A novel germline hyperactivating JAK2 mutation L604F.

Somatic JAK2 mutations are the main molecular cause of the vast majority of polycythemia vera (PV) cases. According to a recent structural model, the prevalent acquired V617F mutation improves the stability of the JAK2 dimer, thereby enhancing the constitutive JAK2 kinase activity. Germline JAK2 mutations usually do not largely alter JAK2 signaling, although they may modulate the impact of V617F. We found an unusual germline JAK2 mutation L604F in homozygous form in a young PV patient, along with a low allele burden JAK2 V617F mutation, and in her apparently healthy sister. Their father with a PV-like disease had L604F in a heterozygous state, without V617F. The functional consequences of JAK2 L604Fmutation were compared with those induced by V617F in two different in vitro model systems: (i) HEK293T cells were transfected with plasmids for exogenous JAK2-GFP expression, and (ii) endogenous JAK2 modifications were introduced into HeLa cells using CRISPR/Cas9. Both mutations significantly increased JAK2 constitutive activity in transfected HEK293T cells. In the second model, JAK2 modification resulted in reduced total JAK2 protein levels. An important difference was also detected: as described previously, the effect of V617F on JAK2 kinase activity was abrogated in the absence of the aromatic residue F595. In contrast, JAK2 hyperactivation by L604F was only partially inhibited by the F595 change to alanine. We propose that the L604F mutation increases the probability of spontaneous JAK2 dimer formation, which is physiologically mediated by F595. In addition, L604F may contribute to dimer stabilization similarly to V617F.

Synthesis and optical properties of N→Ga coordinated gallium boroxines.

Reactions of the N,C,N-chelated organogallium amide LGa(NEt2)2 (1), where L is {2,6-(Me2NCH2)2C6H3}-, with organoboronic acids RB(OH)2 yielded molecular gallium boroxines LGa(O3B2R2) (2: R = OH, 3: R = Ph, 4: R = 4-MeO-C6H4, 5: R = 4-CHO-C6H4, 6: R = Fc), neutral analogues of gallaborates. The molecular structures revealed the presence of a six-membered central GaB2O3 ring. The film forming properties of 5 allowed the deposition of transparent thin films by a spin coating method. The thicknesses, refractive index, energy of the optical gap (Eoptg), activation energy of surface electrical conductivity (Esa) and pre-exponential factor (σ0) of the thin layers of 5 were measured and they are close to those found for related oxygen glass. Finally, GBO 5 was also used as an additive to printing ink and a thin film of 5 was prepared by the gravure printing technique.

Ph- myeloproliferative neoplasms and the related risk factors for stroke occurrence: Results from a registry of patients treated with Anagrelide.

Arterial thrombosis is a common complication in patients with Ph- myeloproliferative neoplasms (MPN). We searched for the risk factors of stroke in MPN patients from anagrelide registry. We analyzed the potential risk factors triggering a stroke/TIA event in 249 MPN patients with previous stroke (n = 168) or Transient Ischemic Attack (TIA) (n = 140), and in 1,193 MPN control subjects (without clinical history of thrombosis). These patients were registered in a prospective manner, providing a follow-up period after Anagrelide treatment. The median age of the patients in the experimental group was of 56 years of age (ranging from 34-76) and of 53 years of age (ranging from 26-74) in the control group (p < 0.001). Using a multivariate model, we determined the following as risk factors: JAK2V617F mutation (OR 2.106, 1.458-3.043, p = 0.006), age (OR 1.017/year, 1.005-1,029, p = 0.006), male gender (OR 1.419, 1.057-1.903, p = 0.020), MPN diagnosis (OR for PMF 0.649, 0.446-0.944, p = 0.024), BMI (OR 0.687 for BMI > 25, 0.473-0.999, p = 0.05) and high TAG levels (OR 1.734, 1.162-2.586, p = 0.008), all of which were statistically significant for CMP development. Concerning the risk factors for thrombophilia, only the antiphospholipid syndrome (OR 1.994, 1.017-3.91, p = 0.048) was noteworthy in a stroke-relevant context. There was no significant difference between the blood count of the patients prior to a stroke event and the control group, both of which were under a cytoreductive treatment. We found that age, male gender, JAK2V617F mutation, previous venous thrombosis, and hypertriglyceridemia represent independent risk factors for the occurrence of a stroke in Ph- MPN patients.

