JAK2V617F-positive Essential Thrombocythemia Research Articles

A rare case of concurrent JAK2V617F-positive essential thrombocythemia, multiple myeloma, and colorectal adenocarcinoma

ABSTRACT Multiple myeloma (MM), essential thrombocythemia (ET), and colorectal adenocarcinoma (CA) are three distinct diseases. The co-occurrence of MM, ET, and CA in a single patient is exceedingly rare. Our study presents a remarkable case involving a 75-year-old patient who was simultaneously diagnosed with these three diseases. The JAK2V617F mutation was detected in the bone marrow with a variant allele frequency (VAF) of 13.38%, whereas the isolated CD138-positive plasma cells exhibited a significantly lower VAF of 0.38%. This notable difference implies the potential for separate origins of MM and ET. Additionally, the patient responded well to a bortezomib-based treatment regimen, despite the absence of specific therapy for ET. We believe that our findings add new understanding to these rare diseases and encourage further research in this area.

Pronounced gut microbiota signatures in patients with JAK2V617F-positive essential thrombocythemia.

Essential thrombocythemia (ET) is part of the Philadelphia chromosome-negative myeloproliferative neoplasms. It is characterized by an increased risk of thromboembolic events and also to a certain degree hypermetabolic symptoms. The gut microbiota is an important initiator of hematopoiesis and regulation of the immune system, but in patients with ET, where inflammation is a hallmark of the disease, it is vastly unexplored. In this study, we compared the gut microbiota via amplicon-based 16S rRNA gene sequencing of the V3-V4 region in 54 patients with ET according to mutation status Janus-kinase 2 (JAK2V617F)-positive vs JAK2V617F-negative patients with ET, and in 42 healthy controls (HCs). Gut microbiota richness was higher in patients with ET (median-observed richness, 283.5; range, 75-535) compared with HCs (median-observed richness, 191.5; range, 111-300; P < 0.001). Patients with ET had a different overall bacterial composition (beta diversity) than HCs (analysis of similarities [ANOSIM]; R = 0.063, P = 0.004). Patients with ET had a significantly lower relative abundance of taxa within the Firmicutes phylum compared with HCs (51% vs 59%, P = 0.03), and within that phylum, patients with ET also had a lower relative abundance of the genus Faecalibacterium (8% vs 15%, P < 0.001), an important immunoregulative bacterium. The microbiota signatures were more pronounced in patients harboring the JAK2V617F mutation, and highly similar to patients with polycythemia vera as previously described. These findings suggest that patients with ET may have an altered immune regulation; however, whether this dysregulation is induced in part by, or is itself inducing, an altered gut microbiota remains to be investigated. IMPORTANCE Essential thrombocythemia (ET) is a cancer characterized by thrombocyte overproduction. Inflammation has been shown to be vital in both the initiation and progression of other myeloproliferative neoplasms, and it is well known that the gut microbiota is important in the regulation of our immune system. However, the gut microbiota of patients with ET remains uninvestigated. In this study, we characterized the gut microbiota of patients with ET compared with healthy controls and thereby provide new insights into the field. We show that the gut microbiota of patients with ET differs significantly from that of healthy controls and the patients with ET have a lower relative abundance of important immunoregulative bacteria. Furthermore, we demonstrate that patients with JAK2V617F-positive ET have pronounced gut microbiota signatures compared with JAK2V617F-negative patients. Thereby confirming the importance of the underlying mutation, the immune response as well as the composition of the microbiota.

Efficacy and safety of peginterferon-α2b for treatment of myeloproliterative neoplasms

