JAK2V617F drives gut microbiota differences in patients with myeloproliferative neoplasms.

BACKGROUND: Essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (MF) are myeloproliferative neoplasms (MPN). Inflammation is involved in the initiation, progression, and symptomology of the diseases. The gut microbiota impacts the immune system, infection control, and steady-state hematopoiesis. METHODS: We analyzed the gut microbiota of 227 MPN patients and healthy controls (HCs) using next-generation sequencing. We expanded our previous results in PV and ET patients with additional PV, pre-MF, and MF patients which allowed us to compare MPN patients collectively, MPN sub-diagnoses, and MPN mutations (separately and combined) vs. HCs (N = 42) and compare within MPN sub-diagnoses and MPN mutation. RESULTS: MPN patients had a higher observed richness (median, 245 [range, 49-659]) compared with HCs (191.5 [range, 111-300; p = .003]) and a lower relative abundance of taxa within the Firmicutes phylum; for example, Faecalibacterium (6% vs. 14%, p < .001). The microbiota of CALR-positive patients (N = 30) resembled that of HCs more than that of patients with JAK2V617F (N = 177). In JAK2V617F-positive patients, only minor differences in the gut microbiota were observed between MPN sub-diagnoses, illustrating the importance of this mutation. CONCLUSION: The gut microbiota in MPN patients differs from HCs and is driven by JAK2V617F, whereas the gut microbiota in CALR patients resembles HCs more.

Interferon alpha-2 treatment reduces circulating neutrophil extracellular trap levels in myeloproliferative neoplasms.

Neutrophil extracellular traps (NETs) may play a pathogenic role in the thrombosis associated with myeloproliferative neoplasms (MPNs). We measured serum NET levels in 128 pretreatment samples from patients with MPNs and in 85 samples taken after 12 months of treatment with interferon alpha-2 (PEG-IFNα-2) formulations or hydroxyurea (HU). No differences in NET levels were observed across subdiagnoses or phenotypic driver mutations. In PV, a JAK2V617F+ allele burden ≥50% associated with increased NET levels (p = 0.006). Baseline NET levels correlated with neutrophil count (r = 0.29, p = 0.001), neutrophil-to-lymphocyte ratio (r = 0.26, p = 0.004) and JAK2V617F allele burden (r = 0.22, p = 0.03), particularly in patients with PV and with allele burden ≥50% (r = 0.50, p = 0.01, r = 0.56, p = 0.002 and r = 0.45, p = 0.03 respectively). In PV, after 12 months of treatment, NET levels decreased on average by 60% in patients with allele burden ≥50%, compared to only 36% in patients with an allele burden <50%. Overall, treatment with PEG-IFNα-2a or PEG-IFNα-2b reduced NETs levels in 77% and 73% of patients, respectively, versus only 53% of HU-treated patients (average decrease across treatments: 48%). Normalization of blood counts did not per se account for these reductions. In conclusion, baseline NET levels correlated with neutrophil count, NLR and JAK2V617F allele burden, and IFNα was more effective at reducing prothrombotic NET levels than HU.

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

BACKGROUND: 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. OBJECTIVES: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

Clonal haematopoiesis of indeterminate potential and impaired kidney function-A Danish general population study with 11 years follow-up.

The myeloproliferative neoplasms are associated with chronic kidney disease but whether clonal haematopoiesis of indeterminate potential (CHIP) is associated with impaired kidney function is unknown. In the Danish General Suburban Population Study (N = 19 958) from 2010 to 2013, 645 individuals were positive for JAK2V617F (N = 613) or CALR (N = 32) mutations. Mutation-positive individuals without haematological malignancy were defined as having CHIP (N = 629). We used multiple and inverse probability weighted (IPW)-adjusted linear regression analysis to estimate adjusted mean (95% confidence interval) differences in estimated glomerular filtration rate (eGFR; ml/min/1.73 m2 ) by mutation status, variant allele frequency (VAF%), blood cell counts, and neutrophil-to-lymphocyte ratio (NLR). We performed 11-year longitudinal follow-up of eGFR in all individuals. Compared to CHIP-negative individuals, the mean differences in eGFR were -5.6 (-10.3, -0.8, p = .02) for CALR, -11.9 (-21.4, -2.4, p = 0.01) for CALR type 2, and -10.1 (-18.1, -2.2, p = .01) for CALR with VAF ≥ 1%. The IPW-adjusted linear regression analyses showed similar results. NLR was negatively associated with eGFR. Individuals with CALR type 2 had a worse 11-year longitudinal follow-up on eGFR compared to CHIP-negative individuals (p = .004). In conclusion, individuals with CALR mutations, especially CALR type 2, had impaired kidney function compared to CHIP-negative individuals as measured by a lower eGFR at baseline and during 11-year follow-up.

