Bone Marrow Remodeling Research Articles

Bone Marrow Remodelling and Topographic Redistribution of CD34 Positive Progenitors Characterize the Progression of Myelodysplastic Syndromes and Their Evolution to AML

Myelodysplastic Syndrome (MDS) is a multifaceted disease of Hematopoietic Stem Cells characterized by myeloproliferation associated with mono- or pan-cytopenia, ultimately leading to an advantageous clonal growth of frankly leukemic cells. Although the disruption of bone marrow (BM) architecture and alterations of differentiated hematopoietic compartments have been repeatedly described, the potential involvement of the structural remodeling and redistribution of hematopoietic progenitors within the BM on the evolution of MDS has been poorly investigated. The aim of our study was to determine whether the number and localization of CD34pos progenitors and vascular structures are differentially affected in MDS subcategories and whether these changes are associated with disease progression. Thus, the morphometric evaluation of the number and linear distance from the endosteal surface (PT: paratrabecular <20mm; IT: intertrabecular >21mm) of capillaries, sinusoids, arterioles and CD34pos progenitors was performed by immunohistochemistry on BM biopsies from 97 consecutive MDS patients (12 RA; 2 RCUD; 19 RCMD; 3 RARS-T; 2 5q-; 20 RAEB-1; 27 RAEB-2; 12 U-MDS) in stable or evolving disease and in 15 cases that progressed to AML. Controls (CTR) were represented by 22 disease-free BM biopsies. Risk stratification was based on IPSS. Compared to CTR, capillary density linearly increased in IT marrow from RA thorough RAEB-1, with a subsequent progressive drop in RAEB-2 and AML. Sinusoid density was reduced in MDS and progressively declined from RAEB-1 through AML. PT and IT arterioles increased by more than 3-fold in MDS vs CTR although a progressive rarefaction associated with an increased thickness was measured from RA thorough RAEB-2. A marked rise in arteriolar number was also observed in AML. As expected, CD34pos cell number progressively increased with the severity of the disease, however, a sharp redistribution from PT to perisinusoid and fat areas within the IT marrow was documented (Figure). Intriguingly, the BM redistribution of vascular structures had higher impact than the increased number of CD34pos progenitors to identify high risk MDS and the subset of MDS patients who subsequently developed AML. Finally, patients responsive to azacytidine displayed a reduced number and a topographic redistribution of CD34pos cells together with an increased BM capillarization. BM structural remodelling and rearrangement of hematopoietic progenitors characterize the evolution of MDS. The prognostic significance and the predictive value of these parameters may be translated in new therapeutic approaches able to improve patient management. [Display omitted] DisclosuresGiuliani:Janssen: Research Funding; Celgene: Research Funding.

Cytokine Expression Pattern in Bone Marrow Microenvironment after Allogeneic Stem Cell Transplantation in Primary Myelofibrosis

The only curative therapy for primary myelofibrosis (PMF) is allogeneic stem cell transplantation (ASCT). However, although we know that patients can benefit from ASCT, we do not know the extent of the changes of the expression profile of cytokines and matrix modulation factors. In this first systematic analysis, we evaluated the expression profile of 103 factors before and after transplantation to identify potential biomarkers. The expression of fibrosis-, inflammation-, and angiogenesis-associated genes was analyzed in a total of 52 bone marrow biopsies: PMF patients (n = 14) before and after ASCT and, for control purposes, post-ASCT multiple myeloma patients (n = 14) and non-neoplastic hematopoiesis (n = 10). In post-ASCT PMF cases, decreased expression of tissue inhibitor of metalloproteinases (TIMP) and platelet-derived growth factor alpha (PDGFA) correlated with bone marrow remodeling and hematological remission. Expression of several other matrix factors remained at high levels and may contribute to post-ASCT remodeling. This is the first systematic analysis of cytokine expression in post-ASCT PMF bone marrow that shows that normalization of bone marrow microenvironment is paralleled by decreased expression of TIMP and PDGFA.

