Philadelphia-negative Chronic Myeloproliferative Neoplasms Research Articles

Retinal Vessel Analysis and Microvascular Abnormalities in Patients with Philadelphia-Negative Chronic Myeloproliferative Neoplasms.

Background: Philadelphia-negative chronic myeloproliferative neoplasms are a group of clonal hematopoietic disorders including polycythemia vera, essential thrombocythemia, and primary myelofi-brosis. These neoplasms are characterized by an increased risk of thrombotic complications. Several studies have highlighted that the study of vessels of the retina offers the opportunity to visualize, in vivo, the damage to microcirculation that is common in various systemic pathologies. Methods: in our study, forty patients underwent an ophthalmological examination, using non-invasive imaging tech-niques, for analyses of their retinal vascularization. The objective was to correlate the findings ob-tained from this study of the retina with different markers of thrombotic risk, to demonstrate the usefulness of studying retinal vessels as a possible new prognostic biomarker of thrombotic risk in patients affected by Philadelphia-negative chronic myeloproliferative neoplasms. Results: retinal imaging demonstrated changes in the microcirculation, with a reduced vascular density of the deep and superficial capillary plexuses with respect to a normal group, and a correlation between retinal changes and blood parameters. Conclusions: additional research will allow us to determine whether retinal changes in individuals with chronic myeloproliferative neoplasms could be predictive of the development of thrombotic events in these subjects.

Acquired Von Willebrand Syndrome, Epidemiology, Laboratory Findings and Molecular Alterations on Classic Myeloproliferative Neoplasms in Mexican Population

Introduction: Acquired von Willebrand syndrome (AvWS) has been associated with Philadelphia-negative chronic myeloproliferative neoplasms (MPN) since 1984. However, the epidemiology of these disorders is not precise and has been underestimated worldwide but prevalence of 10-20% has been described in other series. Determinations of the antigenic von Willebrand factor (VWF:Ag) and its activity (VWF:RCo) are only performed when patients present thrombocytosis over 1,000 x10 3/µL. Essential Thrombocythemia (ET) is the most commonly associated MPN, found in 5-30%. Some cases have been described in patients with polycythemia vera (PV) and primary myelofibrosis (PMF) who develop AvWS even with platelet counts below 1,000 x10 3/µL. The epidemiology of AvWS in MPN and its association between laboratory and molecular parameters among the Mexican population have not been described. Objectives - Describe the clinical, biochemical and molecular characteristics of patients who develop AvWS with the aim of establishing the difference between cut-off scores of platelets to suspect AvWS in Mexican patients. - Establish the association between mutations in JAK2, CALR and MPL, and the appearance of AvWS. Material and methods: Retrospective, descriptive, observational, and single-center analysis. Patients over 18 years of age with a diagnosis of MPN from the INCMNSZ were recruited. The mutational analysis in peripheral blood (PB) for JAK2, CALR and MPL was assessed, with at least one follow-up between 2012 and 2022, excluded whose medical record were incomplete. Information was consecutively collected from 184 patients with MPN diagnosis between 01/01/2000 to 05/31/2022. Thirty patients were identified as having AvWS with a VWF:RCo/VWF:Ag diagnostic ratio of < 0.7. The epidemiology, clinical, laboratory and mutational alterations of the patients who developed AvWS by was analyzed using descriptive statistics with SPSS. This study was approved by the institutional bioethics committee. Results: AvWS developed in 16.4% (n = 30) of patients with MPN with an annual incidence of 2.4 cases per year was identified. Most patients were women in 66% (n = 20), with a median age of 57 years (IQR 20-72 years). Of the thirty cases with AvWS, 46.7% (n =14) corresponded to PMF, 40% (n =12) to PV, and 13.3% to ET (n = 4). The most frequently found mutation was JAK2 exon 14 with 76.7%, followed by CALR in 13.3% and triple negatives in 10%, no patient presented MPL mutations. The global median of VWF:RCo/VWF:Ag was 0.47 [PV 0.47 (IQR 0.31-0.64), ET 0.53 (IQR 0.5-0.64) and PMF 0.48 (0.29-0.65)] (Figure 1) and the median of the VWF:Ag 118 U/dL with FVW:RCo of 44.6 UI/dL (Table 1). The median platelet count for patients with the JAK2 mutation was 987.9 x103/µL (IQR 218 - 2307 x10 3/µL), while those with CALR and triple negatives were 1,033 x10 3/µL (IQR 713 - 1339 x10 3/µL), and 1,325 x10 3/µL (IQR 760 - 2059 x10 3/µL), respectively. Regarding hemoglobin (Hb) and hematocrit (Hto) levels, patients with PV and JAK2 mutation with AvWS presented medians of 17.45 g/d and 54.4% respectively, highlighting that AvWS was generated in some patients with platelet counts below 1,000 x10 3/µL. At diagnosis, bleeding episodes occurred in 13.3%. Lastly, 96.7% of the patients were treated with hydroxyurea, which corrected cellular alterations and laboratory abnormalities found in AvWS. Conclusions: AvWS can be found in patients with platelet counts below 1,000 x10 3/µL, especially among those with PV with elevated Hb and Hct levels. This may deem it necessary to carry out diagnostic tests in patients with MPN and any degree of thrombocytosis. To our knowledge, this is the most comprehensive study among latin american patients that describes the coexistence of PMF and AvWS; even though the mechanism by which some PMF patients develop AvWS is not perfectly understood. In our study, patients with PMF presented AvWS more frequently; this is a contrasting result from other international reports where ET was most attributed. We attribute this finding to changes made to the WHO criteria between 2008 and 2016.

