Primary myelofibrosis (PMF) is a fatal neoplastic disease characterized by clonal myeloproliferation and progressive bone marrow (BM) fibrosis thought to be induced by mesenchymal stromal cells stimulated by overproduced growth factors. However, tissue fibrosis in other diseases is associated with monocyte-derived fibrocytes. Therefore, we sought to determine whether fibrocytes play a role in the induction of BM fibrosis in PMF. In this study, we show that BM from patients with PMF harbors an abundance of clonal, neoplastic collagen- and fibronectin-producing fibrocytes. Immunodeficient mice transplanted with myelofibrosis patients' BM cells developed a lethal myelofibrosis-like phenotype. Treatment of the xenograft mice with the fibrocyte inhibitor serum amyloid P (SAP; pentraxin-2) significantly prolonged survival and slowed the development of BM fibrosis. Collectively, our data suggest that neoplastic fibrocytes contribute to the induction of BM fibrosis in PMF, and inhibiting fibrocyte differentiation with SAP may interfere with this process.

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by the clonal expansion of myeloid cells, notably megakaryocytes (MKs), and an aberrant cytokine production leading to bone marrow (BM) fibrosis and insufficiency. Current treatment options are limited. TGF-β1, a profibrotic and immunosuppressive cytokine, is involved in PMF pathogenesis. While all cell types secrete inactive, latent TGF-β1, only a few activate the cytokine via cell type-specific mechanisms. The cellular source of the active TGF-β1 implicated in PMF is not known. Transmembrane protein GARP binds and activates latent TGF-β1 on the surface of regulatory T lymphocytes (Tregs) and MKs or platelets. Here, we found an increased expression of GARP in the BM and spleen of mice with PMF and tested the therapeutic potential of a monoclonal antibody (mAb) that blocks TGF-β1 activation by GARP-expressing cells. GARP:TGF-β1 blockade reduced not only fibrosis but also the clonal expansion of transformed cells. Using mice carrying a genetic deletion of Garp in either Tregs or MKs, we found that the therapeutic effects of GARP:TGF-β1 blockade in PMF imply targeting GARP on Tregs. These therapeutic effects, accompanied by increased IFN-γ signals in the spleen, were lost upon CD8 T-cell depletion. Our results suggest that the selective blockade of TGF-β1 activation by GARP-expressing Tregs increases a CD8 T-cell-mediated immune reaction that limits transformed cell expansion, providing a novel approach that could be tested to treat patients with myeloproliferative neoplasms.

Primary myelofibrosis (PMF) is a chronic myeloproliferative disorder in human bone marrow. Over 50% of patients with myelofibrosis have mutations in JAK2, MPL, or CALR. However, these mutations are rarely detected in children, suggesting a difference in the pathogenesis of childhood PMF. In this study, we investigated the response to drug treatment of a monozygotic twin pair with typical childhood PMF. The twin exhibited different clinical outcomes despite following the same treatment regimen. The transcriptomic profiles of patient samples after drug treatment (E2 and Y2) were significantly different between the twin pair, which is consistent with the observation that the drug treatment was effective only in the younger brother, despite the twin being genetically identical. Bioinformatics analysis of the drug-responsive genes showed that the JAK-STAT pathway was activated in the cured younger brother, which is opposite to the pathway inhibition observed in adult PMF cases following treatment. Moreover, apoptosis and cell cycle processes were both significantly influenced by drug treatment in the sample of younger brother (Y2), implying their potential association with the pathogenesis of childhood PMF. Gene mutations in JAK2, MPL, or CALR were not observed; however, mutations in genes including SRSF2 and SF3B1 occurred in this twin pair with childhood PMF. Gene fusion events were extensively screened in the twin pair samples and the occurrence of IGLV2-14-IGLL5 gene fusion was confirmed. The current study reported at transcriptomic level the different responses of monozygotic twin brothers with childhood PMF to the same androgen/prednisone treatment regimen providing new insights into the potential pathogenesis of childhood PMF for further research and clinical applications.

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.

