedules may build up even stronger and stable decrease in spleen size.In addition to MRI imaging, we also analyzed the composition of peripheral blood cells. At the start of the experiments on day 1, blood parameters showed the typical abnormalities of myeloproliferative disease in both cohorts. Mean counts in isotype treated animals were: WBC = 14,300/µL, hematocrit = 80.3 %, PLT = 1283/nL. Mean counts in β1/β2 integrin antibody treated animals were: WBC = 13,850/µL, hematocrit = 76.0 %, PLT = 1348/nL. This confirms that the JAK2-V617F mouse model used indeed resembles polycythemia vera. In general, the changes observed upon β1/β2 integrin antibody treatment in blood were subtle: the WBC slightly decreased from 13,850/µL to 12,520/µL on day 15, whereas RBC, HCT and PLT remained nearly unchanged. This indicates tolerability of β1/β2 integrin antibody treatment at the level of the hematopoietic system. In isotype treated mice, peripheral blood counts also stayed stable. Data obtained by FACS analysis of peripheral blood and of spleen tissue (composition in granulocytes, B-cells and T-cells) will be presented.Overall, our results indicate a novel role of JAK2-V617F induced overactivation of β1/β2 integrins in development of splenomegaly. Thus, modulation of integrin activity may have therapeutic potential to reduce the spleen volume in CMN patients insufficiently responding to standard treatment. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Role of NRAS G12D Mutations in the Response to Conventional Chemotherapy and 5-Azacitidine in Secondary AML

Secondary Acute Myeloid Leukemia (sAML) accounts for 10-30% of all AML. It arises from a preexisting clonal disorder of hematopoiesis, such as myelodysplastic syndromes (MDS) or chronic myeloproliferative neoplasia (cMPN) in most cases (60-70%) or from exposure to a leukemogenic agent e.g. chemotherapy. sAML is generally considered to be of unfavorable prognosis, as treatment sensitivity is reduced, compared to de novo AML (dnAML) and overall survival is shortened. The incidence of AML associated NRAS are similar between sAML and dnAML (10 to 15%, Jelena D. Milosevic et al.). Prognostic impact of such mutations have been controversially discussed, but have been linked to favorable response to high dose cytarabine treatment in dnAML patients (Andreas Neubauer et al.), thus providing the first example of an oncogenic mutation impacting drug response in dnAML. This effect, however, has not yet been shown in sAML, therefore the aim of this work is to study the role of mutated NRAS in the response to chemotherapy and the hypomethylating agent (HMA) 5-azacitidine in sAML.We utilized two sAML cell lines SET-2 and HEL (both NRAS wildtype) in which we stably introduced the NRAS WT and the known activating hotspot mutation NRAS G12D using the sleeping Beauty technology. The dose-response assays of conventional chemotherapy and 5-azacitidine were carried out in the parental cell lines (SET-2/HEL) compared to NRAS WT (SET-2 NRAS WT/HEL NRAS WT) and NRAS G12D (SET-2 NRAS G12D/HEL NRAS G12D). In contrast to our expectations, both NRAS G12D mutation harboring cell lines, SET-2 and HEL developed resistance to cytarabine, idarubicin and 5-azacytidine, whereas the ones with wildtype NRAS remained susceptible to the drugs. To reverse the resistance we tested the MEK inhibitors Binimetinib and Trametinib in our SET-2 NRAS G12D cell line model according to recent reports about preclinical efficacy of MEK inhibition in NRAS mutant dnAML cells (Michael R. Burgess et al.). And in fact, single agent Binetimib and Trametinib treatments reduced cell viability by 20% at 48 hours. Strikingly, in combination with 5-azacitidine, Binimetinib and Trametinib treatments led to a viability reduction by 90%. Next we induced necroptosis in our NRAS mutant cell line models through the combination of Birinapant (SMAC mimetics) and Emricasan (Inhibitor of Caspase 8), as recently described by Brumatti et al. and were, in addition, able to reduce the cell viability by 60 %.In summary, we provide first evidence, that in contrast to dnAML, activating NRAS mutations may promote resistance to conventional chemotherapy and 5-azacitidine in sAML cell lines. Furthermore we were able to demonstrate, that the combination of MEK inhibitors (Binimetinib and Trametinib) and 5-azacitidine as well as the induction of necroptosis such as the combination birinapant and emricasan, may provide a potential strategy to overcome the resistance. DisclosuresHaferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

