BCR-ABL Negative Myeloproliferative Neoplasms Research Articles

Myeloproliferative neoplasms (BCR-ABL1 negative) and myelodysplastic/myeloproliferative neoplasms: current diagnosticprinciples and upcoming updates.

Since the publication of the latest World Health Organization (WHO) classification in 2008, there has been a significant effort for clarification of unresolved questions, especially with the help of the rapidly developing field of molecular genetic studies, next-generation sequencing in particular. Numerous entities within the WHO categories of myeloproliferative neoplasms (MPNs) and myelodysplastic (MDS)/MPNs have been extensively studied, with large published series attempting to characterize and better define their morphologic and molecular genetic features. This emerging genetic landscape maintains a robust correlation with the various disease entities recognized by the WHO classification scheme based on a careful integration of detailed clinical information, bone marrow and peripheral blood morphology, immunohistology, and genomics. This brief review summarizes the current guidelines as they apply to diagnosing both the classical BCR-ABL1 negative MPN (polycythemia vera, essential thrombocythemia, and primary myelofibrosis) and the more common subtypes of MDS/MPN overlap syndromes. The more important recent molecular updates as well as the upcoming changes to the current WHO classification, expected to be published in late 2016, will also be briefly reviewed.

Among a panel of polymorphisms in genes related to oxidative stress, CAT-262 C>T, GPX1 Pro198Leu and GSTP1 Ile105Val influence the risk of developing BCR-ABL negative myeloproliferative neoplasms

Objectives: To analyze the relationship between six polymorphisms in genes related to oxidative stress, namely CAT-262 C>T, MnSOD Ala16Val, GPX1 Pro198Leu, GSTM1 and GSTT1 null genotypes, and GSTP1 Ile105Val, and the occurrence of BCR-ABL negative myeloproliferative neoplasms (polycythemia vera, essential thrombocythemia, and primary myelofibrosis).Methods: We genotyped for these polymorphisms 328 patients with a known mutation status for JAK2 V617F, MPL and CALR, and 363 controls, using molecular genetics assays.Results: The CAT-262 C>T and GPX1 Pro198Leu polymorphisms were seen significantly less frequently, while the GSTP1 IleVal105 polymorphism was seen significantly more frequently in patients with BCR-ABL negative myeloproliferative neoplasms, regardless of the molecular sub-type (e.g. JAK2 V617F or CALR mutated).Discussion and conclusion: Our study provides evidence that variation in genes related to oxidative stress might modulate the risk of developing BCR-ABL negative myeloproliferative neoplasms.

Myeloid-derived suppressor cells in patients with myeloproliferative neoplasm

Although BCR-ABL negative myeloproliferative neoplasms (MPN) – and especially myelofibrosis (MF) – are recognized to be associated with autoimmune phenomena, immune derangements in MPN have been much less studied. Myeloid-derived suppressor cells (MDSC) are one type of important immune modulator cell. Therefore, we studied MDSCs in MPN disease. MDSCs were studied in two cohorts: the first cohort was 55 patients including 16 primary myelofibrosis (PMF), 7 post-polycythemia vera (PV)-MF, 2 post-essential thrombocythemia (ET)-MF, 11 ET, 17 PV, 2 undefined MPN disorder, and 23 normal controls; the second cohort included 38 patients: 17 ET, 7 PMF, 3 ET-MF, 2 PV-MF, 9 PV patients, and 20 normal volunteers. The second cohort was studied using freshly collected specimens and a comparable age group as controls. CD11b+, CD14−, and CD33+ cells were defined as MDSCs in both cohorts by flow cytometry. Since there are no differences in MDSC levels among different MPN categories, they were grouped as MPNs. The results showed that MDSCs were significantly elevated in MPNs compared with controls in both cohorts. We also performed RT-PCR and found that MPN patients have significantly elevated arginase-1 mRNA compared with controls, and sorted MDSCs were found to have suppressor T cell activity in MPNs, substantiating the hypothesis that levels of MDSCs are, in fact, deranged in MPNs. MDSC levels were not correlated with JAK2 status, white blood cells, Hb levels, platelet counts, splenomegaly, or the degree of bone marrow fibrosis (in MF). Further studies in immune therapy involving MDSC inhibitors or differentiation may be developed to treat MPN disease.

Comparative study of different methodologies to detect the JAK2 V617F mutation in chronic BCR-ABL1 negative myeloproliferative neoplasms

ObjectivesA mutation in the JAK2 gene, V617F, has been identified in several BCR-ABL1 negative myeloproliferative neoplasms (MPN): polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). Defining the presence or absence of this mutation is an essential part of clinical diagnostic algorithms and patient management. Here, we aimed to evaluate the performance of three PCR-based assays: Amplification Refractory Mutation System (ARMS), High-Resolution Melting analysis (HRM), and Sanger direct sequencing, and compare their results with those obtained by a PCR restriction fragment polymorphism assay (PCR-RFLP). Design and methodsWe used blood samples from 136 patients (PV=20; PMF=20; ET=28, and other MPN suspected cases=68). ResultsComparable results were observed among the four assays in patients with PV, PMF, and MPN suspected cases. In patients with a diagnosis of ET, the JAK2 V617F mutation was detected in 67.8% of them by the PCR-ARMS and PCR-HRM assay and in 64% of them by the conventional Sanger sequence approach. The PCR-ARMS and PCR-HRM assays were 100% concordant. With these tests, only one of the 20 patients with ET and one of the three patients with clinically suspected MPN gave different results compared with those obtained by the PCR-RFLP. ConclusionsOur results have demonstrated that the PCR-ARMS and PCR-HRM assays could detect the JAK2 V617F mutation effectively in MPN patients, but PCR-HRM assays are rapid and the most cost-effective procedures.