To evaluate the clinical efficacy and adverse reactions of peginterferon-α2b for treatment of chronic myeloproliferative neoplasms (MPN). We retrospectively analyzed the data of 107 patients with MPN, including 95 with essential thrombocythemia (ET) and 12 with polycythemia vera (PV), who all received peginterferon-α2b treatment for at least 12 months. The clnical and follow-up data of the patients were analyzed to evaluate the efficacy and adverse reactions of the treatment. After receiving peginterferon- α2b treatment, both ET and PV patients achieved high hematological remission rates, and the total remission rates did not differ significantly between the two groups (86% vs 78%, P>0.05). In the overall patients, the spleen index decreased by 13.5% (95%CI: 8.5%-18.5%) after the treatment. The patients with hematological remission showed a significantly greater reduction of the total symptom score than those without hematological remission (P < 0.01). The median percentage of JAK2V617F allele load of PV patients decreased from 67.23% (49.6%-84.86%) at baseline to 19.7% (0.57%-74.6%) after the treatment, and that of JAK2V617F-positive ET patients decreased from 48.97% (0.45%-74.24%) at baseline to 22.1% (0.33%-65.42%) after the treatment. Mild adverse reactions (grade 1-2) were observed in both ET and PV groups without significant differences between them. The overall incidence of thrombotic events during the treatment was 2.8% in these patients, and no serious adverse reactions were observed. For patients with chronic myelodysplasia, peginterferon-α2b treatment can achieve a high peripheral blood cell remission rate and maintain a long-term stable state with good effect in relieving symptoms such as splenomegaly. Peginterferon- α2b treatment caused only mild adverse reactions, which can be tolerated by most of the patients.

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

Clinical features and outcomes of JAK2 V617F-positive polycythemia vera and essential thrombocythemia according to the JAK2 V617F allele burden.

BackgroundJAK2 mutation status is a well-known risk factor for thrombosis in patients with myeloproliferative neoplasms. However, the clinical usefulness of JAK2 V617F allele burden is under investigation.MethodsWe retrospectively evaluated the impact of the JAK2 V617F allele burden on clinical characteristics and outcomes of JAK2 V617F-positive polycythemia vera (PV) and essential thrombocythemia (ET). The JAK2 V617F allele burden was measured using sequencing.ResultsAltogether, 127 patients with JAK2 V617F mutation (PV, N=61; ET, N=66) were included in this study. JAK2 V617F allele burdens were positively correlated with white blood cell counts, hemoglobin values, lactate dehydrogenase levels, and platelet counts. The median values of JAK2 V617F allele burden in patients with PV and ET were 58% and 30%, respectively. A JAK2 V617F allele burden of ≥30%, older age, and a higher hemoglobin level were risk factors for thrombotic events in ET. In patients with PV, older age was the only thrombotic risk factor. The 8-year probabilities of overall survival (OS) were 82.9% in all patients. A high JAK2 V617F allele burden (≥58%) was associated with poor OS in patients with PV. For the patients with ET, the difference in 8-year OS based on the JAK2 V617F allele burden was not significant.ConclusionThe JAK2 V617F allele burden was correlated with hematologic parameters and clinical outcomes. Assessing the JAK2 V617F allele burden can be helpful in predicting the thrombotic risk and disease course in patients with JAK2 V617F-positive PV and ET.

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.

Comparison of the effects between MPL and JAK2V617F on thrombosis and peripheral blood cell counts in patients with essential thrombocythemia: a meta-analysis

To assess the effects between MPL and JAK2V617F on the thrombosis risk and peripheral blood cell counts in patients with essential thrombocythemia (ET), we identified eligible studies from PubMed, Embase, and the Cochrane Library. Seven studies were ultimately included in this meta-analysis. All studies reported the peripheral blood cell counts of ET patients, and three of them reported the eligible thrombotic events. In comparing the effect of MPL versus JAK2V617F on thrombosis, 1257 ET patients (73 MPL + and 1184 JAK2V617F +) were included. MPL-positive (MPL +) ET patients had a higher risk of thrombosis than JAK2V617F-positive (JAK2V617F +) ET patients [RR = 1.80 (1.08–3.01), P = 0.025]. And 3453 ET patients (138 MPL + and 3315 JAK2V617F +) were included in the comparison of peripheral blood cell counts. Platelet counts of MPL + ET patients were higher than that of JAK2V617F + ET patients [WMD = 81.18 (31.77–130.60), P = 0.001]. MPL + ET patients had lower hemoglobin [WMD = − 11.66 (− 14.32 to − 9.00), P = 0.000] and white blood cell counts [WMD = − 1.01 (− 1.47 to − 0.56), P = 0.000] than JAK2V617F + ET patients. These findings indicate that the MPL mutation is a high-risk factor for thrombosis in ET patients, and it may be rational to include MPL mutation in the revised IPSET as a criterion for thrombosis prediction scores. And given the differences in peripheral blood, it is necessary to further study whether MPL + ET patients differ from JAK2V617F + ET patients in bleeding and survival.

