Higher JAK2V617F Allele Burden Research Articles

Recurrent Stroke with Rapid Development of Intracranial Artery Stenosis and Subsequent Successful Mechanical Thrombectomy in Essential Thrombocythemia.

Essential thrombocythemia is a myeloproliferative neoplasm. Ischemic stroke is frequently the first manifestation of essential thrombocythemia. We herein report a patient with JAK2V617 mutation-positive essential thrombocythemia who developed recurrent ischemic stroke with rapid development of intracranial artery stenosis and subsequently underwent successful mechanical thrombectomy. The high JAK2V617F allele burden in our patient (58.4%) may have affected the patient's condition. We discuss similar reports in the literature and the possible pathophysiologic mechanism of large artery involvement in these patients.

Clinical Impact of JAK2V617F Allele Burden in Philadelphia-Negative Myeloproliferative Neoplasms

The impact of JAK2V617F allele burden on clinical course in Philadelphia-negative (Ph-negative) myeloproliferative neoplasms (MPNs) is not clear. We analyzed the clinical impact of JAK2V617F allele burden in a relatively large series of patients with Ph-negative MPNs and long-term follow-up. A total of 228 patients with Ph-negative MPNs, including 118 with essential thrombocythemia (ET), 84 with primary myelofibrosis (PMF), and 26 with polycythemia vera (PV), were analyzed. The JAK2 MutaScreen assay was used to quantify JAK2V617F allele burden in genomic DNA. In PV cases, high JAK2V617F allele burden was associated with a trend towards inferior overall survival. In ET, high JAK2V617F allele burden was associated with lower hemoglobin and hematocrit levels, higher lactate dehydrogenase (LDH) levels, larger spleen size, and increased bleeding and mortality rates. In PMF, high JAK2V617F allele burden was associated with higher leukocyte counts and larger spleen size. In the entire cohort, high allele burden was associated with higher leukocyte and lower platelet counts, higher LDH levels, larger spleen size, higher percentage of bleeding events, higher death rate, and inferior overall survival. Our results suggest that high JAK2V617F allele burdens are associated with more severe disease in PV and ET. In PMF, high JAK2V617F allele burdens were associated with more pronounced myeloproliferative phenotypes. In Ph-negative MPNs, high allele burdens were associated with more aggressive phenotypes. Our data with a long follow-up period support the possibility of JAK2V617F allele burden being used as a marker for predicting clinical phenotype in cases of Ph-negative MPNs.

A Comparison of Bone Marrow Morphology and Peripheral Blood Findings in Low and High Level JAK2 V617F Allele Burden.

Cases with low level JAK2 V617F mutations are increasingly detected; however, the clinical interpretation of the low allele JAK2 burden may be challenging. The aim of this study is to analyze and compare the bone marrow morphology and peripheral blood findings in the low level JAK2 V617F allele burden (≤15% of JAK2) and high JAK2 V617F mutation burden patients (>15% JAK2). In total, 122 JAK2 V617F positive cases with concomitant bone marrow biopsies and peripheral blood findings were re-evaluated (62 low and 60 high level JAK2 V617F positive). Within the low burden group, normal looking megakaryocytes (p = 0.0005) were more frequently found, compared with those with no atypia (p = 0.0003), their number was more frequently not increased (p = 0.009), and they did not form clusters (p = 0.001). We found statistically significant difference in the number of platelet (p = 0.0003) and hematocrit levels (p = 0.032) when comparing the JAK2 V617F <3% and ≥3% mutation burden. In the high-level burden, the megakaryocytes were more frequently atypical (p = 0.054), and more frequently formed clusters (p = 0.053) with nuclei with maturation defects (p ≤ 0.0001). In conclusion, the JAK2 V617F mutation burden is reflected by morphological changes in the bone marrow and careful follow up of each and every patient with a low JAK2 V617F positivity is mandatory.

High JAK2V617F allele burden is associated with an increased burden of coronary artery calcification in MPNs