Fusion Gene Detection Using Whole Transcriptome Analysis in Patients with Chronic Myeloproliferative Neoplasms and Secondary Acute Myeloid Leukemia

Fusion oncogenes resulting from chromosomal aberrations are common disease drivers in myeloid malignancies. The most prominent example is BCR-ABL1 fusion present in chronic myeloid leukemia, which together with essential thromobocythemia (ET), primary myelofibrosis (PMF) and polycythemia vera (PV) belongs to the classic myeloproliferative neoplasms (MPN). The BCR-ABL1 negative MPNs are driven by somatic mutations in JAK2, MPL and CALR. MPN patients can progress to acute myeloid leukemia (AML) but the transformation process is not well understood. Studies using standard karyotyping and SNP microarrays have shown that disease progression is characterized by an increase in karyotype complexity. We aimed to identify novel fusion oncogenes in patients with BCR-ABL1 negative MPN during chronic phase and disease progression in high-throughput and cost-efficient manner using RNA-seq technology. In addition this approach enabled us to perform RNA-seq variant calling for identification of gene mutations on the same cohort of patients.Whole transcriptome sequencing was performed on 121 patients (112 chronic phase MPN and 9 secondary AML samples) and 23 healthy controls in a 100 base pair paired-end manner. The cohort consisted of 44% PMF, 22% ET, 12% PV and 6% secondary AML patients. The output of three fusion detection tools (Defuse, Tophat-fusion and SOAPfuse) was combined in order to increase sensitivity. Extensive filtering steps were applied in order to enrich for cancer specific fusion events, including filtering for fusions appearing in healthy individuals, filtering for read-throughs and false positives with external databases and manual inspection of sequencing reads. The outcome of analysis for Defuse, Tophat-fusion and SOAPfuse resulted in the total of 52, 54 and 38 candidate fusions, respectively. Candidate fusions were Sanger-sequenced and for Tophat-fusion and Defuse the validation rate was 60%, while for SOAPfuse only 20% could be validated. Approximately 70% of the fusion candidates were not shared among the 3 tools which underlines the importance of selecting the union of all calls from each tool rather than the intersect.We did not observe clustering of breakpoints along the genome. Most fusion candidates could be detected in PMF which corresponds to the disease entity that was most represented in the cohort (44% of patients). No enrichment for fusions was found in 7 triple negative (no JAK2, CALR, MPL mutations) cases. 42% of chromosomal aberrations were translocations, followed by duplication (31%), inversion (14%) and deletion events (11%). Among the intragenic fusions, approximately half had genomic breakpoints less than 1 Mb apart. 70% of validated fusions were out of frame, while 28% were in frame. In the leukemic samples a higher abundance of fusions was found (4/9). Typical fusions for de novo AML were not detected within secondary AML (sAML) samples. We did not detect a recurrent fusion oncogene in our patient cohort.In a PMF patient with JAK2-V617F mutation we identified a BCR-ABL1 fusion, indicating a clonal exchange which was consistent with patient's phenotype. Another PMF patient exhibited an inversion event involving the first exon of CUX1, causing a CUX1 loss of function. Other fusions in chronic MPN patients affected genes involved in histone modifications (SMYD3-AHCTF1, KDM4B-CYHR1). In post-MPN AML patients we identified a somatic in frame-fusion involving INO80D and GPR1 and a fusion truncating the first 3 exons of RUNX2 (XPO5-RUNX2).The high quality of RNA sequencing data, allowed us to set up a variant detection workflow that will be compared with matched samples that have been exome sequenced. Preliminary results could demonstrate that mutations in the JAK2 gene in a cohort of 96 patients were all correctly recalled, emphasizing its sensitivity.Fusion events among patients in chronic phase MPN are rare and the majority of these events imply loss of function of both fusion gene partners. This approach adds valuable information on the true frequency of inactivation of genes such as CUX1 in patients, as small inversions like the one described above would not be detectable by other methods. Detection of a subclone with BCR-ABL1 fusion underlines the strength of the fusion detection workflow for diagnostic purposes. Typical de novo AML fusions were not found in sAML and further suggests that de novo AML and sAML are distinct disease entities on a genetic level. DisclosuresGisslinger:Janssen Cilag: Honoraria, Speakers Bureau; Sanofi Aventis: Consultancy; AOP ORPHAN: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau; Geron: Consultancy. Kralovics:AOP Orphan: Research Funding; Qiagen: Membership on an entity's Board of Directors or advisory committees.

β-arrestin2 Is Necessary for Development of MPLW515L Mutant Primary Myelofibrosis

Background:Primary myelofibrosis (PMF) is a BCR-ABL negative myeloproliferative neoplasm (MPN) characterized by proliferation of myeloid cells and secondary development of marrow fibrosis due to release of growth factors from clonally expanded cells. PMF is characterized by mutations such as JAK2V617F and MPLW515L that lead to cytokine-independent cell growth and constitutive activation of downstream signaling pathways.β-arrestins are multifunctional adaptor proteins that have been identified as mediators of distinct G protein-coupled receptor signaling cascades independent of G proteins. One isoform, β-arrestin2 (βarr2), has been shown to mediate anti-apoptotic signaling and has been implicated in initiation and progression of another MPN, chronic myeloid leukemia. It is also known that long-term hematopoietic stem cell self-renewal may be dependent on βarr2-mediated signaling with significant defects noted in conditions of increased cellular stress. Based on these observations, we hypothesize that βarr2 plays a causal role in the pathogenesis of PMF by mediating anti-apoptotic signaling and cell proliferation and may serve as a therapeutic target in this disease.Methods:We used a standard murine retroviral transduction system to model MPLW515L mutant PMF. KLS cells (Lin-, Sca-1+, c-kit+) were harvested from marrow of donor wild type (WT), β-arrestin1 knockout (βarr1-/-), and β-arrestin2 knockout (βarr2-/-) mice (CD45.2). KLS cells were retrovirally transduced with MSCV-MPLW515L-IRES-GFP and were subsequently injected retro-orbitally into lethally irradiated congenic WT recipient mice (CD45.1). Mice were monitored for leukemia development including splenomegaly and were subject to weekly blood analysis including complete blood counts and flow cytometry for donor cells present in recipient mice. Primary endpoint measured was survival. To elucidate potential pathogenic mechanisms, we harvested KLS cells from marrow of WT, βarr1-/-, and βarr2-/- mice, cultured in vitro and subjected cells to no treatment versus treatment with MPLW515L mutant retrovirus. [3 H]-Thymidine incorporation was performed to assess proliferation and cells were stained for annexin V and propidium iodide (PI) to assess apoptosis.Results:In total, twelve mice each received WT or βarr1-/- donor cells and sixteen mice received βarr2-/- donor cells. Survival of mice receiving βarr2-/- donor cells was dramatically improved compared to mice receiving WT or βarr1-/- donor cells by log-rank (Mantel-Cox) test (p<0.0001). One of sixteen mice who received βarr2-/- cells died by the end of the study period compared to 11/12 and 12/12 mice who received WT or βarr1-/- cells respectively (Figure 1A). Mice who received βarr2-/- cells had lower white blood cell counts over time as well as smaller spleens at death measuring 0.12 ±0.013 grams versus 0.63 ± 0.08 grams or 0.76 ± 0.13 grams for mice receiving WT or βarr1-/- cells respectively (p< 0.0001, one-way ANOVA) (Figure 1B).Increased cell proliferation was observed in KLS cells treated with mutant MPLW515L retrovirus compared to no treatment as assessed by thymidine incorporation. No statistically significant differences in proliferation among WT, βarr1-/- and βarr2-/- cells treated with MPLW515L mutant retrovirus were observed. Increased apoptosis was observed in KLS cells treated with mutant MPLW515L retrovirus compared to no treatment as assessed by annexin V staining in all genotypes. βarr2-/- MPLW515L mutant treated KLS cells demonstrated increased apoptosis at 12.54 ± 0.79 percent compared to untreated KLS cells at 7.22 ± 0.73 percent (p < 0.01, one-way ANOVA) as well as compared to WT treated (p <0.05, one-way ANOVA) and untreated controls (Figure 2).Conclusions:These data demonstrate that βarr2 is necessary for development of MPLW515L mutant PMF. Absence of βarr2 does not affect proliferation although it does lead to increased apoptosis in conditions of increased cell proliferation or stress. Hematopoietic cells lacking βarr2 are more prone to apoptosis when subject to the increased cellular proliferation observed in MPNs. βarr2 may therefore be an attractive potential therapeutic target in PMF. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Decreased Activity of NK Cells in Myeloproliferative Neoplasms