Immune thrombocytopenic purpura with subsequent development of JAK2 V617F-positive essential thrombocythemia: Case Report

The sequential occurrence of Immune Thrombocytopenic Purpura (ITP) and Essential Thrombocythemia (ET) has been reported in the literature on a few occasions, as these are two hematologic disorders with distinct etiologies and patients usually have contrasting clinical presentations.

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.

Open Label Phase I Study of Single Agent Oral RG7388 (idasanutlin) in Patients with Polycythemia Vera and Essential Thrombocythemia

Background: Polycythemia vera (PV) and essential thrombocythemia (ET) are associated with an increased risk of thrombosis, progression to myelofibrosis (MF) and significant symptom burden. While hydroxyurea, interferon, and JAK2 inhibitors are used to treat these myeloproliferative neoplasms (MPNs), hematopoietic stem cell-depleting therapies may result in an alternative mechanism to alter disease course and improve outcome. MDM2, a negative regulator of P53, is overexpressed in CD34+ MPN cells harboring wild type P53. It is the target of idasanutlin (IDA), an oral MDM2 antagonist that results in up-regulation of P53 and down-stream activators of apoptosis. Preclinical data from our laboratory group supports the clinical evaluation of IDA in patients (pts) with ET/PV (Lu et al Blood. 2014;124(5):771-9). We evaluated the safety and tolerability of IDA in a phase I dose escalation trial of pts with JAK2V617F-positive ET or PV [NCT02407080].Methods: 13 pts (1 withdrew prior to treatment) resistant/intolerant to hydroxyurea and/or interferon therapy and without prior JAK2 inhibitor therapy were enrolled in a single institution phase I trial of oral IDA at two dose levels of 100 mg and 150 mg daily for five consecutive days and repeated every 28 day cycles. The first cycle consisted of 56 days for the evaluation of dose limiting toxicities (DLT). A DLT was defined as any non-hematologic AE of grade 3+ or a hematologic AE of grade 2+ thrombocytopenia, or grade 3+ neutropenia or anemia. The absence of DLT in the initial cohort of 100 mg daily in 3 evaluable pts allowed for dose escalation in a new cohort to 150 mg and after a total of 6 pts were treated at this level an additional 3 pts were treated at 100mg to complete the intended study. Pts that did not attain at least a partial response (PR) by modified European LeukemiaNet (ELN) criteria after cycle 6 and met eligibility were able to continue receiving IDA in combination with pegylated interferon-α (PEG) at 45ug weekly. Correlative studies included baseline mutational profiling, MDM2 protein levels, changes in serum MIC-1 levels and JAK2V617F variant allele frequency (VAF) with therapy.Results: 12 pts (6 at 100 mg; 6 at 150 mg) were treated and their baseline characteristics are shown in Table 1. Pts received a median of 7 cycles (range, 1-12) over a median of 33 weeks (range, 8-107) on study. Three pts discontinued treatment due to pt decision (n=2) and physician decision (n=1). A DLT was not identified for either dose level during cycle 1, and no hematologic treatment emergent adverse events (TEAE) were seen at any time point. Table 2 shows the non-hematologic TEAEs that occurred in at least 2 pts regardless of attribution. Grade 3 fatigue (n=1) and grade 3 headache (n=1) were the only significant TEAE noted at any time on study (both in 100mg cohort). The overall response rate by modified ELN criteria by cycle 7 for the 9 evaluable pts was 7/9 (78%). 7 of 10 (70%) evaluable pts achieved a ≥50% improvement in total symptom score (TSS) from baseline. There were no thrombotic or major hemorrhagic episodes, or progression to MF. Bone marrow pathologic response was assessed in 2 pts after 5 cycles. One case showed histological improvement, with normalization of overall marrow cellularity and megakaryocyte number, and less pronounced megakaryocytic atypia. The median JAK2V617F VAF at baseline, 7 cycles, and 9 cycles of therapy was 45% (n=12), 12% (n=6) and 13% (n=6), respectively. One pt was eligible to receive combination therapy and achieved phlebotomy freedom, normalization of palpable spleen (baseline 18cm), leukocyte count (baseline 44 x109/L), and PV-related symptoms by cycle 8. This pt also attained a 20% reduction in JAK2V617F VAF.The percentage of mononuclear cells expressing MDM2 was dramatically increased as compared to normal controls (p=0.01) (Figure 1). Serum levels of MIC-1 was used to assess pharmacodynamic effects of IDA therapy. Serum MIC-1 levels were elevated (~ 5 fold) after 5 days of administration of IDA in 85% of the pts (Figure 2), indicating activation of P53 by the low doses of IDA administered. Persistent elevation in MIC-1 levels was observed on day 15 in 6 of the 12 pts treated.Conclusion: IDA is well tolerated and demonstrates a clear signal of clinical activity in pts with therapy refractory PV. Correlatives support on-target activity. Mature follow up on the entire cohort will be presented. A multicenter phase II trial of IDA at a dose of 150 mg is underway [Display omitted] DisclosuresMascarenhas:CTI Biopharma: Research Funding; Janssen: Research Funding; Promedior: Research Funding; Novartis: Other: DSMB member , Research Funding; Merck: Research Funding; Incyte: Other: Clinical Trial Steering Committee , Research Funding.