Abstract Background Patients with the haematological cancers Philadelphia-negative Myeloproliferative Neoplasms (MPNs), have an increased risk of coronary artery and aortic valve calcification, and an increased risk of myocardial infarctions. The most common acquired mutation in MPNs, the JAK2V617F mutation, has been linked to increased risk of thrombosis. Aims To exam the association between the JAK2V617F mutation, its allele burden, and coronary artery and aortic valve calcification. Methods One hundred and sixty one patients with MPN disease from one specialized haematological outpatient clinic where included. Information on demographics, smoking, alcohol habits and co-morbidities were registered. Blood samples were drawn for determination of the JAK2V617 mutation and allele burden. Patients were examined by cardiac computer tomography in order to determine their coronary artery calcium score (CACS) and aortic valve calcification (AVC) score. The association between the JAK2V617F mutation, its allele burden and coronary artery and aortic calcification was investigated with univariate and multivariate logistic regression analysis, adjusting for age, sex, ischemic heart disease (IHD), stroke, smoking, obesity, hypertension, hypercholesterolemia, diabetes mellitus, and family history of IHD or stroke in the multivariate analysis. Results Of the 161 patients (52% male, mean age 65.5±10.5 years), 137 (85%) were JAK2V617F positives, and the JAK2V617F allele burden was quantified in 120. The median JAK2V617F allele burden was 12% (IQR range 6–33%). There were 42 (26%) patients with a CACS &amp;gt;400, and 93 (58%) patients AVC. Among the JAK2V617F positive patients, 38 (24%) had a CACS &amp;gt;400 and 81 (59%) had AVC. In the 32 patients with a JAK2V617F allele burden&amp;gt;33%, 19 (59%) had a CACS &amp;gt;400 and 26 (81%) had AVC. In the univariate logistic regression analysis the presence of JAK2V617F mutation was not associated with a CACS &amp;gt;400 (Odds Ratio (OR) 1.92, 95% confidence interval (CI) 0.62–5.98, p=0.26). Similar result was found for the analysis on AVC (OR 1.45, 95% CI 0.61–3.45, p=0.41). In contrast, a JAK2V617F allele burden &amp;gt;33% was significantly associated with a CACS &amp;gt;400 (OR 5.31, 95% CI 2.23–12.66, p=0.0002), and similarly with AVC (OR 4.14, 95% CI 1.55–11.05, p=0.0045). In the multivariate adjusted analysis, a JAK2V617F allele burden &amp;gt;33% was significantly associated with a CACS &amp;gt;400 (OR 2.08, 95% CI 0.43–10.10, p=0.36), but not with AVC (OR 0.90, 95% CI 0.27–3.03, p=0.86). In the adjusted analysis the association between a JAK2V617F allele burden &amp;gt;33% and CACS &amp;gt;400 was significant (OR3.86, 95% CI 1.14–13.09, p=0.031), but the analysis on AVC was not (OR 1.59, 95% CI 0.42–6.04, p=0.50). Conclusion There is a significant association between a JAK2V617F allele burden &amp;gt;33% and the burden of coronary calcification in MPNs, measured as a CACS &amp;gt;400. The association remains significant after adjustment for cardiovascular risk factors. Funding Acknowledgement Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): Region Sjællands Sundhedsvidenskabelige ForskningsfondTømrermester Jørgen Holm og hustru Elisa F. Hansens Mindelegat

Low-risk polycythemia vera treated with phlebotomies: clinical characteristics, hematologic control and complications in 453 patients from the Spanish Registry of Polycythemia Vera

Hematological control, incidence of complications, and need for cytoreduction were studied in 453 patients with low-risk polycythemia vera (PV) treated with phlebotomies alone. Median hematocrit value decreased from 54% at diagnosis to 45% at 12 months, and adequate hematocrit control over time (< 45%) was observed in 36%, 44%, and 32% of the patients at 6, 12, and 24 months, respectively. More than 5 phlebotomies per year in the maintenance phase were required in 19% of patients. Worsening thrombocytosis, age > 60 years, and microvascular symptoms constituted the main indications for starting cytoreduction. Median duration without initiating cytoreduction was significantly longer in patients younger than 50 years (< 0.0001). The incidence rate of thrombosis under phlebotomies alone was 0.8% per year and the estimated probability of thrombosis at 10 years was 8.5%. The probability of arterial thrombosis was significantly higher in patients with arterial hypertension whereas there was a trend to higher risk of venous thrombosis in cases with high JAK2V617F allele burden. Rates of major bleeding and second primary neoplasm were low. With a median follow-up of 9 years, survival probability at 10 years was 97%, whereas the probability of myelofibrosis at 10 and 20 years was 7% and 20%, respectively. Progression to acute myeloid leukemia was documented in 3 cases (1%). Current management of low-risk PV patients is associated with low rate of thrombosis and long survival. New treatment strategies are needed for improving hematological control and, in the long term, reducing progression to myelofibrosis.

Impact of Anti-Endothelial Cell Antibodies (AECAs) in Patients with Polycythemia Vera and Thrombosis.

