Elevated levels of oxidized nucleosides in individuals with the JAK2V617F mutation from a general population study.

It is unknown if the somatic mutations in chronic myeloproliferative neoplasms (MPNs), JAK2V617F and Calreticulin, are associated with oxidative stress, or impaired mitochondrial defense against reactive oxygen species. In the Danish General Suburban Population Study (GESUS), including 116 JAK2V617F-mutated, 8 CALR-mutated, and 3310 mutation-negative participants without overt MPN, and in a study of 39 patients with myelofibrosis, the most advances type of MPNs, and 179 matched controls, we compared the urinary concentration of oxidized nucleosides - 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanosine (8-oxoGuo) - as markers of oxidative stress. In GESUS, we performed Mendelian randomization analyses, using the Ala16Val single nucleotide polymorphism in the superoxide dismutase2 (SOD2) gene. In the multivariate analyses in GESUS, the 8-oxodG and 8-oxoGuo concentration were 13% (95%CI: 6-21%, p < 0.001) and 6% (95%CI: 0.4-11%, p = 0.035) higher in mutation-positive than in mutation-negative participants, respectively. Each SOD2 T allele was associated with an odds ratio of being mutation-positive of 1.69 (95%CI: 1.12-2.55, p = 0.013) through 8-oxodG. The 8-oxodG and 8-oxoGuo concentrations were 77% (95%CI: 49-110%, p < 0.001) and 105% (95%CI: 80-133%, p < 0.001) higher in myelofibrosis patients than in controls, respectively. In conclusion, an impaired mitochondrial antioxidative defense, that is causatively associated with markers of oxidative stress, may contribute to the development of mutations associated with MPNs.

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.

B-cell frequencies and immunoregulatory phenotypes in myeloproliferative neoplasms: Influence of ruxolitinib, interferon-α2, or combination treatment.

OBJECTIVE: Given a proposed role for PD-L1+ and IL-10-producing B-cell subsets in promoting certain cancers, we sought to characterize the frequency and phenotype of B cells in patients with chronic myeloproliferative neoplasms (MPNs) and the influence of ruxolitinib and interferon-α2 therapy. METHODS: We analyzed B-cell frequencies and phenotype in patients with MPNs (n = 107), before and during treatment with ruxolitinib (n = 29), interferon-α2 (n = 21), or the two drugs in combination (COMBI; n = 42) and healthy donors (HDs; n = 52) using flow cytometry. RESULTS: Myelofibrosis patients had lower lymphocyte counts and proportions of B cells than patients with essential thrombocythemia or polycythemia vera and HDs. The B-cell count correlated inversely with JAK2-V617F allele burden and spleen size and increased after ruxolitinib or COMBI treatment. The proportions of PD-L1+ B cells and PD-1+ B cells were significantly higher in patients with myelofibrosis or polycythemia vera than in HDs and decreased during ruxolitinib and COMBI treatment. The proportions of TNF-α+ and IL-6+ B cells were elevated in myelofibrosis patients. The proportion of IL-6+ B cells decreased, and the proportion of IL-10+ B cells increased during ruxolitinib treatment. CONCLUSION: B-cell frequency and phenotype were altered in MPN patients. Ruxolitinib therapy had marked effects on both frequency and phenotype.

Ruxolitinib treatment reduces monocytic superoxide radical formation without affecting hydrogen peroxide formation or systemic oxidative nucleoside damage in myelofibrosis.

The role of excess reactive oxygen species (ROS) with consequent DNA/RNA damage is now recognized as a hallmark of cancer. In JAK2V617F mutated myeloproliferative neoplasms, ROS have been suggested to be important factors in disease initiation and progression. Ruxolitinib is the most widely used drug for myelofibrosis, because it improves symptom-score. However, both the anti-clonal potential and improvement in overall survival are limited. We investigated the impact of ruxolitinib on formation of superoxide radical and hydrogen peroxide by monocytes in sequentially acquired blood samples from patients with myelofibrosis. We also investigated the impact on RNA and DNA damage by measuring urinary excretion of 8-oxo-Guo and 8-oxo-d-Guo. The formation of superoxide by monocytes was reduced significantly during ruxolitinib therapy, but no impact on the formation of hydrogen peroxide by monocytes or the systemic amount of oxidatively damaged RNA or DNA could be demonstrated. We conclude that ruxolitinib holds little anti-oxidative potential.