International external quality assurance of JAK2 V617F quantification.

External quality assurance (EQA) programs are vital to ensure high quality and standardized results in molecular diagnostics. It is important that EQA for quantitative analysis takes into account the variation in methodology. Results cannot be expected to be more accurate than limits of the technology used, and it is essential to recognize factors causing substantial outlier results. The present study aimed to identify parameters of specific importance for JAK2 V617F quantification by quantitative PCR, using different starting materials, assays, and technical platforms. Sixteen samples were issued to participating laboratories in two EQA rounds. In the first round, 19 laboratories from 11 European countries analyzing JAK2 V617F as part of their routine diagnostics returned results from in-house assays. In the second round, 25 laboratories from 17 countries participated. Despite variations in starting material, assay set-up and instrumentation the laboratories were generally well aligned in the EQA program. However, EQA based on a single technology appears to be a valuable tool to achieve standardization of the quantification of JAK2 V617F allelic burden.

Recommendations for the diagnosis and treatment of patients with polycythaemia vera.

OBJECTIVES: To present the Central European Myeloproliferative Neoplasm Organisation (CEMPO) treatment recommendations for polycythaemia vera (PV). METHODS: During meetings held from 2015 through 2017, CEMPO discussed PV and its treatment and recent data. RESULTS: PV is associated with increased risks of thrombosis/thrombo-haemorrhagic complications, fibrotic progression and leukaemic transformation. Presence of Janus kinase (JAK)-2 gene mutations is a diagnostic marker and standard diagnostic criterion. World Health Organization 2016 diagnostic criteria for PV, focusing on haemoglobin levels and bone marrow morphology, are mandatory. PV therapy aims at managing long-term risks of vascular complications and progression towards transformation to acute myeloid leukaemia and myelodysplastic syndrome. Risk stratification for thrombotic complications guides therapeutic decisions. Low-risk patients are treated first line with low-dose aspirin and phlebotomy. Cytoreduction is considered for low-risk (phlebotomy intolerance, severe/progressive symptoms, cardiovascular risk factors) and high-risk patients. Hydroxyurea is suspected of leukaemogenic potential. IFN-α has demonstrated efficacy in many clinical trials; its pegylated form is best tolerated, enabling less frequent administration than standard interferon. Ropeginterferon alfa-2b has been shown to be more efficacious than hydroxyurea. JAK1/JAK2 inhibitor ruxolitinib is approved for hydroxyurea resistant/intolerant patients. CONCLUSIONS: Greater understanding of PV is serving as a platform for new therapy development and treatment response predictors.

Analysis of Real-world Data on Postremission Therapy for Acute Myeloid Leukemia With Intermediate Risk Cytogenetics in First Complete Remission.

BACKGROUND: We retrospectively analyzed data from 310 patients with acute myeloid leukemia with intermediate-risk cytogenetics in first complete remission (CR1) to evaluate the usage and efficacy of various types of postremission therapy. PATIENTS AND METHODS: Cox regression with time-dependent covariates, landmark analysis, and competing risk models were used to estimate the outcomes and effects of treatment and patient- and disease-related risk factors. RESULTS: The early relapse rate and early nonrelapse mortality (NRM) were 12.8% and 4.4%, respectively. In our study, 77.2% of patients completed postremission therapy: 44% received allogeneic hematopoietic cell transplantation (HCT), 20% completed treatment with high-dose cytarabine (HIDAC), and 13% completed treatment with intermediate-dose cytarabine. The 3-year overall survival rate was 67.5% for patients treated with HIDAC and 63.4% after HCT (P = .5876). The NRM and relapse rate at 3 years were 0% and 58.9% after HIDAC and 21.9% and 29.3% after HCT, respectively. HCT reduced the risk of relapse (hazard ratio, 0.6; 95% confidence interval, 0.36-0.98). Total body irradiation-based myeloablative conditioning increased NRM compared with busulfan-based conditioning (hazard ratio, 8.33; 95% confidence interval, 2.52-27.45). CONCLUSION: Most patients with acute myeloid leukemia with intermediate-risk cytogenetics received allogeneic HCT, which decreased the risk of relapse but increased NRM, leading to a similar overall survival for patients who received HCT and HIDAC. Our data support the use of allogeneic transplantation for patients in CR1 from a human leukocyte antigen-matched related or unrelated donor after a busulfan-based myeloablative conditioning regimen as a primary strategy of postremission therapy for eligible younger patients.