Primary myelofibrosis (PMF) is a Philadelphia-negative (Ph-) myeloproliferative disorder, showing abnormal CD34+ progenitor cell trafficking, splenomegaly, marrow fibrosis leading to extensive extramedullary haematopoiesis, and abnormal neoangiogenesis in either the bone marrow or the spleen. Monocytes expressing the angiopoietin-2 receptor (Tie2) have been shown to support abnormal angiogenic processes in solid tumors through a paracrine action that takes place in proximity to the vessels. In this study we investigated the frequency of Tie2 expressing monocytes in the spleen tissue samples of patients with PMF, and healthy subjects (CTRLs), and evaluated their possible role in favouring spleen angiogenesis. We show by confocal microscopy that in the spleen tissue of patients with PMF, but not of CTRLs, the most of the CD14+ cells are Tie2+ and are close to vessels; by flow cytometry, we found that Tie2 expressing monocytes were Tie2+CD14lowCD16brightCDL62-CCR2- (TEMs) and their frequency was higher (p = 0.008) in spleen tissue-derived mononuclear cells (MNCs) of patients with PMF than in spleen tissue-derived MNCs from CTRLs undergoing splenectomy for abdominal trauma. By in vitro angiogenesis assay we evidenced that conditioned medium of immunomagnetically selected spleen tissue derived CD14+ cells of patients with PMF induced a denser tube like net than that of CTRLs; in addition, CD14+Tie2+ cells sorted from spleen tissue derived single cell suspension of patients with PMF show a higher expression of genes involved in angiogenesis than that found in CTRLs. Our results document the enrichment of Tie2+ monocytes expressing angiogenic genes in the spleen of patients with PMF, suggesting a role for these cells in starting/maintaining the pathological angiogenesis in this organ.

Philadelphia negative myeloproliferative neoplasms (MPNs) are classically characterized by excess production of terminal myeloid cells in the peripheral blood. They include polycythemia vera, essential thrombocythemia, and primary myelofibrosis. Among this group, primary myelofibrosis is the least common and usually carries the worst prognosis. Bone involvement in primary myelofibrosis has many forms; it affects bone marrow leading to bone marrow fibrosis, it can cause periostitis, in addition to bone and joint pain. A common radiologic finding in primary myelofibrosis is the presence of osteosclerotic lesions. However, the presence of osteolytic lesions in bone imaging was described in few reports. In this review, we searched English literature using the PRISMA guidelines looking for patients with Primary myelofibrosis who had osteolytic bone lesions to assess the impact of such findings on the disease and its effect on prognosis. We found the vast majority of lesions were painful affecting most commonly the vertebral column, pelvis, and ribs, and were detected in patients above 50 years of age with no gender preference, unfortunately they represented advanced disease stages, resulting in inadequate treatment response and poor outcome.

Bone marrow fibrosis is a critical component of primary myelofibrosis (PMF). However, the origin of the myofibroblasts that drive fibrosis is unknown. Using genetic fate mapping we found that bone marrow leptin receptor (Lepr)-expressing mesenchymal stromal lineage cells expanded extensively and were the fibrogenic cells in PMF. These stromal cells downregulated the expression of key haematopoietic-stem-cell-supporting factors and upregulated genes associated with fibrosis and osteogenesis, indicating fibrogenic conversion. Administration of imatinib or conditional deletion of platelet-derived growth factor receptor a (Pdgfra) from Lepr+ stromal cells suppressed their expansion and ameliorated bone marrow fibrosis. Conversely, activation of the PDGFRA pathway in bone marrow Lepr+ cells led to expansion of these cells and extramedullary haematopoiesis, features of PMF. Our data identify Lepr+ stromal lineage cells as the origin of myofibroblasts in PMF and suggest that targeting PDGFRA signalling could be an effective way to treat bone marrow fibrosis.

Primary myelofibrosis (PMF) is a Ph-negative myeloproliferative neoplasm (MPN), characterized by advanced bone marrow fibrosis and extramedullary haematopoiesis. The bone marrow fibrosis results from excessive proliferation of fibroblasts that are influenced by several cytokines in the microenvironment, of which transforming growth factor-β (TGF-β) is the most important. Micromechanics related to the niche has not yet been elucidated. In this study, we hypothesized that mechanical stress modulates TGF-β signalling leading to further activation and subsequent proliferation and invasion of bone marrow fibroblasts, thus showing the important role of micromechanics in the development and progression of PMF, both in the bone marrow and in extramedullary sites. Using three PMF-derived fibroblast cell lines and transforming growth factor-β receptor (TGFBR) 1 and 2 knock-down PMF-derived fibroblasts, we showed that mechanical stress does stimulate the collagen synthesis by the fibroblasts in patients with myelofibrosis, through the TGFBR1, which however seems to be activated through alternative pathways, other than TGFBR2.

