JAK2V617F Essential Thrombocythemia Research Articles

Dysregulated Autophagy in Platelets from JAK2V617F-Positive Polycythemia Vera and Essential Thrombocythemia Underlies Bioenergetic Failure As a Possible Mechanism for Thrombohemorrhagic Complications

Background: JAK2V617F-positive polycythemia vera (PV) and essential thrombocythemia (ET) are the most common Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs). ET/PV patients have high levels of inflammation and are at increased risk of thrombosis. Platelets from patients with ET/PV are characterized by hyperreactivity (low activation threshold), contributing to the increased incidence of thrombosis in the MPNs. We previously described that platelets from PV patients accumulate dysmorphic mitochondria. Autophagy is a housekeeping and an inducible cellular process that eliminates damaged proteins and organelles (such as mitochondria and endoplasmic reticulum), providing biosynthetic and bioenergetic substrates for the cell. Dysregulated autophagy has been described in cancer, aging, and neurodegenerative, cardiac, and muscular diseases. We hypothesized that impairment of the platelet autophagic flux in ET/PV Jak2V617F alters platelet metabolism and function at the early and late stages of platelet activation. Methods: Platelets from sex and age-matched healthy subjects and patients with PV were analyzed. Platelets were incubated for two hours with chloroquine (CQ) [50 and 100 µM] to inhibit autophagy. CQ blocks the late stage of the autophagy process; therefore, functional autophagy was assessed by quantifying the accumulation of autophagic-related proteins by western blot. Oxidative stress (4-hydroxynonenal protein adducts) was measured by western blot. Platelet bioenergetics was studied by the Seahorse extracellular flux analyzer. Semi-quantitative metabolomics was performed using the Vanquish UHPLC system. Clot retraction was assessed by measuring the weight of serum extruded from the clot. Results: Platelets from healthy patients showed basal and inducible autophagy characterized by the accumulation of autophagic markers such as LC3II (p<0.0024), GABARAP, the autophagic cargo receptor p62 and the mitochondrial marker TOM20 (p<0.021). However, ET/PV platelets showed dysfunctional basal and inducible autophagy. Moreover, PV platelets and platelets treated with CQ had higher levels of ROS-damaged proteins (p<0.0001) compared to healthy platelets. Platelets from PV patients exhibited hyperreactivity by flow cytometry (PAC-1 binding, p<0.05) and under flow conditions (p<0.03). Moreover, platelets from ET/PV patients showed marked bioenergetic failure characterized by increased mitochondrial depolarization, higher intracellular calcium, depressed mitochondrial respiration, and lower levels of ADP and ATP. Pharmacological inhibition of autophagy of platelets from healthy individuals with CQ recapitulated the bioenergetic and functional profiles of platelets from patients with ET/PV. Bioenergetically, autophagy inhibition resulted in depressed basal (p<0.03) and ATP-linked respiration (p<0.01). Functionally, pharmacological inhibition of the autophagic flux led to significantly decreased clot retraction, as observed with the platelets from patients with ET/PV. Conclusion: Our results suggest that impairment of autophagy in platelets from ET/PV patients might exert a negative bioenergetic effect on platelet function, promoting platelet hyperreactivity and dysfunctional clot retraction, which would likely favor the formation of unstable clots. Our findings may explain why patients with Jak2V617F ET and PV are at increased risk for both thrombosis and hemorrhage and suggest that targeting autophagy might lower this risk. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

MPN-304: Identification of the Key Genes and Molecular Pathways in Essential Thrombocythemia Associated with JAK2V617F Mutation Using Bioinformatics Approach

Introduction: JAK2V617F mutation is one of the most common driver mutations for essential thrombocythemia (ET), found in approximately 55% of patients. Thrombotic events were reported more often in JAK2V617F-positive patients than in JAK2V617F-negative patients. This study focused on identifying the differentially expressed genes (DEGs) in JAK2V617F ET. Method: The gene expression profiles of 6 ET patients (3 JAK2V617F-positive, 3 JAK2V617F-negative) and 5 healthy individuals (GSE55976) were downloaded from the Gene Expression Omnibus (GEO) database. DEGs were identified using GEO2R. An adjusted p-value 1 were defined as the criteria for DEG. Gene ontology functional and pathway enrichment analyses of DEGs were performed using DAVID, and the protein–protein interaction network was constructed. Results: A total of 58 DEGs were identified in JAK2V617F ET compared to healthy controls. Of the 58 DEGs, 12 genes were commonly overexpressed in both JAK2V617F-positive and JAK1V617F-negative ET; 32 genes were overexpressed in JAK2V617F-positive, with no differences were found between JAK2V617F-negative ET and controls; and the remaining 14 genes were overexpressed only in JAK2V617F-positive ET (ANKRD13A, ATP6V0E1, HERPUD1, HLA-DRB3, IGK, KIAA0930, LTB, PILRA, PYCARD, RAB8B, SNAP23, SON, VCAN, YWHAB). GO analysis revealed that the 14 genes associated with JAK2V617F were significantly enriched in the components of biological processes (BP), including cell communication, leukocyte activation, and immune response. The cellular components (CC) category indicated an enrichment of genes associated with the membrane and cytoplasmic vesicle. The most significant GO terms describing molecular functions (MF) for the enriched genes were protein domain-specific binding and receptor binding. Pathway analysis revealed that most of the genes in this dataset were involved in immune system pathways, membrane trafficking pathways, and cell adhesion molecules (CAMs). Conclusion: This data manifested that the majority of the overexpressed genes in JAK2V617F ET were localized on the cell membrane and involved in cell communication and immune response. The cross-talk between inflammation and thrombosis was previously described. A more interesting finding in this dataset is that SNAP23 was identified as one of the highly connected genes. SNAP23 is known to regulate platelet granule secretion. This gives insight into SNAP23 as a potential therapeutic target in JAK2V617F ET.