High Mobility Group A1 Chromatin Remodeling Proteins Amplify Inflammatory Networks to Drive Leukemic Transformation in Chronic Myeloproliferative Neoplasia in Humans and JAK2V617F Transgenic Mouse Models

Introduction: Myeloproliferative neoplasms (MPN) are clonal hematopoietic stem cell (HSC) disorders characterized by overproduction of mature blood cells and increased risk of transformation to myelofibrosis (MF) and acute myeloid leukemia (AML), although molecular mechanisms driving disease progression remain elusive. While most patients who acquire a JAK2V617F mutation in CD34+ cells present with chronic, indolent Polycythemia Vera (PV), ~25% will progress to MF or AML. High Mobility Group A1/2 (HMGA1/2) genes encode oncogenic chromatin remodeling proteins which are overexpressed in aggressive leukemia where they portend adverse outcomes. In murine models, Hmga1/2 overexpression drives clonal expansion and uncontrolled proliferation. HMGA1/2 genes are also overexpressed in MPN with disease progression. We therefore sought to: 1) test the hypothesis that HMGA proteins are required for leukemic transformation and rational therapeutic targets in MPN progression, and, 2) identify mechanisms mediated by HMGA1/2 during disease progression.Methods: We measured HMGA1/2 in JAK2V617F mutant human AML cell lines from MPN patients (DAMI, SET-2), CD34+ cells from PV patients during chronic and transformation phases, and JAK2V617F transgenic murine models of PV (transgenic JAK2V617F) and PV-AML (transgenic JAK2V617F/MPLSV; Blood 2015;126:484). To elucidate HMGA1/2 function, we silenced HMGA1 or HMGA2 via short hairpin RNA in human MPN-AML cell lines (DAMI, SET-2) and assessed proliferation, colony formation, and leukemic engraftment in immunodeficient mice. To further assess Hmga1 function in vivo, we crossed mice with heterozygous Hmga1 deficiency onto murine models of PV and PV-AML. Finally, to dissect molecular mechanisms underlying HMGA1, we compared RNA-Seq from MPN-AML cell lines (DAMI, SET-2) after silencing HMGA1/2 to that of controls and applied Ingenuity Pathway Analysis.Results:HMGA1/2 mRNA are up-regulated in all JAK2V617F-positive contexts, including primary human PV CD34+ cells and total bone marrow from JAK2V617F mouse models for PV compared to controls. Further, there is a marked up-regulation in both HMGA1/2 in CD34+ cells from PV patients after transformation to MF or AML and in leukemic blasts from our PV-AML mouse model compared to PV mice. Overexpression of HMGA1/2 also correlates with clonal dominance of human JAK2V617F-homozygous stem cells and additional mutations of epigenetic regulators (EZH2, SETBP1). Silencing HMGA1 or HMGA2 in human MPN-AML cell lines (DAMI, SET-2) dramatically halts proliferation, disrupts clonogenicity, and prevents leukemic engraftment in mice. Further, heterozygous Hmga1 deficiency decreases splenic enlargement in PV mouse models with advancing age. Moreover, heterozygous Hmga1 deficiency prolongs survival in the transgenic PV-AML murine model with fulminant leukemia and early mortality. PV-AML mice survived a median of 5 weeks whereas PV-AML mice with heterozygous Hmga1 deficiency survive a median of 12 weeks (P< 0.002). The leukemic burden was also decreased in mice with Hmga1 deficiency. Preliminary RNA-Seq analyses from DAMI and SET-2 cells show that HMGA1 drives pathways involved in Th1/Th2 activation, chemotaxis, cell-cell signaling, myeloid cell accumulation and other immune cell trafficking, inflammation, and injury, suggesting that HMGA1 co-opts immune and inflammatory networks to drive tumor progression. Surprisingly, atherosclerosis pathways are also induced by HMGA1.Conclusions:HMGA1/2 genes are overexpressed in MPN with highest levels in more advanced disease (MF, AML) both in primary human tumors and murine models. Strikingly, silencing HMGA1 or HMGA2 halts proliferation and clonogenicity in vitro and prevents leukemic engraftment in vivo. Further, heterozygous Hmga1 deficiency prolongs survival in a murine model of fulminant MPN AML and decreases tumor burdens. Finally, preliminary RNA-Seq analyses suggest that HMGA1 amplifies transcriptional networks involved in immune cell trafficking and inflammation to drive tumor progression. Unexpectedly, HMGA1 also regulates pathways involved in atherosclerosis, implicating HMGA1 as a novel link between clonal hematopoiesis and cardiovascular disease. Our findings further highlight HMGA1/2 as a key molecular switch for leukemic transformation in MPN and opens the door to novel therapeutic approaches to prevent disease progression. DisclosuresNo relevant conflicts of interest to declare.