Smoking impairs molecular response, and reduces overall survival in patients with chronic myeloproliferative neoplasms: A retrospective cohort study.

The effects of smoking on the molecular response (MR) and overall survival (OS) in patients with chronic myeloproliferative neoplasms (MPNs) have not been investigated before. We analysed a historical cohort of 498 consecutive patients diagnosed with MPNs. Moreover, we analysed a subgroup of 270 consecutive patients with MPNs with > 1 measurement of the JAK2V617F variant allele frequency. The data were analysed using Kaplan-Meier plots and Cox regression analysis, along with linear regression models. In all patients, the rate of MR was significantly higher in never-smokers compared with current smokers in the univariate model (HR, 1·9; 95% CI, 1·1-3·3; P = 0·033) and the multivariate model (HR, 1·9; 95% CI, 1·1-3·5; P = 0·029). Similar findings were observed with different cut-off values for a partial MR. A subgroup analysis including only interferon-α2-treated patients showed similar results. In multivariate analyses, the OS was significantly better for never-smokers (HR, 0·46; 95% CI, 0·29-0·75; P = 0·002) than current smokers. The differences were more pronounced in the pegylated interferon-α2-treated patients. However, no significant interaction of interferon-α2 treatment was observed. In conclusion, we found that tobacco smoking reduced the rate of MR and OS in patients with MPNs. Cessation of smoking should be encouraged.

Data-driven analysis of the kinetics of the JAK2V617F allele burden and blood cell counts during hydroxyurea treatment of patients with polycythemia vera, essential thrombocythemia, and primary myelofibrosis.

BACKGROUND: Hydroxyurea (HU) treatment of patients with essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF) (MPNs) normalizes elevated blood cell counts within weeks in the large majority of patients. Studies on the impact of HU upon the kinetics of the JAK2V617F allele burden, leukocyte, and platelet counts over time are scarce. PURPOSE: Using data-driven analysis as a novel tool to model the kinetics of the JAK2V617F allele burden and blood cell counts over time during treatment with HU. MATERIAL AND METHODS: Using serial measurements of JAK2V617F and correlation analysis of routine hematological values (the Hb-concentration, leukocyte count, platelet count, and lactic dehydrogenase), we present a detailed description and analysis of the kinetics of the JAK2V617F, leukocyte, and platelet counts and lactic dehydrogenase in 27 patients (PV = 18; ET = 7; PMF = 2), who were followed in the Danish randomized trial (DALIAH). To further analyze the JAK2V617F kinetics, we use a machine learning clustering algorithm to group the response patterns. RESULTS: Response patterns were highly heterogeneous, with clustering resulting in 3 groups and 3 outliers. In the large majority of patients, HU treatment was initially associated with a modest decline in the JAK2V617F allele burden in concert with a decline in leukocyte and platelet counts. However, HU did not induce a sustained and continuous decrease in the JAK2V617F allele burden. CONCLUSION: Using data-driven analysis of the JAK2V617F allele burden, leukocyte, and platelet kinetics during treatment with HU, we have shown that HU does not induce a sustained decrease in the JAK2V617F allele burden and neither induces sustained normalization of elevated cell counts in MPN patients. Our results may explain why MPN patients during treatment with HU still have a substantially increased risk of thrombosis.

Effect of a Posture-Cueing Shirt on Sitting Posture During a Functional Task in Healthy Participants: A Randomized Cross-Over Study.

OBJECTIVES: The purpose of this study was to investigate the effect of a posture-cueing shirt on sitting posture during a functional task. METHODS: Thirty healthy male participants were seated at a standardized workstation while completing 3 laptop writing tasks of 15-minute duration wearing either a posture-cueing shirt, a compression shirt, or no shirt. Posture was assessed based on photos taken at minutes 1 and 15 into the writing task from which the head and shoulder angles were measured and extracted for analysis. After each task, participants rated any potential pain they felt during the task on an 11-point numeric rating scale (NRS). RESULTS: The results showed that none of the shirts significantly affected the head or shoulder angles at any time point. Participants reported lower pain levels after using the posture-cueing shirt (NRS 0 [0-1]) compared with no shirt (NRS 1 [0-2]; P = .012). No significant difference in pain levels was observed between shirts. CONCLUSION: Although posture did not change in any conditions for these healthy male participants, the posture-cueing shirt resulted in a lower pain intensity compared with no shirt but not with a compression shirt. Although a significant difference was found for pain intensity favoring the posture-cueing shirt, this difference was negligible, and thus its value to reduce pain or improve posture in healthy participants remains in question.

Ruxolitinib and interferon-α2 combination therapy for patients with polycythemia vera or myelofibrosis: a phase II study.

We report the final 2-year end-of-study results from the first clinical trial investigating combination treatment with ruxolitinib and low-dose pegylated interferon-α2 (PEG-IFNα2). The study included 32 patients with polycythemia vera and 18 with primary or secondary myelofibrosis; 46 patients were previously intolerant of or refractory to PEGIFNα2. The primary outcome was efficacy, based on hematologic parameters, quality of life measurements, and JAK2 V617F allele burden. We used the 2013 European LeukemiaNet and International Working Group- Myeloproliferative Neoplasms Research and Treatment response criteria, including response in symptoms, splenomegaly, peripheral blood counts, and bone marrow. Of 32 patients with polycythemia vera, ten (31%) achieved a remission which was a complete remission in three (9%) cases. Of 18 patients with myelofibrosis, eight (44%) achieved a remission; five (28%) were complete remissions. The cumulative incidence of peripheral blood count remission was 0.85 and 0.75 for patients with polycythemia vera and myelofibrosis, respectively. The Myeloproliferative Neoplasm Symptom Assessment Form total symptom score decreased from 22 [95% confidence interval (95% CI):, 16-29] at baseline to 15 (95% CI: 10-22) after 2 years. The median JAK2 V617F allele burden decreased from 47% (95% CI: 33-61%) to 12% (95% CI: 6-22%), and 41% of patients achieved a molecular response. The drop-out rate was 6% among patients with polycythemia vera and 32% among those with myelofibrosis. Of 36 patients previously intolerant of PEG-IFNα2, 31 (86%) completed the study, and 24 (67%) of these received PEG-IFNα2 throughout the study. In conclusion, combination treatment improved cell counts, reduced bone marrow cellularity and fibrosis, decreased JAK2 V617F burden, and reduced symptom burden with acceptable toxicity in several patients with polycythemia vera or myelofibrosis. #EudraCT2013-003295-12.

Reduced skeletal-muscle perfusion and impaired ATP release during hypoxia and exercise in individuals with type 2 diabetes.

AIMS/HYPOTHESIS: Plasma ATP is a potent vasodilator and is thought to play a role in the local regulation of blood flow. Type 2 diabetes is associated with reduced tissue perfusion. We aimed to examine whether individuals with type 2 diabetes have reduced plasma ATP concentrations compared with healthy control participants (case-control design). METHODS: We measured femoral arterial and venous plasma ATP levels with the intravascular microdialysis technique during normoxia, hypoxia and one-legged knee-extensor exercise (10 W and 30 W) in nine participants with type 2 diabetes and eight control participants. In addition, we infused acetylcholine (ACh), sodium nitroprusside (SNP) and ATP into the femoral artery to assess vascular function and ATP signalling. RESULTS: Individuals with type 2 diabetes had a lower leg blood flow (LBF; 2.9 ± 0.1 l/min) compared with the control participants (3.2 ± 0.1 l/min) during exercise (p < 0.05), in parallel with lower venous plasma ATP concentration (205 ± 35 vs 431 ± 72 nmol/l; p < 0.05). During systemic hypoxia, LBF increased from 0.35 ± 0.04 to 0.54 ± 0.06 l/min in control individuals, whereas it did not increase (0.25 ± 0.04 vs 0.31 ± 0.03 l/min) in the those with type 2 diabetes and was lower than in the control individuals (p < 0.05). Hypoxia increased venous plasma ATP levels in both groups (p < 0.05), but the increase was higher in control individuals (90 ± 26 nmol/l) compared to those with type 2 diabetes (18 ± 5 nmol/l). LBF and vascular conductance were lower during ATP (0.15 and 0.4 µmol min-1 [kg leg mass]-1) and ACh (100 µg min-1 [kg leg mass]-1) infusion in individuals with type 2 diabetes compared with the control participants (p < 0.05), whereas there was no difference during SNP infusion. CONCLUSIONS/INTERPRETATION: These findings demonstrate that individuals with type 2 diabetes have lower plasma ATP concentrations during exercise and hypoxia compared with control individuals, and this occurs in parallel with lower blood flow. Moreover, individuals with type 2 diabetes have a reduced vasodilatory response to infused ATP. These impairments in the ATP system are both likely to contribute to the reduced tissue perfusion associated with type 2 diabetes. TRIAL REGISTRATION: ClinicalTrials.gov NCT02001766.

Bridging blood cancers and inflammation: The reduced Cancitis model.

A novel mechanism-based model - the Cancitis model - describing the interaction of blood cancer and the inflammatory system is proposed, analyzed and validated. The immune response is divided into two components, one where the elimination rate of malignant stem cells is independent of the level of the blood cancer and one where the elimination rate depends on the level of the blood cancer. A dimensional analysis shows that the full 6-dimensional system of nonlinear ordinary differential equations may be reduced to a 2-dimensional system - the reduced Cancitis model - using Fenichel theory. The original 18 parameters appear in the reduced model in 8 groups of parameters. The reduced model is analyzed. Especially the steady states and their dependence on the exogenous inflammatory stimuli are analyzed. A semi-analytic investigation reveals the stability properties of the steady states. Finally, positivity of the system and the existence of an attracting trapping region in the positive octahedron guaranteeing global existence and uniqueness of solutions are proved. The possible topologies of the dynamical system are completely determined as having a Janus structure, where two qualitatively different topologies appear for different sets of parameters. To classify this Janus structure we propose a novel concept in blood cancer - a reproduction ratio R. It determines the topological structure depending on whether it is larger or smaller than a threshold value. Furthermore, it follows that inflammation, affected by the exogenous inflammatory stimulation, may determine the onset and development of blood cancers. The body may manage initial blood cancer as long as the self-renewal rate is not too high, but fails to manage it if an inflammation appears. Thus, inflammation may trigger and drive blood cancers. Finally, the mathematical analysis suggests novel treatment strategies and it is used to discuss the in silico effect of existing treatment, e.g. interferon-α or T-cell therapy, and the impact of malignant cells becoming resistant.

Mathematical modelling of stem and progenitor cell dynamics during ruxolitinib treatment of patients with myeloproliferative neoplasms.

