La Polyglobulie de Vaquez (PV) est une néoplasie myéloproliférative caractérisée par une prolifération clonale des cellules souches hématopoïétiques provoquant une croissance excessive du tissu ...myéloïde. La mutation somatique gain de fonction JAK2V617F, présente dans la lignée myéloïde de plus de 95% des patients PV, provoque une activation constitutive de la protéine JAK2 et une prolifération cellulaire anormale indépendante des cytokines. Cette mutation entraine une augmentation du nombre de globules rouges (GR) et un risque élevé de thromboses, principale cause de morbidité et de mortalité chez ces patients. Les anomalies génétiques ont été largement étudiées dans la PV pour expliquer la prolifération anormale des lignées myéloïdes chez ces patients. Au contraire, les anomalies protéiques restent actuellement peu caractérisées. Dans cette thèse nous avons étudié (1) les effets de la mutation JAK2V617F sur l’expression des protéines à la membrane des GR PV, (2) l’effet des traitements par hydroxycarbamide (HC) et interféron-alpha (IFN-alpha) sur les propriétés des GR PV et (3) l’impact de l’expression de JAK2V617F dans les cellules endothéliales. Une analyse protéomique comparative a montré une expression anormale de protéines du réticulum endoplasmique, telles que la calréticuline et la calnexine, à la membrane des GR PV. Nous avons montré que le traitement par HC augmentait l’expression de plusieurs protéines à la membrane des GR PV, comme la protéine d’adhérence Lu/BCAM qui se retrouve activée par phosphorylation dans ces cellules, augmentant leur adhérence à la laminine. Enfin, au niveau endothélial, nous avons montré que la mutation JAK2V617F induisait un phénotype pro-angiogénique et pro-adhésif de ces cellules par l’activation des voies de signalisation STAT3 et AKT. Ces découvertes révèlent un rôle nouveau de la mutation JAK2V617F dans les globules rouges et les cellules endothéliales en plus de son rôle connu dans la prolifération cellulaire
Polycythemia vera (PV) is a myeloproliferative neoplasm characterized by increased red cell mass and a high risk of thrombosis considered as a major cause of mortality and morbidity in this disease. The vast majority of PV patients exhibit the V617F activating mutation in the tyrosine kinase JAK2. Genetic abnormalities have been widely studied in PV to explain the abnormal proliferation of myeloid cell lines in this disease but protein abnormalities remain poorly characterized. In this thesis, we studied (1) the effect of JAK2V617F on the protein expression at the membrane of PV red blood cells (RBCs) (2) the impact of hydroxycarbamide (HC) and interferon-alpha (IFN-alpha) treatments on PV RBC properties and (3) the influence of the JAK2V617F mutation on endothelial cells. Our comparative proteomic analysis revealed an abnormal expression of endoplasmic reticulum proteins, such as calreticulin and calnexin, in the membrane of PV RBCs. We showed that HC treatment increased the expression of several proteins on the PV RBC surface, including the adhesion molecule Lu/BCAM that we found activated in these cells leading to increased adhesion to laminin. In endothelial cells, we showed that the JAK2V617F mutation mediated pro-angiogenic and pro-adhesive phenotypes of these cells by activating the STAT3 and AKT signaling pathways. These findings reveal a new role of the JAK2V617F mutation in RBCs and endothelial cells in addition to its well-known effect on cell proliferation
Patients with Philadelphia-negative myeloproliferative neoplasms (MPNs), particularly those carrying the JAK2V617F mutation, are at increased risk of thrombosis. While an association of MPNs with ...autoimmune disorders has been established, the prevalence of inherited or acquired thrombophilias in JAK2V617F-positive patients remains obscure. We therefore investigated the coincidence of the JAK2V617F mutation with additional thrombogenic risk factors.
In a retrospective study, we analyzed all patients referred for thrombophilia work-up between 01/2011 and 08/2019, in whom additional JAK2V617F mutation analysis was performed because of thromboembolic events that were recurrent, atypically located and/or associated with abnormal blood counts.