CCL-2 Is a KIT D816V-Dependent Modulator of Bone Marrow Remodeling and Microenvironmental Alterations in Systemic Mastocytosis

Systemic mastocytosis (SM) is a myeloproliferative neoplasm (MPN) characterized by mast cell (MC) infiltration in the bone marrow (BM) and/or other organs. Most patients present with the activating KIT mutation D816V. Increased production of cytokines is typically found in patients with classical MPN and has been linked to alterations of the BM microenvironment (e.g. enhanced angiogenesis and fibrosis) that are also commonly found in SM. However, little is known about mechanisms contributing to microenvironment alterations in the BM in SM and the role of MC-derived cytokines.The aim of our study was to identify cytokines that are produced in neoplastic MC in a KIT-dependent manner and contribute to increased BM angiogenesis and fibrosis in SM. In a first step, we screened for KIT D816V-dependent production of cytokines relevant to inflammation and microenvironment alterations using growth factor-dependent human cell lines (TF-1 and Mo7e). In these experiments, expression of CCL-2, IL-8, OSM, and VEGF mRNA was induced by KIT D816V but not by wild type KIT. Based on its pleiotropic effects, we focused on CCL-2, also referred to as monocyte chemotactic protein 1 (MCP-1), a CC chemokine that recruits inflammatory cells to sites of inflammation and enhances angiogenesis. KIT D816V+ HMC-1.2 cell were found to express and secrete substantial amounts of CCL-2. Midostaurin, a multikinase inhibitor suppressing the kinase activity of KIT D816V, was found to reduce expression of CCL-2 similar to RNAi-mediated knockdown of KIT. Furthermore, knockdown of STAT5, a key transcription factor downstream of KIT D816V, reduced expression of CCL-2 in HMC-1.2 cells. These results confirmed that CCL-2 expression in neoplastic MC is dependent on KIT D816V.Since CCL-2 has been reported to promote angiogenesis, we analyzed effects of conditioned medium obtained from HMC-1.2 cells on human umbilical vein endothelial cells (HUVEC) known to express the CCL-2 receptor CCR-2. Indeed, MC-derived conditioned medium induced migration of HUVEC in a wound healing assay (214% ± 38% of control, mean ± SD, p<0.01) as well as in a boyden chamber assay (126% ± 8% of control, p<0.05). Moreover, pre-incubation with a neutralizing antibody against CCL-2 significantly reduced migratory responses (p<0.01), and RNAi-mediated knockdown of CCL-2 in neoplastic MC reduced the effect of conditioned medium to baseline levels (p<0.01). These results confirmed that the migration was CCL-2 dependent. Furthermore, patients with advanced SM often present with marked eosinophilia; and eosinophils are often located in the vicinity of, or even within, BM MC infiltrates. Therefore, we also studied the effect of CCL-2 on eosinophils. Normal human eosinophils as well as the eosinophilic cell line EOL-1 were found to express CCR-2, and to migrate against recombinant CCL-2 in a modified boyden chamber assay. Conditioned medium from KIT D816V+ MC also induced a migratory response in eosinophils (200% ± 30% of control, p<0.05), and a neutralizing antibody against CCL-2 reduced this effect to baseline levels (p<0.05).Finally, SM-patients were found to have significantly elevated serum CCL-2 levels (n=35, p=0.0048, 407.4 ± 42.3 pg/ml, mean ± SEM) compared to controls (274.3 ± 15.9 pg/ml). The highest serum levels of CCL-2 were detected in patients with advanced SM where tissue remodeling in the BM is often a prominent feature. Moreover, CCL-2 levels were found to correlate with the grade of MC infiltration in the BM (r=0.656, p=0.0002). In summary, KIT D816V induces the expression of various cytokines potentially involved in tissue remodeling and microenvironment alterations in SM. Moreover, we have identified CCL-2 as a critical, KIT D816V-dependent, cytokine-mediator that interacts with structural BM cells and thereby may be involved in disease evolution and progression in SM. Whether CCL-2 may also serve as a therapeutic target in SM is currently being examined.This study was supported by Austrian Science Fund (FWF) grant P26079-B13. DisclosuresSperr:Novartis: Honoraria. Valent:Pfizer: Honoraria; Celgene: Honoraria; Ariad: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Novartis: Consultancy, Honoraria, Research Funding.

Matrix Metalloproteinases 2 and 9 Polymorphism in Patients With Myeloproliferative Diseases: A STROBE-Compliant Observational Study.