Genomic Profiling of Blast Phase in Philadelphia-Negative Myeloproliferative Neoplasms

Background Classic Ph-negative myeloproliferative neoplasms (MPN) include essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF). Clonal evolution can lead MPN patients in chronic phase (CP) to developacute myeloid leukemia (AML), called blast phase (BP), a rare and late event in the natural history of MPN.Aim To evaluate differences in clinical, cytogenetic and molecular features and outcome in 92 patients with BP MPN, according to MPN diagnosis and mutational profile.Methods We identified in our database all patients affected with ET, PV and PMF who developed AML according to 2016 WHO criteria and for whom at least one DNA sample was available to define the mutational status of the 3 MPN driver genes (JAK2, CALR, MPL). Target sequencing was performed using the Illumina TruSight Myeloid Sequencing Panel kit on the HiSeq 2000 platform. For each patient circulating granulocytes or CD34+ cells were analyzed as neoplastic tissue and CD3+ T cells as control tissue; data were analyzed with the pipeline for somatic variant calling implemented at CeMM. Treatment used in BP was classified as palliation (supportive care or low intensity chemotherapy) or induction chemotherapy (de novo AML-like therapy).Results We retrospectively identified 92 patients who progressed to BP, with a known molecular profile: 29 ET (18 JAK2 V617F , 9 CALR, 1 MPL mutated and 1 triple-negative), 39 PV (all JAK2 V617F mutated) and 24 PMF patients (18 JAK2 V617F, 1 CALR, 2 MPL mutated and 3 triple-negative).Median age at BP was 70.8 years (range 46.3-86); age at evolution was not significantly different (P 0.182) in the 3 diagnostic categories (ET, PV, PMF).The complete blood count at leukemic evolution was not influenced by the initial diagnosis.Median time to leukemic evolution was 110 months (range 3.6-367.6) and it was shorter in PMF compared to ET (29.8 vs 153.4 months, P < 0.001) and PV (29.8 vs 129.7 months, P < 0.001). Moreover, time to leukemic evolution was shorter in JAK2 V617F positive ET compared to CALR mutated ET (117.2 vs 210.9 months, P 0.027), but not statistically different in JAK2 mutated compared to CALR mutated PMF (P 0.312).At the time of BP, 35 out of 49 patients (71%) for whom cytogenetic analysis was available showed an abnormal karyotype (22 patients with complex karyotype or high risk aberrations).JAK2 mutated MPN can evolve into JAK2 wild type AML (8 of 32 patients with blast DNA available), whereas CALR and MPL mutations were identified also in AML blasts in all patients for which DNA was available (3 for CALR and 3 for MPL mutated AML).We performed targeted re-sequencing in 53 patients, in 23 studying both the CP and the BP sample. Overall, we identified 176 variants in 39 different genes (68% were missense mutations); the most commonly involved pathways were DNA methylation (22%), transcriptional regulation (20%) and chromatine regulation (18%). The most frequently involved genes were TET2, TP53, RUNX1 and ASXL1 . In the 23 paired cases, we observed a statistically significant increase in the median number of mutations in the BP, compared to CP [4 (range 1-10) vs 2 (range 0-9); P 0.002]. This was observed in JAK2 mutated patients (P 0.006) and in those with PV (P 0.008), but not in subjects with TE or PMF. In our paired-sample cohort, BP evolution was associated with the acquisition of mutations (e.g. IDH1) and/or an increase in mutation occurrence or variant allele frequency (VAF) of genes implicated in leukemogenesis including TET2 (P 0.026), RUNX1 (P 0.003), ETV6 (P 0.068) and TP53 (P 0.003), the latter known to be involved also in JAK2 mutated MPN evolving into JAK2 wild type AML. In addition, VAFs suggested an heterogeneous clonal architecture and evolution of somatic mutations in these patients.Outcome was dismal, with a median OS of 3.8 months (range 3.1-5.3), independently from MPN diagnosis (P 0.603) and treatment during BP (5.6 months with induction chemotherapy vs 3.5 months with palliation; P 0.171).Conclusion Clinical phenotype and outcome of BP is not influenced neither by the diagnosis in CP nor by the type of driver mutation; likewise the outcome is poor irrespective of treatment. PMF patients have a shorter time to BP than ET and PV patients; in ET JAK2 V617F mutation is associated with a shorter time to BP compared to CALR mutation. TP53, RUNX1, TET2 and ETV6 are more frequently involved in leukemic evolution and the number of mutations increases from CP to BP. DisclosuresNo relevant conflicts of interest to declare.

NPM1+ /FLT3- Acute Myeloid Leukemia after JAK2-V617F+ Essential Thrombocythemia. Management and Prognosis

NPM1+ AML after Essential Thrombocythemia (ET) or Myeloproliferative neoplasms is extremely rare. Only 2 cases have been previously reported after Primary Myelofibrosis. Given the extremely poor prognosis of the Blastic phase of Myeloproliferative neoplasms (MPN-BP), the only curative treatment of these patients is allogeneic stem cell transplantation (allo-SCT) after achieving Complete Response (CR). De novo NPM1+/FLT3- AML is considered a good prognosis entity in which allo-SCT is not contemplated as the first option. A 41 year old diagnosed of ET JAK2 V617F+ 4 years before the diagnosis of AML with normal karyotype and NPM1+/FLT3- was treated with conventional AML induction with Cytarabine and Idarubicine and 3 cycles of high dose Cytarabine. At the diagnosis of AML other 2 mutations were noted: EZH2 and IKZ1. After treatment of AML, NPM1+ clone disappeared, and JAK2 V617F clone reappeared. We opted to treat NPM1+ AML as a de novo AML and we decided to follow up during 2 years without allo-SCT. The patient remains in complete response with NPM1 minimal residual disease negative during the follow up. This case exemplifies the nature of NPM1+ AML secondary to MPD as an extremely sensitive disease to induction therapy plus high dose cytarabine and makes that these type of patients perhaps could be managed without the use of allo-SCT.

Distinguishing essential thrombocythemia JAK2V617F from polycythemia vera: limitations of erythrocyte values.