JAK2-V617F promotes venous thrombosis through β1/β2 integrin activation.

JAK2-V617F-positive chronic myeloproliferative neoplasia (CMN) commonly displays dysfunction of integrins and adhesion molecules expressed on platelets, erythrocytes, and leukocytes. However, the mechanism by which the 2 major leukocyte integrin chains, β1 and β2, may contribute to CMN pathophysiology remained unclear. β1 (α4β1; VLA-4) and β2 (αLβ2; LFA-1) integrins are essential regulators for attachment of leukocytes to endothelial cells. We here showed enhanced adhesion of granulocytes from mice with JAK2-V617F knockin (JAK2+/VF mice) to vascular cell adhesion molecule 1- (VCAM1-) and intercellular adhesion molecule 1-coated (ICAM1-coated) surfaces. Soluble VCAM1 and ICAM1 ligand binding assays revealed increased affinity of β1 and β2 integrins for their respective ligands. For β1 integrins, this correlated with a structural change from the low- to the high-affinity conformation induced by JAK2-V617F. JAK2-V617F triggered constitutive activation of the integrin inside-out signaling molecule Rap1, resulting in translocation toward the cell membrane. Employing a venous thrombosis model, we demonstrated that neutralizing anti-VLA-4 and anti-β2 integrin antibodies suppress pathologic thrombosis as observed in JAK2+/VF mice. In addition, aberrant homing of JAK2+/VF leukocytes to the spleen was inhibited by neutralizing anti-β2 antibodies and by pharmacologic inhibition of Rap1. Thus, our findings identified cross-talk between JAK2-V617F and integrin activation promoting pathologic thrombosis and abnormal trafficking of leukocytes to the spleen.

Thrombin generation - a potentially useful biomarker of thrombotic risk in Philadelphia-negative myeloproliferative neoplasms.

The diagnosis of essential thrombocythemia and polycythemia vera is often made during a thrombotic event which can be serious. Philadelphia-negative chronic myeloproliferative neoplasia patients have an increased thrombotic risk. This is assessed using various scoring systems but these are far from ideal and individual risk. The currend trend to personalised medicine requires finding the most useful thrombotic risk biomarker in these patients. Routine tests for coagulation do not take account of both pro- and anti-coagulant factors which is why these tests are not useful in patients with Philadelphia-negative myeloproliferative neoplasms. Thrombin generation reflects more accurately the balance between pro- and anti-coagulant factors. Some parameters of thrombin generation such as the endogenous thrombin potential are higher in Philadelphia-negative myeloproliferative neoplasm patients, especially in JAK2 V617F carriers than in healthy controls. They are even higher in those with reactive thrombocytosis. The JAK2 V617F allele burden correlates more with a higher thrombin generation potential in patients who are not treated with hydroxycarbamidum. Instead, JAK2 V617F-positive patients with Philadelphia-negative myeloproliferative neoplasms were the most sensitive to hydroxycarbamidum, as was reflected in lower values of platelet thrombin generation potential. The use of thrombin generation examination in these patients would enable detection of imminent thrombosis and personalised prophylactic management.