Polycythemia vera (PV) causes thrombosis. Erythrocytosis and cell adhesiveness are responsible for thrombosis. JAK2V617F causes inflammation and autoimmunity; however, whether or not autoimmunity or inflammation causes thrombosis has yet to be proven. In 60 PV patients, we analyzed JAK2V671F and its allele burden, autoimmune Th17 cells, interleukin-17 (IL-17), anti-endothelial cell antibodies (AECAs), endothelial leukocyte adhesion molecule-1 (ELAM-1), intercellular adhesion molecule-1 (ICAM-1), and von Willebrand factor antigen (VWF:Ag). Fifty blood donors were used as the controls. All patients were on phlebotomy-maintaining hematocrit <45% and aspirin. Of the 60 patients, 40 had thrombosis. Those patients with thrombosis had a higher JAK2V617F allele burden than those without thrombosis, andTh17 cells and IL-17 were also higher in patients with thrombosis. Interestingly, we observed a high AECA IgG ELISA ratio (ER) in patients with thrombosis, which was normal in patients without thrombosis. We found high ELAM-1 and ICAM-1 as well as high VWF:Ag in patients with thrombosis compared to patients without thrombosis. AECA-positive sera from patients with thrombosis showed enhanced binding to cytokine-treated HUVEC and a positive antibody-dependent cellular cytotoxicity, suggesting that AECA may contribute to vascular injury. A positive correlation between AECAs, allele burden, and thrombosis was found. These results suggest that autoimmunity may be an additional mechanism in PV thrombogenesis.

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-376: The Clinical Impact of the Burden of JAK2V617F Mutated Allele in Philadelphia-Negative Chronic Myeloproliferative Neoplasms

Context: 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. Objective: A total of 202 consecutive Philadelphia-negative MPN patients, 118 with ET and 84 with PMF, were included in this study. Methods: 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. Results: 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). Conclusion: 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.

Patients with myeloproliferative neoplasms and high levels of systemic inflammation develop age-related macular degeneration.

BackgroundEpidemiological data show that myeloproliferative neoplasms (MPNs) are associated with increased risk of neovascular age-related macular degeneration (AMD). However, knowledge about the retinal findings in these patients is lacking. This study was conducted to examine retinal ageing and the prevalence of a hallmark of AMD; drusen, in patients with MPNs. Further, we examine the role of chronic systemic inflammation, considered central in both AMD and MPNs.MethodsIn this single-centre cross-sectional study, we consecutively enrolled 200 patients with MPNs. The study was divided into three substudies. Firstly, we obtained colour fundus photographs from all patients to evaluate and compare the prevalence of drusen with the published estimates from three large population-based studies. Secondly, to evaluate age-related changes in the various retinal layers, optical coherence tomography images were obtained from 150 of the patients and compared to a healthy control group, from a previous study. Thirdly, venous blood was sampled from 63 patients to determine the JAK2V617F allele burden and neutrophil-to-lymphocyte ratio (NLR), a marker of systemic inflammation, in MPN patients with and without drusen.FindingsPatients with MPNs had an increased risk of having large drusen compared to the three population-based studies OR 5·7 (95%CI, 4·1–8·0), OR 6·0 (95%CI, 4·2–8·4) and OR 7·0 (95%CI, 5·0–9·7). Also, we found that the retinal site of drusen accumulation - the Bruch's-membrane-retinal-pigment-epithelium-complex was thicker compared to healthy controls, 0·43μm (95%CI 0·17–0·71, p = 0·0014), but there was no sign of accelerated retinal ageing in terms of thinning of the neuroretina. Further, we found that MPN patients with drusen had a higher level of systemic inflammation than MPN patients with no drusen (p = 0·0383).InterpretationPatients with MPNs suffer from accelerated accumulation of subretinal drusen and therefore AMD from an earlier age than healthy individuals. We find that the retinal changes are located only between the neuroretina and the choroidal bloodstream. Further, we find that the drusen accumulation is associated with a higher JAK2V617F allele burden and a higher NLR, suggesting that low-grade chronic inflammation is a part of the pathogenesis of drusen formation and AMD.FundingFight for Sight, Denmark and Region Zealand's research promotion fund.

Methylation age as a correlate for allele burden, disease status, and clinical response in myeloproliferative neoplasm patients treated with vorinostat