Clinical Research: Treatment with Warfarin Followed By Hydroxyurea for Thrombosis Caused By JAK2V617F-Positive Essential Thrombocythemia Associated with Mutation in the Thrombomodulin Gene and Deficiency of Protein S

JAK2V617F-positive essential thrombocythemia is rarely associated with mutation in the thrombomodulin gene and deficiency of protein S without being reported in the literature. A case of a previously healthy 50-year-old Chinese female presenting with superior mesenteric vein thrombosis and portal thrombosis showed by enhanced computed tomography(CT) scan is reported. Blood platelet count showed 644×109/L. Molecular testing for genetic mutation found a JAK2V617F mutation by 11089 copies per 100ng DNA revealed by real-time polymerase chain reaction (RT-PCR), with the mutation rate being over ten percent. Level of free protein S decreased to 30.4%(reference:70%-140%). Heterozygous missense mutation(c.404C&amp;gt;G, i.e.nucleotide 404 of encoding region was mutated from cytosine to guanine) in exon 1 in the thrombomodulin gene was detected in peripheral blood, which caused p.Pro135Arg(i.e.amino acid No.135 was changed from proline to arginine). The chromosomal position of thrombomodulin gene was at chr20:23029738. Family investigation showed that a similar mutation in the thrombomodulin gene was detected in peripheral blood in her daughter. Low molecular weight heparin was given at the beginning of treatment, and bleeding was monitored. In the meantime, warfarin was administered orally and the dosage of which was adjusted to maintain the international normalized ratio of prothrombin time at the range of 2 to 3. Adjust the dosage of hydroxyurea according to blood cell count. A month later, no new thrombosis was found in enhanced CT scan. It was an amazing feat that the patient was in a state of remission without new thrombosis being found for a year. JAK2V617F-positive essential thrombocythemia with mutation in the thrombomodulin gene and deficiency of protein S tends to be incurable. Warfarin alone with hydroxyurea may serve as an effective option to those cases. Disclosures No relevant conflicts of interest to declare.

Overexpression of PD-L1 Correlates with JAK2-V617F Mutational Burden and Is Associated with Chromosome 9p Uniparental Disomy in MPN