Thromboembolic Events in Polycythaemia Vera Patients:An Audit of the Hungarian Philadelphia Negative Chronic Myeloproliferative Neoplasia Register

Objective: The Hungarian National Registry for Philadelphia chromosome negative myeloproliferative neoplasms was used to assess the clinical characteristics of Hungarian patients with polycythemia vera. Methods: Data from 351 JAK2 V617F-positive patients diagnosed with PV were collected online from 15 haematology centres reporting clinical characteristics, therapeutic interventions, venous and arterial thromboembolic events, and myelofibrotic or leukaemic transformations. Vascular events (thromboembolic and haemorrhagic) were evaluated before and after diagnosis based upon the Landolfi risk assessment scale. Results: TE were reported on 116 occasions (106 patients) before diagnosis and 152 occasions (102 cases) during follow-up. Compared to before diagnosis, after diagnosis frequency of major arterial events decreased from 11.7% to 2.6% (p<0.0001), and minor venous events increased from 2.0% to 14.2% (p<0.0001); there was no significant change in number of major venous events (from 6.3% to 8.8%; p=0.25) or minor arterial events (from 13.1% to 17.7%; p=0.12). Bleeding events were recorded in 6.4% of patients. Despite treatment, 42.2% of patients with prior thromboembolic events had recurrent thromboembolic complications. After diagnosis age and prior history of thromboembolic events were independent risk factors for arterial events, and white blood cells and diabetes for venous events. Hydroxyurea use in the low+moderate risk Landolfi group slightly, but not significantly, increased thromboembolic event risk (p=0.74). Conclusions: This registry enables characterisation of patients with polycythemia vera. Data suggest the need for accuracy of diagnostic criteria and compliance with risk-adapted therapeutic guidelines.

Diagnosis of del(5q) MDS, 14 years after JAK-2 positive PV appearance: complete remission of both diseases with lenalidomide monotherapy

This is the report of the clinical case of a patient who presents the association of a JAK-2 positive chronic myeloproliferative neoplasia to a subsequent 5q- myelodysplastic syndrome, developed after about 14 years from the first diagnosis. Patient’s symptoms had rapidly worsened, and she became transfusion-dependent. Therapy with low-dose Lenalidomide quickly reduced the splenomegaly, and completely brought white cells counts, haemoglobin and platelets back to normal. After more than one year from start, blood cell count is still normal. As far as we know this is the first case of an effective treatment with Lenalidomide reported in this clinical setting.

Treatment outcome of patients with essential thrombocythemia at our department in Hungary

Essential thrombocythemia is a Philadelphia chromosome-negative chronic myeloproliferative neoplasia with a risk of bleeding and thromboembolic complications during the course of illness. Cytoreductive drugs, such as non-selective hydroxyurea or interferon as first-line and specific, megakaryocyte-thrombocyte reductive anagrelide chosen as second-line treatment in cases of adverse, intolerable effects of hydroxyurea can lower the incidence of bleeding/thrombotic episodes in patients with essential thrombocythemia. In this observational survey the effect of anagrelide was investigated in patients with essential thrombocythemia, who were first treated with hydroxyurea but failed to have clinicopathologic reponse (resistant) or were intolerant (adverse effects). Between 2000 and 2014, 104 patients were diagnosed with essential thrombocythemia and treated first-line with hydroxyurea (weekly median dose of 7500 mg) in the haematologic outpatient department of the authors. Because of intolerance and/or resistance, hydroxyurea was changed to anagrelide (7.5 mg weekly median dose), the doses of hydroxyurea and anagrelide were adjusted to achieve clinicopathological response according to the updated criteria of the European LeukemiaNET. Effect of anagrelide as monotherapy (first- or second-line after hydroxyurea) or in combination with hydroxyurea was followed. Statistical analysis was performed using the Windows Statistical Package Program. Of the 104 patients with essential thrombocythemia (according to the updated WHO-ET classifications 58 patients JAK2V617F mutation positive, 46 patients negative, 15 patients calreticulin mutation negative, 6 patients MPL-1 mutation negative) 87 patients received hydroxyurea in first line, 4 patients interferon, and 13 patients acetylsalycilic acid only. Seven patients who proved to be intolerant and 22 patients who were resistant to hydroxyurea received anagrelide in second line (in 18 patients monotherapy and in 11 patients in combination with hydroxyurea), while other 5 rather young patients in first line therapy (34/104, 32.6%). In the anagrelide first line group 5 patients (100%), in the second line anagrelide monotherapy group 16 patients (88,8%), and in the combined hydroxyurea plus anagrelide group 9 patients (82.1%) achieved complete remission. The 10-year overall survival was 82.1%. In 2 patients treated with anagrelide major bleeding and in one patient myocardial infarction occurred, other serious adverse events due to anagrelide treatment were not detected. Three elder patients died from non-hematologic diseases, but leukaemic transformation was not observed. First or second line anagrelide therapy, combined with hydroxyurea if necessary, was able to reduce the platelet-count and the rate of complications, and to control the course of essential thrombocythemia with tolerable adverse effects.