Antiplatelet therapy versus observation in low-risk essential thrombocythemia with a CALR mutation.

The role of antiplatelet therapy as primary prophylaxis of thrombosis in low-risk essential thrombocythemia has not been studied in randomized clinical trials. We assessed the benefit/risk of low-dose aspirin in 433 patients with low-risk essential thrombocythemia (271 with a CALR mutation, 162 with a JAK2(V617F) mutation) who were on antiplatelet therapy or observation only. After a follow up of 2215 person-years free from cytoreduction, 25 thrombotic and 17 bleeding episodes were recorded. In CALR-mutated patients, antiplatelet therapy did not affect the risk of thrombosis but was associated with a higher incidence of bleeding (12.9 versus 1.8 episodes per 1000 patient-years, P=0.03). In JAK2(V617F)-mutated patients, low-dose aspirin was associated with a reduced incidence of venous thrombosis with no effect on the risk of bleeding. Coexistence of JAK2(V617F)-mutation and cardiovascular risk factors increased the risk of thrombosis, even after adjusting for treatment with low-dose aspirin (incidence rate ratio: 9.8; 95% confidence interval: 2.3-42.3; P=0.02). Time free from cytoreduction was significantly shorter in CALR-mutated patients with essential thrombocythemia than in JAK2(V617F)-mutated ones (median time 5 years and 9.8 years, respectively; P=0.0002) and cytoreduction was usually necessary to control extreme thrombocytosis. In conclusion, in patients with low-risk, CALR-mutated essential thrombocythemia, low-dose aspirin does not reduce the risk of thrombosis and may increase the risk of bleeding.

Thrombosis in thrombocythemic Ph- myeloproliferations is associated with higher platelet count prior to the event: results of analyses of prothrombotic risk factors from a registry of patients treated with anagrelide.

Controversies still exist regarding definition of the thrombotic risks in Ph- (BCR/ABL1-) myeloproliferative disorders with thrombocythemia (MPD-T). Platelet counts at diagnosis are currently not taken as a risk factor of thrombosis. In our cohort of 1179 patients with MPD-T, prospectively registered for anagrelide treatment, we found that the median platelet count prior to the thrombotic event was significantly higher than at time points without any ensuing thrombosis (453 vs. 400 × 10(9)/L, P < 0.001), albeit higher platelet counts at diagnosis tended to be connected with fewer thrombotic events (in contrast to WBC counts at diagnosis). The JAK2(V617F) mutation predicted both arterial and venous events, while age >65 yr, hypertension, diabetes mellitus, smoking, elevated triglyceride and homocysteine levels predicted arterial events only. For venous events, the specific thrombophilic risk factors (factor V 'Leiden' and others), antiphospholipid antibodies, and elevated factor VIII levels played a major role. During anagrelide treatment (± aspirin), we documented a decrease in both venous (6.7-fold) and arterial events (1.8-fold), while bleeding (mostly minor events) increased twofold compared to history. Our results suggest that keeping platelet counts at low levels may be a meaningful therapeutic measure to prevent thrombosis, although their counts at diagnosis lack any prognostic value.

Molecular diagnostics of myeloproliferative neoplasms.

Since the discovery of the JAK2 V617F mutation in the majority of the myeloproliferative neoplasms (MPN) of polycythemia vera, essential thrombocythemia and primary myelofibrosis ten years ago, further MPN-specific mutational events, notably in JAK2 exon 12, MPL exon 10 and CALR exon 9 have been identified. These discoveries have been rapidly incorporated into evolving molecular diagnostic algorithms. Whilst many of these mutations appear to have prognostic implications, establishing MPN diagnosis is of immediate clinical importance with selection, implementation and the continual evaluation of the appropriate laboratory methodology to achieve this diagnosis similarly vital. The advantages and limitations of these approaches in identifying and quantitating the common MPN-associated mutations are considered herein with particular regard to their clinical utility. The evolution of molecular diagnostic applications and platforms has occurred in parallel with the discovery of MPN-associated mutations, and it therefore appears likely that emerging technologies such as next-generation sequencing and digital PCR will in the future play an increasing role in the molecular diagnosis of MPN.

