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.

Expression profile analysis reveals hub genes that are associated with immune system dysregulation in primary myelofibrosis.

OBJECTION: Primary myelofibrosis (PMF) is a familiar chronic myeloproliferative disease with an unfavorable prognosis. The effect of infection on the prognosis of patients with PMF is crucial. Immune system dysregulation plays a central role in the pathophysiology of PMF. To date, very little research has been conducted on the molecular mechanism of immune compromise in patients with PMF. METHODS: To explore potential candidate genes, microarray datasets GSE61629 and 26049 were obtained from the Gene Expression Omnibus (GEO) database. The differentially expressed genes (DEGs) between PMF patients and normal individuals were evaluated, gene function was measured and a series of hub genes were identified. Several significant immune cells were selected via cell type enrichment analysis. The correlation between hub genes and significant immune cells was determined. RESULTS: A total of 282 DEGs were found, involving 217 upregulated genes and 65 downregulated genes. Several immune cells were found to be reduced in PMF, such as CD4+ T cells, CD4+ Tems, CD4+ memory T cells. Gene Ontology (GO) enrichment analysis of DEGs reflected that most biological processes were associated with immune processes. Six hub genes, namely, HP, MPO, MMP9, EPB42, SLC4A1, and ALAS2, were identified, and correlation analysis revealed that these hub genes have a negative correlation with immune cell abundance. CONCLUSIONS: Taken together, the gene expression profile of whole blood cells in PMF patients indicated a battery of immune events, and the DEGs and hub genes might contribute to immune system dysregulation.

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.

Reactivation of tuberculosis following ruxolitinib therapy for primary myelofibrosis: Case series and literature review.

Primary myelofibrosis (PMF) is a subtype of BCR-ABL1 negative myeloproliferative neoplasm. Its characteristic features include clonal myeloproliferation, dysregulation of kinase signaling pathway, abnormal release of cytokines leading to fibrosis in the bone marrow, osteosclerosis, and extramedullary hematopoiesis. Approximately 20% of deaths occur because of disease progression, but death may also result occur because of cardiovascular complications or as a consequence of either infection or bleeding. The only and curative option for PMF is allogeneic hematopoietic stem cell transplant (allo-HSCT); however, the Janus kinase (JAK) 1/2 inhibitor ruxolitinib is highly effective in reducing constitutional symptoms and spleen volume, and has been found to improve survival. Ruxolitinib decreases the activity of type I T-helper cells, leading to decreased release of cytokines including tumor necrosis factor-α, interleukin-1 (IL-1), IL-6, interferon-γ, and production of IL-12, which can be a risk factor for opportunistic infections. In this report, we describe three cases of tuberculosis reactivation shortly after initiation of ruxolitinib therapy followed by a literature review.

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.

Increased CXCL4 expression in hematopoietic cells links inflammation and progression of bone marrow fibrosis in MPN.

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm (MPN) that leads to progressive bone marrow (BM) fibrosis. Although the cellular mutations involved in the pathogenesis of PMF have been extensively investigated, the sequential events that drive stromal activation and fibrosis by hematopoietic-stromal cross-talk remain elusive. Using an unbiased approach and validation in patients with MPN, we determined that the differential spatial expression of the chemokine CXCL4/platelet factor-4 marks the progression of fibrosis. We show that the absence of hematopoietic CXCL4 ameliorates the MPN phenotype, reduces stromal cell activation and BM fibrosis, and decreases the activation of profibrotic pathways in megakaryocytes, inflammation in fibrosis-driving cells, and JAK/STAT activation in both megakaryocytes and stromal cells in 3 murine PMF models. Our data indicate that higher CXCL4 expression in MPN has profibrotic effects and is a mediator of the characteristic inflammation. Therefore, targeting CXCL4 might be a promising strategy to reduce inflammation in PMF.

Fecal microbiota transplantation in the treatment of intestinal steroid-resistant graft-versus-host disease: two case reports and a review of the literature.

Acute graft-versus-host disease (aGvHD) reduces the efficiency and safety of allogeneic hematopoietic stem cell transplantation (allo-HSCT). In recent years, attempts have been made to transplant fecal microbiota from healthy donors to treat intestinal GvHD. This study presented two cases of patients undergoing allo-HSCT who were later selected for fecal microbiota transplantation (FMT). In the first patient, FMT resulted in the complete resolution of symptoms, whereas therapeutic efficacy was not achieved in the second patient. FMT eliminated drug-resistant pathogens, namely very drug-resistant Enterococcus spp., but not multidrug-resistant Acinetobacter baumannii or Candida spp. Further research is needed, particularly on the safety of FMT in patients with intestinal steroid-resistant GvHD and on the distant impact of transplanted microflora on the outcomes of allo-HSCT. FMT appears promising for the treatment of patients with steroid-resistant GvHD.

Immunoproteasome Genes Are Modulated in CD34+ JAK2V617F Mutated Cells from Primary Myelofibrosis Patients.