Distinguishing essential thrombocythemia JAK2V617F from polycythemia vera is difficult because of shared mutation and phenotypic characteristics. The World Health Organization suggested hemoglobin and hematocrit values to diagnose polycythemia vera (PV), but their sensitivity and specificity were not tested. Moreover, red cell values do not accurately predict red cell mass, which we use to discriminate essential thrombocythemia JAK2V617F from PV. Eighty-three PV and 39 essential thrombocythemia JAK2V617F patients were diagnosed based on JAK2V617F positivity, chromium-51 red cell mass, and marrow biopsy findings. Red cell values used to construct a receiver operating characteristic analysis determined optimal thresholds for distinguishing essential thrombocythemia JAK2V617F from PV. Red cell value frequencies were plotted determining if overlap existed. Chromium-51 red cell mass separated PV from essential thrombocythemia JAK2V617F, but red cell values overlapped in 25.0-54.7%. Our data indicate that a significant proportion of PV patients may be underdiagnosed by using only red cell values. A bone marrow biopsy was performed in 199 of 410 (48.5%) and a serum erythropoietin value was measured in 225 of 410 (54.9%) of potential PV patients at our institution. Without isotope studies, marrow biopsies and serum erythropoietin values should improve diagnostic accuracy and become mandatory, but clinical data suggest these tests have not been routinely performed. Therefore, the clinical hematologist must be aware of imperfect accuracy when using only red cell values for distinguishing essential thrombocythemia JAK2V617F from PV.

New and treatment-relevant risk stratification for thrombosis in essential thrombocythemia and polycythemia vera.

New and treatment-relevant risk stratification for thrombosis in essential thrombocythemia and polycythemia vera.

A meta-analysis comparing clinical characteristics and outcomes in CALR-mutated and JAK2V617F essential thrombocythaemia.

A systematic review and meta-analysis were carried out to compare the clinical features and outcomes in calreticulin (CALR)-mutated and JAK2V617F patients of essential thrombocythemia (ET). Compared with JAK2V617F ET patients, CALR-mutated ET was associated with a clear increase in male predominance [OR 1.71 (95 % CI 1.28-2.28), P < 0.001, I(2)) = 51.6] and a significant decrease in thrombosis events [OR 0.40 (95 % CI 0.32-0.50), P < 0.001, I(2) = 0]. No difference was observed in hemorrhagic events [OR 0.86 (95 % CI 0.52-1.42), P = 0.558, I(2) = 0] or splenomegaly [OR 0.8 (95 % CI 0.55-1.14), P = 0.217 I (2) = 42.9]. CALR-mutated ET did not show better overall survival (OS) [HR 1.03 (95 % CI 0.74-1.44) P = 0.854, I(2) = 47.6] but showed better thrombosis-free survival (TFS) [HR 0.62 (0.44-0.87), P = 0.005, I(2) = 0] than JAK2V617F ET. In conclusion, CALR-mutated ET and JAK2V617F ET may represent two different subgroups of essential thrombocythaemia with respect to clinical features and outcomes.

In Vitro Characterization of Peripheral Blood Progenitor Cell Differentiation and Platelet Function in Essential Thrombocythemia (ET)

Bleeding and thrombotic events are major clinical complications of ET, together with progression to myelofibrosis and leukemic transformation (AML). The JAK2 V617F mutation, the thrombopoietin receptor mutation MPL W515K/L and the most recently discovered calreticulin (CALR) mutations are mutually exclusive in ET, account for up to 80-90 % of ET cases and support a novel molecular categorization of ET. However, the link between these driver mutations and ET pathogenesis and complications remains elusive.We have studied megakaryocyte (MK) differentiation of peripheral blood progenitor cells in vitro as well as the platelet aggregation capacity in ET patients. Progenitors cells were grown under standard conditions in serum-free media supplemented with stem cell factor (SCF), FLT3 ligand, erythropoietin (Epo) and thrombopoietin (TPO), and gradually were switched to TPO only media after almost a week of expansion of the hematopoietic progenitors. ET MKs are larger in size, they are more granular and they display higher ploidy levels when compared to healthy MKs. They also co-express the megakaryocytic and erythroid markers, i.e. CD41 and CD71 at higher levels suggesting aberrant lineage specification from multipotent progenitors that can give rise to both the megakaryocytic and erythroid lineage. Remarkably, ET peripheral blood progenitors have a prolonged survival in vitro under the tested culture conditions when compared to healthy MKs. Megakaryocytic cultures from healthy donors last for 12-14 days in vitro while the ET MK cultures can be maintained for up to two extra weeks. Electron microscopy analysis of cultured MKs is consistent with the morphological observations mentioned above regarding size and granularity, which is higher in ET MKs, and hence support aberrant differentiation pathways. However, no differences were found between the molecular subtypes of ET. Therefore, we performed transcriptional analysis by RNA sequencing of megakaryocytic and erythroid cultures of progenitors from JAK2 V617F ET, CALR mutant ET or triple negative ET in order to identify the molecular cues that characterize abnormal megakaryopoiesis in ET subgroups. We have analyzed three biological replicates per genotype and we are currently confirming the results by RT-PCR and functional assays in primary cells and cell lines.In parallel, we analyzed the aggregation capacity of JAK2 V617F, CALR mutant and triple negative ET platelets by a novel flow cytometry-based aggregation assay (De Cuyper IM et al. Blood 2013) in order to dissect potential signaling defects due to single receptor malfunction. We were able to pinpoint a significant defect in the response to collagen mediated by integrin a2b1 in ET platelets, regardless of the driver mutation, in comparison with platelets from healthy donors. However, convulxin-stimulated aggregation mediated by GPVI was normal. We also explored the degranulation capacity of ET platelets upon stimulation with thrombin peptide PAR-1. Platelets from CALR mutant or triple negative ET exhibited a reduced response as measured by the change of surface expression of the alpha granule protein CD62P compared to WT (healthy donors) and ET JAK2 V617F platelets. This was correlated with increased basal CD62P expression, suggesting that platelets in CALRmutant or triple negative ET degranulate spontaneously.In summary, we have developed in vitro assays to explore peripheral blood progenitor cell differentiation and platelet function in ET. Our findings indicate functional differences between JAK2 V617F ET as compared with CALR mutant or triple negative ET. The correlation between these functional differences and the clinical behavior of the molecular subtypes of ET needs to be further investigated.ReferencesCuyper IM, Meinders M, van de Vijver E, de Korte D, Porcelijn L, de Haas M, Eble JA, Seeger K, Rutella S, Pagliara D, Kuijpers TW, Verhoeven AJ, van den Berg TK, Gutiérrez L. A novel flow cytometry-based platelet aggregation assay. Blood 2013 Mar 7;121(10):e70-80 DisclosuresNo relevant conflicts of interest to declare.