The myeloproliferative neoplasms (MPNs) are a heterogeneous group of clonal neoplastic disorders. Driver mutations in JAK2, CALR, and MPL genes have been identified in the majority of cases. Alongside these, an increasing number of genes are repeatedly identified as mutated in MPN. These, including ASXL1, TET2, DMNT3A, and EZH2, have key roles in epigenetic regulation. Dysregulation of epigenetic processes is therefore a key feature of MPN. Vorinostat is a pan histone deacetylase inhibitor (HDACi) that has been investigated in MPN. DNA methylation (DNAm) is a well-defined epigenetic mechanism of transcription modification. It is known to be affected by ageing, lifestyle, and disease. Epigenetic ageing signatures have been previously described allowing calculation of a methylation age (MA). In this study we examined the effect of vorinostat on MA in MPN cell lines and in patients with polycythaemia vera (PV) and essential thrombocythaemia (ET) treated with vorinostat as part of a clinical trial. An older MA was observed in patients with a higher JAK2 V617F allele burden and those with a longer duration of disease. PV patients had a MA older than that predicted whilst MA was younger than predicted in ET. Treatment with vorinostat resulted in a younger MA in PV patients and older MA in ET patients, in both cases a trend towards the normal chronological age. When MA change was compared against response, nonresponse was associated with a younger than predicted MA in ET patients and a higher than predicted MA in PV patients. The link between MA and JAK2 mutant allele burden implies that allele burden has a role not only in clinical phenotype and disease evolution in MPN patients, but also in the overall methylation landscape of the mutated cells.

Increased SLAMF7high monocytes in myelofibrosis patients harboring JAK2V617F provide a therapeutic target of elotuzumab

AbstractMonocyte-derived fibrocytes recently garnered attention because the novel pathogenesis of myelofibrosis (MF), and suppression of fibrocyte differentiation by serum amyloid P remarkably improved MF. We previously revealed that human fibrocytes highly expressed signaling lymphocytic activation molecule F7 (SLAMF7) compared with macrophages and that SLAMF7high monocytes in the peripheral blood (PB) of MF patients were significantly elevated relative to those in healthy controls (HCs). In this study, we evaluated SLAMF7high monocyte percentage in the PB of HCs, myeloproliferative neoplasm (MPN) patients with MF, and MPN patients without MF by using a cross-sectional approach. We found that MPN patients with MF who harbored JAK2V617F had a significantly elevated SLAMF7high monocyte percentage, which correlated positively with the JAK2V617F allele burden. In addition, the serum concentration of interleukin-1ra (IL-1ra) was significantly correlated with the SLAMF7high monocyte percentage and JAK2V617F allele burden. These findings suggest that both SLAMF7high monocytes and IL-1ra could be useful noninvasive markers of MF onset. Furthermore, the JAK2V617F allele burden of SLAMF7high monocytes was significantly higher than that of SLAMF7low monocytes and could be a potential target of elotuzumab (Elo), an anti-SLAMF7 antibody used for treating multiple myeloma. Elo independently inhibited differentiation of fibrocytes derived not only from HCs but also from MF patients in vitro. Elo also ameliorated MF and splenomegaly induced by romiplostim administration in humanized NOG mice. In conclusion, an increase of SLAMF7high monocytes with higher JAK2V617F allele burden was associated with the onset of MF in MPN patients harboring JAK2V617F, and Elo could be a therapeutic agent for MPN patients with MF who harbor JAK2V617F.

PF687 RISK OF THROMBOSIS IN PATIENTS WITH POLYCYTHEMIA VERA AND ESSENTIAL THROMBOCYTHEMIA ACCORDING TO THE JAK2 V617F ALLELE BURDEN

Background: Thrombosis is one of the major causes of morbidity and mortality in patients with chronic phase myeloproliferative neoplasms (MPN) comprising polycythemia vera (PV) and essential thrombocythemia (ET). The JAK2 V617F mutation is the most common driver mutation in BCR-ABL1 negative MPNs and the presence of JAK2 V617F is an independent risk factor of thrombotic events in patients with ET. Aims: In this study, we analyzed whether the JAK2 V617F allele burden is associated with the incidence of thrombosis in JAK2 V617F-positive patients with PV or ET. Methods: This was a retrospective study that assessed the impact of JAK2 V617F allele burden on clinical and laboratory characteristics in JAK2 V617F-positive patients with PV or ET. Patients with a diagnosis of PV or ET according to the WHO criteria at Daegu Catholic University Hospital between 2008 and 2018 were included in this study. JAK2 V617F allele burden were measured by pyrosequencing. Results: A total of 127 patents, median age 68 years, with PV (n = 61) or ET (n = 66) were included in our analyses. The JAK2 V617F allele burden in the entire population of patients was 45.0 ± 25.9%. A higher V617F allele burden was associated with higher white blood cell counts, higher hemoglobin values, lower platelet counts and higher LDH level (p < 0.001, p < 0.001, p = 0.001, and p = 0.001, respectively). There was a statistically significant difference in the JAK2 V617F allele burden between PV and ET (p < 0.001). The median V617F allele burden in PV patients was 58%, and ET was 30%. Thrombotic events were observed in 27.8% and 51.5% in patients with PV and ET, respectively. Multivariate logistic regression analysis revealed that older age (odds ratio, 1.07; 95% CI 1.01–1.18) was associated with higher incidence of thrombosis in patients with PV. In patients with ET, multivariate logistic regression analysis revealed that older age (odds ratio, 1.36; 95% CI 1.14–1.72), higher hemoglobin (odds ratio, 1.26; 95% CI 1.09–1.54), and V617F allele burden≥30% (odds ratio, 1.03; 95% CI 1.00–1.08) was elucidated as risk factors for thrombotic complications. Summary/Conclusion: JAK2 V617F allele burden can represent the disease phenotype. The incidence of thrombotic complications in ET patients with high JAK2 V617F allele burden is significantly higher than patients with low JAK2 V617F allele burden. There was no significant difference of the rate of thrombosis in PV patients according to the JAK2 V617F allele burden.