Myeloproliferative neoplasms (MPN) are characterized by clonal hematopoiesis, hyperproliferation of myeloid cells, hyperinflammation and immune deregulation. The three classical BCR-ABL1-negative MPN are essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF). The disease is driven by JAK2, CALR or MPL somatic mutations in most patients. Drug resistance is a major problem in MPN. Recent data suggest that MPN cells display certain immune checkpoint molecules that may contribute to resistance, including PD-L1. Antibodies targeting the PD1/PD-L1 axis are highly promising anti-cancer drugs. Their potential use in MPN is being explored but it is unclear which MPN subtypes are most suitable for testing in clinical trials. The aim of our project was to assess PD-L1 expression in disease-initiating neoplastic stem cells (SC) and differentiated cells of MPN patients and to develop therapeutic approaches capable of blocking PD-L1 expression in MPN SC. In a first step, PD-L1 expression was assessed by RNA-sequencing of granulocytes of 106 MPN patients and 15 healthy donors (HD). The cohort included 56 PMF, 33 ET and 17 PV patients. For 102 patients data from Human Genome-wide Affymetrix 6.0 SNP arrays were available. We observed a ~5-fold higher expression of PD-L1 mRNA in patients with PV compared to other MPN (P&amp;lt;.01) or HD (P&amp;lt;.01). JAK2-V617F positive ET patients had higher expression of PD-L1 compared to CALR-mutated ET (p&amp;lt;.005) and the same was observed in PMF (p&amp;lt;.01). Other mutations (TET2, DNMT3A) detected by NGS did not affect PD-L1 expression. Since PD-L1 and JAK2 are located on chromosome 9p24, we looked into our previously published dataset of 400 MPN patients analyzed by SNP arrays and found that in all 195 patients with 9p uniparental disomy (UPD) the aberrations covered both genes. As PD-L1 is more centromeric it could represent the second target of 9pUPD which can precede the acquisition of JAK2-V617F in MPN. Granulocytes in JAK2-V617F positive patients with 9pUPD expressed significantly higher levels of PD-L1 compared to patients without 9pUPD (P&amp;lt;.0001; Figure 1A). Moreover, the JAK2-V617F mutational burden significantly correlated with PD-L1 expression (R=.52, P&amp;lt;.0001; Figure 1B). This correlation was lost when cases with 9pUPD were excluded from the analysis (R=.03, P=.9), indicating that the UPD is relevant for PD-L1 upregulation. To investigate PD-L1 surface expression on MPN SC we analyzed CD34+CD45dimCD38- cells isolated from fresh bone marrow (BM) samples of another 51 MPN patients and 7 HD by flow cytometry (FC). MPN patients showed a significantly higher surface expression of PD-L1 on CD34+CD45dimCD38- cells compared to HD (p&amp;lt;.001; Figure 1C). PD-L1 levels on the SC surface were elevated in both JAK2- and CALR-mutated MPN patients compared to HD (p&amp;lt;.001 and P&amp;lt;.005, respectively). PD-L2 was neither expressed in MPN granulocytes nor on MPN SC. CD4+ and CD8+ T-cells from BM samples of 17 MPN patients expressed the PD-L1 receptor PD-1 as assessed by FC. We cultured ex vivo primary MPN cells from 7 JAK2-V617F positive patients and showed that PD-L1 expression on MPN SC spontaneously decreases in culture, that interferon-gamma (IFN-γ) can promote expression of PD-L1 on these cells, and that ruxolitinib and the BRD4-degrader dBET6 block IFN-γ-induced PD-L1 expression in CD34+CD45dimCD38- MPN SC (P&amp;lt;.05). Together, we show that PD-L1 is overexpressed on the surface of disease-initiating MPN SC, that PD-L1 mRNA is overexpressed in granulocytes of MPN patients and that PD-L1 overexpression in granulocytes correlates with the JAK2-V617F mutational burden. In patients with JAK2-V617F positive MPN, 9pUPD leads to further PD-L1 upregulation either through increasing the mutant JAK2 gene dosage, loss of wt-JAK2,or amplification of PD-L1 allele with higher expression. Our data suggest the possibility that 9pUPD and the subsequent elevation of PD-L1 expression may provide an immune escape mechanism and may contribute to positive selection of JAK2-V617F homozygous SC. Ruxolitinib and dBET6 downregulate PD-L1 expression on MPN SC suggesting a role for the JAK2 and BRD4-MYC pathway. As recent studies revealed an immunogenic potential of JAK2 and CALR mutants, overcoming the disease-mediated immune escape may be of particular importance. Further preclinical and clinical studies are now required to examine the value of PD1/PD-L1 inhibitors in patients with MPN. Disclosures Gisslinger: Celgene: Honoraria; MyeloPro Diagnostics and Research: Honoraria; AOP Orphan Pharmaceuticals AG: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; PharmaEssentia: Honoraria; Janssen-Cilag: Honoraria; Roche: Honoraria. Kralovics:AOP Orphan Pharmaceuticals AG: Honoraria; PharmaEssentia: Honoraria; Qiagen: Honoraria; Novartis: Honoraria; MyeloPro Diagnostics and Research: Current equity holder in private company. Valent:Allcyte GmbH: Research Funding; Pfizer: Honoraria; Cellgene: Honoraria, Research Funding.