Natural killer (NK) cells are a subset of lymphocytes that play a critical role in both innate and adaptive immune responses and provide defense against microbial infections and malignant transformation. When stimulated through their activating or cytokine receptors, NK cells rapidly produce cytokines and chemokines, including IFNγ, TNFα, TGFβ, GM-CSF, MIP1α, MIP1β, IL-10, and others, which can affect the function of hematopoietic stem cells (HSC)s. We hypothesized that NK-mediated cytokine production could regulate HSC function and that its deregulation may favor HSC malignant transformation and proliferation. In this regard, a few studies have shown that NK cells are defective in stem cell-derived diseases such as chronic myeloid leukemia (CML), for example, raising the hypothesis that immunological dysfunctions may be involved in initiating or contributing to the pathogenesis of myeloproliferative diseases. In contrast to CML, the role of NK cells in BCR-ABL1 negative-myeloproliferative neoplasms (MPN) is currently unexplored. Therefore, we aimed to study NK cell activity in murine and human primary cells from MPN. NK cells and receptors were quantified by flow cytometry by the use of spleen cells from a conditional knockin Jak2V617F (Jak2VF) murine model, that faithfully resemble the main clinical and laboratorial characteristics of human Polycytemia Vera (PV), and from MPN patients diagnosed and followed at the Clinical Hospital of the Medical School of Ribeirao Preto, University of Sao Paulo. For immunophenotyping of murine lymphocytes, spleen cells were isolated and stained with fluorescence-conjugated antibodies against murine Ter119, CD19, CD4, CD8, CD3, NK1.1 (for NK cells quantification) and NKG2A, NKG2D, Ly49H and Ly49D (for receptors analysis). For analysis of human lymphocytes, peripheral blood mononuclear cells were purified by density gradient centrifugation, resuspended in serum-enriched phosphate buffered saline and stained with fluorescence-labeled anti-human antibodies against the following molecules: CD45, CD19, CD3, CD16, CD56. All the stainings were performed for 20 minutes at 40 C and cells were acquired using a FACScalibur flow cytometer (BD Biosciences). Data was analyzed by the use of the Flojo software version X and the statistical analyses were performed by the use of SPSS version 20. When compared to Jak2WT controls, Jak2VF mice had lower numbers of NK cells (2.09% versus 22.7% of the spleen cells, P = 0.022) and NKG2A (0.57% versus 2.47% of NK cells, P = 0.037) and NKG2D (0.17% versus 0.46% of NK cells, P = 0.020) receptors. For the study of human NK cells, 38 patients diagnosed with MPN and who were not in use of cytoreductive therapy were included and classified according to the WHO 2008 criteria as Polycytemia Vera (PV, n = 11), Essential Thrombocytemia (ET, n = 11), Primary Myelofibrosis (PMF, n = 11) or Unclassified MPN (uMPN, n = 5). Blood samples from 34 healthy donors served as controls. JAK2V617F (JAK2VF) mutated patients had lower numbers of CD16+ CD56dim NK cells as compared to JAK2VF negative and healthy controls (0.73% versus 1.06% versus 1.54%, P = 0.038, respectively). Among the JAK2 mutated patients, those with PV and PMF presented lower numbers of NK cells than ET patients. In agreement, NK cells were reduced in JAK2VF PV patients when compared to JAK2VF negative and controls (0.39% versus 1.42% and 1.54%, P = 0.047). PMF patients had lower NK cell numbers than controls, regardless of JAK2VF mutation (0.35%, 0.49% and 1.54% for JAK2VF positive, negative and controls, respectively, P = 0.025). No significant differences were observed in ET or uMPN. In summary, NK cells were reduced among lymphocytes in MPN patients, especially in the JAK2VF mutated cases, as shown in PV patients and Jak2VF mice. In addition, the expression of NK receptors was reduced in Jak2VF mice, thus suggesting that these cells are defective in MPN. New insights into the pathogenesis of this group of diseases are of great relevance considering that MPN treatment remains non-curative with the strategies available so far despite the development of drugs that inhibit JAK2. Thus, identifying the mechanisms of NK-cell deficiency in MPN and the detailed interaction between these cells and the malignant stem cell may help to establish combination therapeutic strategies that may improve therapy responses in MPN. DisclosuresNo relevant conflicts of interest to declare.

Classic Cardiovascular Risk Factors and Certain Comorbidities May Contribute to the Risk of Thrombembolic and Bleeding Events in Patients with Classical Myeloproliferative Neoplasms

Background. Thrombembolic (TE) and bleeding (B) events pose a severe risk for patients (pts) with myeloproliferative neoplasms (MPN). Multiple disease-related factors have been identified that contribute to these events, such as the interaction between platelets, leucocytes, and endothelial cells. However, not all pts develop thrombosis or severe bleeding, suggesting that further contributing factors may play a role, including specific comorbidities and cardiovascular risk factors.Aims. The aim of our analysis was to evaluate the association between thromboembolic events, specific comorbidities, and cardiovascular risk factors in MPN pts.Methods. We analyzed clinical data frompts treated at the MPN Center at RWTH Aachen University hospital. All pts were aged ≥18 years and carried a diagnosis of classical BCR-ABL negative MPN (polycythemia vera [PV], essential thrombocythemia [ET], primary and secondary myelofibrosis [MF]), as defined by WHO criteria (2008). In addition, data about MPN disease complications, age, cardiovascular risk factors (smoking habits, arterial hypertension, diabetes mellitus, obesity, and hyperlipoproteinemia) and MPN subtype were included. Comorbidities were scored using three different comorbidity scales (CIRS, Charlson comorbidity index, and ACE-27).Results. 75 MPN pts were included in this analysis, 25 with PV, 19 ET pts and 31 pts with MF (19 pts with PMF, 9 Post-PV-MF pts and 3 pts with Post-ET-MF). Characterization of pts regarding age, sex, thrombembolic/bleeding events as well as median scores in comorbidity scales are shown in Table 1. In addition, pts were divided into a group with (Group TE+B) or without (Group no TE+B) thromboembolic or severe bleeding events. 40 patients (53.3%) had previously experienced a thrombembolic or bleeding event. These pts showed significantly higher median comorbidity scores (CIRS of 6.5 vs. 2.0, p< 0,001, Charlson score of 1 vs. 0, p<0,001, and median ACE-27 of 2 vs. 0, p<0,001). Presence of classic cardiovascular risk factors among groups TE+B vs. no TE+B was heterogeneous (10 vs. 11 (ex-)smokers; 18 vs. 9 pts with arterial hypertension; 9 vs. 14 pts with a BMI > 25; 4 vs. 1 pts with diabetes mellitus; 4 vs. 0 pts with hyperlipoproteinemia). TE+B pts had high scores in certain CIRS categories (each category includes a scale of 0-4) especially in categories regarding cardiac diseases, hypertension, vascular disease & endocrinological disease (see Table 2). Furthermore, more than one third of the TE+B pts had also neurological (16/40 pts. (40%) vs. 3/35 (8.6%) in the no TE+B group) and upper-GI-disease (13/40 pts (32.5%) vs. 8/35 (22.9%) in the no TE+B group).Summary and Conclusions. Given the obvious limitations of this analysis due to small sample numbers, this analysis provides an indication that classic cardiovascular risk factors (especially diabetes mellitus, arterial hypertension, and hyperlipoproteinemia) and additional comorbidities contribute to the risk of thromboembolic and bleeding events in pts with classical MPN. Clinical trials should address whether the improvement of such risk factors in addition to anti-MPN treatment can minimize the risk of such events.Table 1Characterization of ptsSubtype of MPNNumber of patientsMedian age at diagnosis (range)Number of patients that developed thromboembolic events (TE) or bleeding (B)*Number of patients without thromboembolic/bleeding eventsMedian CIRS score(Range 0-56)Median Charlson comorbidity index(Range 0-37)Median ACE-27 score(Range 0-3)Polycythemia vera25(11m/14f)58 (31-77)15 (15 TE, 2 B)10611Essential Thrombo-cythemia19(10m/9f)54 (23-64)6 (5 TE, 1 B)13401All Myelofibrosis31(22m/9f)63 (29-77)19 (16 TE, 7 B)12501MyelofibrosisPMF19(16m/3f)61 (29-77)11 (9 TE, 4 B)8401Post-PV-MF9(3m/6f)63 (42-73)7 (6 TE, 3 B)2811Post-ET-MF3(3m/no f)68 (68-73)1 (1 TE)21322· Few Pts had suffered from both thromboembolic and bleeding eventsTable 2Number of pts in the TE+B group in comparison to the no TE+B group in certain CIRS categories subdivided according to scoreCIRS category (TE+B pts vs. no TE+B pts)Score of 0 per categoryScore of 1 per categoryScore of 2 per categoryScore of 3 per categoryScore of 4 per categoryCardiac disease20 vs. 306 vs. 25 vs. 39 vs. 00 vs. 0Hypertension22 vs. 262 vs. 04 vs. 612 vs. 30 vs. 0Vascular disease21 vs. 323 vs. 112 vs. 03 vs. 11 vs. 1Endocrinological disease21 vs. 260 vs.119 vs.70 vs.10 vs.0 DisclosuresIsfort:Pfizer: Honoraria; BMS: Honoraria; Mundipharma: Other: Travel & Accomodation; Roche: Other: Travel & Accomodation; Novartis: Other: Travel & Accomodation; Amgen: Other: Travel & Accomodation; Hexal: Other: Travel & Accomodation. Bruemmendorf:Ariad: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Patent on the use of imatinib and hypusination inhibitors: Patents & Royalties. Koschmieder:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel reimbursement for scientific conferences, Research Funding; Novartis Foundation: Research Funding; Baxalta/CTI: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel reimbursement for scientific conferences; Janssen Cilag: Other: Travel reimbursement for scientific conferences.

Whole Exome Sequencing Reveals Clonal Evolution of Myeloproliferative Neoplasms to Acute Myeloid Leukemia

Disease progression to acute myeloid leukemia (AML) is observed in 7% of the cases with the three classical BCR-ABL1 negative myeloproliferative neoplasms (MPN), polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). According to the WHO, the presence of ≥ 20% blasts in bone marrow or peripheral blood is the diagnostic criterion for establishing leukemic transformation. PMF patients are at the highest risk, while PV and ET patients often develop secondary myelofibrosis prior to the leukemic transformation. Post-MPN AML patients have poor prognosis and clonal evolution of MPN chronic phase to AML is not well understood. Here we aimed to study the clonal evolution from MPN to AML in 7 cases, by performing whole exome sequencing (WES) on samples taken at various disease stages from individual patients.From the 7 post-MPN AML patients included in the study, 4 were diagnosed with PV and 3 with ET during chronic phase of the disease. For all 7 patients WES was performed on DNA samples from the control tissue, chronic phase and/or accelerated phase, and the leukemic phase of the disease. All variants identified by WES were validated using Sanger sequencing. In addition, tumor samples were analyzed for genomic deletions, gains and uniparental disomies (UPD) using SNP microarrays.We identified on average 16 somatic mutations (range 12-27) and 4 chromosomal aberrations (range 0-9) per patient in the leukemic stage of the disease. All patients were JAK2-V617F positive. 115 validated somatic mutations affected a total of 100 different genes. Most mutations were found in genes that were previously not linked to myeloid cancers, however, they were not recurrent. Besides JAK2-V617F, recurrent mutations were found in TP53 (N=3/7), RUNX1 (N=2/7), TET2 (N=2/7) and MPL (N=2/7). Biallelic TP53, RUNX1 and TET2 mutations were present in single patients. Known MPN and AML-related genes such as DNMT3A, SRSF2, U2AF1, IDH2, KIT, and PHF6 were mutated in single patients. We identified 25 chromosomal aberrations in 7 patients. Del5q, del6p, del7q and 9pUPD were recurrent. UPDs and trisomies of chromosomes 9, 12q, 17p and 21 were coupled with mutations in JAK2, SH2B3, TP53 and RUNX1. One patient harbored focal deletions of <1Mb on chromosomes 10 and 12, targeting TET1 and ETV6, respectively.We used variant frequencies detected by WES and copy number ratios and allelic difference values detected by microarrays at various stages of the disease to reconstruct the clonal evolution from chronic phase to AML in the 7 studied cases. Mutations with similar variant frequencies showing changes of allelic frequency in the same direction were assumed to be part of the same clone. Figure 1 illustrates an example of the proposed model for clonal evolution in Patient 6. As in this patient we had WES data from chronic, accelerated and leukemic stage of the disease, we first analyzed the clonal evolution from chronic to the accelerated phase. In the chronic phase the ~60-80% of granulocytes were derived from a single clone carrying 5 somatic mutations (JAK2, CAD, PPFIA2, SCNG, USH2A) and 2 chromosomal aberrations (1q gain and trisomy 9). At the accelerated phase of the disease we could observe that the main clone acquired somatic mutations in ADIPOQ, EYA3 and FAM123C and that there is at least one subclone (~40% of cells) appearing with mutations in OGDH, PHF6, USH2A, del7q and 9qUPD. At the leukemic stage, the clone with 9qUPD was suppressed by the outgrowing clone carrying OGDH and other mutations. We could also show that at the final leukemic stage the dominant clone acquired RUNX1 S400X mutation, amplified with a trisomy of chromosome 21, while the other RUNX1 allele mutated to Q262X.In each of the 7 studied patients the clonal evolution was unique and complex process with a few common features. Loss of TP53 is the most common genetic lesion. TET2 mutations are early events in clonal evolution of MPN and often precede the acquisition of JAK2-V617F, while RUNX1 mutations seem to be late events, leading to differentiation arrest and appearance of blasts. We demonstrated that in 6/7 studied cases the clonal evolution was a linear process, led by sequential acquisition of somatic mutations on the basis of the same clone causing the chronic disease. This finding is in line with the results of our previous study where we showed that the genetic basis of secondary AML is significantly different from de novo AML. [Display omitted] DisclosuresKralovics:AOP Orphan: Research Funding; Qiagen: Membership on an entity's Board of Directors or advisory committees.