Whole blood transcriptional profiling reveals highly deregulated atherosclerosis genes in Philadelphia-chromosome negative myeloproliferative neoplasms.

The Philadelphia-negative chronic myeloproliferative neoplasms (MPNs) are associated with a huge comorbidity burden, including an increased risk of cardiovascular diseases. Recently, chronic inflammation has been suggested to be the driving force for clonal evolution and disease progression in MPN but also potentially having an impact upon the development of accelerated (premature) atherosclerosis. Since chronic inflammation, atherosclerosis, and atherothrombosis are prevalent in MPNs and we have previously shown oxidative stress genes to be markedly upregulated in MPNs, we hypothesized that genes linked to development of atherosclerosis might be highly deregulated as well. Using whole blood gene expression profiling in patients with essential thrombocythemia (ET; n = 19), polycythemia vera (PV; n = 41), or primary myelofibrosis (PMF; n = 9), we herein for the first time report aberrant expression of several atherosclerosis genes. Of 84 atherosclerosis genes, 45, 56, and 46 genes were deregulated in patients with ET, PV, or PMF, respectively. Furthermore, BCL2L1, MMP1, PDGFA, PTGS1, and THBS4 were progressively significantly upregulated and BCL2 progressively significantly downregulated from ET over PV to PMF (all FDR <0.05). We have for the first time shown massive deregulation of atherosclerosis genes in MPNs, likely reflecting the inflammatory state in MPNs in association with in vivo activation of leukocytes, platelets, and endothelial cells being deeply involved in the atherosclerotic process.

Outcomes of SARS-CoV-2 infection in Ph-neg chronic myeloproliferative neoplasms: results from the EPICOVIDEHA registry.

Patients with Philadelphia-negative chronic myeloproliferative neoplasms (MPN) typically incur high rates of infections and both drugs and comorbidities may modulate infection risk. The present study aims to assess the effect of immunosuppressive agents on clinical outcomes of MPN patients affected by the coronavirus disease 2019 (COVID-19). This is an observational study. We specifically searched and analyzed MPN patients collected by EPICOVIDEHA online registry, which includes individuals with hematological malignancies diagnosed with COVID-19 since February 2020. Overall, 398 patients with MPN were observed for a median of 76 days [interquartile range (IQR): 19-197] after detection of SARS-CoV2 infection. Median age was 69 years (IQR: 58-77) and 183 individuals (46%) had myelofibrosis (MF). Overall, 121 patients (30%) of the whole cohort received immunosuppressive therapies including steroids, immunomodulatory drugs, or JAK inhibitors. Hospitalization and consecutive admission to intensive care unit was required in 216 (54%) and 53 patients (13%), respectively. Risk factors for hospital admission were identified by multivariable logistic regression and include exposure to immunosuppressive therapies [odds ratio (OR): 2.186; 95% confidence interval (CI): 1.357-3.519], age ⩾70 years, and comorbidities. The fatality rate was 22% overall and the risk of death was independently increased by age ⩾70 years [hazard ratio (HR): 2.191; 95% CI: 1.363-3.521], previous comorbidities, and exposure to immunosuppressive therapies before the infection (HR: 2.143; 95% CI: 1.363-3.521). COVID-19 infection led to a particularly dismal outcome in MPN patients receiving immunosuppressive agents or reporting multiple comorbidities. Therefore, specific preventive strategies need to be tailored for such individuals. EPICOVIDEHA registry reports inferior outcomes of COVID-19 in patients with Philadelphia-negative chronic myeloproliferative neoplasms receiving immunosuppressive therapies. Patients with Philadelphia-negative chronic myeloproliferative neoplasms (MPN) incur high rates of infections during the course of their disease.The present study was aimed at assessing which patient characteristics predicted a worse outcome of SARS-COV-2 infection in individuals with MPN.To pursue this objective, the researchers analyzed the data collected by EPICOVIDEHA, an international online registry, which includes individuals with hematological malignancies diagnosed with COVID-19 since February 2020.The database provided clinical data of 398 patients with MPN incurring COVID-19:Patients were mostly elderly (median age was 69 years);Forty-six percent of them were affected by myelofibrosis, which is the most severe MPN;Moreover, 32% were receiving immunosuppressive therapies (JAK inhibitors, such as ruxolitinib, steroids, or immunomodulatory IMID drugs, such as thalidomide) before COVID-19.Hospitalization was required in 54% of the patients, and the risk of being hospitalized for severe COVID-19 was independently predicted byOlder age;Comorbidities;Exposure to immunosuppressive therapies.Overall, 22% of MPN patients deceased soon after COVID-19 and the risk of death was independently increased over twofold byOlder age;Comorbidities;Exposure to immunosuppressive therapies before the infection.In conclusion, COVID-19 infection led to a particularly dismal outcome in MPN patients receiving immunosuppressive agents, including JAK inhibitors, or reporting multiple comorbidities. Therefore, specific preventive strategies need to be tailored for such individuals.

Coexistence of JAK2V617F and CALR mutation in Tunisian cohort with Philadelphia-negative chronic myeloproliferative neoplasms

Coexistence of JAK2V617F and CALR mutation in Tunisian cohort with Philadelphia-negative chronic myeloproliferative neoplasms

Philadelphia-negative chronic myeloproliferative neoplasm follow-up: when the phone rings. Changes during the COVID-19 pandemicand patient satisfaction. Experience in 30 health centers in Spain.