Progression of coronary calcium burden and carotid stiffness in patients with essential thrombocythemia associated with JAK2 V617F mutation

Background and aimsPatients with myeloproliferative neoplasms often succumb to cardiovascular events, but little is known on the early stages of their vascular disease. We studied how patients with JAK2 V617F positive essential thrombocythemia (ET) without overt atherosclerotic disease differed from control subjects in the progression of carotid artery stiffness and preclinical atherosclerosis. MethodsThirty-six patients with JAK2 V617F positive ET and 38 age-, gender- and Framingham coronary heart disease (CHD) -matched control subjects were examined twice within 4 years. Clinical and laboratory testing, echo-tracking ultrasound of carotid arteries, coronary calcium measurement and digital plethysmography were performed (ClinTrials.gov NCT03828422). ResultsCoronary calcium correlated with the Framingham CHD risk score at the first examination in the control group (rs = 0.410), but not among the ET patients (rs = 0.116). Both groups progressed in coronary calcium, but the outliers were more prominent among ET patients. Carotid artery stiffness increased with time in the ET patients much more than in the control group: the increase in β-index 1.95 (SD 2.18) vs. 0.22 (SD 1.99), p < 0.001, and the increase in carotid pulse wave velocity 0.72 (SD 0.92) vs. 0.08 (SD 0.72) m/s, p = 0.001. There was no correlation between carotid stiffness and Framingham CHD risk in either group. Digital endothelial function did not change. ConclusionCarotid artery stiffness progressed faster in patients with JAK2 V617F positive ET than in control subjects. Coronary calcium correlated with the Framingham CHD risk only in control subjects. This indicates that JAK2 V617F positive ET acted as a non-classical risk factor for vascular disease.

JAK2-negative acute monocytic leukemia with TET2 mutation in essential thrombocythemia with JAK2 mutation with literature review

Essential thrombocythemia (ET) is an indolent myeloproliferative neoplasm (MPN) with a transformation to acute myeloid leukemia in <5% of patients. A 79-year-old man with JAK2V617F-positive ET exhibited leukocytosis with an increase in monoblastic cells, leading to a diagnosis of acute monoblastic and monocytic leukemia. Leukemic cells carried a TET2 mutation but not JAK2V617F mutation. We concluded that the TET2 mutation occurred in MPN-initiating cells and overcame JAK2-mutated cells. The absence of a JAK2 mutation in the leukemic cells in this case suggests the leukemia emerged from a JAK2-negative MPN cell clone carrying the TET2 mutation.

Clinical Characteristics and Cardioavscular Events in Patients with Esential Thrombocythemia with