JAK2V617F mutation and TNF-α expression in myeloproliferative neoplasms and their correlation

This study was aimed to explore the JAK2V617F mutation and TNF-α expression in patients with myeloproliferative neoplasm (MPN), and the relation between them so as to provide theoretical basis for clinical practice and target therapy. Sixty-two confirmed BCR-ABL-negative MPN patients and 15 healthy adults were enrolled in this study. The peripheral blood mononuclear cells of the patients and healthy controls were divided into two parts, one part was used to extract DNA, the other one was used to extract mRNA and reverse-transcribe into cDNA. Real-time fluorescent quantitative PCR was used to detect JAK2V617F mutation proportion and the expression level of TNF-α. The results showed that the positive rate of JAK2V617F mutation in MPN patients was 64.52% (40/62) , including 54.28% in essential thrombocythemia (ET) patients (19/35), 94.74% in polycythemia vera (PV) patients (18/19) and 37.50% in myelofibrosis (MF) (3/8) patients. Mutation proportions of JAK2V617F in ET, PV and MF patients were 0.838 ± 0.419, 4.417 ± 0.658, 2.746 ± 2.009 respectively. The expression of TNF-α in ET, PV and MF patients were higher than that in healthy controls: 1.7, 7.0, 8.2-fold (P < 0.05) respectively. In addition, TNF-α expression was correlated with JAK2V617F allele burden (Pearson r = 0.610,R(2) = 0.372,P = 0.005). It is concluded that TNF-α plays an important role in the pathogenesis of MPN, the TNF-α expression increases and is different in ET,PV and MF patients,which correlates with JAK2V617F allele burden.

Discriminating between essential thrombocythemia and masked polycythemia vera in JAK2 mutated patients

In patients not meeting the required hematocrit (HCT) or hemoglobin (Hb) thresholds according to BCSH and the WHO diagnostic criteria, the diagnosis of masked polycythemia vera (mPV) has been proposed. A comparison of HCT or Hb values with the expression of JAK2V617F, JAK2 exon 12, and CALR mutations in strictly WHO-defined 257 overt PV and 140 mPV (59 mPV according to BCSH) and 397 patients with essential thrombocythemia (ET) was performed. Hb and HCT thresholds of mPV patients were significantly higher than JAK2V617F ET (P < 0.0001). The best cut-off for Hb to discriminate JAK2-mutated ET from PV was 16.5 g/dL for males and 16.0 g/dL for females. For HCT, this was 49% in males and 48% in females. The proportion of patients correctly classified as ET or PV when regarding Hb or HCT levels was 95% in males and 93% in females and 94% in both males and females, respectively.

Metalloproteases Could Be Involved in Erythroid Differenciation in MPN

Abstract 5161 BackgroundSeveral hypotheses have been developed to explain how a unique mutation is related to three different phenotypes in Philadephia negative myeloproliferative neoplasms (MPN). Metalloproteases are a group of proteins involved in matrix remodelling, migration and differentiation processes. Although their role has never been studied in MPN, our group has found a differential expression of Matrix metalloproteinase-14 (MMP14) and CD44 between PV and ET JAK2V617F patients (E. Albizua et al, Ann Hematol. 2011Feb). AimsThe purpose of the present study is to validate MMP14 and CD44 differential gene expression profile between PV and ET using a proteomic-based approach and an in vitro model, with the aim of confirming MMP14 and CD44 as MPN biomarkers as well as novel targeted therapy. MethodsSeventy MPN patients diagnosed by WHO criteria were included in the study: 24 Polycythemia Vera (PV) 24 Essential Thrombocythemia (ET) JAK2V617F, 11 ET JAK2 wild type (JAK2WT) and 11 Primary Myelofibrosis (PMF). In addition 24 healthy donors were used as controls.Peripheral blood was analyzed by flow cytometry (FCM) using anti-CD44 and anti-CD45 antibodies in 10 PV and 10 ET. Fifty-five bone marrow biopsies (11 PV, 11 ET JAK2V617F, 11 ET JAK2WT, 11 PMF and 11 healthy controls) were processed to perform immunohistochemistry (IHC) with anti-MMP14 and anti-CD44 antibodies (R&D). Finally an in vitro MPN model was performed. Mononuclear cells from 10 MPN (5 PV and 5 ET) and 5 healthy controls were extracted and seeded in Methocult with IL-3, SCF and EPO. MMP14 was inhibited by Marimastat at 50μM, 25μM and 10μM; and CD44, with anti-CD44 antibody at 10mg/ml, 1mg/ml and 0.1mg/ml. Results were analyzed by BFU-E count, viability study by trypan blue and FCM employing anti-CD45, anti-CD41, anti-CD34, anti-CD71, anti-CD44, and Annexin antibodies. BFU-E DNA was extracted to analyze JAK2V617F allele burden by RT-PCR.Intracellular proteins, including phospho-proteins p38, P-p38, MEK, P-MEK, STAT1, P-STAT1, AKT and P-AKT were studied by cytometric bead array multiplexed bead-based immunoassay (CBAs) technique.The Mann-Whitney non-parametrical statistical hypothesis test was used to assess the statistical significance of our results. ResultsFirst of all, FCM of the granulocytes showed a slight over-expression of CD44 in PV population versus ET.Secondly, IHC also showed MMP14 over-expression in megakaryocites of PV (100% positive patients, 60% positive megakaryocites), and ET (100% positive patients 73% positive megakaryocites) compared to controls (36% positive subjects, 2% positive megakaryocites). Additionally 45% ET JAK2WT were MMP14 positive (20% positive megakaryocites).Significant differences in inhibition of BFU-E growth and cell proliferation were found comparing cultures either under anti-MMP14, Marimastat (50 μM and 25 μM) or under anti-CD44 (10 mg/ml and 1 mg/ml-) to cultures under no inhibitory treatment (P=0.05 and P=0.037 respectively). FCM of BFU-E cultures pointed to a significant decrease of CD71 (erythroid population) with any of the inhibitors.Over-expression of CD44 in PV compared to ET was observed and confirmed in BFU-E by FCM.All phospho-proteins studied halved after anti-CD44 (1 mg/ml) and Marimastat treatments (25 μM). ConclusionsOur results suggest that MMP14 and CD44 could play a role in erythroid differentiation by JAK-STAT, MAPK and AKT pathways. Differences between ET and PV may be caused by both molecules, which may contribute to phenotypic divergence. Additionally, MMP14 IHC could become a useful diagnostic tool in, at least, 45% of ET JAK2WT. Our results suggest that MMP14 and CD44 could become future therapeutic targets and could help to MPN diagnosis. Disclosures:No relevant conflicts of interest to declare.