Safety and efficacy of combination therapy of interferon-α2 and ruxolitinib in polycythemia vera and myelofibrosis.

Interferon-α2 reduces elevated blood cell counts and splenomegaly in patients with myeloproliferative neoplasms (MPN) and may restore polyclonal hematopoiesis. Its use is limited by inflammation-mediated toxicity, leading to treatment discontinuation in 10-30% of patients. Ruxolitinib, a potent anti-inflammatory agent, has demonstrated benefit in myelofibrosis (MF) and polycythemia vera (PV) patients. Combination therapy (CT) with these two agents may be more efficacious than monotherapy with either, potentially improving tolerability of interferon-α2 as well. We report the preliminary results from a phase II study of CT with pegylated interferon-α2 and ruxolitinib in 50 MPN patients (PV, n = 32; low-/intermediate-1-risk MF, n = 18), the majority (n = 47) being resistant and/or intolerant to interferon-α2 monotherapy. Objectives included remission (2013 revised criteria encompassing histologic, hematologic, and clinical responses), complete hematologic response (CHR), molecular response, and toxicity. Follow-up was 12 months. Partial remission (PR) and sustained CHR were achieved in 9% and 44% of PV patients, respectively. In MF patients, complete or partial remission was achieved in 39%, and sustained CHR in 58%. The median JAK2V617F allele burden declined significantly in both groups. Hematologic toxicity was the most common adverse event and was managed by dose reduction. Thirty-seven serious adverse events were recorded in 23 patients; the discontinuation rate was 20%. We conclude that CT with interferon-α2 and ruxolitinib is efficacious in patients with low-/intermediate-1-risk MF and, to a lesser extent, in patients with PV. These preliminary results encourage phase III studies as well as a study with CT in newly diagnosed MPN patients.

Reactive oxygen species inhibit catalytic activity of peptidylarginine deiminase.

Protein citrullination catalysed by peptidylarginine deiminase (PAD) may play an important pathogenic role in several chronic inflammatory diseases and malignancies. PAD2, PAD4, and citrullinated proteins are found in the synovium of rheumatoid arthritis patients. PAD activity is dependent on calcium and reducing conditions. However, reactive oxygen species (ROS) have been shown to induce citrullination of histones in granulocytes. Here we examine the ability of H2O2 and leukocyte-derived ROS to regulate PAD activity using citrullination of fibrinogen as read-out. H2O2 at concentrations above 40 µM inhibited the catalytic activity of PAD2 and PAD4 in a dose-dependent manner. PMA-stimulated leukocytes citrullinated fibrinogen and this citrullination was markedly enhanced when ROS formation was inhibited by the NADPH oxidase inhibitor diphenyleneiodonium (DPI). In contrast, PAD released from stimulated leukocytes was unaffected by exogenously added H2O2 at concentrations up to 1000 µM. The role of ROS in regulating PAD activity may play an important part in preventing hypercitrullination of proteins.

Minimal residual disease or cure in MPNs? Rationales and perspectives on combination therapy with interferon-alpha2 and ruxolitinib.

INTRODUCTION: The therapeutic landscape of the Philadelphia-negative myeloproliferative neoplasms (MPNs) is markedly changing consequent to the development of JAK-inhibitors and the use of ruxolitinib (RUX) in patients with myelofibrosis (MF) and patients with polycythemia vera (PV) who develop refractoriness or intolerance to hydroxyurea. The use of Interferon-alpha2 (IFN) is rapidly expanding in several countries, based upon favourable safety and efficacy profiles in several single-arm studies during the last 30 years, displaying complete hematological remissions in a large proportion of patients, a reduction in the JAK2V617 F and CALR mutational burden and in a subset of patients with PV with normalisation of the bone marrow after long-term treatment - even being sustained for several years after discontinuation of IFN. To this end the concept of chronic inflammation as the driving force for MPN disease progression is being increasingly recognized. This novel concept has initiated phase II studies in patients with PV and MF of combination therapy with IFN and RUX. Areas covered and Expert commentary: Herein we highlight the background, the rationales and perspectives for this novel combinatorial approach which is foreseen as the most encouraging and promising treatment for patients with MPNs - hopefully with the potential of cure - at least operational cure - in a subset of patients.