[Ph-negative myeloproliferative diseases with thrombocythemia in the context of Thromboreductin® treatment, data from registry 2013]. / Ph-negativní myeloproliferativní onemocnení s trombocytemií ve svetle úcinku lécby prípravkem Thromboreductin® v datech registru ke konci roku 2013.

Czech Working Group for Ph-negative Myeloproliferative diseases (CZEMP) recommends anagrelid (Thromboreductin®) for the treatment of Ph-negative chronic myeloproliferative disease (MPO) with thrombocythemia accompanying. To evaluate the efficacy of this treatment, the patient registry with essential thrombocythemia and/or thrombocytosis accompanying other Ph-negative myeloproliferative diseases was established. The beginnings of data collection go back to 2001, registry itself is maintained from 2005 and the aim is to archive the medical records with detailed physical and laboratory examination, safety patient profile included. The longest follow-up monitors 150 months period. Registry database contained 1,325 patients in the end of 2013, with an annual increase of anagrelid therapy as a drug of first choice in accordance with CZEMP guidelines approved by the Czech Society of Hematology of Czech Medical Association of J. E. Purkyne. Indication criteria contribute to this trend as anagrelid is the first choice agent in 65 years old patients, instead previous 60 years of age. Often, we can observe the combined treatment, especially, in older patients and in patients with primary myelofibrosis and polycythemia vera. There have been founded 543 thrombotic events in 413 patients and 63 bleeding events in 58 patients of study group by the end of 2013. During treatment, thrombosis was diagnosed 225 times in 171 patients and bleeding was observed 139 times in 104 patients. The therapeutic response is achieved after 3 months in 77% and after 6 months in 83% of subjects, but after 12 months, the treatment still fails in 12,5% of patients. It might be caused by slow titration of Thromboreductin®. One of the most important indicators of treatment success is the effect on clinical symptoms presentation, especially the occurrence of thrombotic events. The proof of a good treatment efficacy is demonstrated by 1.8 fold decrease in arterial thrombosis, more than 1.5 fold decrease in microvascular thrombosis and even 6.2 fold decrease in venous thromboembolism events. Bleeding is observed in about double more patients in comparison to the period before inclusion in the systematic monitoring, but the bleedings are clinically insignificant.Key words: anagrelid (Thromboreductin®) - Ph-myeloproliferative diseases - registry - thrombosis.

CBFB-MYH11 hypomethylation signature and PBX3 differential methylation revealed by targeted bisulfite sequencing in patients with acute myeloid leukemia.

BACKGROUND: Studying DNA methylation changes in the context of structural rearrangements and point mutations as well as gene expression changes enables the identification of genes that are important for disease onset and progression in different subtypes of acute myeloid leukemia (AML) patients. The aim of this study was to identify differentially methylated genes with potential impact on AML pathogenesis based on the correlation of methylation and expression data. METHODS: The primary method of studying DNA methylation changes was targeted bisulfite sequencing capturing approximately 84 megabases (Mb) of the genome in 14 diagnostic AML patients and a healthy donors' CD34+ pool. Subsequently, selected DNA methylation changes were confirmed by 454 bisulfite pyrosequencing in a larger cohort of samples. Furthermore, we addressed gene expression by microarray profiling and correlated methylation of regions adjacent to transcription start sites with expression of corresponding genes. RESULTS: Here, we report a novel hypomethylation pattern, specific to CBFB-MYH11 fusion resulting from inv(16) rearrangement that is associated with genes previously described as upregulated in inv(16) AML. We assume that this hypomethylation and corresponding overexpresion occurs in the genes whose function is important in inv(16) leukemogenesis. Further, by comparing all targeted methylation and microarray expression data, PBX3 differential methylation was found to correlate with its gene expression. PBX3 has been recently shown to be a key interaction partner of HOX genes during leukemogenesis and we revealed higher incidence of relapses in PBX3-overexpressing patients. CONCLUSIONS: We discovered new genomic regions with aberrant DNA methylation that are associated with expression of genes involved in leukemogenesis. Our results demonstrate the potential of the targeted approach for DNA methylation studies to reveal new regulatory regions.