Proteomic Analysis Identifies HSP70 As a Novel Target Therapy to Polycythemia Vera

Abstract 2827Background Proteomic screening is a useful tool to find new therapeutic targets. Genetic molecular studies are frequent in myeloproliferative neoplasms (MPN), however proteomic screening studies are limited.HSP70 is a Heat Shock protein related to apoptosis supression and erythroid differenciation through GATA-1. Additionally, other HSP, as HSP90. have been found as novel therapies in Polycythemia Vera (PV) and Essential thrombocythemia (ET) (Marubayashi et al, Journal of Clinical Investigation, 2010Oct).Aims To analyze the phenotypic divergence between PV and ET by a proteomic screening, and to validate these results by protein expression analysis and in vitro model of MPN, with the aim of identifying alternative routes for targeted therapy.Methods Seventy-one MPN diagnosed by WHO criteria were included in the study: 25 PV, 24 ET JAK2V617F, 12 ETJAK2 Wild Type (JAK2WT) negative and 11 Primary Myelofibrosis (PMF). Additionally 24 healthy subjects were used as controls.First of all, granulocytes from whole venous peripheral blood were isolated and the corresponding cytosolic protein fraction was extracted. Cytosolic proteomes of 10 PV, 10 ET and 10 healthy subjects were analyzed using 2D-DIGE gels followed by MALDI-TOFTOF mass spectrometry (MS) analysis of the spots of interest. Results were analyzed with DeCyder v7.0 and Mascot software. Secondly, bone marrow biopsies (BMB) of 55 MPN patients (11 PV, 11 ET JAK2V617F, 11 ET JAK2WT, 11 PMF and 11 healthy controls) were selected to perform immunohistochemistry (IHC) with anti-HSPA1A (HSP70), anti-SERPINB1 and anti-LTA4H. Afterwards, western blot of these three proteins was performed.Finally, an in vitro model of MPN was employed. Mononuclear cells from 4 PV, 4 ET and 3 healthy donors were extracted and seeded in Methocult with IL-3, SCF and EPO. A HSP70 inhibition assay was performed by the drug KNK437 at 100μM, 50μM and 10μM. Results were analyzed by BFU-E count, viability study by trypan blue and flow cytometry (FCM) employing anti-CD45, anti-CD41, anti-CD34, anti-CD71, and Annexin antibodies.Intracellular proteins including phospho-proteins p38, P-p38, MEK, P-MEK, STAT1, P-STAT1, AKT1 and P-AKT were studied by cytometric bead array multiplexed bead-based immunoassay (CBAs) technique. The Mann-Whitney non-parametrical statistical hypothesis test was used to assess the statistical significance of our results.Results 2D-DIGE analysis found 112 spots with statistically significant differences in protein expression between PV and ET samples. On the other hand, 241 spots showed differential expression between ET and healthy donors, and 229 between PV and healthy donors. We identified, by MS, 67 proteins differentially expressed between PV and ET, 20 between PV and controls and 6 between ET and controls. A large number of these proteins were metabolic and citoskeleton proteins, as Lactotranferrin, Enolase, Actin, etc. However, three spots were especially interesting according to our hypothesis: SERPINB1, LTA4H and HSPA1A (HSP70); the last, chaperone related with GATA-1 and erythroid differentiation. IHC showed over-expression of HSPA1A in granulocytes of PV (72% positives patients, 80% positive granulocytes) compared to ET (50% positive patients, 30% positive granulocytes). Finally, significant differences in inhibition of BFU-E growth and cell proliferation were found between cultures treated with HSP70 inhibitor, KNK437 (100μM and 50μM) versus cultures without treatment (P=0.029). FCM of BFU-E cultures pointed to a dramatic increase of erythroid apoptotic cells. CBAs analysis showed a decrease of P-STAT1 and P-MEK in cells of cultures under KNK437 treatment.Conclusions Our results suggest that HSP70 could play a key role in erythroid survival and differentiation through JAK-STAT and MAPK pathways. These molecules might contribute to their phenotypic divergence. Finally, HSP70 could be a new therapeutic target, and KNK437 a novel PV treatment. Disclosures:No relevant conflicts of interest to declare.

