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To the Editor: Vannucchi et al. (Jan. 29 issue) 1 report that ruxolitinib was superior to standard therapy in patients with polycythemia vera who had an inadequate response to or had unacceptable side effects from hydroxyurea. The primary end point included hematocrit control and a reduction in spleen size. We wonder why the latter was included, because spleen size influences neither the risk of thrombosis or hemorrhage nor the prognosis in patients with polycythemia vera. We also wonder why the leukocyte and platelet counts were excluded in the composite assessment, when they significantly influence the risk of thrombosis and hemorrhage, respectively. . . .

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.

This document updates the recommendations on the management of Philadelphia chromosome-negative myeloproliferative neoplasms (Ph-neg MPNs) published in 2011 by the European LeukemiaNet (ELN) consortium. Recommendations were produced by multiple-step formalized procedures of group discussion. A critical appraisal of evidence by using Grades of Recommendation, Assessment, Development and Evaluation (GRADE) methodology was performed in the areas where at least one randomized clinical trial was published. Seven randomized controlled trials provided the evidence base; earlier phase trials also informed recommendation development. Key differences from the 2011 diagnostic recommendations included: lower threshold values for hemoglobin and hematocrit and bone marrow examination for diagnosis of polycythemia vera (PV), according to the revised WHO criteria; the search for complementary clonal markers, such as ASXL1, EZH2, IDH1/IDH2, and SRSF2 for the diagnosis of myelofibrosis (MF) in patients who test negative for JAK2V617, CALR or MPL driver mutations. Regarding key differences of therapy recommendations, both recombinant interferon alpha and the JAK1/JAK2 inhibitor ruxolitinib are recommended as second-line therapies for PV patients who are intolerant or have inadequate response to hydroxyurea. Ruxolitinib is recommended as first-line approach for MF-associated splenomegaly in patients with intermediate-2 or high-risk disease; in case of intermediate-1 disease, ruxolitinib is recommended in highly symptomatic splenomegaly. Allogeneic stem cell transplantation is recommended for transplant-eligible MF patients with high or intermediate-2 risk score. Allogeneic stem cell transplantation is also recommended for transplant-eligible MF patients with intermediate-1 risk score who present with either refractory, transfusion-dependent anemia, blasts in peripheral blood > 2%, adverse cytogenetics, or high-risk mutations. In these situations, the transplant procedure should be performed in a controlled setting.

The Philadelphia-negative chronic myeloproliferative neoplasms - essential thrombocythemia (ET), polycythemia vera (PV), and myelofibrosis (MF) (MPNs) - have recently been shown to be associated with chronic inflammation, oxidative stress and accumulation of reactive oxygen species (ROS). Using whole blood transcriptional profiling, we report that several oxidative stress and anti-oxidative stress genes are significantly deregulated in MPNs. Among the twenty most up- and downregulated genes, ATOX1, DEFB122, GPX8, PRDX2, PRDX6, PTGS1, and SEPP1 were progressively upregulated from ET over PV to PMF, whereas AKR1B1, CYBA, SIRT2, TTN, and UCP2 were progressively downregulated in ET, PV and PMF (all FDR <0.05). The gene Nrf2, encoding the transcription factor nuclear factor erythroid 2-related factor 2 (NFE2L2 or Nrf2) was significantly downregulated in all MPNs. Nrf2 has a key role in the regulation of the oxidative stress response and modulates both migration and retention of hematopoietic stem cells (HSCs) in their niche. The patogenetic importance of Nrf2 depletion in the context of expansion of the hematopoietic progenitor pool in MPNs is discussed with particular focus upon the implications of concomitant downregulation of Nrf2 and CXCR4 for stem cell mobilization.

Discovery of somatic mutations in the calreticulin gene (CALR) has identified a subgroup of Philadelphia-negative chronic myeloproliferative neoplasms (MPN) with separate haematological characteristics and prognosis. CALR mutations serve as novel markers both of diagnostic value and as targets for monitoring molecular responses during therapy. Interferon-α (IFN) selectively targets the malignant clone in a subset of MPN patients and can induce both haematological and molecular remissions in CALR mutated essential thrombocythemia (ET) patients. We investigated the response to IFN in a cohort of 21 CALR mutated MPN patients including ET, prefibrotic primary myelofibrosis (pre-PMF), and primary myelofibrosis (PMF) with a median follow-up of 31 months. For evaluation of a molecular response, we developed highly sensitive quantitative PCR (qPCR) assays for monitoring the mutant allele burden of the two most prevalent CALR mutations (type 1 and type 2). Thirteen patients (62%) experienced a decrease in the mutant allele burden with a median decline of 29% from baseline. However, only four patients, including patients with ET, pre-PMF, and PMF diagnosis, achieved molecular responder (MR) status with >50% reduction in mutant allele burden according to European LeukemiaNet (ELN) guidelines. MR patients displayed significant differences in the dynamics of the CALR mutant load with regard to time to response and dynamics in mutant allele burden after discontinuation of IFN treatment. Furthermore, we highlight the prognostic value of the CALR mutant allele burden by showing a close association with leucocyte- and platelet counts, hemoglobin concentration, in addition to plasma lactate dehydrogenase (LDH) irrespective of molecular response and treatment status.