[The place of JAK2 inhibitors in the treatment of myelofibrosis. An amendment to the recommendations for diagnosis and treatment of Ph negative myeloproliferations of the Czech group for Ph- myeloproliferative disorders (CZEMP)]. / JAK2 inhibitory v lécbe primární myelofibrózy. Dodatek k doporucením pro diagnostiku a lécbu Ph negativních myeloproliferativních onemocnení Ceské pracovní skupiny pro Ph negativní myeloproliferativní onemocnení Ceské hematologické spolecnosti CLS JEP (CZEMP).

Primary myelofibrosis (PMF) belongs to Ph- myeloproliferative diseases. The only curative treatment is hematopoietic stem cell transplantation (HSCT). Conservative treatment options comprise supportive care, especially administration of red blood cell and platelet transfusions, and medication. Hydroxyurea, interferon α, anagrelide, corticosteroids, androgens, or inhibitors of angiogenesis (thalidomide, lenalidomide, pomalidomide) may be used for treatment of PMF, depending on the clinical stage and disease symptoms present. Also splenectomy or radiotherapy of enlarged spleen have palliative potential. JAK2 kinase inhibitors represent a novel class of drugs with a very dynamic development. Ruxolitinib, an oral selective inhibitor of JAK1 and JAK2 kinases, has shown high efficacy in patients with high-risk PMF (or with myelofibrosis following polycythemia vera or essential thrombocythemia) to ameliorate disease symptoms and to reduce splenomegaly in randomized trials COMFORT-I and COMFORT-II. Long-term monitoring of the enrolled patients demonstated prolongation of overall survival. The drug is well-tolerated, the most common side effects of treatment with ruxolitinib being deepening of thrombocytopenia and temporary worsening of anemia. The current review deals with the place of JAK2 inhibitors (and the only drug already approved for clinical use - ruxolitinib) in the management of PMF, as an addendum to the Summary of recommendations for the diagnosis and therapy of BCR/ABL-negative myeloproliferations of the Czech Hematological Societys CZEMP.

Erythrocytosis in children and adolescents-classification, characterization, and consensus recommendations for the diagnostic approach.

During recent years, the increasing knowledge of genetic and physiological changes in polycythemia vera (PV) and of different types of congenital erythrocytosis has led to fundamental changes in recommendations for the diagnostic approach to patients with erythrocytosis. Although widely accepted for adult patients this approach may not be appropriate with regard to children and adolescents affected by erythrocytosis. The "congenital erythrocytosis" working group established within the framework of the MPN&MPNr-EuroNet (COST action BM0902) addressed this question in a consensus finding process and developed a specific algorithm for the diagnosis of erythrocytosis in childhood and adolescence which is presented here.

Estimation of molecular upper remission limit for monitoring minimal residual disease in peripheral blood of acute myeloid leukemia patients by WT1 expression.

To date, approximately one half of acute myeloid leukaemia (AML) patients do not have a suitable specific molecular marker for monitoring minimal residual disease (MRD). The Wilm's tumour gene (WT1) has been suggested as a possible molecular marker of MRD in AML. The expression of WT1 in peripheral blood (PB) was measured using quantitative real-time reverse transcription-polymerase chain reaction in peripheral leukocytes from 151 patients with AML at diagnosis. WT1 expression was significantly elevated, i.e. up to 3 orders of magnitude in the majority (80%) of AML patients at diagnosis compared to the PB of healthy donors. Sequence samples of the long-term followed-up AML patients treated with chemotherapy and/or allogeneic bone marrow transplantation were analysed for WT1 expression. The results revealed that the hematological relapses were preceded (median, 1.8 months) by an increase in WT1 gene expression. For the practical utility of this gene as a molecular marker of relapse, it was necessary to determine an upper remission limit, crossing which would signal hematological relapse. The upper remission limit was determined in our set of patients to be 0.02 WT1/ABL. The AML patients who consequently relapsed crossed this upper remission limit; however, those in permanent remission did not. Therefore, this upper remission limit could be taken as the border of molecular relapse of AML patients. Moreover, insufficient decline of WT1 expression under the upper remission limit following induction and/or consolidation therapy was associated with markedly high risk of relapse. The results show that our upper remission limit can be taken as the border of molecular relapse of AML patients and WT1 levels following initial therapy as a beneficial prognostic marker.