355 5q deletion acquisition was associated with myelodysplastic evolution in patient with essential thrombocythemia JAK2 V617F mutation

355 5q deletion acquisition was associated with myelodysplastic evolution in patient with essential thrombocythemia JAK2 V617F mutation

High Rates of Molecular Response After Long-Term Follow-up of Patients with Advanced Essential Thrombocythemia (ET) or Polycythemia Vera (PV) Treated with Pegylated Interferon-ALFA-2A (PEG-IFN-α-2A; PEGASYS)

AbstractAbstract 461 Background:The use of IFN-α in polycythemia vera (PV) and essential thrombocythemia (ET) has been hampered by poor tolerance and inconvenient dosing schedules. The covalent attachment of polyethylene glycol to IFN-α renders a molecule with prolonged serum half-life, which can be administered weekly. Objectives:We conducted a phase II study of subcutaneous PEG-IFN-α-2a (Pegasys) in 84 patients (pts) with high-risk PV (n=44) or ET (n=40). We performed high throughput mutational analysis of JAK2, MPL, TET2, and ASXL1 in all pts. Patients and Therapy:Median age was 51 years (range, 18–79), time from diagnosis to PEG-IFN-α-2a 51 months (range, 0–355), and number of prior therapies was 1 (range, 0–6), including hydroxyurea (HU; n=47), anagrelide (AG; n=26), IFN-α (n=12: 5 oral and 7 sc), imatinib (n=7), and dasatinib (n=1). PEG-IFN-α-2a was initial therapy in 16 (19%) pts (7 PV) that refused HU. JAK2V617F was detected in 19/40 (48%) ET and in 42/44 (95%) PV pts. Nine (11%) pts had abnormal cytogenetics. Initial PEG-IFN-α-2a starting dose was 450 mcg/wk, but that was modified to the current starting dose of 90 mcg/wk. Results:After a median follow-up of 40 months (range, 8–62), 66/83 (80%) assessable pts have responded. Median time to response was 4 weeks (range, 0.5–26). Complete response (CR) was achieved by 62 (75%) pts (for ET: platelets <440×109/L, in the absence of thromboembolic events; for PV: Hb <15 g/dL, no phlebotomy, disappearance of splenomegaly) whereas 4 (5%) pts (2 PV, 2 ET) had a partial response ([PR]; no phlebotomy, off HU and AG, still palpable spleen). Of 5 pts with abnormal karyotype at study entry who were evaluable for response, 2 reverted to diploid cytogenetics.JAK2V617F to total JAK2 ratio was determined by quantitative pyrosequencing assay in all 84 pts prior to PEG-IFN-α-2a. Sixty-one (73%) pts carried the JAK2V617F mutation, which was quantitated at least once on therapy in 54 (64%). Overall, 29 (54%) had >20% reduction in JAK2V617F allele burden, including 10 (19%) in whom the mutation became undetectable (complete molecular response [CMR]) and 15 (28%) who had a >50% reduction (partial molecular response). Molecular responses have not yet reached a plateau among pts with PV.We also analyzed pts for mutations in exon 12 of JAK2, MPL, and the tumor suppressors TET2 and ASXL1 to determine their impact on response to PEG-IFN-α-2a. No pts carried JAK2 exon 12 mutations. One JAK2V617F−negative pt with ET had a MPLW515L mutation, achieved CHR but did not achieve a molecular response. Full length resequencing of all exons of TET2 and ASXL1 genes identified somatic TET2 mutations in 9/71 (13%) and somatic ASXL1 mutations in 3/71 (4%) pts; we identified TET2 (3 JAK2V617F− ET, 2 JAK2V617F+ ET, 3 JAK2V617F+ PV, 1 JAK2V617F− PV) and ASXL1 (1 pt with ET JAK2V617F+, ET JAK2V617F−, and PV JAK2V617F+) mutations in PV and ET pts who were JAK2V617F–positive and negative. TET2 or ASXL1 mutational status did not impact the likelihood of achievement of JAK2 molecular responses, and there was no difference in JAK2V617F allele burden with PEG-IFN-α-2a according to TET2 or ASXL1 mutational status. One pt with baseline mutations in JAK2, TET2, and ASXL1 became JAK2V617F–negative on PEG-IFN-α-2a.Most pts had grade 1–2 toxicities but at doses ≤90 mcg/wk, grade 3–4 toxicity was infrequent. Twenty-five (30%) patients were taken off study after a median of 9 months (range, 3–36) but only 13 (15%) of them due to therapy-related toxicity: g3 neutropenia, anorexia, depression (n=3), ischemic retinopathy, g2 fatigue (n=5), dyspnea, g2 neuropathy. The remaining 59 pts are currently receiving 450 mcg/wk (n=1), 360 mcg/wk (n=1), 240 mcg/wk (n=1), 180 mcg/wk (n=2), 135 mcg/wk (n=3), 90 mcg every 1 (n=8), 2 (n=12), 3 (n=2), or 4 wks (n=1), 45 mcg every 1 (n=9), 2 (n=5), 3 (n=6), or 4 wks (n=8). Conclusion:PEG-IFN-α-2a is remarkably active and acceptably safe in advanced, previously treated PV and ET. Clinical responses are frequently accompanied by significant reduction of JAK2V617F allele burden, which becomes undetectable in a proportion of them suggesting selective targeting of the JAK2V617F clones. Quantitative analysis of ASXL1 and TET2 mutational allele burden during PEG-IFN-α-2a therapy to determine clonal evolution, and methylcellulose-based clonogenic assays in pts who achieved CMR to assess for the presence of erythropoietin independent colony formation are ongoing and will be presented. Disclosures:No relevant conflicts of interest to declare.

Sequence Analysis of MPL Exon 10 of ET and MF JAK2 V617F Negative Patients Reveals a New Mutation