Mutational subtypes of JAK2 and CALR correlate with different clinical features in Japanese patients with myeloproliferative neoplasms.

The majority of patients with Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) harbor JAK2, CALR, or MPL mutations. We compared clinical manifestations of different subtypes of JAK2 and CALR mutations in Japanese patients with MPNs. Within our cohort, we diagnosed 166 patients as polycythemia vera (PV), 212 patients as essential thrombocythemia (ET), 23 patients as pre-primary myelofibrosis (PMF), 65 patients as overt PMF, and 27 patients as secondary myelofibrosis following the 2016 WHO criteria. Compared to patients with JAK2V617F-mutated PV, JAK2 exon 12-mutated PV patients were younger, showed lower white blood cell (WBC) counts, lower platelet counts, higher red blood cell counts, and higher frequency of thrombotic events. Compared to JAK2-mutated ET patients, CALR-mutated ET patients were younger, showed lower WBC counts, lower hemoglobin levels, higher platelet counts, and fewer thrombotic events. CALR type 1-like mutation was the dominant subtype in CALR-mutated overt PMF patients. Compared with JAK2V617F-mutated ET patients, JAK2V617F-mutated pre-PMF patients showed higher LDH levels, lower hemoglobin levels, higher JAK2V617F allele burden, and higher frequency of splenomegaly. In conclusion, Japanese patients with MPNs grouped by different mutation subtypes exhibit characteristics similar to those of their Western counterparts. In addition, ET and pre-PMF patients show different characteristics, even when restricted to JAK2V617F-mutated patients.

Low Mean Cell Haemoglobin is a Valuable Parameter of Thrombotic Risk Stratification in Patients with Polycythemia Vera

Objectives: Thrombosis is a leading cause of morbidity and mortality in patients with Philadelphia negative chronic myeloproliferative neoplasms (MPNs). There are many thrombosis risk stratifications used in this patient group taking into consideration the age, thrombosis history and cardiovascular factors (hypertension, hypercholesterinaemia, hyper-trigliceridaemia, thrombocytosis, smoking and diabetes mellitus). In this work we evaluated the possible role of iron deficiency in thrombotic events (TE) of the polycythaemia vera (PV) patients. Methods: We considered the low mean cell haemoglobin (MCH <28 pg) value as a parameter to assess the iron deficiency in the multicentre database (15 Hungarian haematology centres) of our HUMYPRON GROUP (Hungarian MPN Working Group). The MCH values, recorded at the time of diagnosis of 296 patients with polycythemia vera, were retrospectively analysed.Results: The low MCH, at the diagnosis, was found to be a risk factor for thrombotic events occurring after diagnosis (OR: 1.966). It was also shown as an additive and independent parameter in the Tefferi high-risk patient groups, and combining it with Tefferi risk stratification an extremely high thrombotic risk group could be determined (Nagelkerke R square: 0.084). We have supposed that low MCH in PV reveals a disease form featured with a high proliferation activity. Our hypothesis was confirmed with a sub-study (n=52) showing that the high JAK2V617F allele burden was significantly correlated with the low MCH (p=0.005) and the high white blood cell count (WBC) (p<0.001).Conclusions: Iron deficiency, existing at the time of diagnosis of PV, was proven to be a risk factor for imminent thrombotic events. The low MCH was found to be a strong additive factor when it was combined with the known thrombotic risk stratification systems. The low MCH showed significant correlation with the high JAK2V617F allele burden.

The clinical and prognostic relevance of driver mutations in 203 Taiwanese patients with primary myelofibrosis

AimsWe investigated the clinical and prognostic relevance of the mutational status of driver genes with allele burden and endogenous erythroid colony (EEC) growth in 203 Taiwanese patients with primary myelofibrosis...