Introduction The clinical characteristics, treatment, cardiovascular events (CVE) and evolution of patients diagnosed with JAK2 V617F positive essential thrombocythemia (ET) with low allele burden (LAB) are scarcely studied. Its presence in people without a confirmed diagnosis of malignant hemopathy is called clonal hematopoiesis of uncertain significance (CHIP) and confers higher risk of developing CVE. The objective of this study was to compare the clinical characteristics and CVE of a series of JAK2 V617F-positive ET patients with &lt;10% (LAB) vs. ≥10% allele burden (HAB), from the GEMFIN (Grupo Español de Enfermedades Mieloproliferativas Crónicas Filadelfia Negativas) Group. Methods From the database of the GEMFIN group, 410 ET patients were JAK2 V617F positive, 89 (21.7 %) with LAB and 321 (78.3%) with HAB. The clinical characteristics, treatment (cytoreduction, antiagregation, anticoagulation, JAK inhibitor), CVE (before, at and after diagnosis) and evolution to myelofibrosis (MF) or acute myeloid leukemia (AML) of these two groups of patients were compared. Results LAB and HAB groups did not significantly differ regarding the main clinical characteristics (i.e cardiovascular risk factors [CVRF] and International Prognostic Score for Thrombosis in Essential Thrombocythemia [IPSET] score) except for the median platelet count: LAB 636 x109/L [436- 2500] vs HAB 687 x109/L[440-1980L], p=0.035). CVE after diagnosis of ET were more frequent in patients with HAB (41/137, 30%) than in patients with LAB (5/48, 10%), p=0.007. Only one LAB patient with CVE had JAK2 allele burden &gt;5%. Treatments received by both groups were not significantly different. None of the patients from both groups progressed to AML, whereas 1/48 vs. 6/137 of patients evolved to MF. Median follow-up of patients with LAB and HAB was 3.4 years [0.1-17.7] and 4.3 years [0.1-27.8], respectively (Table 1). Conclusions In these series of ET patients from the GEMFIN group, patients with LAB had significantly lower median platelet count at diagnosis and less CVE after diagnosis than patients with HAB, although CVRF and IPSET scores and treatment approach were similar. The clinical behavior of LAB patients may resemble that of individuals with CHIP. The therapeutic algorithm of ET patients with LAB may be somehow different than that of patients with HAB and therefore, might be revised. Disclosures Bellosillo: Astra-Zeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees; Biocartis: Honoraria; Merck-Serono: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Qiagen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Hoffman â€"La Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; ThermoFisher: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria; BMS: Honoraria. Hernandez Boluda:Incyte: Other: Travel expenses paid. Pérez:Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Oxidative Stress Levels, JAK2V617F Mutational Status and Thrombotic Complications in Patients with Essential Thrombocythemia

Essential thrombocythemia (ET) is a BCR-ABL1-negative myeloproliferative neoplasm associated with thrombotic and haemorrhagic complications. Reactive oxygen species (ROS) overexpression induces a growth advantage to JAK2V617F-positive clones and, in association with a higher number of immature platelets, leukocytosis, and additional cardiovascular risk factors, leads to an increased risk for thrombotic events. We evaluated oxidative stress by measuring ROS levels and the total antioxidant capacity (TAC) in 62 ET patients and investigated the relationship between oxidative stress, JAK2V617F mutational status and the development of thrombotic events. We found higher oxidative stress levels in JAK2V617F-positive vs. JAK2V617F-negative ET cases with no significant differences between homozygous and heterozygous genotypes. Increased ROS levels and thrombotic events were more frequent in ET patients with old age at diagnosis, higher haematocrit levels or leukocytosis.

Multilevel defects in the hematopoietic niche in essential thrombocythemia.

The role of the bone marrow niche in essential thrombocythemia (ET) remains unclear. Here, we observed multilevel defects in the hematopoietic niche of patients with JAK2V617F-positive ET, including functional deficiency in mesenchymal stromal cells (MSC), immune imbalance, and sympathetic-nerve damage. Mesenchymal stromal cells from patients with JAK2V617F-positive essential thrombocythemia had a transformed transcriptome. In parallel, they showed enhanced proliferation, decreased apoptosis and senescence, attenuated ability to differentiate into adipocytes and osteocytes, and insufficient support for normal hematopoiesis. Additionally, they were inefficient in suppressing immune responses. For instance, they poorly inhibited proliferation and activation of CD4-positive T cells and the secretion of the inflammatory factor soluble CD40-ligand. They also poorly induced formation of mostly immunosuppressive T-helper 2 cells (Th2) and the secretion of the anti-inflammatory factor interleukin-4 (IL-4). Furthermore, we identified WDR4 as a potent protein with low expression and which was correlated with increased proliferation, reduced senescence and differentiation, and insufficient support for normal hematopoiesis in MSC from patients with JAK2V617F-positive ET. We also observed that loss of WDR4 in MSC cells downregulated the interleukin-6 (IL-6) level through the ERK–GSK3β–CREB signaling based on our in vitro studies. Altogether, our results show that multilevel changes occur in the bone marrow niche of patients with JAK2V617F-positive ET, and low expression of WDR4 in MSC may be critical for inducing hematopoietic related changes.

