COVID-19 as a mediator of interferon deficiency and hyperinflammation: Rationale for the use of JAK1/2 inhibitors in combination with interferon.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) elicits an interferon (IFN) deficiency state, which aggravates the type I interferon deficiency and slow IFN responses, which associate with e.g. aging and obesity. Additionally, SARS-CoV-2 may also elicit a cytokine storm, which accounts for disease progression and ultimately the urgent need of ventilator support. Based upon several reports, it has been argued that early treatment with IFN-alpha2 or IFN-beta, preferentially in the early disease stage, may prohibit disease progression. Similarly, preliminary studies have shown that JAK1/2 inhibitor treatment with ruxolitinib or baricitinib may decrease mortality by dampening the deadly cytokine storm, which - in addition to the virus itself - also contributes to multi-organ thrombosis and multi-organ failure. Herein, we describe the rationale for treatment with IFNs (alpha2 or beta) and ruxolitinib emphasizing the urgent need to explore these agents in the treatment of SARS-CoV-2 - both as monotherapies and in combination. In this context, we take advantage of several safety and efficacy studies in patients with the chronic myeloproliferative blood cancers (essential thrombocythemia, polycythemia vera and myelofibrosis) (MPNs), in whom IFN-alpha2 and ruxolitinib have been used successfully for the last 10 (ruxolitinib) to 30 years (IFN) as monotherapies and most recently in combination as well. In the context of these agents being highly immunomodulating (IFN boosting immune cells and JAK1/2 inhibitors being highly immunosuppressive and anti-inflammatory), we also discuss if statins and hydroxyurea, both agents possessing anti-inflammatory, antithrombotic and antiviral potentials, might be inexpensive agents to be repurposed in the treatment of SARS-CoV-2.

Distinguishing myelofibrosis from polycythemia vera and essential thrombocythemia: The utility of enumerating circulating stem cells with aberrant hMICL expression by flow cytometry.

INTRODUCTION: Diagnosing BCR-ABL negative myeloproliferative neoplasms (MPN) may be challenging due to overlapping features and lack of robust discriminatory parameters, especially between essential thrombocythemia (ET) and prefibrotic myelofibrosis (MF). Circulating immature hematopoietic cells are variably present in polycythemia vera (PV), ET, and MF. The C-type lectin hMICL is aberrantly expressed on hematopoietic stem cells in the majority of acute myeloid leukemia patients. However, the hMICL expression in MPN, having varying propensity of leukemic transformation, is unsettled. We hypothesized that enumeration of immature cells by flow cytometry (FCM) could be a discriminatory tool in MPN diagnostics. METHODS: By FCM, we quantified circulating stem cells with aberrant hMICL expression in 39 MPN patients, 10 age-matched controls, and in leukapheresis products from 10 patients with lymphoproliferative neoplasms. The utility of the FCM assay for discriminating MPN entities was evaluated by applying ROC curve analysis. RESULTS: While hMICL was absent in control samples, MF patients had significantly more hMICL+ stem cells (median 15.2%) than PV and ET (0.0%, P = .001 and 0.0%, P = .002, respectively). By ROC curve analysis, the presence of hMICL+ stem cells (>0 cells) in peripheral blood reliably discriminates MF from ET and PV with a sensitivity of 80% and a specificity of 97%. CONCLUSION: Enumeration of circulating hMICL+ stem cells by FCM can discriminate between MPN phenotypes and holds potential for monitoring disease evolution.

The calreticulin (CALR) exon 9 mutations are promising targets for cancer immune therapy.