The SARS-CoV-2 pandemic has favored the expansion of telemedicine. Philadelphia-negative chronic myeloproliferative neoplasms (Ph-MPN) might be good candidates for virtual follow-up. In this study, we aimed to analyze the follow-up of patients with Ph-MPN in Spain during COVID-19, its effectiveness, and acceptance among patients. We present a multicenter retrospective study from 30 centers. Five hundred forty-one patients were included with a median age of 67years (yr). With a median follow-up of 19months, 4410 appointments were recorded. The median of visits per patient was 7 and median periodicity was 2.7months; significantly more visits and a higher frequency of them were registered in myelofibrosis (MF) patients. 60.1% of visits were in-person, 39.5% were by telephone, and 0.3% were videocall visits, with a predominance of telephone visits for essential thrombocythemia (ET) and polycythemia vera (PV) patients over MF, as well as for younger patients (< 50yr). The proportion of phone visits significantly decreased after the first semester of the pandemic. Pharmacological modifications were performed only in 25.7% of the visits, and, considering overall management, ET patients needed fewer global treatment changes. Telephone contact effectiveness reached 90% and only 5.4% required a complementary in-person appointment. Although 56.2% of the cohort preferred in-person visits, 90.5% of our patients claimed to be satisfied with follow-up during the pandemic, with an 83% of positive comments. In view of our results, telemedicine has proven effective and efficient, and might continue to play a complementary role in Ph-MPN patients' follow-up.

Interferon-alpha2 Treatment Reduces Circulating Neutrophil Extracellular Trap Levels in Myeloproliferative Neoplasms

Background and aim Arterial and venous thrombosis are major life-threatening events for patients with Philadelphia-negative chronic myeloproliferative neoplasms (MPNs). Neutrophil activation and, in particular, the formation of neutrophil extracellular traps (NETs) have been implicated in the pathogenesis of MPNs and thrombosis, particularly in patients with the most common MPN phenotypic driver mutation, JAK2V617F. Moreover, ruxolitinib treatment reduces ex vivo NET formation in JAK2V617F-positive patients with MPNs. In this study, we aimed to clarify the effect of interferon alpha (IFNα) and hydroxyurea (HU) treatment on the levels of circulating NETs in patients with MPNs. Methods Serum NET (myeloperoxidase-DNA complexes) levels were measured using an in-house ELISA in pre-treatment samples collected from 128 patients with MPNs enrolled in the DALIAH trial (NCT01387763), a randomized controlled phase 3 clinical study. NETs were likewise quantified in 85 paired samples collected after 12 months of therapy with HU or either of two different formulations of pegylated IFNα (PEG-IFNα2a or PEG-IFNα2b). Results At baseline, NET levels did not vary significantly between patients with different MPN sub-diagnoses (ET: 48, PV: 61, Pre-MF: 10, PMF: 9), phenotypic driver mutations (JAK2V617F, CALR, MPL, or triple negative), or between those with or without a history of major thrombosis. However, patients with PV and a JAK2V617F allele burden ≥ 50% at baseline had significantly higher baseline NET levels than those with an allele burden < 50% (p=0.006). NET levels at baseline correlated significantly with neutrophil count (r=0.29, p=0.001), neutrophil-to-lymphocyte ratio (NLR)(r=0.26, p=0.004) and JAK2V617F allele burden (r=0.22, p=0.03), particularly in patients with PV and allele burden ≥ 50% (r=0.50, p=0.01, r=0.56, p=0.007 and r=0.45, p=0.03 respectively), while the same was not observed at follow-up. Furthermore, after 12 months of treatment, NET levels decreased in 70% of patients, with an average decrease of 48%, compared to baseline levels. The effect of treatment was particularly pronounced in patients with PV with an allele burden ≥ 50%, of whom 76% showed decreased NET levels, with an average decrease of 60%, while 67% of patients with allele burden < 50% showed an average reduction in NET levels of only 36% (Figure 1). While 77% and 73% of patients treated with PEG-IFNα-2a or PEG-IFNα-2b, respectively, had decreased NET levels, the same applied to only 53% of patients treated with HU (Figure 2). Discussion and conclusion The increased NET levels in patients with PV with a JAK2V617F allele burden ≥ 50% indicate that the JAK/STAT pathway is involved in NET formation and/or clearance. In keeping with this, the highest proportion of patients with decreased NET levels after treatment and the highest average decrease were seen in patients with PV and a baseline JAK2V617F allele burden ≥ 50%. NET levels at baseline correlated with neutrophil count and NLR, however normalization of blood counts alone did not account for the observed decrease in NET levels. Accordingly, treatment with IFNα, which has previously been shown to target the JAK2V617F clone preferentially, effectively reduced circulating NET levels, and to a greater extent than HU treatment. This may relate to our previous finding that three months' IFNα2 treatment downregulates the expression of PAD4, which is implicated in NET formation. Although, we found no difference in NET levels in patients with and without a history of major thrombotic events, these data suggest that IFNα2 treatment, by reducing levels of potentially prothrombotic NET, may contribute to reduced risk of this complication in patients with MPNs, and that it is an advantageous treatment compared to HU in this respect. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Combination Therapy with Ruxolitinib and Interferon in Newly Diagnosed Patients with Polycythemia Vera