The aim of the present study was to predict pathogenic genes for primary myelofibrosis (PMF) using a system‑network approach by combining protein‑protein interaction (PPI) network and gene expression data with known pathogenic genes. PMF gene expression profiles, known pathogenic genes and protein‑protein interactions were obtained. Using these data, differentially expressed genes (DEGs) were identified between PMF and normal conditions using significance analysis of microarrays, and seed genes were determined based on the intersection of known pathogenic genes and the PMF gene expression profile. A new network was constructed using the seed genes and their adjacent DEGs within the PPI network. Subsequently, a pathogenic network was extracted from the new network, and contained genes that interacted with at least two seed genes, and the candidate pathogenic genes were predicted based on the cohesion with seed genes. Cluster analysis was performed to mine the pathogenic modules from the pathogenic network, and functional analysis was performed to identify the putative biological processes of the candidate pathogenic genes. Results from the present study identified 845 DEGs between PMF and normal conditions, and 45 seed genes in PMF were screened. Subsequently, a pathogenic network comprising 103 nodes and 265 interactions was constructed, and 4 pathogenic modules (modules A‑D) were mined from the pathogenic network. There were nine candidate pathogenic genes contained within Module A and four potential pathogenic genes, including E1A‑binding protein p300, RAS‑like proto‑oncogene A, von Willebrand factor and RAF‑1 proto‑oncogene, serine/threonine kinase, were identified that may be involved in the same biological process with the seed genes. This study predicted 10 candidate pathogenic genes and several signaling pathways that may be related to the pathogenesis of PMF using a system‑network approach. These predictions may shed light on the PMF pathogenesis and may provide guidelines for future experimental verification.

The molecular pathogenesis of primary mielofibrosis (PMF) is still largely unknown. Recently, single-nucleotide polymorphism arrays (SNP-A) allowed for genome-wide profiling of copy-number alterations and acquired uniparental disomy (aUPD) at high-resolution. In this study we analyzed 20 PMF patients using the Genome-Wide Human SNP Array 6.0 in order to identify novel recurrent genomic abnormalities. We observed a complex karyotype in all cases, detecting all the previously reported lesions (del(5q), del(20q), del(13q), +8, aUPD at 9p24 and abnormalities on chromosome 1). In addition, we identified several novel cryptic lesions. In particular, we found a recurrent alteration involving cytoband 20p13 in 55% of patients. We defined a minimal affected region (MAR), an amplification of 9,911 base-pair (bp) overlapping the SIRPB1 gene locus. Noteworthy, by extending the analysis to the adjacent areas, the cytoband was overall affected in 95% of cases. Remarkably, these results were confirmed by real-time PCR and validated in silico in a large independent series of myeloproliferative diseases. Finally, by immunohistochemistry we found that SIRPB1 was over-expressed in the bone marrow of PMF patients carrying 20p13 amplification. In conclusion, we identified a novel highly recurrent genomic lesion in PMF patients, which definitely warrant further functional and clinical characterization.

To compare the mutational profiles of patients with primary myelofibrosis (PMF), polycythemia vera (PV), and essential thrombocytosis (ET).

Myelofibrosis is a chronic and progressive myeloproliferative neoplasm characterized by anemia, splenomegaly, debilitating symptoms and leukemic transformation. Ruxolitinib, an oral JAK1/2 inhibitor, is highly effective in ameliorating systemic symptoms and reducing splenomegaly. Current clinical research is focused on the evaluation of agents based on pre-clinical rationale that can result in disease course modification. Panobinostat is a pan-histone deacetylase inhibitor that has demonstrated clinical activity as a single agent in early phase trials of myelofibrosis. We previously conducted a phase I trial of panobinostat monotherapy in patients with myelofibrosis and determined 25mg thrice weekly as the recommended phase II dose. We then completed an investigator initiated, Simon 2-stage, phase II trial of 22 myelofibrosis patients at our single institution. After 6 cycles of therapy, the overall response rate by IWG-MRT criteria was 36% (8/22; 95% CI: 16-56%). The median percent reduction in spleen volume was 34% (range, 1.6%-73%) in eight evaluable patients. The average reduction in JAK2V617F allele burden was 6.8% (Range; -4.0% to 20.2%) and one patient obtained a complete molecular response. Six patients remained on therapy in the extension phase for a median of 18 months (range, 7-44). Treatment discontinuation was frequent due to patient/physician perception of therapy ineffectiveness. The optimal dosing of panobinostat for the treatment of MF remains somewhat ill-defined but appears to be most effective and better tolerated when administered at lower doses over a prolonged duration of therapy.