Primary myelofibrosis: Older age and high JAK2V617F allele burden are associated with elevated plasma high-sensitivity C-reactive protein levels and a phenotype of progressive disease

We measured plasma levels of high-sensitivity C-reactive protein (hs-CRP) in 526 subjects with primary myelofibrosis (PMF). Thirty-eight percent had an elevated hs-CRP level (≥0.3mg/dL). Elevated hs-CRP levels were associated with a progressive disease phenotype, including anemia, high white blood cell count, low platelet count, increased splenomegaly, increased risk of blast transformation, and worse survival. Age≥52years, but no other demographic characteristics, was associated with an elevated hs-CRP level in multivariable logistic regression (odds ratio [OR], 4.29; 95% CI, 2.73-6.77; P <0.001). Subjects with JAK2V617F mutation and an allele burden≥50% had an age-independent higher incidence of elevated hs-CRP level (OR=1.97; 95% CI,1.21–3.22; P=0.006) compared with a combined cohort of subjects with JAK2V617F <50% allele burden, CALR, MPL mutations, or no detectable driver mutations. Neither ASXL1 or EZH2 sub-clonal mutations, nor JAK2 46/1 haplotype or the A3669G polymorphism of glucocorticoid receptor were significantly associated with increased hs-CRP levels. Subjects with age≥52years and JAK2V617F with≥50% allele burden had a phenotype of progressive disease. Our data indicate that older age and high JAK2V617 allele burden are major determinants of inflammation in PMF, and are associated with disease progression.

Thromboses and hemorrhages are common in MPN patients with high JAK2V617F allele burden.

The most common causes of morbidity and mortality in myeloproliferative neoplasms (MPN) are thrombotic and hemorrhagic complications. The JAK2V617F mutation, commonly found in MPN, correlates with several clinical and laboratory characteristics even if the relevance of JAK2V617F allele burden in the natural history of these diseases is unclear. In this study we searched, a relation between thrombotic and hemorrhagic complications and JAK2V617F allele burden level in MPN patients. We evaluated 253 consecutive MPN [121 essential thrombocythemia (ET), 124 polycythemia vera (PV), and 8 primary myelofibrosis (PMF)] patients in whom the JAK2V617F allele burden was available, all studied and followed (median 8.8years) in our department. Patients were stratified accordingly to their JAK2V617F allele burden, into four quartiles (1st <25%, 2nd 26-50%, 3rd 51-75%, and 4th >75%). Significantly higher incidence of thromboses (p=0.001) and hemorrhages (p<0.001) during follow-up has been observed in higher quartiles when compared to lower ones. Thrombosis- and hemorrhage-free survivals were poorer in patients belonging to the highest quartile. Our data suggest that MPN patients with JAK2V617F allele burden higher than 75% have to be considered as high risk patients, being prone to develop thrombo-hemorrhagic complications during the disease course.

High Levels of High Sensitivity-C Reactive Protein (hs-CRP) Are Associated with Older Age, Chromosomal Abnormalities and JAK2V617F Mutation with High Allele Burden in Primary Myelofibrosis (PMF)