The calreticulin (CALR) exon 9 mutations are found in ∼30% of patients with essential thrombocythemia and primary myelofibrosis. Recently, we reported spontaneous immune responses against the CALR mutations. Here, we describe that CALR-mutant (CALRmut)-specific T cells are able to specifically recognize CALRmut cells. First, we established a T-cell culture specific for a CALRmut epitope. These specific T cells were able to recognize several epitopes in the CALRmut C terminus. Next, we established a CALRmut-specific CD4+ T-cell clone by limiting dilution. These CD4+ T cells recognized autologous CALRmut monocytes and hematopoietic stem cells, and T-cell recognition of target cells was dependent on the presence of CALR. Furthermore, we showed that the CALRmut response was human leukocyte antigen (HLA)-DR restricted. Finally, we demonstrated that the CALRmut-specific CD4+ T cells, despite their phenotype, were cytotoxic to autologous CALRmut cells, and that the cytotoxicity was mediated by degranulation of the T cells. In conclusion, the CALR exon 9 mutations are targets for specific T cells and thus are promising targets for cancer immune therapy such as peptide vaccination in patients harboring CALR exon 9 mutations.

Myeloproliferative neoplasms and inflammation: whether to target the malignant clone or the inflammatory process or both.

The Philadelphia-negative myeloproliferative neoplasms (MPNs) are clonal disorders involving hematopoietic stem and progenitor cells and are associated with myeloproliferation, splenomegaly and constitutional symptoms. Similar signs and symptoms can also be found in patients with chronic inflammatory diseases, and inflammatory processes have been found to play an important role in the pathogenesis and progression of MPNs. Signal transduction pathways involving JAK1, JAK2, STAT3 and STAT5 are causally involved in driving both the malignant cells and the inflammatory process. Moreover, anti-inflammatory and immune-modulating drugs have been used successfully in the treatment of MPNs. However, to date, many unresoved issues remain. These include the role of somatic mutations that are present in addition to JAK2V617F, CALR and MPL W515 mutations, the interdependency of malignant and nonmalignant cells and the means to eradicate MPN-initiating and -maintaining cells. It is imperative for successful therapeutic approaches to define whether the malignant clone or the inflammatory cells or both should be targeted. The present review will cover three aspects of the role of inflammation in MPNs: inflammatory states as important differential diagnoses in cases of suspected MPN (that is, in the absence of a clonal marker), the role of inflammation in MPN pathogenesis and progression and the use of anti-inflammatory drugs for MPNs. The findings emphasize the need to separate the inflammatory processes from the malignancy in order to improve our understanding of the pathogenesis, diagnosis and treatment of patients with Philadelphia-negative MPNs.

Corrigendum to "Combination therapy with interferon and JAK1-2 inhibitor is feasible. Proof of concept with rapid reduction in JAK2V617F-allele burden in Polycythemia Vera" [Leuk. Res. Rep. 3 (2) (2014) 73-75].

[This corrects the article DOI: 10.1016/j.lrr.2014.05.003.].

Combination therapy with interferon and JAK1-2 inhibitor is feasible: Proof of concept with rapid reduction in JAK2V617F-allele burden in polycythemia vera.

We report a 55 year old woman with post-ET PV for 12 years, who experienced resolution of severe constitutional symptoms within 3 days, a marked reduction in splenomegaly and a rapid decline in the JAK2V617F allele burden during combination therapy with interferon-alpha2a and ruxolitinib. Within 4 weeks the patient achieved complete hematological remission with normalization of peripheral blood counts and within 10 months the JAK2V617F-allele burden was reduced from 90% to 28%. Such a rapid decline in the JAK2V617F allele burden is highly unusual in PV-patients during low-dose IFN-alpha2 monotherapy and this finding warrants a prospective study with combination therapy.

Characterization of blood donors with high haemoglobin concentration.