Background: In the COMBI-I trial, we have shown that the combination treatment of Interferon-alpha-2a (IFN) and ruxolitinib is safe and efficacious in patients with polycythemia vera (PV) and myelofibrosis, who were mostly intolerant or refractory to IFN monotherapy. Interferon-alpha2 (IFN) has been used successfully for decades in the treatment of Philadelphia-negative chronic myeloproliferative neoplasms. However, 20-30% of patients are intolerant or show limited response to IFN treatment. Ruxolitinib is an anti-inflammatory agent and is used as first-line treatment in patients with myelofibrosis as well as second-line treatment in patients with PV. Combination therapy with the two agents may be more efficacious than monotherapy. Several reports suggest that after achieving a deep hematological and molecular response, treatment pause for several months to years is safe after IFN treatment. Therefore, an initial high-intensity regimen may help to control the disease allowing for treatment pause or low-dose IFN monotherapy. Herein, we report preliminary safety and efficacy data from the COMBI-II trial in patients with newly diagnosed PV. Aims: To evaluate the safety and efficacy of combination therapy with IFN and ruxolitinib in newly diagnosed patients with PV. Methods: A preplanned interim analysis after at least one year of treatment. Twenty-five patients with newly diagnosed PV were included. They were pretreated according to normal clinical standard with phlebotomies and, if high risk, hydroxyurea before inclusion and initiation of combination treatment. Hydroxyurea was withheld 1 week before initiation of treatment. Patients with severe kidney-, liver-, or heart disease were excluded. Initial therapy was IFN (Pegasys®) 45 μg sc/week and ruxolitinib (Jakavi®) 10 mg BID. The study duration was planned to be two years. Safety was assessed by registering all adverse events graded according to the CTCAE V 5.0. Efficacy was evaluated using the 2013 ELN and IWG-MRT response criteria. The JAK2V617F allele burden was measured using high-sensitivity real-time qPCR on whole blood with a lower detection limit of below 0.1%. Histological response, QoL data, and bone marrow MRI results will be presented in the final report. Results: Twenty-five patients with PV were included in 2019-2021. They had a median age of 70 years; 14 were females and 11 were males. Of the 25 patients, 19 patients were high-risk and 6 were low-risk; 5 patients had previous thrombosis. The median time from accepted referral to initiation of treatment was 67 days. The median follow-up time was 21 months. At time of data evaluation, 13 patients had completed 24 months, while 24 patients had completed 12 months of treatment. One patient was withdrawn from the study early due to the development of myelofibrosis 10 months after beginning treatment. One patient was diagnosed with follicular lymphoma 9 months after initiation of treatment. One patient had an acute myocardial infarction. No other thromboembolic events occurred. No grade 3-4 infections were observed. At follow-up, 19 patients were still receiving combination therapy. One patient was receiving concomitant hydroxyurea; 1 ruxolitinib monotherapy; 1 IFN monotherapy; 1 combination of hydroxyurea and IFN; and 1 hydroxyurea monotherapy. Grade 1-2 anemia was frequent and managed with dose reductions. The median doses of ruxolitinib and IFN at follow-up were 5 mg BID and 45 μg sc/2.week, respectively. High rates of complete hematological response were observed after 1 month of treatment (Figure 1). Three patients required phlebotomy after the first 2 weeks of treatment. The JAK2V617F allele burden was significantly reduced 3 months after the start of treatment, and continued to decline from a median of 47% (95%CI 35-59) at baseline to 6% (95%CI 3-12) after 24 months (figure 2);4 patients had a JAK2V617F allele burden < 1%, and 7 patients had a JAK2V617F allele burden below 5% at the latest measurement. No significant difference in JAK2V617F allele burden was observed between referral and initiation of the combination treatment. Conclusion: Combination therapy with IFN and ruxolitinib is relatively well tolerated and is highly efficacious in newly diagnosed PV patients with frequent normalization of cell counts and a marked reduction of the JAK2V617F allele burden. This therapy could lead to a subsequent long-term approach with low-dose interferon or treatment pauses. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Recombinant Interferon-β in the Treatment of Polycythemia Vera and Related Neoplasms: Rationales and Perspectives.

About 30 years ago, the first clinical trials of the safety and efficacy of recombinant interferon-α2 (rIFN-α2) were performed. Since then, several single-arm studies have shown rIFN-α2 to be a highly potent anticancer agent against several cancer types. Unfortunately, however, a high toxicity profile in early studies with rIFN-α2 -among other reasons likely due to the high dosages being used-disqualified rIFN-α2, which was accordingly replaced with competitive drugs that might at first glance look more attractive to clinicians. Later, pegylated IFN-α2a (Pegasys) and pegylated IFN-α2b (PegIntron) were introduced, which have since been reported to be better tolerated due to reduced toxicity. Today, treatment with rIFN-α2 is virtually outdated in non-hematological cancers, where other immunotherapies-e.g., immune-checkpoint inhibitors-are routinely used in several cancer types and are being intensively investigated in others, either as monotherapy or in combination with immunomodulatory agents, although only rarely in combination with rIFN-α2. Within the hematological malignancies, rIFN-α2 has been used off-label for decades in patients with Philadelphia-negative chronic myeloproliferative neoplasms (MPNs)-i.e., essential thrombocythemia, polycythemia vera, and myelofibrosis-and in recent years rIFN-α2 has been revived with the marketing of ropeginterferon-α2b (Besremi) for the treatment of polycythemia vera patients. Additionally, rIFN-α2 has been revived for the treatment of chronic myelogenous leukemia in combination with tyrosine kinase inhibitors. Another rIFN formulation-recombinant interferon-β (rIFN-β)-has been used for decades in the treatment of multiple sclerosis but has never been studied as a potential agent to be used in patients with MPNs, although several studies and reviews have repeatedly described rIFN-β as an effective anticancer agent as well. In this paper, we describe the rationales and perspectives for launching studies on the safety and efficacy of rIFN-β in patients with MPNs.

