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.

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.

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.

[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.

[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.

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.

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.

Modern and conventional prognostic markers of chronic lymphocytic leukaemia in the everyday haematological practice.

OBJECTIVES: The impact of modern prognostic markers on clinical course of chronic lymphocytic leukaemia (CLL) in everyday practice has been not yet well defined, especially in large series of patients. Therefore, the goal of this study was to assess the influence of conventional as well as modern prognostic factors on overall survival (OS) and time to therapy (TTT) of patients with CLL. METHODS: We retrospectively analysed data of all patients consecutively entered into the databases of five large academic centres in the Czech Republic. The total of 1300 patients was included in the analysis. RESULTS AND CONCLUSION: Through the use of uniparametric analysis, it was determined that gender, clinical stage Rai II-IV, unmutated IgVH status, deletion 17p (for both 5% and 20% cut-off), deletion 11q, ZAP-70 positivity and high expression of CD38 had significant negative influence on OS. TTT was significantly influenced by gender, Rai stage, IgVH status, deletion 11q, deletion 17p, deletion 13q and CD38 expression. Multiparametric analysis revealed that OS was significantly influenced by gender, age, IgVH status and deletion 17p. If only patients who died of CLL were included, gender, age, Rai stage, IgVH status and deletion 17p had significant influence on OS. Based on our results, the examination of biological prognostic markers can give an insight into the possible disease evolution in daily clinical practice. Biological prognostic markers are, however, not ready (maybe except deletion 17p in younger patients) to be used for guidance of therapy at least outside of clinical trials.

Unusually long survival of a 67-year-old patient with near-tetraploid acute myeloid leukemia m0 without erythroblastic and megakaryocytic dysplasia.

Patients with near-tetraploid acute myeloid leukemia (NT-AML) typically have poor survival. We present the case of a 67-year-old Caucasian male with NT-AML M0 who had an unusually long first complete remission of 51 months and an overall survival of 80 months. The only characteristic distinguishing him from other previously described patients with NT-AML was the absence of erythroblastic and/or megakaryocytic dysplasia (EMD) at diagnosis. Molecular-genetic testing for AML fusion transcripts associated with a favorable prognosis (PML/RARα,AML1/ETO, and CBFß/MYH11) were negative, as were other prognostic markers like MLL-PTD,FLT3-ITD, or mutations of FLT3-D835,NPM1, or CEBPA. Expression studies of ERG,MN1, and EVI1 revealed overexpression of ERG only. The absence of EMD may be a useful prognostic/diagnostic feature of this new rare subtype of NT-AML.

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.

Prognosis of children with mixed phenotype acute leukemia treated on the basis of consistent immunophenotypic criteria.

BACKGROUND: Mixed phenotype acute leukemia (MPAL) represents a diagnostic and therapeutic dilemma. The European Group for the Immunological Classification of Leukemias (EGIL) scoring system unambiguously defines MPAL expressing aberrant lineage markers. Discussions surrounding it have focused on scoring details, and information is limited regarding its biological, clinical and prognostic significance. The recent World Health Organization classification is simpler and could replace the EGIL scoring system after transformation into unambiguous guidelines. DESIGN AND METHODS: Simple immunophenotypic criteria were used to classify all cases of childhood acute leukemia in order to provide therapy directed against acute lymphoblastic leukemia or acute myeloid leukemia. Prognosis, genotype and immunoglobulin/T-cell receptor gene rearrangement status were analyzed. RESULTS: The incidences of MPAL were 28/582 and 4/107 for children treated with acute lymphoblastic leukemia and acute myeloid leukemia regimens, respectively. In immunophenotypic principal component analysis, MPAL treated as T-cell acute lymphoblastic leukemia clustered between cases of non-mixed T-cell acute lymphoblastic leukemia and acute myeloid leukemia, while other MPAL cases were included in the respective non-mixed B-cell progenitor acute lymphoblastic leukemia or acute myeloid leukemia clusters. Analogously, immunoglobulin/T-cell receptor gene rearrangements followed the expected pattern in patients treated as having acute myeloid leukemia (non-rearranged, 4/4) or as having B-cell progenitor acute lymphoblastic leukemia (rearranged, 20/20), but were missing in 3/5 analyzed cases of MPAL treated as having T-cell acute lymphobastic leukemia. In patients who received acute lymphoblastic leukemia treatment, the 5-year event-free survival of the MPAL cases was worse than that of the non-mixed cases (53+/-10% and 76+/-2% at 5 years, respectively, P=0.0075), with a more pronounced difference among B lineage cases. The small numbers of MPAL cases treated as T-cell acute lymphoblastic leukemia or as acute myeloid leukemia hampered separate statistics. We compared prognosis of all subsets with the prognosis of previously published cohorts. CONCLUSIONS: Simple immunophenotypic criteria are useful for therapy decisions in MPAL. In B lineage leukemia, MPAL confers poorer prognosis. However, our data do not justify a preferential use of current acute myeloid leukemia-based therapy in MPAL.

Nature of frequent deletions in CEBPA.