Chronic myeloproliferative disorders such as polycythemia vera (PV), essential thrombocytosis (ET), and idiopathic myelofibrosis arise from clonal proliferation of neoplastic stem cells in the bone marrow. Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that have potential to degrade all types of extracellular matrix (ECM) and also play a role in remodeling of the ECM. It is known that MMPs play a role in bone marrow remodeling.The primary goal of our study is to explore the relationship between chronic myeloproliferative diseases and some of MMP gene polymorphisms. The demonstration of a relationship will help to understand whether these polymorphisms may be a potential early diagnosis marker of the diseases.Patients were selected from outpatient clinics of Turgut Ozal University Hospital, Ankara, Turkey, between December 2010 and May 2011. Twenty-eight patients that previously diagnosed and followed-up with PV, 17 with secondary polycythemia (SP), and 12 with ET were enrolled in the study, along with a control group of 22 healthy people.DNA was isolated from peripheral blood. Using polymerase chain reaction–restriction fragment length polymorphism method, MMP2 and MMP9 gene polymorphisms were analyzed with agarose gel electrophoresis. There was a statistically significant difference between the study groups and the control group in terms of Gln279Arg polymorphisms rates of MMP9. The highest MMP9 Gln279Arg polymorphism rate was observed in the ET group. But nobody from the control group had polymorphic MMP9. There was no statistically significant difference between the groups in terms of MMP2-735 C > T polymorphism rates.In conclusion, MMP9 gene Gln279Arg polymorphism was associated with ET, SP, and PV diseases. Hence, we believe that these gene polymorphisms may play a role in the mechanism of bone marrow fibrosis and may be a factor that increases the risk of thrombosis. Illumination of the molecular basis of the relationship between MMP-thrombosis and MMP-fibrosis provides a better understanding of the pathophysiology of PV and ET diseases and will allow new approaches to diagnosis and treatment.

Melioidosis in a European traveler without comorbidities: a case report and literature review

Melioidosis is an endemic disease in Southeast Asia and northern Australia. It habitually affects immune-depressed hosts and may have a wide range of clinical manifestations. The use of positron emission tomography-computed tomography (PET-CT) has not been described previously for this disease. We report the case of a European traveler without comorbidities who developed melioidosis with pulmonary and bone marrow involvement 1 year after exposure. Antibiotic treatment was managed by taking into account the evolution on PET-CT. We review the literature and suggest the use of PET-CT for the initial evaluation of melioidosis, especially to look for a bone location, and to manage the length of antibiotic therapy.

Identification of oncostatin M as a JAK2 V617F‐dependent amplifier of cytokine production and bone marrow remodeling in myeloproliferative neoplasms

The JAK2 mutation V617F is detectable in a majority of patients with Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs). Enforced expression of JAK2 V617F in mice induces myeloproliferation and bone marrow (BM) fibrosis, suggesting a causal role for the JAK2 mutant in the pathogenesis of MPNs. However, little is known about mechanisms and effector molecules contributing to JAK2 V617F-induced myeloproliferation and fibrosis. We show that JAK2 V617F promotes expression of oncostatin M (OSM) in neoplastic myeloid cells. Correspondingly, OSM mRNA levels were increased in the BM of patients with MPNs (median 287% of ABL, range 22-1450%) compared to control patients (median 59% of ABL, range 12-264%; P < 0.0001). OSM secreted by JAK2 V617F+ cells stimulated growth of fibroblasts and microvascular endothelial cells and induced the production of angiogenic and profibrogenic cytokines (HGF, VEGF, and SDF-1) in BM fibroblasts. All effects of MPN cell-derived OSM were blocked by a neutralizing anti-OSM antibody, whereas the production of OSM in MPN cells was suppressed by a pharmacologic JAK2 inhibitor or RNAi-mediated knockdown of JAK2. In summary, JAK2 V617F-mediated up-regulation of OSM may contribute to fibrosis, neoangiogenesis, and the cytokine storm observed in MPNs, suggesting that OSM might serve as a novel therapeutic target molecule in these neoplasms.

Identification of Oncostatin M as a STAT5-Dependent Mediator of Bone Marrow Remodeling in KIT D816V-Positive Systemic Mastocytosis

Systemic mastocytosis is a neoplastic disease of mast cells harboring the activating KIT mutation D816V. In most patients, mast cell infiltration in the bone marrow is accompanied by marked microenvironment alterations, including increased angiogenesis, osteosclerosis, and sometimes fibrosis. Little is known about the mast cell-derived molecules contributing to these bone marrow alterations. We show here that neoplastic mast cells in patients with systemic mastocytosis express oncostatin M (OSM), a profibrogenic and angiogenic modulator. To study the regulation of OSM expression, KIT D816V was inducibly expressed in Ba/F3 cells and was found to up-regulate OSM mRNA and protein levels, suggesting that OSM is a KIT D816V-dependent mediator. Correspondingly, KIT D816V(+) HMC-1.2 cells expressed significantly higher amounts of OSM than the KIT D816V(-) HMC-1.1 subclone. RNA interference-induced knockdown of STAT5, a key transcription factor in KIT D816V(+) mast cells, inhibited OSM expression in HMC-1 cells, whereas a constitutively activated STAT5 mutant induced OSM expression. Finally, OSM secreted from KIT D816V(+) mast cells stimulated growth of endothelial cells, fibroblasts, and osteoblasts, suggesting that mast cell-derived OSM may serve as a key modulator of the marrow microenvironment and thus contribute to the pathology of systemic mastocytosis.