Introduction: The Philadelphia chromosome-negative myeloproliferative neoplasms are a group of slowly progressing haematological malignancies primarily characterised by an overproduction of myeloid blood cells. Patients are treated with various drugs, including the JAK1/2 inhibitor ruxolitinib. Mathematical modelling can help propose and test hypotheses of how the treatment works. Materials and methods: We present an extension of the Cancitis model, which describes the development of myeloproliferative neoplasms and their interactions with inflammation, that explicitly models progenitor cells and can account for treatment with ruxolitinib through effects on the malignant stem cell response to cytokine signalling and the death rate of malignant progenitor cells. The model has been fitted to individual patients' data for the JAK2 V617F variant allele frequency from the COMFORT-II and RESPONSE studies for patients who had substantial reductions (20 percentage points or 90% of the baseline value) in their JAK2 V617F variant allele frequency (n = 24 in total). Results: The model fits very well to the patient data with an average root mean square error of 0.0249 (2.49%) when allowing ruxolitinib treatment to affect both malignant stem and progenitor cells. This average root mean square error is much lower than if allowing ruxolitinib treatment to affect only malignant stem or only malignant progenitor cells (average root mean square errors of 0.138 (13.8%) and 0.0874 (8.74%), respectively). Discussion: Systematic simulation studies and fitting of the model to the patient data suggest that an initial reduction of the malignant cell burden followed by a monotonic increase can be recapitulated by the model assuming that ruxolitinib affects only the death rate of malignant progenitor cells. For patients exhibiting a long-term reduction of the malignant cells, the model predicts that ruxolitinib also affects stem cell parameters, such as the malignant stem cells' response to cytokine signalling.

Neutrophil-to-lymphocyte ratio and all-cause mortality with and without myeloproliferative neoplasms-a Danish longitudinal study.

The neutrophil-to-lymphocyte ratio(NLR) is increased in chronic inflammation and myeloproliferative neoplasms (MPN). We hypothesize that NLR is associated with all-cause mortality and mortality by comorbidity burden in the general population and individuals with MPN. We included 835,430 individuals from The Danish General Suburban Population Study, general practitioners, and outpatient clinics. We investigated NLR on mortality stratified by prevalent and incident MPN, essential thrombocythemia (ET), polycythemia vera (PV), myelofibrosis (MF), comorbidity burden (CCI-score), and the Triple-A risk score using hazard ratio (HR) and 95% confidence interval (95%CI). NLR 1-1.9 was the reference level. During a median follow-up of 11.2 years, 197,802 deaths were recorded. All-cause mortality increased for a stepwise increasing NLR with a HR (95%CI) for NLR ≥ 6 of 2.06(2.03-2.09) for the whole population and 2.93(2.44-3.50) in prevalent MPN. ET, PV, and MF had a HR (95%CI) for NLR ≥ 2 of 2.14(1.71-2.69), 2.19(1.89-2.54), and 2.31(1.91-2.80). Results were similar for incident MPN. Mortality was higher for stepwise increasing NLR and CCI-score(pinteraction < 2×10-16), with a HR for NLR ≥ 6 of 2.23(2.17-2.29), 4.10(4.01-4.20), and 7.69(7.50-7.89), for CCI-score 0, 1-2, or ≥3. The Triple-A risk score demonstrated alignment with NLR. Increasing NLR and comorbidity burden were associated with lower survival in individuals without MPN but were even worse in prevalent and incident MPN, ET, PV, and MF.

A novel integrated biomarker index for the assessment of hematological responses in MPNs during treatment with hydroxyurea and interferon-alpha2.

BACKGROUND: Conventional cytoreductive therapy for patients with chronic Philadelphia-negative myeloproliferative neoplasms (MPNs) includes hydroxyurea (HU), interferon-alpha2 (IFN), and anagrelide. HU is worldwide the most used cytoreductive agent, which lowers elevated blood cell counts within days in the large majority of patients. However, some patients may experience rebound cytosis when HU is reduced due to cytopenia, thereby potentially giving rise to fluctuating cell counts during therapy. Such rapid oscillations may be harmful and potentially elicit thrombosis. Treatment with IFN gradually lowers elevated cell counts within weeks and when the dosage is reduced, the cell counts do not rapidly increase but are sustained within the normal range in the large majority of patients. Conventional hematological response criteria are among others based upon single absolute cell count values and do not take into account the relative decreases toward normal for each cell count. MATERIALS, METHODS & RESULTS: Using serial data from the Danish DALIAH trial, we herein describe a novel integrated biomarker index for the assessment of hematological and molecular (JAK2V617F) responses in patients with MPNs during treatment with IFN or HU. DISCUSSION: This novel tool convincingly displays the superiority of IFN versus HU in normalizing elevated cell counts. Our results need to be validated in larger studies but already now call for studies of the safety and efficacy of combination therapy during the initial treatment of patients with MPNs.