Mutational Analysis of SH2B3 in Korean Patients With BCR-ABL1 Negative Myeloproliferative Neoplasm

Dear Editor, Src homology 2 B3 (SH2B3), previously named LNK, has been reported as a new genetic abnormality in BCR-ABL negative myeloproliferative neoplasms (MPN) [1,2,3]. In western patients, the mutational hot spot of SH2B3 is located in exon 2, within a pleckstrin homology (PH) domain. However, SH2B3 mutations in Korean MPN patients can occur in several other regions of the SH2B3 gene, including exons 7 and 8 [4]. In this study, we performed sequencing analyses of SH2B3 in Korean patients with BCR-ABL1 negative MPN and compared the results with previous studies. In total, 75 patients were enrolled in the study, comprising 32 patients with essential thrombocytosis (ET), 25 patients with polycythemia vera (PV), 10 patients with primary myelofibrosis (PMF), and eight patients with unclassifiable MPN. All patients were diagnosed between June 2007 and March 2012 at Pusan National University Hospital in Busan, Korea. The patients comprised 40 males and 35 females with a median age of 57.3 yr. This research was reviewed and approved by full committee review of the Institutional Review Board at Pusan National University Yangsan Hospital (No. 05-2014-058). We designed the primers and performed the mutation analyses by direct sequencing of the following loci: SH2B3 exons 2, 7, and 8; Janus kinase 2 (JAK2) exon 12 and V617F mutation; and casitas B-lineage lymphoma proto-oncogene (CBL) exons 8 and 9. Alterations in the CBL gene have been identified in AML, MPN, and chronic myelomonocytic leukemia patients [5]. We identified two different SH2B3 mutations (2.7%) in exon 8 (Fig. 1 and Table 1). A novel p.Q571* (c.1711C>T) mutation which results in a premature stop codon and the known p.I568T (c.1703T>C) missense mutation were identified. In addition, two patients have a known polymorphism, p.A356T (c.1606G>A) [4]. The JAK2 V617F mutation was detected in 48 of 75 patients (64.0%); however, no mutation of JAK2 exon 12 or CBL exons 8 or 9 was identified. One of the two patients with a SH2B3 mutation also harbored a JAK2 V617F mutation (Table 1). Fig. 1 Nucleotide sequence and chromatogram of SH2B3 mutations in Korean patients with BCR-ABL1 negative myeloproliferative neoplasm. (A) Nonsense mutation (c.1711C>T; p.Q571*), (B) missense mutation (c.1703T>C; p.I568T). Table 1 The characteristics of two patients with SH2B3 mutations The mutations in SH2B3 were described in approximately 6.1-25.0% of patients with chronic phase MPN in western countries [1,2,3,6]. In this study, the frequency of SH2B3 mutation was 2.7%, and the mutations were found only in exon 8. Recently, Ha et al. [4] demonstrated that mutational frequency of the SH2B3 gene was 7.1% in exons 7 and 8 in 42 Korean patients with chronic phase MPNs. They reported that three types of SH2B3 mutation accompanied by JAK2 V617F mutation, including p.Q423* located on exon 7, and p.R551W and p.I568T located on exon 8; the p.I568T mutation was also detected in this study. Including this data, mutations of SH2B3 are discovered in exon 7 and 8 in the Korean population, and no mutation was identified in exon 2 of SH2B3 gene. The SH2B3 is known to bind JAK2 and perform a critical role in negative regulation of downstream signal transduction [7]. Pardanani et al. reported the co-occurrence frequency of SH2B3 mutations and the JAK2 V617F mutation and found that SH2B3 mutations occur at similar frequencies in both JAK2 V617F negative and positive patients [6]. The effect of co-occurrence of SH2B3 and JAK2 V671F is still unknown, but an animal model study suggested that a more severe phenotype may result in cases with both mutations [8]. In this study, there were no significant differences in prognosis in patients with SH2B3 mutation according to the presence of JAK2 V617F mutation (Table 1). Therefore, the clinical significance might be investigated in a large-scale study. This study has some limitations, such as the small investigation size and the low detection sensitivity of the direct sequencing method. In addition, since we did not search the entire coding region, there is a possibility of SH2B3 mutations in other regions outside exons 2, 7, and 8. For further study of SH2B3, mutational analysis of either the entire coding region or additional exons needs to be considered. In conclusion, racial differences can cause variances not only in the prevalence but also in the mutational hot spot region of SH2B3. Our study suggests that SH2B3 mutations occur infrequently, and exon 8 in SH2B3 may be the most frequent mutational area in BCR-ABL negative MPN patients in Korea.

Oncogenic Drivers in Myeloproliferative Neoplasms: From JAK2 to Calreticulin Mutations.