Background. Diagnosis and classification of myeloproliferative disorders was greatly enhanced by reports describing JAK2V617F mutation in Polycythemia Rubra Vera (PRV) patients and approximately 50% of Essential Thrombocythemia (ET) and Myelofibrosis (MF) patients. More recently mutations mapping to the juxtamembrane region of the thrombopoietin receptor MPL were described in 5–10% of the patients with MF and 1% of the patients with ET. The reported data imply the Exon 10 MPL mutations lead to receptor activation in the absence of thrombopoietin binding and constitutive phosphorylation of JAK2, STAT3, STAT5, AKT and ERK. However, thus far only mutations mapping to codons 515 and 505 have been published. The aim of this study was to determine the frequency of JAK2V617F and MPL exon 10 mutations in MF and ET patients seen at the Hematology Department, Centro Hospitalar de Coimbra.Methods. Sixty three patients with ET and 12 with MF, diagnosed according to the Polycythemia Vera Study Group (PVSG) criteria, were studied. Genomic DNA was extracted from peripheral blood samples collected into EDTA. Samples were tested for JAK2 V617F mutation by allele specific polymerase chain reaction (ASO-PCR). Subsequently these samples were screened for MPL exon 10 mutations by direct sequencing.Results. 45 (71.4%) of the 63 patients with ET and 6 (50%) of the 12 MF patients had JAK2V617F mutation. Of the remaining 6 MF patients 3 had MPL mutations; two with W515L and one with W515K amino acid substitutions. Direct sequencing failed to detect exon 10 MPL mutations in 17 of the 18 ET JAK2 V617F negative patients. The remaining patient had a previously unreported MPL mutation mapping to codon 524 where C was substituted by T, predicting a replacement of arginine residue by cysteine (R524C).Conclusions. JAK2 V617F mutation was detected in 71% of the ET patients and in 50% of the MF. MPL mutations were found in 2 out of 12 MF patients (17%) and in one of the 63 ET patients (1.6%). The MPL codon 515 mutations are reported to activate mpl protein independent of ligand binding. The prevalence of MPL mutations among MF patients is higher than the described in previous reports, however this maybe due to the small number of patients studied. Whilst there is genotype and phenotype correlation between MPD and MPL mutations described the precise functional role in the development of MPD remain unclear. Similarly, whether the previously unreported MPL R524C mutant allele presented here leads to activation of mpl protein like codon 515 mutations is unclear. But nonetheless such mutations assist in accurate diagnosis and classification of Philadelphia negative MPD patients with the view to optimization of clinical management of these patients.

Impact of V617F JAK2 Mutation on Monocyte Tissue Factor and Procoagulant Activity in Patients with Essential Thrombocythemia(ET) or Polycythemia VERA (PV)

Clinical data suggest an increased thrombotic risk in patients with ET or PV carrying the JAK2V617F mutation. Laboratory data from our group show that ET patients carrying the JAK2V617F mutation are characterized by an enhanced platelet and neutrophil activation status (Falanga et al, Exp Hem 2007) and blood coagulation activation (Marchetti et al, Blood 2008), as compared to JAK2 wild-type ET. Since monocytes significantly contribute to blood coagulation activation as an important source of circulating tissue factor (TF), in this study we aimed to characterize the prothrombotic phenotype of monocytes from ET and PV patients and to evaluate whether and to what extent it is influenced by the JAK2V617F mutation. Twenty-four ET patients (10 JAK2 wild-type; 14 JAK2V617F carriers with 2%–35% mutant allele burden), 27 PV patients (all JAK2V617F carriers, 16 with 9%– 44% and 11 with 60%–100% allele burden, respectively), and 20 age-matched healthy subjects (controls, C) were enrolled into the study. Monocyte-associated TF antigen was measured on the cell surface by whole blood flow-cytometry, in both basal condition and after in vitro stimulation by bacterial endotoxin (lypopolysaccharide, LPS), as well as in cell lysates by ELISA. Monocyte procoagulant activity was evaluated by the Calibrated Automated Thrombogram (CAT) as the capacity of isolated monocyte lysates to induce thrombin generation in normal pool plasma. In basal conditions, significantly (p<0.05) higher surface levels of TF were measured on monocytes from ET (17.1±3.2% positive cells) and PV (24.4±3.7% positive cells) patients compared to C (8.2±1.9% positive cells). Similarly, the total TF antigen content of cell lysates was significantly increased in patients compared to C. The analysis of the data according to JAK2V617F mutational status, showed a gradient of increased TF expression starting from JAK2V617F negative patients (11.7±2.5%), versus JAK2V617F ET and PV subjects with <50% allele burden (20.3±3.6% and 23.2±2.8%, respectively), versus JAK2V617F PV patients with >50% allele burden (26.1±4.2%). The in vitro LPS stimulation significantly increased TF expression on monocytes from all study subjects and C compared to non-stimulated monocytes (p<0.05 for all groups), with a more elevated expression by monocytes from PV and ET patients compared to C. However, the relative increase in TF expression was greater in C (=3.7 fold) compared to both ET (=2.2 fold) and PV (=2 fold) patients. As observed in basal conditions, LPS-induced TF was higher in JAK2V617F positive patients as compared to negative, with the highest expression in JAK2V617F PV carriers with >50% allele load. Thrombin generation induced by monocytes from ET and PV patients was significantly increased compared to controls, as determined by significantly higher thrombin peaks (ET=145±12, PV=142±17, C=72.2±5 nM), and quantity of thrombin generated in time, i.e. the endogenous thrombin potential (ETP) (ET=1143±34, PV=1074±64, C=787±58 nM*min). The JAK2V617F PV subjects with >50% allele burden presented with the highest thrombin generation capacity (peak= 184±34 nM; ETP= 1268±32 nM). Our data indicate that the expression of the JAK2V617F mutation in ET and PV patients may confer to monocytes a different hemostatic phenotype in terms of increased expression of surface TF and thrombin generation capacity. These findings are in agreement with the previous observation of a hypercoagulable state associated with this mutation and suggest a new mechanism linking hemostatic cellular phenotypic alteration to genetic dysfunction in patients with myeloproliferative disease.

Different involvement of the megakaryocytic lineage by the JAK2V617F mutation in Polycythemia vera, essential thrombocythemia and chronic idiopathic myelofibrosis

Atypical megakaryocytes provide the histomorphological hallmark of all Philadelphia-chromosome negative chronic myeloproliferative disorder (Ph(-) CMPD) subtypes and have not been studied so far for the JAK2(V617F) mutation. The mutant gene dosage was determined in isolated megakaryocytes from 68 cases of JAK2(+)/Ph(-) CMPD by a pyrosequencing assay. Megakaryocytes from essential thrombocythemia (ET) showed significantly lower levels of mutated JAK2 alleles compared to patients with chronic idiopathic myelofibrosis (cIMF) with manifest fibrosis and polycythemia vera (PV) but not to prefibrotic cIMF. Solely, ET JAK2V617F in megakaryocytes is associated with a PV-like phenotype, and at least in one patient, the JAK2 mutation was exclusively acquired within the megakaryocytic lineage. The overt differences between prefibrotic and fibrotic cIMF suggested a causative role of the gene dosage of mutant JAK2 in fibrotic progression. Megakaryocyte analysis of a follow-up of eight individual cases with sequential biopsies, however, showed that progression to homozygosity of V617F mutated JAK2 and onset of manifest fibrosis appeared to be independent events. We conclude that megakaryocytes might be the predominant or even the exclusive lineage that acquires the JAK2(V617F) mutation in ET and that the JAK2(V617F) evolution to higher gene dosages represents a dynamic and complex process substantially involving megakaryocytes.