Correlation of Thrombosis and Clinicohematological Parameters with JAK2V617F Mutation in Philadelphia-Negative CMPNs: A Study from India.

Objective Philadelphia-negative chronic myeloproliferative neoplasms (CMPNs), which include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), are characterized by the presence of JAK2V617F (exon 14) mutation, and this occurs in 90 to 95% cases of PV and 50 to 60% cases of ET and PMF. Still, this is a matter of debate regarding the correlation of this mutation with thrombosis and clinicohematological parameters in CMPNs. So, we conducted this study to ascertain the association of JAK2V617F mutation with thrombotic complications and clinicohematological parameters of these patients. Materials and Methods This prospective and retrospective study was conducted during 2018 to 2019 at the Department of Laboratory Sciences and Molecular Medicine of a tertiary care hospital, and 160 CMPN patients were enrolled. Complete hemogram was done and DNA was extracted, followed by real-time qualitative polymerase chain reaction to check for JAK2V617F mutation. This mutation was then correlated with complications, mainly thrombosis, hematological parameters, and clinical parameters such as age and splenomegaly. Results Among 160 CMPN patients, 60 were females and 100 were males, with male to female ratio of 1:0.6, and age range of 27 to 85 years. Total 91 (56.9%) patients were JAK2V617F positive and the remaining 69 (43.1%) were negative for this mutation. We observed statistically significant correlation of leukocyte count, splenomegaly, and thrombosis in JAK2V617F-mutated patients as compared to unmutated patients. Conclusion This study emphasizes the importance of JAK2V617F mutation in CMPNs and stresses on its association with clinical, hematological phenotype, and thrombotic complications, which may open new horizons in prognostication and management protocol.

Triple-Negativity Identifies a Subgroup of Patients with Better Overall Survival in Essential Thrombocythemia.

Essential thrombocythemia, as defined by the WHO in 2016, is a Philadelphia-negative chronic myeloproliferative neoplasm showing a better prognosis than polycythemia vera and myelofibrosis. In a variable percentage, patients with essential thrombocythemia show none of the known driver-gene mutations that may occur on JAK2, CALR, and MPL genes. Such patients are classified as triple-negative and their clinical features and prognosis have not been described with precision yet. In this study, we evaluated some of the characteristics of this population by comparing them with those of patients with driver-gene mutated ET. Data from 266 consecutive essential thrombocythemia patients were analysed. Triple-negative patients had a significantly lower symptom load and a lower frequency of splenomegaly at diagnosis. The results show that the rate of thrombosis was equal in the two subgroups. Overall survival was slightly better in the triple-negative group of patients.

A Novel Morphological Parameter Predicting Fibrotic Evolution in Myeloproliferative Neoplasms: New Evidence and Molecular Insights.

Philadelphia-negative chronic myeloproliferative neoplasms (MPNs) represent a group of hematological disorders that are traditionally considered as indistinct slow progressing conditions; still, a subset of cases shows a rapid evolution towards myelofibrotic bone marrow failure. Specific abnormalities in the megakaryocyte lineage seem to play a central role in this evolution, especially in the bone marrow fibrosis but also in the induction of myeloproliferation. In this review, we analyze the current knowledge of prognostic factors of MPNs related to their evolution to myelofibrotic bone marrow failure. Moreover, we focused the role of the megakaryocytic lineage in the various stages of MPNs, with updated examples of MPNs in vitro and in vivo models and new therapeutic implications.

Spleen and Liver Fibrosis Is Associated to Treatment Response and Prognosis in Philadelphia-Negative Chronic Myeloproliferative Neoplasms

Spleen and Liver Fibrosis Is Associated to Treatment Response and Prognosis in Philadelphia-Negative Chronic Myeloproliferative Neoplasms

Philadelphia-Negative Chronic Myeloproliferative Neoplasms during the COVID-19 Pandemic: Challenges and Future Scenarios.

Simple SummaryLittle information has been reported about the impact of the COVID-19 pandemic in Philadelphia-negative chronic myeloproliferative neoplasms (MPN). In this review, we summarize the knowledge about MPN clinical management, including cytoreductive and antiplatelet/anticoagulant therapy, thrombotic risk, prognosis, and vaccination strategies at the time of COVID-19.An outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) started in December 2019 in China and then become pandemic in February 2020. Several publications investigated the possible increased rate of COVID-19 infection in hematological malignancies. Based on the published data, strategies for the management of chronic Philadelphia-negative chronic myeloproliferative neoplasms (MPNs) are provided. The risk of severe COVID-19 seems high in MPN, particularly in patients with essential thrombocythemia, but not negligible in myelofibrosis. MPN patients are at high risk of both thrombotic and hemorrhagic complications and this must be accounted in the case of COVID-19 deciding on a case-by-case basis. There are currently no data to suggest that hydroxyurea or interferon may influence the risk or severity of COVID-19 infection. Conversely, while the immunosuppressive activity of ruxolitinib might pose increased risk of infection, its abrupt discontinuation during COVID-19 syndrome is associated with worse outcome. All MPN patients should receive vaccine against COVID-19; reassuring data are available on efficacy of mRNA vaccines in MPNs.

Increased Plasma Levels of lncRNAs LINC01268, GAS5 and MALAT1 Correlate with Negative Prognostic Factors in Myelofibrosis.