Acute transmural myocardial infarction by coronary embolism in a patient with JAK2 V617F-positive essential thrombocythemia.

Acute transmural myocardial infarction by coronary embolism in a patient with JAK2 V617F-positive essential thrombocythemia -

Bone Marrow Histology Characteristics in MPL515 Mutated Thrombocythemia with Various Degrees of Myelofibrosis: A Cross Sectional Follow-up Study in Eight Cases

MPLW515l/K mutated Essential Thrombocythemia (ET) usually present with increased platelet counts around 1000 x 109/L as the only abnormal laboratory finding with normal values for hemoglobin and leukocytes and minor splenomegaly on palpation only in a few. Early stage MPL515 mutated ET show the presence of clustered small and giant megakaryocytes with pronounced deeply lobulated nuclei, which are not seen in JAK2V617F positive ET, prodromal PV, and classical PV. Indicating that MPL515 mutated ET has no clinical, laboratory and bone marrow features of prodromal PV. Clustering of large mature large to giant megakaryocytes with pronounced hyper lobulated nuclei in a normocellular bone marrow is the hallmark of JAK2-wild type MPL515 mutated thrombocythemia. Bone marrow histology in MPL515 mutant patients revealed isolated megakaryocytic proliferation in a normocellular bone marrow at diagnosis with a reduction of erythropoiesis. MPL515 Thrombocythemia do not have PV features at diagnosis, do not evolve into PV during follow-up, have normal LAP score, serum EPO and ferritin levels. In contrast to JAK2V617F positive ET, no spontaneous endogenous erythroid colonies (EEC) was found in any of evaluated MPLW515L cases. Spontaneous megakaryocyte growth in culture with an overall normal response to thrombopoietin (TPO) has been demonstrated in two MPL515 mutated cases.

A phase 1 study of the Janus kinase 2 (JAK2)V617F inhibitor, gandotinib (LY2784544), in patients with primary myelofibrosis, polycythemia vera, and essential thrombocythemia

Mutations in Janus kinase 2 (JAK2) are implicated in the pathogenesis of Philadelphia-chromosome negative myeloproliferative neoplasms, including primary myelofibrosis, polycythemia vera, and essential thrombocythemia. Gandotinib (LY2784544), a potent inhibitor of JAK2 activity, shows increased potency for the JAK2V617F mutation. The study had a standard 3+3 dose-escalation design to define the maximum-tolerated dose. Primary objectives were to determine safety, tolerability, and recommended oral daily dose of gandotinib for patients with JAK2V617F-positive myelofibrosis, essential thrombocythemia, or polycythemia vera. Secondary objectives included estimating pharmacokinetic parameters and documenting evidence of efficacy by measuring clinical improvement. Thirty-eight patients were enrolled and treated (31 myelofibrosis, 6 polycythemia vera, 1 essential thrombocythemia). The maximum-tolerated dose of gandotinib was 120mg daily, based on dose-limiting toxicities of blood creatinine increase or hyperuricemia at higher doses. Maximum plasma concentration was reached 4h after single and multiple doses, and mean half-life on day 1 was approximately 6h. Most common treatment-emergent adverse events were diarrhea (55.3%) and nausea (42.1%), a majority of which were of grade 1 severity. Best response of clinical improvement was achieved by 29% of myelofibrosis patients. A ≥50% palpable spleen length reduction was observed at any time during therapy in 20/32 evaluable patients. Additionally, ≥50% reduction in the Total Symptom Myeloproliferative Neoplasm Symptom Assessment Form Score was seen in 11/21 (52%) and 6/14 patients (43%) receiving ≥120mg at 12 and 24 weeks respectively. Gandotinib demonstrated an acceptable safety and tolerability profile, and findings at the maximum-tolerated dose of 120mg supported further clinical testing. Clinicaltrials.gov identifier: NCT01134120.