Revisiting Circulating Extracellular Matrix Fragments as Disease Markers in Myelofibrosis and Related Neoplasms.

Philadelphia chromosome-negative chronic myeloproliferative neoplasms (MPNs) arise due to acquired somatic driver mutations in stem cells and develop over 10-30 years from the earliest cancer stages (essential thrombocythemia, polycythemia vera) towards the advanced myelofibrosis stage with bone marrow failure. The JAK2V617F mutation is the most prevalent driver mutation. Chronic inflammation is considered to be a major pathogenetic player, both as a trigger of MPN development and as a driver of disease progression. Chronic inflammation in MPNs is characterized by persistent connective tissue remodeling, which leads to organ dysfunction and ultimately, organ failure, due to excessive accumulation of extracellular matrix (ECM). Considering that MPNs are acquired clonal stem cell diseases developing in an inflammatory microenvironment in which the hematopoietic cell populations are progressively replaced by stromal proliferation-"a wound that never heals"-we herein aim to provide a comprehensive review of previous promising research in the field of circulating ECM fragments in the diagnosis, treatment and monitoring of MPNs. We address the rationales and highlight new perspectives for the use of circulating ECM protein fragments as biologically plausible, noninvasive disease markers in the management of MPNs.

The gut microbiota in patients with polycythemia vera is distinct from that of healthy controls and varies by treatment.

Chronic inflammation is believed to play an important role in the development and disease progression of polycythemia vera (PV). Because an association between gut microbiota, hematopoiesis, and inflammation is well established, we hypothesized that patients with PV have a gut microbiota distinct from healthy control participants (HCs). Recombinant interferon alfa 2 (IFN-α2)-treatment of patients with PV is reportedly disease modifying in terms of normalization of elevated blood cell counts in concert with a reduction in the JAK2V617F allelic burden. Therefore, we hypothesized that patients treated with IFN-α2 might have a composition of the gut microbiota toward normalization. Herein, via amplicon-based next-generation sequencing of the V3 to V4 regions of the 16S ribosomal RNA gene, we report on an abnormal gut microbiota in 102 patients with PV compared with 42 HCs. Patients with PV had a lower alpha diversity and a lower relative abundance of several taxa belonging to Firmicutes (45%) compared with HCs (59%, P <.001). Furthermore, we report the composition of the gut microbiota to differ between the treatment groups (IFN-α2, hydroxyurea, no treatment, and combination therapy with IFN-α2 and ruxolitinib) and the HCs. These observations are highly interesting considering the potential pathogenetic importance of an altered gut microbiota for development of other diseases, including chronic inflammatory diseases. Our observations call for further gut microbiota studies to decipher potential causal associations between treatment and the gut microbiota in PV and related neoplasms.

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

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

Case Report: First longitudinal study of a patient with CALR positive clonal hematopoiesis of indeterminate potential developing into pre-fibrotic myelofibrosis.

Initial diagnosis of overt myeloproliferative neoplasms (MPNs) represents the juncture during clonal evolution when symptoms or complications prompt an afflicted individual to seek medical attention. In 30-40% of the MPN subgroups essential thrombocythemia (ET) and myelofibrosis (MF), somatic mutations in the calreticulin gene (CALR) are drivers of the disease resulting in constitutive activation of the thrombopoietin receptor (MPL). In the current study, we describe a healthy CALR mutated individual during a 12 year follow-up from initial identification of CALR clonal hematopoiesis of indeterminate potential (CHIP) to the diagnosis of pre-MF. The pre-diagnostic exponential development dynamics of the malignant clone demonstrated close correlation with the platelet counts, neutrophil-to-lymphocyte (NLR) ratio, and inversely correlated to hemoglobin and erythrocyte counts. Backward extrapolation of the growth rate indicated the potential for discovery of the malignant clone many years prior to presentation of overt disease, opening a window of opportunity for early treatment intervention. We did not find any additional mutations associated with MPNs and the current case report provides novel information regarding the development of a driver mutation and the association with blood cell counts prior to clinical manifestation of symptoms suggesting that pre-diagnostic dynamics may supplement future diagnostic criteria for early diagnosis and intervention in MPN patients.