Simple SummaryMyelofibrosis (MF) displays the worst prognosis among Philadelphia-negative chronic myeloproliferative neoplasms. There is no curative therapy for MF, except for bone marrow transplantation, which however has a consistent percentage of failure. There is thus an urgent need of novel biomarkers to complement current stratification models and to enable better management of patients. To address this issue, we herein measured the plasma levels of several long noncoding RNAs (lncRNAs). Circulating lncRNAs has been already largely described as potential non-invasive biomarkers in cancers. In our study we unveiled that LINC01268, MALAT1 (both p < 0.0001) and GAS5 (p = 0.0003) plasma levels are significantly higher in MF patients if compared with healthy donors, and their increased plasma levels correlate with several detrimental features in MF. Among them, LINC01268 is an independent variable for both OS (p = 0.0297) and LFS (p = 0.0479), thus representing a putative new biomarker suitable for integrate contemporary prognostic models.Long non-coding RNAs (lncRNAs) have been recently described as key mediators in the development of hematological malignancies. In the last years, circulating lncRNAs have been proposed as a new class of non-invasive biomarkers for cancer diagnosis and prognosis and to predict treatment response. The present study is aimed to investigate the potential of circulating lncRNAs as non-invasive prognostic biomarkers in myelofibrosis (MF), the most severe among Philadelphia-negative myeloproliferative neoplasms. We detected increased levels of seven circulating lncRNAs in plasma samples of MF patients (n = 143), compared to healthy controls (n = 65). Among these, high levels of LINC01268, MALAT1 or GAS5 correlate with detrimental clinical variables, such as high count of leukocytes and CD34+ cells, severe grade of bone marrow fibrosis and presence of splenomegaly. Strikingly, high plasma levels of LINC01268 (p = 0.0018), GAS5 (p = 0.0008) or MALAT1 (p = 0.0348) are also associated with a poor overall-survival while high levels of LINC01268 correlate with a shorter leukemia-free-survival. Finally, multivariate analysis demonstrated that the plasma level of LINC01268 is an independent prognostic variable, suggesting that, if confirmed in future in an independent patients’ cohort, it could be used for further studies to design an updated classification model for MF patients.

Abnormal Expression Of WT1-As, MEG3 and ANRIL Long Non-Coding RNAs In Primary Myelofibrosis and Their Clinical Correlates

A growing body of evidence suggests that long non-coding RNAs (lncRNAs) are involved in the regulation of gene expression in normal and cancer cells by recruiting chromatin remodeling complexes to specific genomic loci [Rinn JL, et al. Cell. 2007; Mattick JS, et al. Bioessays 2009]. For this reason, lncRNAs deregulation can play key roles in malignant transformation and cancer cell behavior. [Kogo R, et al. Cancer Res 2011; Yang F, et al. Hepatology 2011].Despite the increasing number of studies on lncRNAs expression and their involvement in some solid tumors, lncRNAs are far from being extensively characterized in malignant hematopoiesis [Heuston EF, Front Genet 2011]. In particular, lncRNAs expression has never been investigated in cells from primary myelofibrosis (PMF) patients.PMF is a Philadelphia negative chronic Myeloproliferative Neoplasm (MPN) that originates from deregulated clonal proliferation of hematopoietic stem cell associated with overproduction of mature blood cells. Molecular mechanisms underlying MPN pathogenesis were partially unraveled in 2005-2006 with the identification of somatic gain-of-function mutations of JAK2 [James C, et al. Nature 2005; Kralovics R, et al. N Engl J Med 2005; Levine RL, et al. Cancer Cell 2005] and MPL [Pardanani AD, et al. Blood 2006] after which many other mutations were identified. Recently, several new molecular pathogenetic mechanisms were proposed, such as the aberrant expression of coding and non-coding RNAs.After extensive screening of the long non-coding RNA database (lncRNAdb), which is a database providing comprehensive annotations of eukaryotic lncRNAs (http://www.lncrnadb.org) [Amaral PP, Nucleic Acids Res 2011], we identified some previously described lncRNAs as potentially involved in hematological malignancies, such as CDKN2B-antisense (ANRIL), MEG3 and WT1-antisense lncRNAs.With the aim to describe possible abnormalities of lncRNAs in MPN, we investigated the expression of those lncRNAs in CD34+ cells from PMF patients. Since the perturbation of the antisense RNA could alter the expression of the sense gene, we also analyzed the expression of the ANRIL and WT1-as coding genes, called CDKN2B and WT1, to evaluate the correlation of each sense/antisense couple.This study included a total of 26 PMF patients and 16 healthy donors. The diagnosis of PMF was made according to World Health Organization (WHO) criteria.The results evidenced that the majority of PMF samples (74%) displayed a co-upregulation of WT1 and its antisense RNA compared to healthy controls. We found that the large part (61%) of patients harboring concordant upregulation of WT1 sense and antisense belongs to the intermediate-2 and high risk categories of DIPSS-plus scoring system (57.4% and 43.1% in the intermediate-1 and -2, respectively versus 0% in the high-risk category; P = .006) and presents low Hb level, higher percentage of circulating CD34+ cells and a blast count > 1% .Noteworthy, almost 90% of patients with increased expression of WT1 and its antisense showed concurrent upregulation of MEG3 and shared the same clinical variables associated with poor outcome. In fact, also patients harboring higher levels of MEG3 cluster in higher DIPSS-plus scoring system categories, suffer from anemia, have high levels of circulating CD34+ cells and a blast count >1%. These data suggest that not only high levels of WT1-as we previously described [Guglielmelli P, Stem Cells 2007], but also its antisense and MEG3 could identify PMF patients with more severe disease.Finally, we could observe a correlation between CDKN2B upregulation and the grade of BM fibrosis. In fact, the majority of samples having CDKN2B upregulation clustered in categories 2 and 3 (87.3%) as compared to only 4.5% in case of samples showing CDKN2B downregulation (P = .043). Noteworthy, 82% of patients overexpressing CDKN2B were JAK2V617F positive as compared to 22.1% among those with CDKN2B downregulation (P = .004)To our knowledge, this is the first study describing the expression profiles of human lncRNAs in CD34+ cells of PMF patients. Our data demonstrate that WT1-as, MEG3 and ANRIL lncRNAs are aberrantly expressed in PMF CD34+ cells compared to healthy controls. Moreover, different patterns of expression are correlated with some patient' clinical features. Therefore, our results suggest that a deregulated expression of these lncRNAs could play a role in PMF pathogenesis and progression. Disclosures:No relevant conflicts of interest to declare.