Pegylated interferon-α (peg-IFNa) treatment induces molecular responses (MR) in patients with myeloproliferative neoplasms (MPNs), including partial MR (PMR) in 30–40% of patients. Here, we compared the efficacy of IFNa treatment in JAK2V617F- vs. calreticulin (CALR)-mutated cells and investigated the mechanisms of differential response. Retrospective analysis of MPN patients treated with peg-IFNa demonstrated that patients harboring the JAK2V617F mutation were more likely to achieve PMR than those with mutated CALR ( p  = 0.004), while there was no significant difference in hematological response. In vitro experiments confirmed an upregulation of IFN-stimulated genes in JAK2V617F-positive 32D cells as well as patient samples (peripheral blood mononuclear cells and CD34+ hematopoietic stem cells) compared to their CALR-mutated counterparts, and higher IFNa doses were needed to achieve the same IFNa response in CALR− as in JAK2V617F-mutant 32D cells. Additionally, Janus-activated kinase-1 (JAK1) and signal transducers and activators of transcription 1 (STAT1) showed constitutive phosphorylation in JAK2V617F-mutated but not CALR-mutated cells, indicating priming towards an IFNa response. Moreover, IFN-induced growth arrest was counteracted by selective JAK1 inhibition but enhanced by JAK2 inhibition. In conclusion, our data suggest that, clinically, higher doses of IFNa are needed in CALR-mutated vs. JAK2V617F-positive patients and we suggest a model of JAK2V617F-JAK1/STAT1 crosstalk leading to a priming of JAK2V617F-positive cells to IFNa resulting in differential sensitivity

Peripheral T cell CXCR3 expression has been found uniquely lower in patients having neovascular age-related macular degeneration (nAMD) than in healthy individuals. The CXCR3-axis has been shown to have angiostatic and antifibrotic properties. We have recently investigated systemic markers in patients with myeloproliferative neoplasms (MPNs) because of their higher prevalence of AMD, and we have observed higher systemic chronic low-grade inflammation and immunosenescence signs in MPNs with drusen (MPNd) compared to those with normal retinas (MPNn). The MPNs evolve in a biological continuum from early cancer-stages (essential thrombocytosis, polycythemia vera) to the advanced myelofibrosis stage. Especially myelofibrosis is characterized by bone marrow angiogenesis and fibrosis, similarly to retinal observations in nAMD. We speculate if we can find lower CXCR3 expression in MPNs, particularly myelofibrosis and if differences are seen between MPNd and MPNn. We also wanted to compare expression in nAMD and intermediate (i)AMD. Patients in this cross-sectional study were 29 nAMD, 28 iAMD, 35 MPNd, and 27 MPNn. We performed flowcytometry on blood to measure CXCR3 expression. CD8+CXCR3 expression in nAMD was 6,1%, significantly lower than in iAMD 16%, MPNd 11%, MPNn 12% (p-values<0.05). Similar results were seen for CD4+CXCR3 expression. We also found CXCR3 expression decreasing over the MPN-continuum. For instance, in myelofibrosis, intermediate monocytes expression was 6.2%, significantly lower than 18% in ET and 18% in PV (p-values<0.05). We find CXCR3 downregulation on T-cells and some monocyte subset in nAMD compared to iAMD, MPNd, and MPNn, in line with previous nAMD studies. We also find CXCR3 downregulation in most monocyte subsets over the MPN continuum. Systemic leukocyte CXCR3 expression could both be involved in changes seen in the retina and the bone marrow. Further understanding the CXCR3-axis in AMD and MPNs may elucidate underlying pathogenic mechanisms and reveal new targets for treatment.

The chronic Philadelphia-negative myeloproliferative neoplasms (MPNs) are acquired stem cell neoplasms which ultimately may transform to acute myelogenous leukemia. Most recently, chronic inflammation has been described as an important factor for the development and progression of MPNs in the biological continuum from early cancer stage to the advanced myelofibrosis stage, the MPNs being described as \"A Human Inflammation Model for Cancer Development\". This novel concept has been built upon clinical, experimental, genomic, immunological and not least epidemiological studies. Only a few studies have described the development of MPNs by mathematical models, and none have addressed the role of inflammation for clonal evolution and disease progression. Herein, we aim at using mathematical modelling to substantiate the concept of chronic inflammation as an important trigger and driver of MPNs.The basics of the model describe the proliferation from stem cells to mature cells including mutations of healthy stem cells to become malignant stem cells. We include a simple inflammatory coupling coping with cell death and affecting the basic model beneath. First, we describe the system without feedbacks or regulatory interactions. Next, we introduce inflammatory feedback into the system. Finally, we include other feedbacks and regulatory interactions forming the inflammatory-MPN model. Using mathematical modeling, we add further proof to the concept that chronic inflammation may be both a trigger of clonal evolution and an important driving force for MPN disease progression. Our findings support intervention at the earliest stage of cancer development to target the malignant clone and dampen concomitant inflammation.