Recombinant Interferon-ß 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-ß (rIFN-ß)-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-ß 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-ß in patients with MPNs.

Interferon-alpha2 treatment of patients with polycythemia vera and related neoplasms favorably impacts deregulation of oxidative stress genes and antioxidative defense mechanisms.

Chronic inflammation is considered a major driving force for clonal expansion and evolution in the Philadelphia-negative myeloproliferative neoplasms, which include essential thrombocythemia, polycythemia vera and primary myelofibrosis (MPNs). One of the key mutation drivers is the JAK2V617F mutation, which has been shown to induce the generation of reactive oxygen species (ROS). Using whole blood gene expression profiling, deregulation of several oxidative stress and anti-oxidative defense genes has been identified in MPNs, including significant downregulation of TP53, the NFE2L2 or NRF2 genes. These genes have a major role for maintaining genomic stability, regulation of the oxidative stress response and in modulating migration or retention of hematopoietic stem cells. Therefore, their deregulation might give rise to increasing genomic instability, increased chronic inflammation and disease progression with egress of hematopoietic stem cells from the bone marrow to seed in the spleen, liver and elsewhere. Interferon-alpha2 (rIFNα) is increasingly being recognized as the drug of choice for the treatment of patients with MPNs. Herein, we report the first gene expression profiling study on the impact of rIFNα upon oxidative stress and antioxidative defense genes in patients with MPNs (n = 33), showing that rIFNα downregulates several upregulated oxidative stress genes and upregulates downregulated antioxidative defense genes. Treatment with rIFNα induced upregulation of 19 genes in ET and 29 genes in PV including CXCR4 and TP53. In conclusion, this rIFNα- mediated dampening of genotoxic damage to hematopoietic cells may ultimately diminish the risk of additional mutations and accordingly clonal evolution and disease progression towards myelofibrotic and leukemic transformation.

Genomic profiling of a randomized trial of interferon-α vs hydroxyurea in MPN reveals mutation-specific responses.

Although somatic mutations influence the pathogenesis, phenotype, and outcome of myeloproliferative neoplasms (MPNs), little is known about their impact on molecular response to cytoreductive treatment. We performed targeted next-generation sequencing (NGS) on 202 pretreatment samples obtained from patients with MPN enrolled in the DALIAH trial (A Study of Low Dose Interferon Alpha Versus Hydroxyurea in Treatment of Chronic Myeloid Neoplasms; #NCT01387763), a randomized controlled phase 3 clinical trial, and 135 samples obtained after 24 months of therapy with recombinant interferon-alpha (IFNα) or hydroxyurea. The primary aim was to evaluate the association between complete clinicohematologic response (CHR) at 24 months and molecular response through sequential assessment of 120 genes using NGS. Among JAK2-mutated patients treated with IFNα, those with CHR had a greater reduction in the JAK2 variant allele frequency (median, 0.29 to 0.07; P < .0001) compared with those not achieving CHR (median, 0.27 to 0.14; P < .0001). In contrast, the CALR variant allele frequency did not significantly decline in those achieving CHR or in those not achieving CHR. Treatment-emergent mutations in DNMT3A were observed more commonly in patients treated with IFNα compared with hydroxyurea (P = .04). Furthermore, treatment-emergent DNMT3A mutations were significantly enriched in IFNα-treated patients not attaining CHR (P = .02). A mutation in TET2, DNMT3A, or ASXL1 was significantly associated with prior stroke (age-adjusted odds ratio, 5.29; 95% confidence interval, 1.59-17.54; P = .007), as was a mutation in TET2 alone (age-adjusted odds ratio, 3.03; 95% confidence interval, 1.03-9.01; P = .044). At 24 months, we found mutation-specific response patterns to IFNα: (1) JAK2- and CALR-mutated MPN exhibited distinct molecular responses; and (2) DNMT3A-mutated clones/subclones emerged on treatment.