C/EBPalpha (CCAAT/enhancer binding protein alpha) belongs to the family of leucine zipper transcription factors and is necessary for transcriptional control of granulocyte, adipocyte and hepatocyte differentiation, glucose metabolism and lung development. C/EBPalpha is encoded by an intronless gene. CEBPA mutations cause a myeloid differentiation block and were detected in acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), multiple myeloma and non-Hodgkin's lymphoma (NHL) patients. In this study we identified in 41 individuals from 824 screened individuals (290 AML patients, 382 MDS patients, 56 NHL patients and 96 healthy individuals) a single class of 23 deletions in CEBPA gene which involved a direct repeat of at least 2 bp. These mutations are characterised by the loss of one of two same repeats at the ends of deleted sequence. Three most frequent repeats included in these deletions in CEBPA gene are CGCGAG (493-498_865-870), GCCAAGCAGC (508-517_907-916) and GG (486-487_885-886), all according to GenBank accession no. NM_004364.2. A mechanism for deletion formation between two repetitive sequences can be recombination events in the repair process. Double-stranded cut in DNA can initiate these recombination events of adjacent DNA sequences.

CEBPA polymorphisms and mutations in patients with acute myeloid leukemia, myelodysplastic syndrome, multiple myeloma and non-Hodgkin's lymphoma.

The transcription factor CCAAT/enhancer binding protein (C/EBP)alpha is a myeloid-specific transcription factor which is required for normal myeloid differentiation. C/EBPalpha is encoded by an intronless gene that is 2783 bp long and maps to human chromosome 19q13.1. C/EBPalpha is a member of the basic region leucine zipper (bZIP) class of DNA-binding proteins. The loss of function of C/EBPalpha has leukemogenic potential. Four types of polymorphisms and 25 mutations (3 already known mutations and 22 novel mutations) were detected in CEBPA (gene for the transcription factor CCAAT/enhancer binding protein (C/EBP) alpha) in analysed samples from 390 patients with myelodysplastic syndrome (MDS) and hematologic malignancies. CEBPA mutations were found in 14/152 (9.2%) of acute myeloid leukemia (AML) patients' samples, 6/143 (4.2%) of MDS patients' samples, 2/56 (3.6%) of non-Hodgkin's lymphoma (NHL) patients' samples and 2/39 (5.1%) of multiple myeloma (MM) patients' samples. No C/EBPalpha mutations were detected in healthy donors (41 individuals). We discuss how these mutations can affect the cellular function of C/EBPalpha and block the myeloid differentiation.

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.

WHO bone marrow features and European clinical, molecular, and pathological (ECMP) criteria for the diagnosis of myeloproliferative disorders.

The bone marrow criteria defined by the World Health Organization (WHO) are based on characteristic increase and clustering of morphologically abnormal enlarged megakaryocytes as a pathognomonic clue to describe three distinct phenotypic entities of myeloproliferative disorders (MPDs): (1) essential thrombocythemia (ET), (2) early and overt polycythemia vera (PV) and (3) prefibrotic, early fibrotic, and fibrotic chronic idiopathic myelofibrosis (CIMF-0, 1, 2 and 3). Based on established WHO bone marrow features, and the use of new molecular and laboratory markers including JAK2(V617F) mutation, endogenous erythroid colony (EEC) formation and serum erythropoietin (EPO), we present updated European clinical, molecular and pathological (ECMP) criteria for the differential diagnosis of true ET, PV and CIMF. As compared to the WHO bone marrow features, each of the laboratory and molecular markers are not sensitive enough for the diagnosis and classification of the three prefibrotic MPDs. The proposed WHO/ECMP criteria reduce the platelet count to the upper limit of normal (>400x10(9)l(-1)) as inclusion criterion for the diagnosis of thrombocythemia in true ET, early stages of PV and prefibrotic CIMF. The combined use of WHO and ECMP criteria differentiate PV from congenital and acquired erythrocytosis, true ET from reactive thrombocytosis and separates true ET from CIMF-0/1 mimicking ET. Only half of the patients with true ET and CIMF carry the JAK2(V617F) mutation (sensitivity 50%). Early PV mimicking ET is featured by the presence of JAK2(V617F) mutation, EEC, low serum EPO levels, normal hematocrit, and increased bone marrow cellularity due to increased erythropoiesis ("forme fruste" PV) when WHO/ECMP criteria are applied. The combination of JAK2(V617F) PCR test and increased hematocrit is diagnostic for PV (sensitivity 95%, specificity 100%). The degree of JAK2(V617F) positivity of granulocytes is related to disease stage: heterozygous in true ET and early PV and mixed hetero/homozygous to homozygous in overt and advanced PV and CIMF. Bone marrow histology assessment should remain the gold standard criterion for the diagnosis and staging of the MPDs true ET, PV and CIMF and its differentiation from primary or secondary erythrocytosis, reactive thrombocytosis and thrombocythemias associated with atypical MPD, myelodysplastic syndromes, and chronic myeloid leukemia,. The proposed WHO/ECMP criteria allow a cross talk between clinicians, pathologists and scientists to much better characterize the nature and natural history of each of the WHO/CMP defined early and overt MPDs.