▪A number of evidences indicate that inflammation and deranged innate immunity intervene in the pathobiology of PMF. In this study we aimed to analyze the inflammatory components of PMF by measuring hs-CRP in a vast population of patients. hs-CRP can detect minimal variations of serum CRP levels even in the range of normal limits, and thus is a sensitive marker of low grade systemic inflammation. We included PMF patients from the data base of the Center for the Study of Myelofibrosis in Pavia with available archived plasma. We excluded patients who had been treated with disease-modifying agents at any time before or on the date of base-cohort entry (hydroxyurea, interferon, ruxolitinib, corticosteroids) and patients with acute inflammatory or autoimmune disease, malignancy, severe liver or renal dysfunction. In this study we report the results on the determinants of levels of circulating hs-CRP in PMF in an analysis in which we used parameters that could be hypothesized with a biological plausibility to influence the level of hs-CRP. Selected predictors were: age, sex, the five different genotypes reflecting the presence or absence of the driver mutations (JAK2V617F with low allele burden (<50%), JAK2V617F mutation with high allele burden (≥ 50%), CALR mutations, MPL mutations, triple negative genotype), the accessory subclonal mutations in the ASXL1 and EZH2 genes, and abnormal karyotype. 152 subjects were analyzed at diagnosis, while 353 after (median time from diagnosis, 43 months, range, 4-396 months). 86 patients (17%) had increased hs-CRP (>1 mg/L). The mean age of the patients was 54.4 years. Increasing age was correlated with increasing hs-CRP (R=0.45;P<0.001). By dividing the PMF population into that younger or older than 50 years (this cut-off value was obtained from the ROC curve analysis),3.7% in the former category had increased levels of hs-CRP with respect to 27.4% in the latter (P<0.001). 227 patients were females and 279 males. There were significant differences in plasma hs-CRP between males and females (0.86 mg/L vs. 0.44 mg/L; P<0.001). While 21,5% of males had increased levels of hs-CRP, this proportion was 11.4 in females (P=0.002). There was a significant difference in plasma hs-CRP between subjects with different PMF genotypes (Figure). This was due to an exceedingly higher hs-CRP in patients bearing JAK2V617F mutation with high allele burden (N=82) with respect to those with low allele burden (N=190) (1.09 mg/L vs. 0.46 mg/L; P<0.001). By dividing the PMF population into that with high JAK2V617F allele burden and other genotypes, 29.2% in the former had increased levels of hs-CRP while it was 12.2% in the latter (P<0.001). Among patients with JAK2V617F mutation, there was a significant association between the level of hs-CRP and the allele burden (R=0.12; P=0.018). Cytogenetic characteristics at diagnosis were available in 83 patients and 32 (38.5%) of them had abnormal karyotype. There was a significant difference in hs-CRP between patients bearing a chromosomal aberration and those without (mean 0.90 mg/L vs. 0.35 mg/L P=0.003). By dividing chromosomal abnormalities in unfavorable and favorable, there was no difference in hs-CRP levels between the two groups. Assay of subclonal mutations ASXL1 and EZH2 was available in 99 patients, 8.8% of them had ASXL1 mutations and 4.4% had EZH2 mutation. No difference in hs-CRP was revealed between patients bearing or not ASXL1 or EZH2 or both. We investigated the collinearity of the variables significantly associated with hs-CRP. Age was correlated with the allele burden of JAK2V627F (R=0.18; P=<0.001) and higher age was associated with higher frequency of chromosomal abnormalities (P=0.023). At multivariable analysis, older age (P=0.009) and presence of chromosomal abnormalities (P=0.010) were identified as independently correlated variables, and were considered the best predictors of hs-CRP increase. In conclusion, our results highlighted the association between older age/ presence of chromosomal abnormalities and inflammation in PMF. These results were coherent with the hypothesis that chronic inflammation is a promotor of mutagenesis in MF (Hasselbalch HC. Leuk Res. 2013;37:214), but also that genomic instability (older age, high JAK2V617F allele burden, chromosomal abnormalities) is a determinant of inflammation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Impact of JAK2V617F Mutation Burden on Disease Phenotype in Chinese Patients with JAK2V617F-positive Polycythemia Vera (PV) and Essential thrombocythemia (ET).

Most patients with polycythemia vera (PV) and half of essential thrombocythemia (ET) possess an activating JAK2V617F mutation. The objective of this study was to better define the effect of JAK2V617F mutant allele burden on clinical phenotypes in Chinese patients, especially thrombosis. By real-time polymerase chain reaction (RT-PCR), the JAK2V617F mutation burden was detected in 170 JAK2V617F-positive patients, including 54 PV and 116 ET. The results showed that JAK2V617F allele burden was higher in PV than in ET (P< 0.001). Higher percentage of patients had JAK2V617F allele burden over 20% in PV than in ET (68.5% VS 26.7%) (P< 0.001). In PV patients, higher JAK2V617F allele burden was observed in female (P< 0.05) and leukocytosis patients (WBC above 10×109/L) (P< 0.001). Meanwhile, ET patients showed increased JAK2V617F allele burden in the group with higher hemoglobin (HGB above 150g/L) (P< 0.05), leukocytosis (WBC above 10×109/L) (P< 0.001), splenomegaly (P< 0.05) and thrombosis (P< 0.05). In conclusion, the JAK2V617F mutation allele burden is higher in Chinese patients with PV than ET. In PV patients, JAK2V617F mutation burden had influence on WBC counts. And the clinical characteristics of ET patients, such as WBC counts, hemoglobin level, splenomegaly and thrombosis, were influenced by JAK2V617F mutation burden. Male, high hemoglobin (HGB above 150g/L), and increased JAK2V617F mutation burden (JAK2V617F allele burden ≥16.5%) were risks of thrombosis (P< 0.05) for ET patients by Logistic Regression.