Of 472 tested patients, 49 (10.4%) were JAK2V617F-positive. While the frequency of inherited thrombophilias (factor V Leiden and prothrombin G20210A mutation, deficiency of antithrombin, protein C, protein S) was not different between the two groups, the prevalence of definite antiphospholipid syndrome (APS), mostly associated with a moderate- or high-risk antibody profile, was significantly higher in patients with (22.4%) than in those without (8.4%) JAK2V617F mutation (p < 0.01). All evaluable JAK2V617F-positive patients with APS were subsequently diagnosed with MPN. In patients with JAK2V617F mutation, presence of concomitant APS was associated with a significantly younger age (49 ± 14 vs. 60 ± 15 years) at the time of thrombophilia work-up (p < 0.05).
We found a significant association between JAK2V617F-positive MPN and definite APS. The presence of concomitant APS in patients carrying the JAK2V617F mutation may lead to earlier manifestation of thromboembolic events and may warrant more aggressive antithrombotic treatment strategies to prevent recurrence.
•The JAK2V617F (JAK2) mutation confers an increased risk of thromboembolism (TE).•The role of additional thrombophilias in JAK2-positive (JAK2+) patients is unclear.•We retrospectively analyzed 472 TE patients, 49 of whom carried a JAK2 mutation.•Diagnosis of JAK2 was associated with definite antiphospholipid syndrome (APS).•JAK2+ patients with APS were significantly younger than JAK2+ patients without APS.
Myeloproliferative neoplasms (MPN) are a heterogeneous group of hematopoietic stem-cell diseases with excessive proliferation of one or more blood cell lines. In this study, we evaluated the effect ...of different oxygen concentrations on HIF-1α and NOS3 gene expression to determine the effect of the bone marrow microenvironment on JAK2V617F positive Philadelphia chromosome negative (Ph−) MPNs.
Peripheral blood mononuclear cells (MNC) of 12 patients with Ph− MPN were collected. The presence of JAK2V617F allele status was determined with allele-specific nested PCR analysis. MPN CD34+ and CD34depleted populations were isolated from MNC by magnetic beads. Separate cell cultures of CD34+/depleted populations were managed at different oxygen concentrations including anoxia (∼0%), hypoxia (∼3%), and normoxia (∼20%) conditions for 24 h. HIF-1α and NOS3 gene expression changes were examined in each population related to JAK2V617F status with real time RT-PCR.
It was revealed that relative HIF-1α and NOS3 expressions were significantly increased in response to decreased oxygen concentration in all samples. Relative HIF-1α and NOS3 expressions were found to be higher especially in CD34+ and CD34depleted populations carrying JAK2V617F mutations compared to MPN patients carrying wild-type JAK2.
JAK2V617F might have specific role in HIF-1α and NOS3 regulations with respect to low oxygen concentrations in Ph− MPN. Further evaluations might reveal the effect of JAK2V617F on Ph− MPN pathogenesis in bone marrow microenvironment.
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.
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 <0.05 and |log2FC|>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.
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).
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.