BACKGROUND AND OBJECTIVES: The literature contains little on the prevalence and causes of high predonation haemoglobin levels among blood donors. This study aimed to characterize and develop an algorithm to manage would-be donors with polycythaemia. MATERIALS AND METHODS: Between November 2009 and November 2011, we offered haematology consultations to blood donors with repeated haemoglobin concentration (Hb) above the WHO limit for polycythaemia vera (PV) (10·2 and 11·5 mm/16·5 and 18·5 g/dl for women and men, respectively). Investigation of such donors included Hb, haematocrit, mean cell volume, erythropoietin, ferritin, platelet count and leucocyte count, JAK2 V617 and JAK2 exon12 analysis, as well as other routine measurements. RESULTS: Among 46 such donors, 39 had a history of smoking, which contributes to erythrocytosis. Two had PV, five had severe hypertension, one of them because of renal artery stenosis, and two had diabetes mellitus. Thus, we found a high morbidity among such donors. Of the 36 others, 30 donated again before May 2012, at which time the Hb was significantly lower. CONCLUSION: We recommend JAK2 V617 and JAK2 exon12 screening and clinical investigation for donors with concurrently high Hb, high haematocrit and iron deficiency. We also recommend that they stop or cut down on smoking to reduce the risk of thrombosis in general. We disqualified 10 of the donors.

Inheritance of the chronic myeloproliferative neoplasms. A systematic review.

This systematic review investigated the inheritance of the classical chronic myeloproliferative neoplasms (MPNs) including polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (PMF) and chronic myelogenous leukemia (CML). Sixty-one articles were included and provided 135 families with a total of 341 participants distributed to various subtypes of MPN: 50% PV, 23% ET, 14% PMF, 10% CML and 3% non-MPN hematological disorder. Women developed the disease earlier than men (43.1 years vs 47.3 years; p = 0.074), while the general average age of onset was 46 years, notably younger than sporadic cases. The clinical phenotype of the families showed a homogenous (67%) and a heterogeneous (33%) pattern, with the majority being PV-PV pairs (36%) and PV-PMF pairs (17%), respectively. This observation suggests that the susceptibility gene (or genes) is not restricted to one subtype supporting the hypothesis of a mutation in an early multipotent stem cell. Furthermore, a major subgroup of families provided evidence of an autosomal dominant (AD) inheritance with reduced penetrance. This study suggests that the origin of MPNs may occur in at least three different settings: (i) a sporadic, (ii) genetic heterogeneity with polygenetic and environmental impact and (iii) a familial phenotype following an AD inheritance.

Systemic adverse events following rituximab therapy in patients with Graves' disease.

BACKGROUND AND AIM: Rituximab (RTX) therapy has shown promising results in Graves' disease (GD), with or without ophthalmopathy. We examined the occurrence of adverse events in GD patients treated with RTX. SUBJECTS AND METHODS: Ten patients received RTX and methimazole, while 10 patients received methimazole only. Adverse events were recorded, and the presence of circulating immune complexes (CIC) was measured as IgG, IgM and complement component 3 (C3) depositing on normal monocytes following incubation with patient plasma. RESULTS: Five patients had benign infusion-related adverse events at first infusion. Two patients developed a serum sickness-like reaction 11 days after the first RTX-infusion. One of these patients developed diarrhea, raised orosomucoid levels, lowgrade inflammation in colonoscopic biopsies, and iridocyclitis 1 yr later. At day 14, the most pronounced immunoglobulin/ C3-adherent to the test monocytes, indicative of CIC, was observed in the presence of plasma from these 2 patients (p=0.003 to p=0.01 vs asymptomatic patients). A 3rd patient had recurrent fever and symmetric polyarthritis from day 38, and colonoscopy-verified ulcerative colitis at day 68. This patient had the 3rd highest increase in Ig deposition on monocytes by day 14. The arthralgias persisted in 2 of the patients, despite glucocorticoid rescue therapy. CONCLUSIONS: We report articular adverse events in 3 and gastrointestinal symptoms in 2 out of 10 GD patients who received RTX without concurrent immunosupression. The joint symptoms were related to CIC formation.

Sustained major molecular response on interferon alpha-2b in two patients with polycythemia vera.