During the past 10 years, major progress has been accomplished with the discovery of activating mutations that are associated with the vast majority of BCR-ABL negative human myeloproliferative neoplasms (MPNs). The identification in 2005 of JAK2 V617F triggered great interest in the JAK2-STAT5/STAT3 pathway. Discovery in 2006 of mutants of thrombopoietin receptor (TPO-R/MPL) and later on of mutants in negative regulators of JAK-STAT pathway led to the notion that persistent JAK2 activation is a hallmark of MPNs. In 2013, mutations in the gene coding for the chaperone calreticulin were reported in 20-30% of essential thrombocythemia and primary myelofibrosis patients. Here, we will address the question: what do we know about calreticulin that could help us understand its role in MPNs? In addition to oncogenic driver mutations, certain MPNs also exhibit epigenetic mutations. Targeting of both oncogenic drivers and epigenetic defects could be required for effective therapy.

Research progress of JAK1/2 inhibitors in the treatment of myeloproliferative neoplasms

The bcr-abl negative myeloproliferative neoplasms (MPN) include polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). Along with the detecting of JAK2 V617F gene mutation in MPN patients, a series of small molecules drugs that targeted to the mutation have been investigated in clinical trials. Ruxolitinib is the inhibitor of JAK1/2, which has been proved by USA Food and Drug Administration (FDA) and the European Medicines Agency (EMA) to treat patients with intermediate-2 or high-risk PMF or post-PV MF or post-ET MF and PV patients who are intolerant or resistant to hydroxycarbamide, attributed to a number of clinical trials, especially in COMFORT-Ⅰ and COMFORT-Ⅱ. As a result, the MPN patients have new hope for the treatment. Key words: Myeloproliferative neoplasms; Polycythemia vera; Primary myelofibrosis; Ruxolitinib

Clinical analysis of JAK2 V617F gene mutation in 120 patients with myeloproliferative neoplasms

Objective To investigate the JAK2 V617F gene mutations in myeloproliferative neoplasms (MPN) patients and its clinical significance. Methods 120 patients were detected bone marrow pathological conditions with bone marrow cytology and biopsy analysis,and monitored the Ph chromosome and bcr-abl fusion gene. Genomic DNA from bone marrow cells was extracted from 120 patients, JAK2 V617F gene mutations were tested by fluorescence quantitative PCR. Results All patients of each MPN type presented their typical characteristics. Ph chromosome and bcr-abl fusion gene were negative. The positive rate of JAK2 V617F gene mutations was 66.7 % (80/120), among them the positive rate in polycythemia vera (PV) was 72.7 % (16/22), the positive rate in essential thrombocythemia (ET) was 66.0 % (62/94), and among 4 patients primary myelofibrosis (PMF) JAK2 V617F was positive in 2 cases. In JAK2 V617F positive PV patients, the white blood cell count (P = 0.001) and platelet count (P = 0.01) were significantly higher than those in negative patients. In JAK2 V617F positive ET patients, the white blood cell count was significantly higher than that in negative patients (P = 0.006). In PMF patients, there was no significant difference (P> 0.05). Conclusion JAK2 V617F gene mutations testing is helpful to the diagnosis and definite diagnosis of bcr-abl negative MPN, which promote the early detection and treatment. Key words: Myeloproliferative neoplasms; JAK2 V617F gene mutation; Polycythemia vera; Essential thrombocythemia; Primary myelofibrosis

Analysis of CALR, JAK2 and MPL gene mutations in BCR-ABL negative myeloproliferative neoplasms

To explore the mutational status of CALR, JAK2 and MPL genes in BCR-ABL negative myeloproliferative neoplasms (MPN) patients and the clinical features of MPN patients with these mutations. A total of 246 patients with a definite diagnosis of BCR-ABL negative MPN were enrolled from January 2009 to January 2014 into this study. Among them, there were 48 cases of polycythemia vera (PV) patients, 171 cases of essential thrombocythemia (ET) patients and 27 cases of primary myelofibrosis (PMF) patients. And CALR, JAK2 V617F, 12 exons of JAK2 and MPL W515L/K genes were amplified by PCR and sequenced directly. Clinical features were also analyzed in patients. Among 246 cases of BCR-ABL-negative MPN patients, 52 cases (21.1%) had CALR mutation, 121 cases (49.2%) JAK2 V617F mutation, 0 case (0) 12 exons of JAK2 mutation, and 2 cases (0.8%) MPL W515L/K mutation, respectively. These mutations were found existing exclusively. In PV patients, the white blood cell and platelet counts in JAK2 V617F mutated group were higher than those in wild-type JAK2 V617F group, while the level of hemoglobin was higher in wild-type JAK2 V617F group (all P<0.05). In ET patients, the white blood cell count, the level of hemoglobin, the frequency of thromboembolic events and risk stratification in JAK2 V617F mutated group were higher than those in CALR mutated group (all P<0.05). In PMF patients, the level of hemoglobin in JAK2 V617F mutated group were significantly higher than those in CALR mutated group (P<0.05). The proliferative level of bone marrow, risk of thromboembolic events and stratification are lower in CALR mutated patients than those in JAK2 V617F mutated patients. The pathogenic mechanism of mutated gene should be further investigated in future.

Research progress of myeloproliferative neoplasms

The classic BCR-ABL negative myeloproliferative neoplasm (MPN) comprise polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). These three disorders are characterized by stem cell-derived clonal myeloproliferation and presence of somatic mutations. The WHO diagnostic criteria for the classic BCR-ABL negative MPN has been revised in the 2008 edition by incorporating new information about their molecular pathogenesis. Robust prognostic system for PMF has already done, and those for PV and ET are under discussion. Treatment with novel drugs is promising, and allo-stem cell transplantation (allo-ASCT) is the only curative treatment for myelofibrosis, however, the patient selection and management before transplant have been discussed for a long time.

Standardization of real-time quantitative polymerase chain reaction for detection of the JAK2V617F mutation in BCR-ABL1 negative myeloproliferative neoplasms: A tertiary care centre experience

Background: Identification of JAK2V617F mutations has led to a significant development in our understanding of the pathogenesis and therapy of BCR-ABL1 negative myeloproliferative neoplasms (MPNs). However, not all cases of BCR-ABL1 negative MPNs carry JAK2V617F mutations. The present study was undertaken with an aim to standardize the real-time quantitative polymerase chain reaction (PCR) for the detection of JAK2V617F mutations and to find the prevalence of Janus kinase 2 (JAK2) mutations in MPNs in the Indian scenario. Materials and Methods: Real-time quantitative PCR was used to detect the JAK2V617F mutation. Standardization of the detection procedure was carried out using recommended guidelines to ensure the accuracy and reproducibility of results. Forty-nine patients of BCR-ABL1 negative MPNs were included in the study. Results: The JAK2V617F mutation was detected in 63.3% patients of BCR-ABL1 negative MPNs. On classification of these BCR-ABL1 negative MPNs, JAK2V617F mutation was detected in 78.3% patients with polycythemia vera (PV), 62.5% patients with essential thrombocythemia (ET), and 44.4% patients with Primary myelofibrosis (PMF). Conclusion: Role of detection of JAK2 mutations in BCR-ABL1 negative MPN has begun to be described and can be used in the diagnosis of PV, ET, and PMF along with other criterias. In future, it may be suitable for treatment monitoring and prognostication.