Primary myelofibrosis (PMF) is a rare myeloproliferative neoplasm characterized by stem-cell-derived clonal over-proliferation of mature myeloid lineages, bone marrow fibrosis, osteosclerosis, defective erythropoiesis, and pro-inflammatory cytokine over-expression. The aim of the present study was to highlight possible differences in the transcriptome among CD34+ cells from peripheral blood (PB) of PMF patients. Therefore, we merged two microarray datasets of healthy control subjects and PMF (34 JAK2V617F MUTATED and 28 JAK2 wild-type). The GO analysis of upregulated genes revealed enrichment for JAK2/STAT1 pathway gene set in PB CD34+ cells of PMF patients with and without the JAK2V617F mutation comparing to the healthy control subjects, and in particular a significant upregulation of immunoproteasome (IP)-belonging genes as PSMB8, PSMB9, and PSMB10. A more detailed investigation of the IFN-gamma (IFNG) pathway also revealed that IFNG, IRF1, and IFNGR2 were significantly upregulated in PB CD34+ cells of PMF patients carrying the mutation for JAK2V617F compared to JAK2 wild-type PMF patients. Finally, we showed an upregulation of HLA-class I genes in PB CD34+ cells from PMF JAK2V617F mutated patients compared to JAK2 wild-type and healthy controls. In conclusion, our results demonstrate that IPs and IFNG pathways could be involved in PMF disease and in particular in patients carrying the JAK2V617F mutation.

Primary myelofibrosis marrow-derived CD14+/CD34- monocytes induce myelofibrosis-like phenotype in immunodeficient mice and give rise to megakaryocytes.

To confirm that neoplastic monocyte-derived collagen- and fibronectin-producing fibrocytes induce bone marrow (BM) fibrosis in primary myelofibrosis (PMF), we injected PMF BM-derived fibrocyte-precursor CD14+/CD34- monocytes into the tail vein of NOD-SCID-γ (NSG) mice. PMF BM-derived CD14+/CD34- monocytes engrafted and induced a PMF-like phenotype with splenomegaly, myeloid hyperplasia with clusters of atypical megakaryocytes, persistence of the JAK2V617F mutation, and BM and spleen fibrosis. As control we used normal human BM-derived CD14+/CD34- monocytes. These monocytes also engrafted and gave rise to normal megakaryocytes that, like PMF CD14+/CD34--derived megakaryocytes, expressed HLA-ABC and human CD42b antigens. Using 2 clonogenic assays we confirmed that PMF and normal BM-derived CD14+/CD34- monocytes give rise to megakaryocyte colony-forming cells, suggesting that a subpopulation BM monocytes harbors megakaryocyte progenitor capacity. Taken together, our data suggest that PMF monocytes induce myelofibrosis-like phenotype in immunodeficient mice and that PMF and normal BM-derived CD14+/CD34- monocytes give rise to megakaryocyte progenitor cells.

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.

Cytokine production in myelofibrosis exhibits differential responsiveness to JAK-STAT, MAP kinase, and NFκB signaling.

The distinct clinical features of myelofibrosis (MF) have been attributed in part to dysregulated inflammatory cytokine production. Circulating cytokine levels are elevated in MF patients; a subset of which have been shown to be poor prognostic indicators. In this study, cytokine overproduction was examined in MF patient plasma and in MF blood cells ex vivo using mass cytometry. Plasma cytokines measured following treatment with ruxolitinib remained markedly abnormal, indicating that aberrant cytokine production persists despite therapeutic JAK2 inhibition. In MF patient samples, 14/15 cytokines measured by mass cytometry were found to be constitutively overproduced, with the principal cellular source for most cytokines being monocytes, implicating a non-cell-autonomous role for monocyte-derived cytokines impacting disease-propagating stem/progenitor cells in MF. The majority of cytokines elevated in MF exhibited ex vivo hypersensitivity to thrombopoietin (TPO), toll-like receptor (TLR) ligands, and/or tumor necrosis factor (TNF). A subset of this group (including TNF, IL-6, IL-8, IL-10) was minimally sensitive to ruxolitinib. All TPO/TLR/TNF-sensitive cytokines, however, were sensitive to pharmacologic inhibition of NFκB and/or MAP kinase signaling. These results indicate that NFκB and MAP kinase signaling maintain cytokine overproduction in MF, and that inhibition of these pathways may provide optimal control of inflammatory pathophysiology in MF.

Autoimmune myelofibrosis: a rare haematological involvement in systemic lupus erythematosus.

Autoimmune myelofibrosis is a distinct clinicopathological entity that occurs with autoimmune disorders. We report the case of a 44-year-old woman admitted with pancytopenia and clinical features of systemic lupus erythematosus, Sjogren's syndrome and antiphospholipid antibodies syndrome. Bone marrow biopsy showed decreased global cells and an increase of reticulin fibres on argentic coloration, consistent with myelofibrosis. The JAK2 V617, Myeloproliferative leukemia (MPL) and calreticulin mutations were negative. The patient's condition improved after treatment with hydroxychloroquine, vitamin K antagonists and prednisone.

A rare combination: chylous polyserositis and autoimmune myelofibrosis as a presentation of systemic lupus erythematosus.