Thromboembolic events and bleeding episodes are the main complications of myeloproliferative neoplasms (MPNs). JAK2V617Fmutation is a known thrombotic risk factor in MPNs, while few data correlate ...JAK2V617F with bleeding risk. Therefore, the thrombosis/hemorrhage risk reassessment in MPN patients should be reappraised. The aim of our study is to evaluate the impact of JAK2V617V mutation positivity on bleeding complications in MPNs. A cross-sectional study was performed at two tertiary centers in upper Egypt, Assiut and Qena University Hospitals, from January 2019 to December 2020. Newly diagnosed MPN patients underwent history taking, physical examination, basic laboratory work, and molecular assessment of both the JAK2V617Vmutation and Philadelphia chromosome. The cohort included 78 Philadelphia-negative MPN patients, 55% of whom were female, and the median age was 57 (range 40–85). Half of the patients were polycythemia rubra vera (PRV; n=40, 51%), while one-third were primary myelofibrosis (PMF; n=28, 36%), and only 13% were essential thrombocytosis (ET; n=10). The median total leukocytic count (TLC) was 9.6 (range: 0.3–190) × 109/L, median hemoglobin level (HB) was 15 (range: 4–21) g/dl, and median platelet count was 359 (range: 3–2960) × 109/L. Almost half of the patients (n=35; 45%) harbor JAK2V617F. Regarding patients’ clinical criteria based on JAK2V617V mutation, there was no significant difference in itching (6% for JAK2V617F-positive group vs 9% JAK2V617F-negative group, P=0.6) and splenomegaly (71% vs 67%, P=0.8). Erythema was significantly higher in the JAK2V617F-positive group (34% vs 14%; P=0.0008). The main observation was that thrombosis occurred significantly more often in the JAK2V617V-positive group (49% vs 16%; P=0.0003). On the contrary, there was no significant difference in bleeding between the JAK2V617F-positive (23%) and JAK2V617F-negative groups (28%; P=0.6). A multivariate model was performed to identify the significant independent variables that lead to bleeding, which showed that only platelets (odds ratio=1) and splenomegaly (odds ratio=8.5) had significant effects. In conclusion, the data confirmed that JAK2V617V mutation is associated with an increased risk of thrombosis in MPNs, but it does not have a role in increased bleeding risk in MPNs. Large prospective studies are needed to confirm the role of JAK2V617V in bleeding during the course of MPNs and its relation to different lines of treatment.
Bilateral adrenal infarction is an extremely rare disease, and it has been reported that some coagulation abnormalities, including essential thrombocythemia (ET), exist in the background. We herein ...report a 76-year-old patient in whom the platelet count had been in the normal range at the onset of adrenal infarction but subsequently increased to 102×104/μL at 7 months later, leading to the diagnosis of JAK2V617F-positive ET. As the presence of the JAK2V617F mutation increases the risk of thrombosis, JAK2 genetic testing should be considered in some cases of nonspecific unknown thrombosis, even if there are no obvious hematological findings, such as clonal hematopoiesis of indeterminate potential (CHIP).
von Willebrand factor ristocetin cofactor (vWF activity) and platelet count (PLT) are negatively correlated in patients with polycythemia vera (PV) and essential thrombocythemia (ET). However, vWF ...activity does not always normalize upon controlling PLT in those patients. To address this issue, we investigated the correlation between vWF activity and PLT in PV and ET patients. The negative correlation between vWF activity and PLT was stronger in calreticulin mutation‐positive (CALR+) ET than in Janus kinase 2 mutation‐positive (JAK2+) PV or ET groups. When PLT were maintained at a certain level (<600 × 109/L), low vWF activity (<50%) was more frequently observed in JAK2+ PV patients than in JAK2+ ET (p = .013) or CALR+ ET (p = .013) groups, and in PV and ET patients with ≥50% JAK2+ allele burden than in those with allele burden <50% (p = .015). High vWF activity (>150%) was more frequent in the JAK2+ ET group than in the CALR+ ET group (p = .005), and often associated with vasomotor symptoms (p = .002). This study suggests that some patients with JAK2+ PV or ET have vWF activity outside the standard range even with well‐controlled PLT, and that the measurement of vWF activity is useful for assessing the risk of thrombosis and hemorrhage.