Quantitative assessment of the JAK2 V617F allele burden during disease evolution and ongoing myelosuppressive treatment is likely to be implemented in the future clinical setting. Interferon alpha has demonstrated efficacy in treatment of both chronic myeloid leukemia and the Philadelphia chromosome negative chronic myeloproliferative disorders. Reductions in the JAK2 V617F allele burden in patients treated with pegylated interferon alpha-2a (Peg-IFN-2a) have been demonstrated, although follow-up was relatively short. We report here the first profound and sustained molecular responses with a JAK2 V617F allele burden below 1.0% in two patients with polycythemia vera treated with interferon alpha-2b (IFN-2b). Discontinuation of IFN-2b in one of the patients was followed by a sustained long-lasting (12 months of follow-up) major molecular response.

Anagrelide treatment in 52 patients with chronic myeloproliferative diseases.

In this retrospective multi-centre study, we report our experience with anagrelide in the treatment of thrombocytosis in patients with chronic myeloproliferative diseases. Our study included 52 patients (age 20-78 years). The initial anagrelide dose was, in general, 0.5 mg once daily and mean maintenance dosage was 1.7 mg/day. The overall response rate was 79% including 75% complete remission and 4% partial remission. Forty-two patients (81%) had adverse effects and in 29% of the study population, the adverse effects necessitated cessation of anagrelide. The most common adverse effect was moderate anaemia (50%). Two patients experienced erectile dysfunction which has been described only once previously in association with anagrelide treatment. One patient progressed to acute leukaemia. However, this patient had been pre-treated with two potentially leukaemogenic drugs and had only been in short-term treatment with anagrelide. Furthermore, a total of 13 events were recorded. More than 25% of these events occurred in patients with platelet counts between 400 and 600 x 10(9)/l and almost 40% of all events occurred in patients with platelet counts above 400 x 10(9)/l. This observation supports the hypothesis that aggressive control of thrombocytosis to a platelet count <400 x 10(9)/l might reduce the number of thrombohaemorrhagic events. Anagrelide is safe and effective in reducing the platelet counts, but a high proportion of the patients discontinue treatment because of the adverse effects of the drug.

SU6668 in idiopathic myelofibrosis--a rational therapeutic approach targeting several tyrosine kinases of importance for the myeloproliferation and the development of bone marrow fibrosis and angiogenesis.

Idiopathic myelofibrosis is a chronic myeloproliferative disorder being featured by progressive accumulation of connective tissue in concert with marked neovascularization (angiogenesis) of the bone marrow. Both fibrogenesis and angiogenesis are considered to develop consequent to the intramedullary release of various growth-promoting factors from rapidly proliferating and dysplastic megakaryocytes. Among these growth factors are platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), transforming growth factor beta (TGF-beta) and vascular endothelial growth factor (VEGF). The protein kinase inhibitor SU6668 is a potent antiangiogenic inhibitor of receptor tyrosine kinases, including those of VEGFR, PDGFR, bFGFR, and c-kit. The hypothesis is that SU6668 may be an effective agent in the treatment of idiopathic myelofibrosis. This compound has an inhibitory target profile on several tyrosine kinases involved in the myeloproliferation, the development of myeloid metaplasia (bFGFR, PDGFR, VEGFR, and c-kit) and the development of the major stromal changes in the bone marrow - fibrosis and angiogenesis (bFGFR, PDGFR, and VEGFR).

Sustained remission of platelet counts following monoclonal anti-CD20 antibody therapy in two cases of idiopathic autoimmune thrombocytopenia and neutropenia.

Idiopathic autoimmune thrombocytopenia and neutropenia (ITN) is a primary haemocytopenic disorder clinically characterised by recurrent mucocutaneous bleeding episodes and infections. Unlike in simple idiopathic thrombocytopenic purpura, the platelet deficiency of ITN tends to be chronic and difficult to treat. We describe two patients with ITN who obtained sustained remission of their platelet counts after therapy with the chimeric monoclonal anti-CD20 antibody Rituximab. In one of two cases, Rituximab also induced prolonged normalisation of the neutrophil count and disappearance of auto-antibodies. Our observations indicate that disturbed B-cell function plays a central role in the pathogenesis of ITN. Anti-CD20 antibody therapy seems to constitute a safe and efficient alternative to corticosteroids for the management of ITN patients with chronic thrombocytopenia.

Increased circulating platelet-leukocyte aggregates in myeloproliferative disorders is correlated to previous thrombosis, platelet activation and platelet count.

Platelet-leukocyte adhesion may occur as a consequence of platelet activation and possibly plays a key role in the deposition of activated platelets and fibrin in the thrombotic plug. The aim of the present study was to assess by whole blood flow cytometry the presence of circulating platelet-leukocyte aggregates (PLA) and the platelet-leukocyte response to platelet agonist stimulation (ADP and TRAP) in 50 patients with chronic myeloproliferative disorders (MPD) and 30 controls. PLA were identified as platelet-granulocyte/monocyte aggregates (PGMA), platelet-monocyte aggregates (PMA) and defined as the percentage of leukocytes coexpressing the platelet-specific marker glycoprotein Ib. Compared to controls the mean percentage of PGMA and PMA was increased in unstimulated whole blood from patients with MPD (7.98 vs. 1.76%; p<0.001 and 12.34 vs. 3.2%; p<0.001, respectively). The percentage of PGMA was correlated to the platelet count (r=0.46; p<0.001), percentage of P-selectin (r=0.69; p<0.001) and thrombospondin (r=0.58; p<0.001) positive platelets and platelet expression of GPIV (r=0.33; p=0.02). The mean percentage of PGMA and PMA was significantly increased in ADP-stimulated whole blood of patients (57.14 vs. 47.92%; p=0.009 and 54.91 vs. 45.89%; p<0.001, respectively). Compared to patients without a history of thrombosis, patients having experienced microvascular disturbances or a thrombotic event had a higher mean percentage of PGMA and PMA in non-stimulated whole blood (10.07 vs. 6.34%; p=0.025 and 14.81 vs. 10.48%; p=0.021, respectively) and a higher percentage of PGMA in ADP stimulated whole blood (64.32 vs. 51.50%; p<0.01). These data document an increased frequency of PLA in non-stimulated whole blood in MPD associated with a previous history of thrombosis or microvascular disturbances.

[The chronic Philadelphia chromosome-negative myeloproliferative syndrome I. Pathogenesis and pathophysiology]. / Det kroniske Philadelphia-kromosom-negative myeloproliferative syndrom I. Patogenese og patofysiologi.

In polycythaemia vera (PV) the erythroid progenitors proliferate autonomously independently of the circulating erythropoietin. The progenitors are hypersensitive to various growth factors, including insulin-like growth factor 1, which inhibits apoptosis in erythroid and myeloid progenitor cells. No change has been found in the erythropoietin (EPO) receptor. Thrombopoietin (Tpo) regulates the production of haematopoietic progenitor cells, particularly of platelets. By inhibiting apoptosis, this growth factor may be responsible for the autonomous proliferation of the megakaryocyte cell lineage in PV and idiopathic myelofibrosis (IMF), which are featured by highly elevated circulating Tpo levels. Thrombopoietin may also be involved in the pathogenesis of myelofibrosis and development of extramedullary haematopoiesis. Both fibrogenesis and angiogenesis in the bone marrow, spleen, and liver develop secondary to the release of various growth-promoting factors from the megakaryocyte cell lineage. The lesion of the pluripotent stem cell in PV and IMF seems to imply several defects, including lack of or decreased expression of the Tpo receptor, alterations in the sensitivity of progenitor cells to various growth factors, and alterations in important gene systems (Bcl-2), which govern cell survival. Essential thrombocytosis seems to be a heterogeneous disease entity, as about 50% of the patients have polyclonal haematopoiesis.