Chylous polyserositis and autoimmune myelofibrosis occurring concomitantly inn a case of SLE are a rare phenomenon. We here report a case of a 38-year-old woman who was admitted with a history of cough and shortness of breath for 1½ months along with fever and abdominal distension for 1 month. She also had arthralgias, weight loss and pancytopenia. She was diagnosed as a case of SLE with Chylous polyserositis and autoimmune myelofibrosis. She was started on steroids and immunosuppressive therapy, to which she responded. To summarize, this is the first case report where chylous polyserositis and pancytopenia due to autoimmune myelofibrosis occurred which was responsive to steroids and immunosuppressive therapy.

Primary myelofibrosis but not autoimmune myelofibrosis accompanied by Sjögren's syndrome and primary biliary cirrhosis in a patient with trisomy 8 mosaic: a case report and literature review.

Bone marrow fibrosis has been found to be associated with autoimmune disorders, and autoimmune myelofibrosis (AIMF) has been defined. Primary myelofibrosis (PMF), a clonal myeloproliferative disorder, should be distinguished from AIMF which has a good response to steroids, as the former has a high mortality and very bad response to conventional treatment. This case report describes a rare case of PMF accompanied with Sjögren's syndrome (SJS) and primary biliary cirrhosis (PBC), in a patient with trisomy 8 mosaic. Careful clinical assessment, gene mutation screening, and bone marrow evaluation can lead to an accurate diagnosis.

HSP27 is a partner of JAK2-STAT5 and a potential therapeutic target in myelofibrosis.

Heat shock protein 27 (HSP27/HSPB1) is a stress-inducible chaperone that facilitates cancer development by its proliferative and anti-apoptotic functions. The OGX-427 antisense oligonucleotide against HSP27 has been reported to be beneficial against idiopathic pulmonary fibrosis. Here we show that OGX-427 is effective in two murine models of thrombopoietin- and JAKV617F-induced myelofibrosis. OGX-427 limits disease progression and is associated with a reduction in spleen weight, in megakaryocyte expansion and, for the JAKV617F model, in fibrosis. HSP27 regulates the proliferation of JAK2V617F-positive cells and interacts directly with JAK2/STAT5. We also show that its expression is increased in both CD34+ circulating progenitors and in the serum of patients with JAK2-dependent myeloproliferative neoplasms with fibrosis. Our data suggest that HSP27 plays a key role in the pathophysiology of myelofibrosis and represents a new potential therapeutic target for patients with myeloproliferative neoplasms.

Targeting JAK2 reduces GVHD and xenograft rejection through regulation of T cell differentiation.

Janus kinase 2 (JAK2) signal transduction is a critical mediator of the immune response. JAK2 is implicated in the onset of graft-versus-host disease (GVHD), which is a significant cause of transplant-related mortality after allogeneic hematopoietic cell transplantation (allo-HCT). Transfer of JAK2-/- donor T cells to allogeneic recipients leads to attenuated GVHD yet maintains graft-versus-leukemia. Th1 differentiation among JAK2-/- T cells is significantly decreased compared with wild-type controls. Conversely, iTreg and Th2 polarization is significantly increased among JAK2-/- T cells. Pacritinib is a multikinase inhibitor with potent activity against JAK2. Pacritinib significantly reduces GVHD and xenogeneic skin graft rejection in distinct rodent models and maintains donor antitumor immunity. Moreover, pacritinib spares iTregs and polarizes Th2 responses as observed among JAK2-/- T cells. Collectively, these data clearly identify JAK2 as a therapeutic target to control donor alloreactivity and promote iTreg responses after allo-HCT or solid organ transplantation. As such, a phase I/II acute GVHD prevention trial combining pacritinib with standard immune suppression after allo-HCT is actively being investigated (https://clinicaltrials.gov/ct2/show/NCT02891603).

CD8+ T cells and IFN-γ induce autoimmune myelofibrosis in mice.

Myelofibrosis usually occurs either as a part of a myelodysplastic syndrome or in conjunction with neoplasia. It is not commonly thought of an autoimmune disease. We reported that p40-/-IL-2Rα-/- (interleukin-12p40 and interleukin-2 receptor alpha double knockout) mice, a mouse model of human primary biliary cholangitis, exhibited features consistent with autoimmune myelofibrosis, including anemia associated with bone marrow fibrosis, and extramedullary hematopoiesis (EMH) including LSK (Lineage-c-Kit+Sca-1+) cells in spleen, liver and peripheral blood. There were also increased LSK cells in bone marrow but they demonstrated impaired hematopoiesis. Importantly effector memory T cells that infiltrated the bone marrow of p40-/-IL-2Rα-/- mice manifested a higher ability to produce IFN-γ. CD8+ T cells, already known to play a dominate role in portal inflammation, were also key for bone marrow dysregulation and EMH. IFN-γ was the key cytokine that induced bone marrow fibrosis, bone marrow failure and EMH. Finally anti-CD8α antibody therapy fully protected p40-/-IL-2Rα-/- mice from autoimmune myelofibrosis. In conclusion, our results demonstrate that CD8+ T cells and IFN-γ are associated with autoimmune myelofibrosis, a finding that may allow targeting of CD8+ T cells and IFN-γ as a therapeutic targets.

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.