The Role of the Axis TGF-β1/Mir-382-5p/SOD2 in the Induction of Oxidative Stress in CD34+ Hematopoietic Stem/Progenitor Cells from Primary Myelofibrosis

Philadelphia negative chronic myeloproliferative neoplasms (MPNs) are acquired clonal disorders of hematopoietic stem cell. Among MPNs, Primary Myelofibrosis (PMF) carries the worst prognosis with severe clinical hallmarks, including bone marrow failure, extramedullary hematopoiesis and splenomegaly. MNPs are intimately associated with high level of pro-inflammatory cytokines (i.e., transforming growth factor-β1, TGF-β1) and excess generation of Reactive Oxygen Species (ROS) that are, at least in part, responsible for disease progression. Recently, extensive studies have provided unequivocal evidences that differentially expressed microRNAs (miRNAs) may play a role in MNP's pathogenesis. The miRNA expression profile performed on CD34+ hematopoietic stem/progenitor cells (HSPCs) obtained from PMF patients (R. Norfo et al., Blood, 2014), allowed us to identify miR-382-5p as up-regulated miRNA. Overexpression experiments using miRNA mimic in normal CD34+ cells have demonstrated its central role in granulocyte fate decision (R. Zini et al., Stem Cell and Dev, 2016).In this study, to establish the role of miR-382-5p in PMF pathogenesis, we performed a gene expression profile (GEP) in cord blood CD34+ cells overexpressing miR-382-5p by means of Affymetrix HG-U219 microarray platform. We identified 75 downregulated genes in miR-382-5p overexpressing cells compared to controls. Then, we pointed out 6 genes downregulated both upon miRNA overexpression and in PMF CD34+ cells compared to healthy donor. Among them, we selected the anti-oxidant superoxide dismutase 2 (SOD2), depicted as the most favorable predicted target according to different prediction algorithms such as Target Scan, miRNA.org and miRDB. Luciferase reporter assay, performed in K562 cells, confirmed SOD2 as a real miR-382-5p target. Then, we showed that miR-382-5p overexpression leads to SOD2 down-regulation both at mRNA and protein level and reduced SOD activity in CB CD34+ cells. Furthermore, we demonstrated that miR-382-5p induced ROS accumulation as compared to control (42.1±1.5% vs 31.8±1.7%, p<0.05), increased DNA damages (measured by 8-OHdG level) and enhanced CD34+ cells proliferation. Afterwards, to confirm that miR-382-5p play a role in PMF pathogenesis, we performed inhibition experiments in PMF CD34+ cells using miR-382-5p antagomiR. Our data revealed that miR-382-5p silencing restored SOD2 expression and activity, induced ROS disposal (50.1±3.8% vs 62.1±3.2%, p<0,05), decreased DNA oxidation and impaired cell proliferation.Then, to verify whether miR-382-5p could be related to chronic inflammatory state typical of PMF, we treated CD34+ cells with the pro-inflammatory cytokine TGF-β1, already described up-regulated cytokine in PMF serum, and involved in PMF pathogenesis (A. Agarwal et al., Stem Cell Investig, 2016). We showed that TGF-β1 treatment induced miR-382-5p expression, which in turn suppresses SOD2 both at mRNA and protein level and impaired its activity leading to ROS over-production (54.7±1.1% vs 36.7±1.3% 24h after TGF-β1 treatment, p<0.05, 72.4±0.7% vs 51.9±2.0% 48h after TGF-β1 treatment, p<0.01). Finally, to further confirm the involvement of the axis TGF-β1/miR-382-5p/SOD2 in the induction of oxidative stress, we inhibited TGF-β1 pathway in PMF CD34+ cells using 500nM of Galunisertib, a specific TGF-β-receptor I kinase inhibitor, already demonstrated to be capable of attenuating the fibrotic phenotype in MPN mouse model (L.Yue et al., Blood, 2015). As a result, TGF-β1 inhibition reduced miR-382-5p expression, increasing SOD activity and its expression both at mRNA and protein level. Of note, the TGF-β-receptor I kinase inhibitor significantly reduced ROS accumulation 24h after Galunisertib treatment, in PMF CD34+ cells as compared to untreated sample (33.1±7.2% vs 45.3±6.3%, p< 0.05).Taken together, our data showed that miR-382-5p enhanced oxidative stress and oxidative-DNA-damages in normal and PMF CD34+ cells through the modulation of its target SOD2. We showed for the first time that the axis TGF-β1/miR-382-5p/SOD2 is responsible, at least in part, for ROS over-production that contribute to a state of chronic oxidative stress and inflammation, which are typical features of PMF. Furthermore, our results suggested that Galunisertib might be an effective therapy to reduce abnormal oxidative stress sustained by increased level of TGF-β1 in PMF CD34+ cells. [Display omitted] DisclosuresVannucchi:Novartis: Honoraria, Speakers Bureau; Shire: Speakers Bureau.