Correlation of JAK2V617F mutational status in primary myelofibrosis with clinico-hematologic characteristics and international prognostic scoring system scoring: a single center experience.

Somatic mutation in the exon 14 of Janus Kinase 2 gene is an established diagnostic marker in bcr-abl negative myeloproliferative neoplasms, especially primary idiopathic myelofibrosis (PIMF). Our primary aim was to find out the correlation between the JAK2V617F mutational status and the clinico-hematologic characteristics, as well as the international prognostic scoring system (IPSS) scoring of patients with PIMF. Clinical and hematologic features were reviewed for 68 patients with primary idiopathic myelofibrosis (PIMF). JAK2V617F mutation status was analyzed by amplification refractory mutation screening-polymerase chain reaction. The patients were further stratified into low, intermediate-1, intermediate-2 and high-risk groups on the basis of IPSS scoring. The JAK2V617F mutation was detected in 58.8% patients. Univariate analysis of variables at presentation identified that JAK2V617F negative patients were significantly associated with more severe anemia (P = 0.045), younger age (P = 0.008), higher transfusion requirement (P = 0.017), and thrombocytopenia (P = 0.015). Patients who were homozygous for JAK2V617F mutation were associated with thrombocytosis (P = 0.014) and also had higher median total leucocyte count (P = 0.20) than the other groups. No significant correlation was detected between JAK2V617F mutational status and the presence of constitutional symptoms, spleen size, grade of bone marrow fibrosis or prognostic risk stratification of the PIMF patients. The variations in the prognostic implication of PIMF patients with mutation status as stated by various publications worldwide, reinstates the need for larger prospective studies using standardized JAK2V617F quantification methods as well as estimation of other newer molecular markers to develop deeper insight into various molecular alterations involving PIMF patients in India as well as worldwide.

Transformation of an Unclassified Myeloproliferative Neoplasm with a Rare BCR-JAK2 Fusion Transcript Resulting from the Translocation (9;22)(p24;q11).

BCR-ABL1 negative myeloproliferative neoplasms (MPNs) are known to contain alterations of the tyrosine kinase JAK2 (located on 9p24) that result in constitutive activation of the encoded protein. JAK2 fusions are reported in acute and chronic leukemias of myeloid and lymphoid phenotypes. Here, we report an unclassified case of MPN (MPN-U) showing a t(9;22)(p24;q11), which generates a BCR-JAK2 fusion gene by fusing the BCR at intron 13 to JAK2 at intron 17 on the derivative chromosome 22. Most reported JAK2 fusions cases reveal an aggressive clinical course and long-term remissions have only been achieved after allogeneic stem cell transplantation (ASCT). To the best of our knowledge, this is the thirteenth case reported worldwide to describe a BCR-JAK2 fusion transcript in MPN-U. The present report revealed a sustained complete clinical, hematologic, and cytogenetic remission 35 months after diagnosis and ~24 months after ASCT. Regarding BCR-ABL1 negative MPN patients this case report provides strong support for a role of JAK2 activation in the oncogenesis and suggests a possible diagnostic and therapeutic target that should be investigated.

Comprehensive Molecular Analyses of BCR-ABL1 Negative MPN Show That PMF Is Genetically Much More Heterogeneous Than ET and PV

Introduction: In the WHO classification (2008) JAK2 and MPL mutations are major criteria for the diagnosis of myeloproliferative neoplasms (MPN): polycythemia vera (PV), primary myelofibrosis (PMF), and essential thrombocythemia (ET). Cytogenetic aberrations are rare in these entities. Although the prognostic impact of JAK2 mutations beside some other gene mutations has been shown in PMF patients, the driving events for establishing accelerated phase or blast crises are unknown. In recent years, novel molecular markers such as ASXL1, SRSF2, and CALR were identified and PMF was investigated in several studies. However, comprehensive mutational analyses of MPN entities in comparison to each other are still rare.Aim: To identify gene mutations beyond JAK2, CALR, and MPL using a 28 gene panel, and to compare mutational data with clinical data and prognostic information in order to identify a risk profile.Patients and Methods: We in the first step investigated 56 patients (19 ET, 18 PMF, and 19 PV; 21 females, 35 males) diagnosed by cytomorphology following WHO criteria and accompanied by genetic studies. All patients underwent mutation analyses by a 28 gene panel containing: ASXL1, BCOR, BRAF, CALR, CBL, DNMT3A, ETV6, EZH2, FLT3-TKD, GATA1, GATA2, IDH1, IDH2, JAK2, KIT, NRAS, KRAS, MPL, NPM1, PHF6, RUNX1, SETBP1, SF3B1, SRSF2, TET2, TP53, U2AF1, and WT1. The library was generated with the ThunderStorm (RainDance Technologies, Billerica, MA) and sequenced on MiSeq instruments (Illumina, San Diego, CA). BCR-ABL1 fusion transcripts were shown to be negative in all cases by PCR. Not yet described genetic variants (n=6) were excluded from statistical analyses. Cytogenetics was available in 55/56 cases and grouped in normal karyotype (n=45, 82%) or aberrant karyotype (n=10, 18%).Results: In the total cohort JAK2 (44/56, 79%) was the most frequently mutated gene, followed by TET2 (13/56, 23%), ASXL1 (11/56, 20%), SRSF2 (7/56, 13%), and CALR (6/56, 11%). All other analyzed genes showed mutation frequencies below 10% (10 genes) or even no mutation (13 genes). Analyzing the number of mutations per patient revealed that only 4 patients showed no mutation (4/56, 7%), the great majority had 1 mutation (19/56, 34%) and 2 mutations (23/56, 41%), while 5 patients showed 3 mutations (5/56, 9%), 4 patients had 4 (4/56, 7%) and 1 patient even 5 mutations (1/56, 2%). Accordingly, the mean number of mutations per patient was 1.9. Summing up the mutations in JAK2, CALR, and MPL resulted in 52/56 (93%) patients that had a mutation in at least 1 of these genes, indicating that most of the patients had just 1 or 2 additional gene mutations to one of the 3 known key player MPN genes (mean: 1.3 additional mutations).Cytogenetically there were no significant differences between the 3 entities in frequencies of normal (65-90%) and aberrant karyotypes (11-35%), although in the PMF cohort there were more aberrant karyotypes (6/17, 35%) in comparison to ET and PV (for each 2/19, 11%).Addressing the mutation patterns of these 3 MPN entities revealed similar frequencies of TET2 mutations. In contrast, as expected JAK2 was more often mutated in PV (18/19, 95%) compared to ET (12/19, 63%, p=0.042) and PMF (14/18, 78%) and CALR was more often mutated in ET (5/19, 26%) in comparison to PMF (1/18, 6%) and PV (0/19, 0%, p=0.046). In PMF ASXL1 (8/18, 44%) and SRSF2 (6/18, 33%) were more often mutated compared to ET (1/19, 5%, p=0.008; 1/19, 5%, p=0.042) and PV (2/19, 11%; p=0.029; 0/19, 0%; p=0.008), respectively.Investigating the numbers of mutated genes per patient resulted in a significantly different distribution within MPN entities: in the ET and PV cohorts patients carried mostly 1 or 2 mutations (36/38, 95%; mean: 1.5), while in PMF 9/18 (50%) patients carried >2 mutations (mean: 2.5; p=0.045). Looking at the affected genes besides JAK2 and CALR showed that in ET and PV 4 more genes were affected, while in PMF 11 different additional genes showed mutations, indicating that PMF is genetically much more heterogeneous than ET or PV. This nicely matches to the finding that PMF is also marked by the highest cytogenetic aberration rate of these 3 BCR-ABL1 negative MPN (24-42%).Conclusions: 1)JAK2 is the most and TET2 the second most frequently mutated gene in BCR-ABL1 negative MPN. 2) Most patients carry only 1 or 2 gene mutations. 3) However, PMF patients are genetically much more heterogeneous than ET and PV patients regarding both cytogenetic and molecular alterations. DisclosuresMeggendorfer:MLL Munich Leukemia Laboratory: Employment; Novartis: Research Funding. Alpermann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Prevalence,Type, and Risk Factors for Bleeding in a Large Cohort of Myeloproliferative Neoplasm Patients