Reduced glutathione as a physiological co-activator in the activation of peptidylarginine deiminase.

BACKGROUND: Citrullination catalysed by peptidylarginine deiminases (PADs) plays an important pathogenic role in anti-citrullinated protein antibody (ACPA)-positive rheumatoid arthritis (RA) and, possibly, several other inflammatory diseases. Non-physiological reducing agents such as dithiothreitol (DTT) are normally added to the reaction buffer when determining PAD activity in vitro. We investigated the ability of reduced glutathione (GSH), the most abundant intracellular small-molecule thiol in vivo, to activate PADs. METHODS: Activity of recombinant human (rh) PAD2 and PAD4, PADs contained in synovial fluid (SF) samples from RA patients and PADs released from phorbol 12-myristate 13-acetate (PMA)-stimulated cells was measured using an in-house PAD activity assay detecting citrullination of fibrinogen. RESULTS: No activity of rhPAD2, rhPAD4 or PADs within SF was observed without addition of an exogenous reducing agent. Activity of both recombinant and SF PAD was observed in the presence of 1 mM DTT or 10-15 mM GSH. Following stimulation with PMA, human isolated leucocytes, but not mononuclear cells, released enzymatically active PAD, the activity of which was abolished upon pre-incubation of the cells with the glutathione reductase inhibitor 2-AAPA. No PAD activity was observed in the corresponding supernatants, but addition of exogenous GSH restored activity. CONCLUSIONS: Catalytic activity of PAD requires reducing conditions. GSH meets this requirement at concentrations comparable with those found within cells. Active PAD, reduced by GSH, is released from PMA-stimulated granulocytes, but becomes inactivated in the extracellular space.

Ruxolitinib is manageable in patients with myelofibrosis and severe thrombocytopenia: a report on 12 Danish patients.

We report 12 Danish myelofibrosis patients who have been treated successfully with ruxolitinib despite having low platelet counts (< 50 × 10(9)/L) during their treatment-course. The majority of the patients experienced marked clinical improvement. Serious side effects were only recorded in a single patient. It is concluded that JAK-inhibition with ruxolitinib is manageable in patients with low platelet counts and should be considered in symptomatic patients who otherwise might not be candidates for treatment.

MPNs as Inflammatory Diseases: The Evidence, Consequences, and Perspectives.

In recent years the evidence is increasing that chronic inflammation may be an important driving force for clonal evolution and disease progression in the Philadelphia-negative myeloproliferative neoplasms (MPNs), essential thrombocythemia (ET), polycythemia vera (PV), and myelofibrosis (MF). Abnormal expression and activity of a number of proinflammatory cytokines are associated with MPNs, in particular MF, in which immune dysregulation is pronounced as evidenced by dysregulation of several immune and inflammation genes. In addition, chronic inflammation has been suggested to contribute to the development of premature atherosclerosis and may drive the development of other cancers in MPNs, both nonhematologic and hematologic. The MPN population has a substantial inflammation-mediated comorbidity burden. This review describes the evidence for considering the MPNs as inflammatory diseases, A Human Inflammation Model of Cancer Development, and the role of cytokines in disease initiation and progression. The consequences of this model are discussed, including the increased risk of second cancers and other inflammation-mediated diseases, emphasizing the urgent need for rethinking our therapeutic approach. Early intervention with interferon-alpha2, which as monotherapy has been shown to be able to induce minimal residual disease, in combination with potent anti-inflammatory agents such as JAK-inhibitors is foreseen as the most promising new treatment modality in the years to come.