The Impact of Allogeneic Hematopoietic Cell Transplantation (HCT) and Treatment with Ruxolitinib on Survival in Patients with Myelofibrosis

Allogeneic HCT is curative in myelofibrosis (MF) with a survival (OS) advantage compared to non-JAK-inhibitor treatments in patients (pts) with Int-2/high-risk DIPSS (Kroeger et al, Blood 2015). On the other hand, data demonstrate a OS benefit of the JAK1/JAK2 inhibitor ruxolitinib (RUX) in intermediate (Int)-2/high-risk IPSS (Cervantes et al, Blood 2013; Verstovsek et al, Haematologica 2015). Yet, comparative data to the impact of RUX and HCT on OS are lacking. This was pursued retrospectively at the University of Leipzig in light of clinical-, cytogenetic-, phenotype-driver-, and somatic mutations-based prognostic variables.Patient and Methods: RUX became available in November, 2009. MF pts (n=137; median age 58y) seen afterwards were included with the exception of MPL + pts (small number). Characteristics are shown in table 1. RUX was given for spleenomegaly and/or constitutional symptoms [n=76 (55%)]. Irrespective of RUX, a donor search was initiated in int-2 and high-risk IPSS pts < 70 y and HCT performed if a suitable donor identified. Based on this biologic randomization, 50 (37%) received HCT, 58 (42%) continued with RUX (median exposure 19 months), and 29 (21%) pts had other options. The RUX- and HCT-groups were balanced in terms of IPSS, platelets, cytogenetic-, and epigenetic molecular-risks. Treatment was initiated within the first year (median 10 months), thus probability of OS 5 years after diagnosis was calculated (SPSS20 software). Cytogenetic-risk was conducted according to Caramazza et al. Leukemia 2011. Somatic mutations detected by NGS were used to calculate molecular-risk (Vannucchi et al. Leukemia 2013).Results: Median OS time was 6.8 y. The RUX-group was older (median age 64 y) with profound spleenomegaly and a higher JAK2 V617F allele burden compared to the HCT group (median age 55 y) (p<0.0005). The 56% OS at 5 years in the RUX group was comparable to that after HCT (60%; p=0.5). In the transplant setting, prior RUX-treatment (n=21; OS 65%) had no negative impact compared to up-front HCT (n=29; OS 55%) (p=0.9). Whilst IPSS low-/int-1-risk retained its survival impact (p=0.04), OS at 5 years was comparable for int-2 and high-risk. OS in the HCT and RUX groups for int-2 was 59% and 62% respectively (p=0.4) and for high-risk IPSS 54% and 50% respectively (p=0.2). Advanced age was associated with an inferior OS (p=0.002) and correlated with higher IPSS, transfusion-dependency and thrombocytopenia. In the HCT group, OS in pts <50 y was 91% vs. 49% in pts >50 y (p=0.001). In the RUX group, age <60 and not <50 y was OS-relevant [79% in pts <60y vs. 43% in those >60 y (p=0.02)]. No prognostic implication of anemia, WBC, peripheral blasts, and constitutional symptoms could be detected with both treatments. OS in JAK2 + pts was similar to CALR + pts in both therapy cohorts (p=0.3). Triple negativity retained its detrimental influence (p<0.0001) even after HCT, with frequent AML-typical mutations (transcription and/or signaling genes) and a leukemic transformation (LT) rate of 35%. Although spliceosome mutations were associated with anemia (p=0.004)/transfusion-dependency (p=0.006) and mutations in chromatin modifiers (ASXL1, EZH2) with splenomegaly(p=0.02),high-molecular epigenetic risk had no impact on OS or LT in the treatment groups. Unfavourable cytogenetics were associated with LT (p=0.04) but not OS in the two groups. Of the 24 pts with a LT, 15 (62%) received no RUX and no HCT.Conclusions: Our data imply that, besides allogeneic HCT, a prolonged JAK1/JAK2 inhibition could, at least partly, attenuate most of the prognostic detrimental parameters. Pre-treatment with ruxolitinib prior to HCT does not seem to have a negative impact on survival. It is the first time where outcome with a non-transplant procedure is shown to be comparable to that after HCT in MF, even in high-risk disease. This needs to be verified in prospective randomized trials to define the role of allogeneic HCT in the era of JAK1/JAK2 inhibition.Table 1Patients characteristics ( n = 137 )VariableImpact on OSImpact on LTMedian age (years)580.0020.6JAK2+/CALR+/Double negative (%)63 / 20 / 17<0.00050.04Median number of MF-related mutations (range)1 (0-5)0.40.07High-molecular risk profile (%)(ASXL1, EZH2, SRSF2, IDH1/2)33.60.80.7Mutations in transcription/signaling genes (%)280.30.008Unfavorable cytogenetics (%)300.10.04IPSS (Int-2 / high-risk) (%)34 / 410.040.2 DisclosuresRoskos:Novartis: Honoraria. Al-Ali:Celgene: Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding.