A journey through infectious risk associated with ruxolitinib.

Ruxolitinib has proved to be effective for the treatment of patients with myelofibrosis (either primary or secondary) and polycythaemia vera, and its approval led to a significant change in the current treatment algorithm. Despite its efficacy and beyond its well described haematological toxicity, a peculiar immunosuppressive effect emerged as our clinical experience grew, both within and outside of a clinical trial setting. Definite and negative interactions with multiple pathways of the immune system of patients have been reported so far, involving both adaptive and innate immune responses. These pathophysiological mechanisms may contribute to the increased risk of reactivation of silent infections (e.g., tuberculosis, hepatitis B virus and varicella zoster virus) that have been associated with the drug. Even though such infectious events may be fatal or may lead to significant impairment of organ function, compromising the eligibility of patients for an allotransplant procedure, there are no dedicated guidelines that may help us in assessing and managing the risk of developing serious infections. On this basis, our aim for the present work was to review the current knowledge on the pathophysiological mechanisms through which ruxolitinib may exert its immunosuppressive effect, and to illustrate our personal approach to the management of three peculiar clinical scenarios, for which a risk-based algorithm is suggested.

Distinctive CD8+ T cell and MHC class I signatures in polycythemia vera patients.

Polycythemia vera (PV) is a myeloproliferative neoplasm characterized by overproduction of red blood cells. We have performed a comprehensive characterization of blood immune cells for expression of naïve and memory receptors as well as ß2m-associated and ß2m-free MHC class I heavy chains, also known as closed and open conformers, respectively, in PV patients and age-matched controls (CTR). We show that the peripheral CD3+CD8+ T cell pool in PV patients is clearly divided into two discrete populations, a more granular CD3+CD8high T cell population enriched in effector-memory CD45RA+ T cells (CD8+ TEMRA) when compared to CTR (P < 0.001), and a less granular CD3+CD8int T cell population that is completely absent in the CTR group (78 vs. 0%, P < 0.001) and is a mixture of naïve (CD8+ TN) and CD8+ TEMRA cells expressing intermediate levels of CD28, i.e., CD3+CD8intCD28int. While the percentage of CD3+CD8int TN cells correlated positively with the number of erythrocytes, the percentage of CD3+CD8int TEMRA correlated negatively with the number of platelets. Finally, we report that PV patients' lymphocytes and monocytes display lower levels of closed (W6/32+) MHC-I conformers at the cell surface while exhibiting increased amounts of open (HC-10+) MHC-I conformers. The implications of this distinctive immune signature are discussed.

Allogeneic Stem Cell Transplantation in Myelofibrosis.

Myeloproliferative neoplasm (MPN) is a category in the World Health Organization classification of myeloid tumors. BCR-ABL1-negative MPN is a subcategory that includes primary myelofibrosis (MF), post-essential thrombocythemia MF, and post-polycythemia vera MF. These disorders are characterized by stem cell-derived clonal myeloproliferation. Clinically, these diseases present with anemia and splenomegaly and significant constitutional symptoms such as severe fatigue, symptoms associated with an enlarged spleen and liver, pruritus, fevers, night sweats, and bone pain. Multiple treatment options may provide symptom relief and improved survival; however, allogeneic stem cell transplantation (HCT) remains the only potentially curative option. The decision for a transplant is based on patient prognosis, age, comorbidities, and functional status. This review describes the recent data on various peritransplantation factors and their effect on outcomes of patients with MF and new therapeutic areas, such as the use and timing of Janus kinase inhibitors with HCT and gives overall conclusions from the available data in the published literature.

Nosocomial Co-Transmission of Avian Influenza A(H7N9) and A(H1N1)pdm09 Viruses between 2 Patients with Hematologic Disorders.

A nosocomial cluster induced by co-infections with avian influenza A(H7N9) and A(H1N1)pdm09 (pH1N1) viruses occurred in 2 patients at a hospital in Zhejiang Province, China, in January 2014. The index case-patient was a 57-year-old man with chronic lymphocytic leukemia who had been occupationally exposed to poultry. He had co-infection with H7N9 and pH1N1 viruses. A 71-year-old man with polycythemia vera who was in the same ward as the index case-patient for 6 days acquired infection with H7N9 and pH1N1 viruses. The incubation period for the second case-patient was estimated to be <4 days. Both case-patients died of multiple organ failure. Virus genetic sequences from the 2 case-patients were identical. Of 103 close contacts, none had acute respiratory symptoms; all were negative for H7N9 virus. Serum samples from both case-patients demonstrated strong proinflammatory cytokine secretion but incompetent protective immune responses. These findings strongly suggest limited nosocomial co-transmission of H7N9 and pH1N1 viruses from 1 immunocompromised patient to another.

Expansion of circulating CD56bright natural killer cells in patients with JAK2-positive chronic myeloproliferative neoplasms during treatment with interferon-α.

In recent years, major molecular remissions have been observed in patients with JAK2-positive chronic myeloproliferative neoplasms (MPNs) after therapy with IFN-α. IFN-α is known to have altering effects on immune cells involved in immune surveillance and might consequently enhance anti-tumor immune response against the JAK2-mutated clone. The objective of this study was to investigate circulating levels and phenotype of natural killer cells in 29 JAK2-positive MPN patients during IFN-α treatment. Furthermore, functional studies of NK cells upon target-cell recognition and cytokine stimulation were performed. The CD56(bright) and CD56(dim) NK cell subtypes display different properties in terms of cytokine production and cytotoxicity, respectively. Our results show a significant increase in the proportion of CD56(bright) NK cells and a decreasing CD56(dim) population during treatment with IFN-α compared to patients that are untreated, treated with hydroxyurea and healthy controls, P < 0.0001. Furthermore, an overall increase in cytokine-dependent (IL-12 and IL-15) IFN-γ expression by CD56(dim) NK cells during IFN-α treatment was observed. In contrast, our data indicate a compromised NK cell response to target-cell recognition during treatment with IFN-α in four patients. We also report low levels of circulating NK cells in untreated patients compared to healthy donors, patients treated with hydroxyurea and IFN-α, P = 0.02. Based on our findings, one might speculate whether treatment with IFN-α skews the human NK population toward a helper type that may assist in CD8(+) T cell priming in lymphoid tissues at the expense of their immediate cytotoxic functions in peripheral blood and tissues.

Proinflammatory Cytokine IL-6 and JAK-STAT Signaling Pathway in Myeloproliferative Neoplasms.

The recent JAK1/2 inhibitor trial in myeloproliferative neoplasms (MPNs) showed that reducing inflammation can be more beneficial than targeting gene mutants. We evaluated the proinflammatory IL-6 cytokine and JAK-STAT signaling pathway related genes in circulating CD34(+) cells of MPNs. Regarding laboratory data, leukocytosis has been observed in polycythemia vera (PV) and JAK2V617F mutation positive versus negative primary myelofibrosis (PMF) patients. Moreover, thrombocytosis was reduced by JAK2V617F allele burden in essential thrombocythemia (ET) and PMF. 261 significantly changed genes have been detected in PV, 82 in ET, and 94 genes in PMF. The following JAK-STAT signaling pathway related genes had augmented expression in CD34(+) cells of MPNs: CCND3 and IL23A regardless of JAK2V617F allele burden; CSF3R, IL6ST, and STAT1/2 in ET and PV with JAK2V617F mutation; and AKT2, IFNGR2, PIM1, PTPN11, and STAT3 only in PV. STAT5A gene expression was generally reduced in MPNs. IL-6 cytokine levels were increased in plasma, as well as IL-6 protein levels in bone marrow stroma of MPNs, dependent on JAK2V617F mutation presence in ET and PMF patients. Therefore, the JAK2V617F mutant allele burden participated in inflammation biomarkers induction and related signaling pathways activation in MPNs.

Immunophenotype of myeloid granulocytes in Chinese patients with BCR::ABL1-negative myeloproliferative neoplasms.

Typical BCR::ABL1-negative myeloproliferative neoplasms (MPN) are mainly referred to as polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofbrosis (PMF). Granulocytes in MPN patients are involved in their inflammation and form an important part of the pathophysiology of MPN patients. It has been shown that the immunophenotype of granulocytes in MPN patients is altered. We used flow cytometry to explore the immunophenotype of MPN patients and correlate it with clinical parameters. The results showed that PMF patients and PV patients had higher CD15+CD11b+ granulocytes than ET patients and normal controls. When grouped by gene mutation, changes in the granulocyte immunophenotype of MPN patients were independent of the JAK2V617F and CALR mutations. There was no significant heterogeneity in immunophenotype between ET patients and Pre-PMF, and between Overt-PMF and Pre-PMF patients. Granulocytes from some MPN patients showed an abnormal CD13/CD16 phenotype with a significant increase in mature granulocytes on molecular and cytomorphological grounds, and this abnormal pattern occurred significantly more frequently in PMF patients than in ET patients. CD15-CD11b- was negatively correlated with WBC and Hb and positively correlated with DIPSS score, whereas high CD10+ granulocytes were significantly and negatively associated with prognostic system IPSS and DIPSS scores in PMF patients. In conclusion, this study demonstrates the landscape of bone marrow granulocyte immunophenotypes in MPN patients. MPN patients, especially those with PMF, have a significant granulocyte developmental overmaturation phenotype. CD10+ granulocytes may be involved in the prognosis of PMF patients.

Increase in circulating CD4⁺CD25⁺Foxp3⁺ T cells in patients with Philadelphia-negative chronic myeloproliferative neoplasms during treatment with IFN-α.

Recent reports have described complete or major molecular remission in patients with polycythemia vera after long-term treatment with the immunomodulatory agent IFN-α2. Accordingly, there are reasons to believe that the immune system is a key player in eradicating the JAK2 mutated clone in these patients. Foxp3(+) regulatory T cells play a pivotal role in maintaining immune homeostasis and, importantly, preventing immune reactivity to self-antigens; however, their suppressive activity can compromise an effective antitumor immune response, and high frequencies of regulatory T cells in peripheral blood have been reported in both hematologic and solid cancers. We have analyzed the number, phenotype, and function of circulating CD4(+)CD25(+)Foxp3(+) T cells in patients with chronic myeloproliferative neoplasms. Surprisingly, we found a marked expansion of this subset of lymphocytes in patients treated with IFN-α2 (13.0%; 95% confidence interval [CI] 10.8% to 15.2%) compared with healthy donors (6.1%; 95% CI 4.9% to 7.2%), patients with untreated chronic myeloproliferative neoplasms (6.9%; 95% CI 5.8% to 7.4%), or patients treated with hydroxyurea (5.8%; 95% CI 4.3% to 7.4%; P < .0001).

Clearance of circulating activated platelets in polycythemia vera and essential thrombocythemia.

Essential thrombocythemia (ET) and polycythemia vera (PV) are characterized by persistent platelet activation. The mechanisms involved in their clearance are poorly characterized. In the present study, we report that leukocytes were actively involved in platelet disposal in 51 patients with ET and 30 with PV, but not in 70 age- and sex-matched controls. The fraction of circulating neutrophils and monocytes that had phagocytosed platelets, as assessed by flow cytometry, was significantly higher in patients with PV or ET, independently of hydroxyurea treatment, than in controls. Platelet phagocytosis by circulating leukocytes was confirmed by confocal and electron microscopy. The lack of effect of hydroxyurea, which disrupts the P-selectin/P-selectin glycoprotein ligand 1 (PSGL-1) interaction, suggests a P-selectin-independent mechanism. This hypothesis was confirmed in an ad hoc animal model based on the in vivo injection of activated platelets from P-selectin(+/+) and P-selectin(-/-) mice. P-selectin expression was associated with an earlier and effective clearance of platelets by neutrophils. A second delayed, P-selectin-independent phase actively involved monocytes. Our results suggest that phagocytic clearance of platelets by leukocytes occurs in PV and ET, possibly involving P-selectin-dependent and -independent pathways, thus representing a novel mechanism to remove activated platelets from the circulation.

Allogeneic hematopoietic cell transplantation for advanced polycythemia vera and essential thrombocythemia.

Allogeneic hematopoietic cell transplantation (HCT) is curative for selected patients with advanced essential thrombocythemia (ET) or polycythemia vera (PV). From 1990 to 2007, 75 patients with ET (median age 49 years) and 42 patients with PV (median age 53 years) underwent transplantations at the Fred Hutchinson Cancer Research Center (FHCRC; n = 43) or at other Center for International Blood and Marrow Transplant Research (CIBMTR) centers (n = 74). Thirty-eight percent of the patients had splenomegaly and 28% had a prior splenectomy. Most patients (69% for ET and 67% for PV) received a myeloablative (MA) conditioning regimen. Cumulative incidence of neutrophil engraftment at 28 days was 88% for ET patients and 90% for PV patients. Acute graft-versus-host disease (aGVHD) grades II to IV occurred in 57% and 50% of ET and PV patients, respectively. The 1-year treatment-related mortality (TRM) was 27% for ET and 22% for PV. The 5-year cumulative incidence of relapse was 13% for ET and 30% for PV. Five-year survival/progression-free survival (PFS) was 55%/47% and 71%/48% for ET and PV, respectively. Patients without splenomegaly had faster neutrophil and platelet engraftment, but there were no differences in TRM, survival, or PFS. Presence of myelofibrosis (MF) did not affect engraftment or TRM. Over 45% of the patients who undergo transplantations for ET and PV experience long-term PFS.

Evaluation of interleukin-23 plasma levels in patients with polycythemia vera and essential thrombocythemia.

Essential thrombocythemia (ET), polycythemia vera (PV) and myelofibrosis share the same acquired genetic lesion, JAK2V617F. It is believed that cytokines participate in the activation of JAK2V617F. In this study, we analyzed the plasma levels of interleukin (IL)-23, IL-10 and IL-22 in patients with PV and ET. In the same subjects we also performed analysis of the JAK2(V617F) mutation, and evaluated a possible relationship between interleukin levels and thrombotic complications or with the symptom pruritus. Plasma levels of IL-23 were significantly increased in all patients with MPN in comparison to controls. Moreover, there was a significant difference between the levels of IL-23 in patients affected by PV and those measured in controls (8.57±3.69pg/mL vs. 6.55±4.125pg/mL; p<0.03). No difference was found between IL-23 levels in ET patients and controls. No statistically significant differences were found between the levels of IL-23, Il-22 or IL-10 in PV or ET subjects with or without thrombosis, in patients with or without pruritus, or according the JAK2V617F burden. In PV patients the JAK2 burden and Hb levels correlated with occurrence of pruritus. Our study seems to point out a possible involvement of IL-23 in the pathogenesis of PV.

Plasma cytokines in polycythemia vera: phenotypic correlates, prognostic relevance, and comparison with myelofibrosis.

Plasma cytokine milieu is abnormal in primary myelofibrosis (PMF) and correlates with disease phenotype and prognosis. In this study, we show that several plasma cytokines are also abnormally expressed in polycythemia vera (PV; n = 65), compared to normal controls (n = 35), but with a significantly different pattern than that of PMF (n = 127). Direct phenotypic correlation in PV included levels of IL-12 with hematocrit; IL-1b, IL-2, IL-7, FGF-b, and HGF with leukocytosis; and IFN-α and IFN-γ with thrombocytosis. In univariate analysis, levels of 13 cytokines (out of 30 analyzed) correlated with survival but only MIP-1ß remained significant on multivariable analysis that included the other cytokines as covariates. Increased level of MIP-1ß (P < 0.01), older age (P < 0.01), and leukocytosis (P = 0.03) maintained their association with shortened survival, on multivariable analysis. This study provides preliminary observations that warrant a larger scale study and suggests the value of plasma cytokines as prognostic biomarkers in PV.

Whole-blood transcriptional profiling of interferon-inducible genes identifies highly upregulated IFI27 in primary myelofibrosis.

Gene expression profiling studies have unraveled deregulation of several genes that might be of pathogenetic importance for the development and phenotype of the Philadelphia-negative chronic myeloproliferative neoplasms. In the context of interferon-alpha2 as a promising therapeutic agent, we focused upon the transcriptional profiling of interferon-associated genes in patients with essential thrombocythemia (ET) (n = 19), polycythemia vera (PV) (n = 41), and primary myelofibrosis (PMF) (n = 9). Using whole-blood transcriptional profiling and accordingly obtaining an integrated signature of genes expressed in several immune cells (granulocytes, monocytes, B cells, T cells, platelets), we have identified a number of interferon-associated genes to be significantly deregulated but with a highly significant deregulation of interferon-inducible gene 27 (IFI27) (ET, PV, and PMF, fold change 8, 16, and 30, respectively). The striking deregulation of IFI genes may reflect a hyperstimulated but insufficient immune system being most enhanced in patients with advanced myelofibrosis, in whom the IFI27 gene displayed an exceedingly high expression. The interferon signature may reflect primary myelofibrosis as the burn-out phase of chronic inflammation which ultimately elicits clonal evolution and expansion owing to an exaggerated but incompetent antitumor immune response. Finally, IFI27 may be a novel biomarker of disease activity and tumor burden in patients with CMPNs.

Lower response to BNT162b2 vaccine in patients with myelofibrosis compared to polycythemia vera and essential thrombocythemia.

In a population of 42 Philadelphia negative myeloproliferative neoplasm patients, all on systemic active treatment, the likelihood of responding to anti-SARS-CoV-2 BNT162b2 vaccine at 2 weeks after the second dose was significantly lower in the ten patients with myelofibrosis compared to the 32 with essential thrombocythemia (n = 17) and polycythemia vera (n = 15) grouped together, both in terms of neutralizing anti-SARS-CoV-2 IgG titers and seroprotection rates (32.47 AU/mL vs 217.97 AU/mL, p = 0.003 and 60% vs 93.8%, p = 0.021, respectively). Ruxolitinib, which was the ongoing treatment in five patients with myelofibrosis and three with polycythemia vera, may be implicated in reducing vaccine immunogenicity (p = 0.076), though large prospective study is needed to address this issue.

The Power of Extracellular Vesicles in Myeloproliferative Neoplasms: "Crafting" a Microenvironment That Matters.

Myeloproliferative Neoplasms (MPN) are acquired clonal disorders of the hematopoietic stem cells and include Essential Thrombocythemia, Polycythemia Vera and Myelofibrosis. MPN are characterized by mutations in three driver genes (JAK2, CALR and MPL) and by a state of chronic inflammation. Notably, MPN patients experience increased risk of thrombosis, disease progression, second neoplasia and evolution to acute leukemia. Extracellular vesicles (EVs) are a heterogeneous population of microparticles with a role in cell-cell communication. The EV-mediated cross-talk occurs via the trafficking of bioactive molecules such as nucleic acids, proteins, metabolites and lipids. Growing interest is focused on EVs and their potential impact on the regulation of blood cancers. Overall, EVs have been suggested to orchestrate the complex interplay between tumor cells and the microenvironment with a pivotal role in "education" and "crafting" of the microenvironment by regulating angiogenesis, coagulation, immune escape and drug resistance of tumors. This review is focused on the role of EVs in MPN. Specifically, we will provide an overview of recent findings on the involvement of EVs in MPN pathogenesis and discuss opportunities for their potential application as diagnostic and prognostic biomarkers.

Novel and combination therapies for polycythemia vera and essential thrombocythemia: the dawn of a new era.

INTRODUCTION: Essential thrombocythemia (ET) and polycythemia vera (PV) belong to the BCR-ABL1-negative myeloproliferative neoplasms and are characterized by the clonal proliferation of hematopoietic stem and progenitor cells. The contribution of aberrant immune regulation within the bone marrow microenvironment to ET and PV pathogenesis as well as the underlying molecular landscape is becoming increasingly understood. AREAS COVERED: Authors searched PubMed and conference abstracts in August 2020 for preclinical and clinical studies to provide an overview of the immune pathobiology in ET and PV and the rationale for several novel agents. A discussion of recent clinical trials on interferon and ruxolitinib in ET and PV patients is provided followed by an outline of the future challenges in the field particularly for novel therapeutics and an increasingly individualized, molecularly driven approach to treatment selection. Several novel agents are currently being actively evaluated and are reviewed herein as well. EXPERT OPINION: While hydroxyurea remains the first-line treatment for cytoreduction in most high-risk ET and PV patients, the disease-modifying potential of IFN is promising and could make it a preferred option for selected patients. Advances in molecular testing will enable a more individualized approach to prognostication and treatment selection.

Cytokine Profiling in Myeloproliferative Neoplasms: Overview on Phenotype Correlation, Outcome Prediction, and Role of Genetic Variants.

Among hematologic malignancies, the classic Philadelphia-negative chronic myeloproliferative neoplasms (MPNs) are considered a model of inflammation-related cancer development. In this context, the use of immune-modulating agents has recently expanded the MPN therapeutic scenario. Cytokines are key mediators of an auto-amplifying, detrimental cross-talk between the MPN clone and the tumor microenvironment represented by immune, stromal, and endothelial cells. This review focuses on recent advances in cytokine-profiling of MPN patients, analyzing different expression patterns among the three main Philadelphia-negative (Ph-negative) MPNs, as well as correlations with disease molecular profile, phenotype, progression, and outcome. The role of the megakaryocytic clone as the main source of cytokines, particularly in myelofibrosis, is also reviewed. Finally, we report emerging intriguing evidence on the contribution of host genetic variants to the chronic pro-inflammatory state that typifies MPNs.

The JAK2V617 mutation induces constitutive activation and agonist hypersensitivity in basophils from patients with polycythemia vera.

BACKGROUND: The JAK2V617F mutation has been associated with constitutive and enhanced activation of neutrophils, while no information is available concerning other leukocyte subtypes. DESIGN AND METHODS: We evaluated correlations between JAK2V617F mutation and the count of circulating basophils, the number of activated CD63(+) basophils, their response in vitro to agonists as well as the effects of a JAK2 inhibitor. RESULTS: We found that basophil count was increased in patients with JAK2V617F -positive myeloproliferative neoplasms, particularly in those with polycythemia vera, and was correlated with the V617F burden. The burden of V617F allele was similar in neutrophils and basophils from patients with polycythemia vera, while total JAK2 mRNA content was remarkably greater in the basophils; however, the content of JAK2 protein in basophils was not increased. The number of CD63(+) basophils was higher in patients with polycythemia vera than in healthy subjects or patients with essential thrombocythemia or primary myelofibrosis and was correlated with the V617F burden. Ultrastructurally, basophils from patients with polycythemia vera contained an increased number of granules, most of which were empty suggesting cell degranulation in vivo. Ex vivo experiments revealed that basophils from patients with polycythemia vera were hypersensitive to the priming effect of interleukin-3 and to f-MLP-induced activation; pre-treatment with a JAK2 inhibitor reduced polycythemia vera basophil activation. Finally, we found that the number of circulating CD63(+) basophils was significantly greater in patients suffering from aquagenic pruritus, who also showed a higher V617F allele burden. CONCLUSIONS: These data indicate that the number of constitutively activated and hypersensitive circulating basophils is increased in polycythemia vera, underscoring a role of JAK2V617F in these cells' abnormal function and, putatively, in the pathogenesis of pruritus.

Peripheral arterial thrombosis with presence of anti-centromere antibodies and polycythemia vera.

Digital gangrene due to arterial thrombosis can occur due to a variety of causes. There have been reports of patients who have presented with digital gangrene with no known predisposing factors, but with positive anti-centromere antibodies (ACA). Despite that they did not have features suggestive of disorders like Scleroderma or CREST syndrome. We report a patient with digital gangrene, who was asymptomatic before this episode and was found to have both ACA positivity and Polycythemia Vera.