Risk of thrombosis in patients with essential thrombocythemia and polycythemia vera according to JAK2 V617F mutation status

We compared the laboratory and clinical findings of 179 patients with essential thrombocythemia (ET) and 77 with polycythemia vera (PV) classified according to the presence of the JAK2 V617F mutation. A gradient was observed in laboratory values between patients with JAK2 wild-type ET, JAK2 V617F ET and PV (all of whom carried the JAK2 mutation). The rate of thrombotic complications in JAK2-positive ET patients was significantly higher than that in wild-type ET patients and not statistically different from that in PV patients.

JAK2 V617F Mutation in Essential Thrombocythemia: Clinical Associations and Long-Term Prognostic Relevance.

Background: In essential thrombocythemia (ET), homozygosity for the JAK2 V617F mutant allele is rare despite the frequent (23–57%) occurrence of the heterozygous state. This is in direct contrast with the situation in polycythemia vera (PV) where homozygosity for the mutation is seen in 25–33% of the cases. The particular observation is consistent with what has been observed in vitro as well as in animal studies where JAK2 V617F has been associated with induction of erythropoietin hypersensitivity in cell lines and erythrocytosis in mice (James et al. Nature 2005; 434:1144). Therefore, regardless of whether or not JAK2 V617F represents a primary event in myeloproliferative disorders, it is probable that it promotes a PV phenotype. In the current study, we explore this hypothesis as well as investigate the long-term prognostic relevance of the mutation in a well defined cohort of patients with ET seen at a single institution.Methods: The diagnosis of ET was according to the WHO classification of tumors (Jaffe ES et al. Lyon, IARC press, 2001). All patients were seen at the Mayo Clinic and patient characteristics and subsequent clinical events were determined both retrospectively (chart review) and prospectively (by means of a questionnaire sent both to patients and their primary doctors). In addition date and cause of death were confirmed by probing both the social security index and the national death index. DNA for mutation screening was derived and sequenced from either archived unsorted cells from the bone marrow or prospectively collected peripheral blood granulocytes according to previously published methods (Levine et al. Cancer Cell , 2005; 7:387).Results: The study cohort consisted of 150 consecutive patients with ET including 99 females and 51 males (median age 49.8 years; range 16.8–98). Overall, JAK2 V617F was detected in 73 patients (48.7%) but none were homozygous for the mutant allele. At diagnosis, ET patients with JAK2 V617F were significantly older (median age of 53.5 vs. 42.3; p=0.02) and also displayed higher readings of both hemoglobin level (p=0.0002) and leukocyte count (p=0.005).All study patients have now been followed for a median of 11.4 years from diagnosis (range 0–32.7) and follow-up period was similar between the two mutational categories (p=0.53). During this period, 32 patients have died and the median survival for the entire group was 19.3 years; 21.9 and 17.7 years in the absence and presence of the mutation, respectively (p=0.09). Disease transformation into acute leukemia, myelofibrosis, and PV occurred in 6 (4%), 15 (10%), and 8 (5.3%) patients, respectively, and the only significant difference between the two mutational categories in this regard was the higher incidence of transformation into PV in patients with the mutation (p=0.02). Multivariate analysis identified age, hemoglobin level, and thrombosis history at diagnosis but not JAK2 V617F mutational status as independent risk factors for survival.Conclusion: Although the presence of JAK2 V617F in ET appears to promote a PV phenotype, it might not carry treatment-relevant information.

X-Linked Clonality Analysis and Quantitative JAK2V617F Assessment Reveals a Strong Association between Clonality and JAK2V617F in PV but Not in ET/MMM, and Identifies a Subset of JAK2V617F Negative ET and MMM Patients with Clonal Hematopoiesis.

Polycythemia vera (PV), essential thrombocythemia (ET), and myeloid metaplasia with myelofibrosis (MMM) are categorized as a set of phenotypically related myeloproliferative disorders (MPD). Recent work has identified a somatic mutation in the JAK2 tyrosine kinase (JAK2V617F) in the majority of PV and in a subset of ET and MMM. As most, but not all, patients with MPD have clonal granulocytes, we sought to determine if there was a relationship between granulocyte clonality and JAK2V617F mutational status using a quantitative clonality assay for the human androgen receptor gene (HUMARA) and a quantitative real-time PCR assay for JAK2V617F. 168 of 190 female MPD patients were informative for the HUMARA polymorphism, and 121 patients (72%) had allele skewing consistent with clonal granulopoiesis including 80% of MMM, 75% of PV, and 67% of ET. The quantitative real-time PCR assay for JAK2V617F detected the JAK2V617F allele in 98.8% of PV, 69% of ET, and 33% of MMM, and was more sensitive than direct sequencing. A significant correlation between the degree of allele skewing and quantitative JAK2V617F allelic ratio was observed in PV (p<0.0001) but not in ET (p= 0.2) or MMM (p=0.771). In addition, a subset of patients with ET and MMM with clonal granulocytes had JAK2V617F present in some, but not all, clonal granulocytes. These data suggest acquisition of the JAK2V617F mutation may be sufficient for the development of PV, but additional genetic events are necessary to cooperate with JAK2V617F in ET and MMM. Most importantly, we identified a subset of patients with ET (23%) and MMM (17%), but not PV, with clonal granulocytes who were JAK2V617F negative. A high-throughput screen for novel activating mutations in signal transduction pathways in JAK2V617F negative ET and MMM is warranted.