Impact of Molecular Genetics on Disease-Free Survival in Myelofibrosis Patients Following Allogeneic Stem Cell Transplantation

IntroductionPrimary or post-ET/post-PV myelofibrosis is one of the Philadelphia chromosome-negative chronic myeloproliferative neoplasia characterized by significantly reduced overall survival. More recently several mutated genes have been detected, which allow, in addition to clinical factors, to identify patients with a significantly shorter overall survival and a higher risk of transformation into acute leukemia.Allogeneic stem cell transplantation is still the only curative treatment option for patients with myelofibrosis, and due to the inherent risk of the treatment procedure careful selection of the patients is required. Molecular genetics may help to select patients for allogeneic stem cell transplantation.Patients and methodsTo determine the impact of mutated genes in patients with myelofibrosis who underwent allogeneic stem cell transplantation we analyzed samples from 169 patients with a median age of 58 years (r: 18 - 75) who received allogeneic stem cell transplantation either from related (n = 36) or unrelated (n = 133) donor. Stem cell source were more often peripheral blood stem cells (n = 165) than bone marrow (n = 4). The intensity of conditioning was mainly reduced intensity (n = 166), rather than myeloablative conditioning (n = 3). Patients suffered from primary myelofibrosis (n = 110), post-ET/PV myelofibrosis (n = 46), while 13 patients were in acceleration or had transformed into acute myeloid leukemia. According to dynamic IPSS (DIPSS) (n = 165) the patients were either low (n = 7), intermediate-1 (n = 35), intermediat-2 (n = 91), or high risk (n = 32). Regarding molecular genetics we found JAK2V617F mutations in 62%, calreticulin (CALR) mutations in 20%, MPL mutations in 4%, U2AF1 in 7%, SRSF2 in 10%, SF3B1 in 4%, ASXL1 in 29%, IDH1 in 2%, IDH2 in 3%, CBL in 1%, DNMT3A in 4%, TET2 in 10%, EZH2 in 4%, while none of the patients showed mutations in ETV6 and PTPN11. Overall, only in 11 patients no mutation could be detected. One mutation could be detected in 41%, 2 mutations in 30%, 3 mutations in 11%, 4 mutations in 5%.ResultsDuring follow-up 39 patients experienced relapse and 46 patients experienced non-relapse mortality.From the non-molecular factors regarding disease-free survival in univariate analysis age < 58 (p < 0.01), intermediate-1 and low risk according to DIPPS (p = 0.002), HLA-matched vs. mismatched (p = 0.04) were significant factors for improved disease-free survival. Regarding molecular markers improved disease-free survival was seen for patients with mutations in CALR (p = 0.005), while negative impact on disease-free survival was seen for mutations in U2AF1 (p = 0.035), ASXL1 (p = 0.05), IDH2 (p = 0.006), DNMT3A (p = 0.029). No significant difference could be seen for patients with EZH2, IDH1, SRSF2, and SF3B1 mutations. There was no difference in disease-free survival for patients without any mutation vs. 1, and more than 1 mutation (p = 0.12). Regarding the previously described unfavorable mutations ASXL1, SRSF2, EZH2, IDH1, and IDH2, we found 40 patients who had at least 1 of these unfavorable mutations, 11 had 2 of these mutation, and 1 had 3 of these unfavorable mutations. However, the estimated 5-year disease-free survival did not differ significantly between patients without any of these unfavorable mutations, with 1 or with 2 of them (47 vs. 40 vs. 41%, p = 0.5).ConclusionsThese results suggest that some molecular marker such as ASXL1, U2AF1, IDH2 and DNMT3A negatively influence DFS in myelofibrosis after allogeneic stem cell transplantation in a univariate analysis. In contrast, the poor prognosis of the recently described unfavorable mutated genes SRSF2, EZH2, and IDH1 was not observed and may therefore be overcome by allogeneic stem cell transplantation. DisclosuresNo relevant conflicts of interest to declare.