It has been postulated that the changes in the molecular characteristics of the malignant clone(s) and the abnormal activation of JAK-STAT signaling are responsible for myeloproliferative neoplasm ...progression to more advanced disease phases and the immune escape of the malignant clone. The continuous JAK-STAT pathway activation leads to enhanced activity of the promoter of CD274 coding programmed death-1 receptor ligand (PD-L1), increased PD-L1 level, and the immune escape of MPN cells. The aim of study was to evaluate the
PDL1
mRNA and
JAK2
mRNA level in molecularly defined essential thrombocythaemia (ET) patients (pts) during disease progression to post-ET- myelofibrosis (post-ET-MF). The study group consisted of 162 ET pts, including 30 pts diagnosed with post-ET-MF. The
JAK2
V617F,
CALR
, and
MPL
mutations were found in 59.3%, 19.1%, and 1.2% of pts, respectively. No copy-number alternations of the
JAK2
,
PDL1
, and
PDCDL1G2
(
PDL2
) genes were found. The level of PD-L1 was significantly higher in the
JAK2
V617F than in the
JAK2
WT,
CALR
mutation-positive, and triple-negative pts. The PD-L1 mRNA level was weakly correlated with both the
JAK2
V617F variant allele frequency (VAF), and with the JAK2V617F allele mRNA level. The total
JAK2
level in post-ET-MF pts was lower than in ET pts, despite the lack of differences in the
JAK2
V617F VAF. In addition, the PD-L1 level was lower in post-ET-MF. A detailed analysis has shown that the decrease in
JAK2
and
PDL1
mRNA levels depended on the bone marrow fibrosis grade. The
PDL1
expression showed no differences in relation to the genotype of the
JAK2
haplotype
GGCC_46/1
, hemoglobin concentration, hematocrit value, leukocyte, and platelet counts. The observed drop of the total
JAK2
and
PDL1
levels during the ET progression to the post-ET-MF may reflect the changes in the
JAK2
V617F positive clone proliferative potential and the PD-L1 level–related immunosuppressive effect. The above-mentioned hypothesis is supported by The Cancer Genome Atlas (TCGA) data, confirming a strong positive association between
CD274
(encoding PD-L1),
CXCR3
(encoding CXCR3), and
CSF1
(encoding M-CSF) expression levels, and recently published results documenting a drop in the CXCR3 level and circulating M-CSF in patients with post-ET-MF.
Several studies reported the impact of JAK2V617F allele burden on phenotypic properties and clinical course in Philadelphia-negative myeloproliferative neoplasms (Ph-negative MPNs). We aimed to ...analyze the association of mutated JAK2V617F allele burden with clinical phenotype and laboratory characteristics in essential thrombocythemia (ET) and primary myelofibrosis (PMF) patients.
A total of 202 consecutive Philadelphia-negative MPN patients, 118 with ET and 84 with PMF, were included in this study.
We used a real-time, semi-quantitative polymerase chain reaction (PCR) with JAK2 MutaScreen assay (Ipsogen, Luminy Biotech, Marseille, France) to screen JAK2V617 mutations and mutant allele burden.
According to the JAK2V617F status, ET patients were stratified into three groups: JAK2V617F mutation-negative (n=54) and JAK2V617F-positive with mutant allele burden in the lower quartile (n=59) and upper quartile (n=5) ranges. ET patients with upper quartile JAK2V617F allele burden had significantly lower Hgb levels (p=0.001), lower Htc levels (p=0.001), and higher LDH levels (p=0.008) as compared to the other two groups. Also, ET patients with upper quartile JAK2V617F allele burden had significantly increased spleen size and higher bleeding complications (p=0.034; 0.002; respectively). We observed significant association between ET patients with high JAK2V617F allele burden and higher death rates (p=0.007). There was a trend towards higher incidence of venous thrombosis in ET patients with upper quartile JAK2V617F allele burden (p=0.057). According to JAK2V617F status, PMF patients were stratified into three groups: JAK2V617F mutation-negative (n=25) and JAK2V617F-positive with mutant allele burden in the lower quartile (n=41) and upper quartile (n=18) ranges. Comparison across all three groups revealed significant associations between upper quartile allele burden and higher leukocyte counts (p=0.001). Also, PMF patients with upper quartile JAK2V617F allele burden had significantly increased spleen size compared to the other two groups (p=0.018). We observed significant association between PMF patients with low JAK2V617F allele burden and higher incidence of venous thrombosis (p=0.035).
Our study reported that presence of higher V617F allele burden in ET is associated with defined haematological and clinical markers indicative of a more aggressive behavior while, in PMF, low allele burden was found to be associated with higher incidence of venous thrombosis.