The Role of Reactive Oxygen Species in Myelofibrosis and Related Neoplasms.

Reactive oxygen species (ROS) have been implicated in a wide variety of disorders ranging between traumatic, infectious, inflammatory, and malignant diseases. ROS are involved in inflammation-induced oxidative damage to cellular components including regulatory proteins and DNA. Furthermore, ROS have a major role in carcinogenesis and disease progression in the myeloproliferative neoplasms (MPNs), where the malignant clone itself produces excess of ROS thereby creating a vicious self-perpetuating circle in which ROS activate proinflammatory pathways (NF-κB) which in turn create more ROS. Targeting ROS may be a therapeutic option, which could possibly prevent genomic instability and ultimately myelofibrotic and leukemic transformation. In regard to the potent efficacy of the ROS-scavenger N-acetyl-cysteine (NAC) in decreasing ROS levels, it is intriguing to consider if NAC treatment might benefit patients with MPN. The encouraging results from studies in cystic fibrosis, systemic lupus erythematosus, and chronic obstructive pulmonary disease warrant such studies. In addition, the antioxidative potential of the widely used agents, interferon-alpha2, statins, and JAK inhibitors, should be investigated as well. A combinatorial approach using old agents with anticancer properties together with novel JAK1/2 inhibitors may open a new era for patients with MPNs, the outlook not only being "minimal residual disease" and potential cure but also a marked improvement in inflammation-mediated comorbidities.

The impact of ruxolitinib treatment on inflammation-mediated comorbidities in myelofibrosis and related neoplasms.

The inflammation-mediated comorbidities in myelofibrosis (MF) and related neoplasms (MPNs) likely reflect the concurrent immune deregulation and systemic inflammatory nature of the MPNs, emphasizing the link between chronic systemic inflammation, immune deregulation, and the malignant clone. JAK1-2 inhibitors in MF-patients reduce constitutional symptoms and splenomegaly, but also taget autoimmune and inflammation-mediated comorbidities.

Circulating YKL-40 in patients with essential thrombocythemia and polycythemia vera treated with the novel histone deacetylase inhibitor vorinostat.

YKL-40 regulates vascular endothelial growth factors and induces tumor proliferation. We investigated YKL-40 before and after treatment with vorinostat in 31 polycythemia vera (PV) and 16 essential thrombocythemia (ET) patients. Baseline PV patient levels were 2 times higher than in healthy controls (P<0.0001) and 1.7 times higher than in ET (P=0.02). A significant correlation between YKL-40 at baseline and neutrophils, CRP, LDH, JAK2V617F and platelets in PV patients was observed, as well as a significantly greater reduction of YKL-40 levels in PV patients responding to therapy. YKL-40 might be a novel marker of disease burden and progression in myeloproliferative neoplasms.

Circulating YKL-40 in myelofibrosis a potential novel biomarker of disease activity and the inflammatory state.

Chronic myeloproliferative neoplasms (MPN), encompassing essential thrombocythaemia (ET), polycythaemia vera (PV) and myelofibrosis (PMF), are featured by a chronic inflammatory state which is pronounced in myelofibrosis The value of YKL-40 as a biomarker of disease burden has been demonstrated in several different diseases, including cancer, diabetes mellitus and cardiovascular diseases. A state of chronic inflammation is shared by them all, YKL-40 also being involved in the severity of chronic endothelial inflammation, which today is considered of crucial importance for the development of atherosclerosis. The MPNs being cancers with a heavy burden of cardiovascular diseases we hypothesised that circulating YKL-40 might reflect the inflammatory process and potentially serve as a novel disease marker. Using ELISA, we measured YKL-40 in 15 patients with ET, 16 patients with PV, 17 patients with PMF and 30 healthy controls. YKL-40 was significantly elevated in PMF vs. control subjects, PMF levels median 43 ng/mL vs. controls median 28 ng/mL, P = 0.033. An increase from ET over PV may reflect the integrated impact of disease processes in MPNs.