Growth differentiation factor 15 (GDF15) is a pleiotropic cytokine that belongs to the transforming growth factor-β superfamily. Elevated serum concentrations of this cytokine have been reported in patients with various malignancies. To assess the potential roles of GDF15 in hematologic malignancies, we measured its serum levels in patients with these diseases. We found that serum GDF15 levels were elevated in almost all these patients, particularly in patients with primary myelofibrosis (PMF). Immunohistochemical staining of bone marrow (BM) specimens revealed that GDF15 was strongly expressed by megakaryocytes, which may be sources of increased serum GDF15 in PMF patients. Therefore, we further assessed the contribution of GDF15 to the pathogenesis of PMF. Recombinant human (rh) GDF15 enhanced the growth of human BM mesenchymal stromal cells (BM-MSCs), and it enhanced the potential of these cells to support human hematopoietic progenitor cell growth in a co-culture system. rhGDF15 enhanced the growth of human primary fibroblasts, but it did not affect their expression of profibrotic genes. rhGDF15 induced osteoblastic differentiation of BM-MSCs in vitro, and pretreatment of BM-MSCs with rGDF15 enhanced the induction of bone formation in a xenograft mouse model. These results suggest that serum levels of GDF15 in PMF are elevated, that megakaryocytes are sources of this cytokine in BM, and that GDF15 may modulate the pathogenesis of PMF by enhancing proliferation and promoting osteogenic differentiation of BM-MSCs.

Myeloproliferative neoplasms (MPNs) are characterized by upregulation of proinflammatory cytokines and immune dysregulation, which provide a reasonable basis for immunotherapy in patients. Megakaryocytes are crucial in the pathogenesis of primary myelofibrosis (PMF), the most clinically aggressive subtype of MPN. In this study, we aimed to explore PD-L1 (programmed death-ligand 1) expression in megakaryocytes and its clinical implications in PMF. We analyzed PD-L1 expression on megakaryocytes in PMF patients by immunohistochemistry and correlated the results with clinicopathological features and molecular aberrations. We employed a two-tier grading system considering both the proportion of cells positively stained and the intensity of staining. Among the 85 PMF patients, 41 (48%) showed positive PD-L1 expression on megakaryocytes with the immune-reactive score ranging from 1 to 12. PD-L1 expression correlated closely with higher white blood cell count (p = 0.045), overt myelofibrosis (p = 0.010), JAK2V617F mutation (p = 0.011), and high-molecular risk mutations (p = 0.045), leading to less favorable overall survival in these patients (hazard ratio 0.341, 95% CI 0.135-0.863, p = 0.023). Our study provides unique insights into the interaction between immunologic and molecular phenotypes in PMF patients. Future work to explore the translational potential of PD-L1 in the clinical setting is needed.

The detection of molecular and cytogenetic alterations is important for the diagnosis, prognosis and classification of myeloproliferative neoplasms.

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 (PMF) is associated with morbidity and mortality. Ruxolitinib gained US FDA approval for treatment of intermediate/high-risk PMF in November 2011. We evaluated differences in survival and second primary malignancy (SPM) incidence among US PMF patients in the years before and after ruxolitinib approval.