Background:The BCR-ABL negative myeloproliferative neoplasms (MPN), including polycythemia vera (PV), essential thrombocythemia (ET) and myelofibrosis (MF), share an increased risk of thrombotic and hemorrhagic complications. The risk of bleeding and thrombosis is less defined in MPN-Unclassifiable (MPN-U). Risk factors for hemorrhage are less defined compared to thrombosis, but acquired von Willebrand disease (aVWD) and thrombocytosis have been implicated. Because patients (pts) with CALR mutations have higher platelet counts compared to JAK2 V617F mutated pts, bleeding rates may be increased in the former group. Our aim was to define the prevalence and bleeding type and to evaluate whether aVWD, thrombocytosis, mutational status or treatment history associated with bleeding in a large cohort of MPN pts.Methods:The Northwestern University Electronic Data Warehouse identified MPN pts ≥18 years, last seen between 2005 and 2013. MPN was diagnosed based on clinical features, histopathology, and mutational status. A significant bleeding event was defined by a need for medical evaluation. Mutations in exon 9 of CALR were evaluated by PCR and fragment analysis. aVWD was defined by vW antigen (ag) and/or ristocetin cofactor activity below the laboratory reference range for blood type (<40% for blood group O or <53% for non blood group O; type defined by vW ag: activity ratio). Associations were tested using Fisher’s exact test; p<0.05 was considered statistically significant.Results I: Baseline characteristics351 MPN pts were identified: 142 with ET (40.4%), 118 with PV (33.6%), 62 with MF (17.7%), 4 with MDS/MPN (1.1%), and 25 with MPN-U (7%). Median follow-up was 5 years, median age at diagnosis was 54.7 years, and 58.1% were female. 73.3% were treated with aspirin, and 51.6% were treated with hydroxyurea. JAK2 V617F was identified in 178/288 patients (61.8%). Among 110 JAK2 V617F-negative samples, residual DNA was available in 46 and 15/46 (32.6%) were CALR mutated, including primarily type 1 (52-bp deletion; c.1092_1143del, N=11), type 2 (5-bp insertion; c.1154_1155insTTGTC, N=3) mutations, and 1 with a 69-bp deletion. CALR mutated patients included 10/29 ET (34.5%), 2/7 MF (28.6%), and 3/9 MPN-U (33%). To date, 34 patients in the cohort (9.8%) progressed to MF and 18 (5.2%) had leukemic transformation.Results II: Bleeding complications55 pts (15.6%) experienced 62 bleeding events, while thrombotic events were reported in 84 cases (23.9%). 19% of cases had both bleeding and thrombosis. In bleeding pts, the mean white blood count (WBC) at the time of diagnosis was 15.0 x 109/L, mean platelet count 803 x 109/L, and mean hemoglobin 12.6 x 109/L. Bleeding occurred at a median of 2 years after diagnosis. Gastrointestinal bleeding (GIB) was the most common type in 28/55 pts (50.9%), including 10 upper GIBs, 9 lower GIB, and 9 cases involving an unspecified location. Bleeding episodes from mucocutaneous sites were noted in 17/55 pts (30.9%; 9 with epistaxis, 1 with gum bleeding, and 7 with vaginal/uterine bleeding). There were 6 pts with intracranial hemorrhage. The mean platelet count at the time of bleeding was 473 x 109/L. Bleeding events were significantly more common in MPN-U (32%) than MF (19%), ET (10.6%), or PV (15.3%), (p=0.0163). Bleeding was associated with older age at diagnosis (59.4 yrs in bleeding pts vs 53.9 yrs in non-bleeders, p=0.03). There was no association between bleeding and mutational status (JAK2 V617F, CALR), gender, or aspirin use. The median ristocetin activity was 94% (N=38), vW ag 110% (N=37) and factor VIII activity 94% (N=28). Of 38 pts, there were 7 (18.4%) cases of VWD; 6 were acquired (5 with type 2, 1 with type 1) and 1 was congenital, respectively. Two of 7 pts with VWD (28.6%) had bleeding events.ConclusionsConsistent with prior reports, bleeding was less prevalent than thrombosis, associated with older age and, uniquely MPN-U, an entity with a less defined natural history compared to ET, PV, or MF. VWD was infrequently tested for, even less commonly identified, and less represented in bleeding cases. The role of routine testing for aVWD requires further definition. We did not find any association between bleeding and aspirin use or mutational status. However, a limited number of CALR mutated patients may have precluded recognition of any association, and future studies with a larger sample of CALR mutated patients are needed to identify its impact on bleeding. DisclosuresNo relevant conflicts of interest to declare.