Epidemiology and Clinical Significance of Secondary and Therapy-Related Acute Myeloid Leukemia: A National Population-Based Cohort Study.

Secondary and therapy-related acute myeloid leukemia (sAML and tAML, respectively) remain therapeutic challenges. Still, it is unclear whether their inferior outcome compared with de novo acute myeloid leukemia (AML) varies as a result of previous hematologic disease or can be explained by differences in karyotype and/or age. In a Danish national population-based study of 3,055 unselected patients with AML diagnosed from 2000 to 2013, we compared the frequencies and characteristics of tAML, myelodysplastic syndrome (MDS) -sAML, and non-MDS-sAML (chronic myelomonocytic leukemia and myeloproliferative neoplasia) versus de novo AML. Limited to intensive therapy patients, we compared chance of complete remission by logistic regression analysis and used a pseudo-value approach to compare relative risk (RR) of death at 90 days, 1 year, and 3 years, overall and stratified by age and karyotype. Results were given crude and adjusted with 95% CIs. Overall, frequencies of sAML and tAML were 19.8% and 6.6%, respectively. sAML, but not tAML, was associated with low likelihood of receiving intensive treatment. Among intensive therapy patients (n = 1,567), antecedent myeloid disorder or prior cytotoxic exposure was associated with decreased complete remission rates and inferior survival (3-year adjusted RR for MDS-sAML, non-MDS-sAML, and tAML: RR, 1.14; 95% CI, 1.02 to 1.32; RR, 1.27; 95% CI, 1.16 to 1.34; and RR, 1.16; 95% CI, 1.03 to 1.32, respectively) compared with de novo AML. Among patients ≥ 60 years old and patients with adverse karyotype, previous MDS or tAML did not impact overall outcomes, whereas non-MDS-sAML was associated with inferior survival across age and cytogenetic risk groups (adverse risk cytogenetics: 1-year adjusted RR, 1.47; 95% CI, 1.23 to 1.76; patients ≥ 60 years old: 1-year adjusted RR, 1.31; 95% CI, 1.06 to 1.61). Our results support that de novo AML, sAML, and tAML are biologically and prognostically distinct subtypes of AML. Patients with non-MDS-sAML have dismal outcomes, independent of age and cytogenetics. Previous myeloid disorder, age, and cytogenetics are crucial determinants of outcomes and should be integrated in treatment recommendations for these patients.

To the problem of thrombus-hemorrhagic complications in chronic myeloproliferative neoplasias. Complicated diagnosis. Clinical case conference.

Disturbances in the system of he­mostasis and vascular complications is the topical clinical problem, requiring profound study with the development of treatment and prophylaxis algorithm. Thrombus-hemorrhagic complications in patients with chronic myeloproliferative (CMPN) neoplasias are caused by the presence of plethoric and myeloproliferative syndromes, findings of hemogram (level of erythrocytes, thrombocytes, leukocytes, hematocrit index) and hemostasiogram testify to this. A case of thrombus-hemorrhagic complication in a female patient with chronic myeloproliferative neoplasia was considered. Carrying out differential diagnostics requires performing additional investigations: USI, CT with defining of hemostasiogram, hemogram findings, their thorough monitoring when surgical intervention is needed. This diagnosis for the first time was made in conditions of surgical in – patient unit. In pre- and postoperative periods as well as for prevention of vascular complicatients in the patient with CMPN it is necessary to mandarory administer agents which correct hematologic status and rheologic indices. This clinical case was analyzed with the aim to draw doctors’ aftention to complexities in diagnostics and defining treatment tactics for patients with thrombus-hemorrhagic complications: necessity to establish all possible risk factors of vascular events development and to carry out constant hemostasiologic menitoring for preventing lethal outcomes.

The Evaluation of Conditions of JAK2V617F Mutation and Thromboembolism with Chronic Myeloproliferative Neoplasia Patients;Single Center Experience

The Evaluation of Conditions of JAK2V617F Mutation and Thromboembolism with Chronic Myeloproliferative Neoplasia Patients;Single Center Experience