Cigarette Smoking, Complete Blood Count, and Myeloproliferative Neoplasms — a Meta-Analysis

IntroductionThe classic chronic Philadelphia-negative chronic myeloproliferative neoplasms (MPN) are acquired stem cell diseases and include essential thrombocythemia, polycythemia vera, primary myelofibrosis and unclassifiable MPN. Recently, these disorders have been described as “A Human Inflammation Model for Cancer Development”, reflecting chronic inflammation to be a major driving force for clonal evolution and disease progression in MPNs.The objectives of this meta-analysis were to investigate if cigarette smoking is associated with increased blood cell counts (CBC) and incidence of MPN as proof of concept.MethodA systematic review and meta-analysis was undertaken using databases Pubmed, EMBASE, Web of Science and World Health Organization Global Health Library for all relevant papers published until 15th February 2017 on smoking and complete blood counts and on smoking and myeloproliferative neoplasms according to a formal protocol that followed the PRISMA guidelines.Following data extraction, STATA 13.0 was used to calculate raw and standardized mean differences for CBC (in SI units) and pooled odds ratios for MPN with 95% confidence intervals using random and fixed effects models. Main analyses included analyses of current smokers, ex-smokers, and ever smokers vs. non- or never smokers. Heterogeneity was measured using I2 -statistical analysis and Cochrane Q statistic, and reported corresponding p-values and sensitivity analyses included among others gender and smoke dosage(heavy (>=20 cigarettes/day) and light(<20 cigarettes/day)). Funnel plots, Egger's test and Begg's tests were used to assess publication bias.ResultsComplete Blood Count (CBC): A total of 88 references published from 1974-2017 in 10 different languages and including 90 study populations with 533,242 individuals, 222,887 current smokers, 92,001 ex-smokers, and 222,538 non-smokers between the ages of 15 and 89 from 34 countries were analyzed.Meta-analyses showed that the mean of most CBCs were higher in smokers and ex-smokers compared to non-nonsmokers. Differences were larger comparing smokers to non-smokers than comparing ex-smokers to non-smokers, and also larger comparing heavy smokers to non-smokers than comparing light smokers to non-smokers. Furthermore, differences in most CBCs were higher in men compared to women.All analyses presented high heterogeneity in main and sensitivity analyses but no overall publication bias.Myeloproliferative Neoplasms (MPN): We included 5 studies with a total of 1,368,738 individuals and 2017 MPN cases. 2 studies were prospective and 3 studies case-control. Pooled odds ratio for MPN were 1.44 (95%CI: 1.33-1.56; p-value=5*10(-20); I-square=0%, p(heterogeneity)=0.56) comparing smokers to non-smokers, 1.10(0.86-1.41; p-value=0.46; I-square=71%, p(heterogeneity)=0.03) comparing ex-smokers to non-smokers, and 1.30(1.14-1.49; p-value=8*10(-5); I-square=45%, p(heterogeneity)=0.16) comparing ever-smokers to never smokers.ConclusionCurrent smokers, ex-smokers, and ever-smokers have higher mean CBCs and an increased risk of MPN compared to non-smokers. [Display omitted] DisclosuresHasselbalch:AOR Orphan: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Genetics and Pathogenetic Role of Inflammasomes in Philadelphia Negative Chronic Myeloproliferative Neoplasms: A Narrative Review.

The last decade has been very important for the quantity of preclinical information obtained regarding chronic myeloproliferative neoplasms (MPNs) and the following will be dedicated to the translational implications of the new biological acquisitions. The overcoming of the mechanistic model of clonal evolution and the entry of chronic inflammation and dysimmunity into the new model are the elements on which to base a part of future therapeutic strategies. The innate immune system plays a major role in this context. Protagonists of the initiation and regulation of many pathological aspects, from cytokine storms to fibrosis, the NLRP3 and AIM2 inflammasomes guide and condition the natural history of the disease. For this reason, MPNs share many biological and clinical aspects with non-neoplastic diseases, such as autoimmune disorders. Finally, cardiovascular risk and disturbances in iron metabolism and myelopoiesis are also closely linked to the role of inflammasomes. Although targeted therapies are already being tested, an increase in knowledge on the subject is desirable and potentially translates into better care for patients with MPNs.

A NEW TYPE-2-LIKE CALR MUTATION IN ESSENTIAL THROMBOCYTHEMIA

CALR mutations, together with JAK-2 and MPL ones, are recognized as “driver” mutations in Philadelphia-negative chronic myeloproliferative neoplasms (MPNs). Most frequent CALR mutations are Type-1 deletions (45-55% of cases) and type-2 insertion (32-42% of cases). These mutations are usually associated with younger age, higher platelet counts, lower leukocyte counts, lower hemoglobin levels and a higher incidence of transformation from ET to MF. Recognizing and describing cases with different mutations can be useful to create a database that might help clinicians to include these patients in risk categories and to guide the appropriate therapeutic choices. We report a case of a 77-years old woman who presented a new type-2 like CALR mutation.