The aim of the present study was to identify potential therapeutic target genes and miRNAs for primary myelofibrosis (PMF). The dataset GSE53482 was downloaded from the Gene Expression Omnibus database. The differentially expressed genes (DEGs) and differentially expressed miRNAs (DEMs) of peripheral blood (PB) cluster of differentiation (CD)34+ cells from PMF patients (PB-PMF group) and peripheral blood CD34+ cells from healthy individuals (PB-control group) were analyzed using the Linear Models for Microarray Data package in R. The Kyoto Encyclopedia of Genes and Genomes was used for pathway enrichment analysis. MiRNA-gene joint enrichment analysis was performed by ENViz and a miRNAs-gene regulatory network was constructed. A total of 1,182 DEGs (773 upregulated and 109 downregulated) and 48 DEMs (28 upregulated and 20 downregulated) were identified. According to the pathway enrichment analysis, a number of DEGs were enriched in metabolic pathways, including IDH1 and DNMT1. Other DEGs were enriched in the citrate cycle (tricarboxylic acid cycle; IDH1 and IDH3A) and certain DEGs were enriched in pyrimidine metabolism, including CARD8. For downregulated genes, certain DEGs were enriched in the spliceosome, including SF3B1 and CDC40. Furthermore, hsa-miR-127-3p, hsa-miR-140-3p and hsa-miR345 were associated with cell cycle-related biological processes, signal transduction and cell surface receptor signaling pathway. The DEM-DEG regulatory network indicated that hsa-miR-543 regulated 113 genes, including CARD8 and TIFA. The present study identified a number of genes, including IDH1, DNMT1, SF3B1 and CARD8, and miRNAs, including hsa-miR-127-3p and hsa-miR-140-3p, which may be therapeutic targets in the treatment of PMF.

Past studies described interactions between normal megakaryocytes, the platelet precursors, and bone cell precursors in the bone marrow. This relationship has also been studied in context of various mutations associated with increased number of megakaryocytes. The current study is the first to examine the effects of megakaryocytes from transgenic mice carrying the most common mutation that causes primary myelofibrosis (PMF) in humans (JAK2V617F) on bone cell differentiation. Organ level assessments of mice using micro-computed tomography showed decreased bone volume in JAK2V617F males, compared to matching controls. Tissue level histology revealed increased deposition of osteoid (bone matrix prior mineralization) in these mutated mice, suggesting an effect on osteoblast differentiation. Mechanistic studies using a megakaryocyte-osteoblast co-culture system, showed that both wild type or JAK2V617F megakaryocytes derived from male mice inhibited osteoblast differentiation, but JAK2V617F cells exerted a more significant inhibitory effect. A mouse mRNA osteogenesis array showed increased expression of Noggin, Chordin, Alpha-2-HS-glycoprotein, Collagen type IV alpha 1 and Collagen type XIV alpha 1 (mostly known to inhibit bone differentiation), and decreased expression of alkaline phosphatase, Vascular cell adhesion molecule 1, Sclerostin, Distal-less homeobox 5 and Collagen type III alpha 1 (associated with osteogenesis) in JAK2V617F megakaryocytes, compared to controls. This suggested that the mutation re-programs megakaryocytes to express a cluster of genes, which together could orchestrate greater suppression of osteogenesis in male mice. These findings provide mechanistic insight into the effect of JAK2V617F mutation on bone, encouraging future examination of patients with this or other PMF-inducing mutations.

Previous data established that plitidepsin, a cyclic depsipeptide, exerted activity in a mouse model of myelofibrosis (MF). New preclinical experiments reported herein found that low nanomolar plitidepsin concentrations potently inhibited the proliferation of JAK2V617F-mutated cell lines and reduced colony formation by CD34+ cells of individuals with MF, at least in part through modulation of p27 levels. Cells of MF patients had significantly reduced p27 content, that were modestly increased upon plitidepsin exposure. On these premise, an exploratory phase II trial evaluated plitidepsin 5 mg/m(2) 3-h intravenous infusion administered on days 1 and 15 every 4 weeks (q4wk). Response rate (RR) according to the International Working Group for Myelofibrosis Research and Treatment consensus criteria was 9.1% (95% CI, 0.2-41.3%) in 11 evaluable patients during the first trial stage. The single responder achieved a red cell transfusion independence and stable disease was reported in nine additional patients (81.8%). Eight patients underwent a short-lasting improvement of splenomegaly. In conclusion, plitidepsin 5 mg/m(2) 3-h infusion q4wk was well tolerated but had a modest activity in patients with primary, post-polycythaemia vera or post-essential thrombocythaemia MF. Therefore, this trial was prematurely terminated and we concluded that further clinical trials with plitidepsin as single agent in MF are not warranted.