Decreased DNA methylation in acute myeloid leukemia patients with DNMT3A mutations and prognostic implications of DNA methylation.

We examined 79 acute myeloid leukemia (AML) patients for DNA methylation of 12 tumor suppressor genes (TSG) and 24 homeobox domain (Hox) genes, and additionally for mutations in DNMT3A gene. We observed lower levels of DNA methylation (P<0.0001) as well as smaller numbers of concurrently hypermethylated genes (P<0.0001) in patients with DNMT3A mutations. Our study of the impact of DNA methylation on prognosis in intermediate and high risk AML patients revealed a relation between higher DNA methylation and better patients' outcome. Lower DNA methylation was linked with higher relapse rates and an inferior overall survival.

Expression of four major WT1 splicing variants in acute and chronic myeloid leukemia patients analyzed by newly developed four real-time RT PCRs.

Although the mechanism of action of leukemic oncogene Wilms' tumor gene 1 (WT1) remains unclear, WT1 has already been used in monitoring of patients with acute myeloid leukemia (AML) and it is being tested for immunotherapy. More detailed understanding of the role of WT1 in leukemia may improve its utilization. At least 36 isoforms may be produced. Four major variants denoted as -5/-KTS, -5/+KTS, +5/-KTS and +5/+KTS are produced by combining splicing of exon 5 and KTS sequence. In this study, we report applicability of newly developed real-time RT PCRs enabling for the first time full quantification of the four major WT1 splicing variants. Following careful optimization and testing of quantification reliability of four assays, we analyzed 34 samples of patients with AML and 12 samples of patients with chronic myeloid leukemia (CML) at the time of diagnosis. Analyses of five more CML patients provided insight into WT1 variants expression kinetics. We found predominance of +5/+KTS in both diagnoses. Comparison of WT1 variant expression in AML and CML patients' groups differing in response to therapy suggested possible importance of particular WT1 variant levels as markers of further disease course.

Prognostic impact of DNMT3A mutations in patients with intermediate cytogenetic risk profile acute myeloid leukemia.

OBJECTIVES: Recently, mutations in DNMT3A gene have been described in about 25% acute myeloid leukemia (AML) cases, preferentially in monocytic AML. They were found to predict worse overall survival (OS) of mutated patients. PATIENTS AND METHODS: RT-PCR followed by direct sequencing was used to test the presence of DNMT3A mutations in 226 AML patients with an intermediate-risk (IR) cytogenetics. RESULTS: Sixty-seven patients of 226 (29.6%) carried a mutation in the DNMT3A gene. Occurrence of DNMT3A mutations was associated with female sex (P = 0.027) and with the presence of FLT3/ITD (P = 0.003), but not with particular FAB subtypes. Patients with DNMT3A mutation had higher initial WBC counts than those without it (P = 0.064) only because of higher incidence of FLT3/ITD within these cases. There was no difference between mutated and wild-type groups in reaching complete remission (CR) (P = 0.380). OS was not affected by DNMT3A mutation (P = 0.251), but OS of patients who reached CR was longer in DNMT3A negative cases (P = 0.025). Patients with DNMT3A mutation had a higher relapse rate (P = 0.007). Patients carrying both the DNMT3A mutation and FLT3/ITD relapsed more often than either patients with single DNMT3A mutation (P = 0.044) or patients with FLT3/ITD only (P = 0.058). DNMT3A mutations were associated with higher relapse rate even within the FLT3/ITD-negative group (P = 0.072). After reaching CR, these two genetic factors were independent predictors of relapse at multivariate analysis (P < 0.001). Only three of 30 'double-mutated' (FLT3/ITD+, DNMT3A+) patients are still alive, all of them having undergone hematopoietic stem cell transplant. CONCLUSIONS: We have confirmed the high incidence of DNMT3A mutations in patients with AML with IR cytogenetics. Patients with DNMT3A mutations relapse more often and have inferior OS when only patients achieving CR are analyzed. 'Double-mutated' patients have a very poor prognosis.