Thrombospondin-1 (TSP-1) in primary myelofibrosis (PMF) — a megakaryocyte-derived biomarker which largely discriminates PMF from essential thrombocythemia

Primary myelofibrosis (PMF) is a chronic myeloproliferative neoplasm showing aberrant bone marrow remodeling with increased angiogenesis, progressive matrix accumulation, and fibrosis development. Thrombospondins (TSP) are factors sharing pro-fibrotic and anti-angiogenic properties, and have not been addressed in PMF before. We investigated the expression of TSP-1 and TSP-2 in PMF related to the stage of myelofibrosis (n = 51) and in individual follow-up biopsies by real-time PCR, immunohistochemistry, and confocal laser scanning microscopy (CLSM). TSP-1 was significantly overexpressed (p < 0.05) in all stages of PMF when compared to controls. Individual follow-up biopsies showed involvement of TSP-1 during progressive myelofibrosis. TSP-2 was barely detectable but 40% of cases with advanced myelofibrosis showed a strong expression. Megakaryocytes and interstitial proplatelet formations were shown to be the relevant source for TSP-1 in PMF. Stroma cells like endothelial cells and fibroblasts showed no TSP-1 labeling when double-immunofluorescence staining and CLSM were applied. Based on its dual function, TSP-1 in PMF is likely to be a mediator within a pro-fibrotic environment which discriminates from ET cases. On the other hand, TSP-1 is a factor acting (ineffectively) against exaggerated angiogenesis. Both features suggest TSP-1 to be a biomarker for monitoring a PMF-targeted therapy.

Identification of new target moleculesPTK2,TGFBR2andCD9overexpressed during advanced bone marrow remodelling in primary myelofibrosis

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by remodelling of the bone marrow, including progressive myelofibrosis and exaggerated angiogenesis. Advanced PMF frequently shows a full-blown fibre meshwork, which avoids aspiration of cells, and the expression profile of genes related to stroma pathology at this stage remains largely undetermined. We investigated bone marrow core biopsies in PMF showing various degrees of myelofibrosis by custom-made low density arrays (LDA) representing target genes with designated roles in synthesis of extracellular matrix, matrix remodelling, cellular adhesion and motility. Among a set of 11 genes up-regulated in advanced stages of PMF (P < or = 0.01) three candidates, PTK2 protein tyrosine kinase 2 (PTK2), transforming growth factor beta type II receptor (TGFBR2) and motility-related protein-1 (CD9 molecule, CD9), were investigated in more detail. PTK2, TGFBR2 and CD9 were significantly overexpressed in larger series of advanced PMF stages (P < or = 0.01 respectively). Endothelial cells of the increased microvessel network in PMF could be identified as a predominant source for PTK2, TGFBR2 and CD9. CD9 also strongly identified activated fibroblasts in advanced myelofibrosis. We conclude that PTK2, TGFBR2 and CD9 represent new target molecules involved in bone marrow remodelling of PMF and warrant further investigation for potential targeted therapy.

EFFECT OF INTERTROCHANTERIC OSTEOTOMY ON THE PROXIMAL FEMUR OF RABBITS: ASSESSMENT WITH POWER DOPPLER SONOGRAPHY AND SCINTIGRAPHY

In bone injury, repair results in local increased vascularity and bone marrow remodeling. Characterizing the vascular and metabolic imaging patterns of the proximal femur following an intertrochanteric osteotomy may help clinicians decide proper management of the patient. Our objective was to measure Doppler sonography and scintigraphy interval changes in the proximal femur following intertrochanteric osteotomy and compare imaging and histomorphometric measurements in the late post-operative stage (6 weeks after surgery) in a rabbit model of bone injury. Both hips of 12 adult rabbits were imaged with power Doppler sonography and scintigraphy prior to and after (7 days and 6 weeks) unilateral osteotomy. Accuracy of the imaging methods was evaluated using hip operative status and histomorphometric results (vascular fractional area and number of vessels per area unit) as reference standard measures. A significant difference in the mean number of pixels was noted between operated and non-operated femura in late post-operative power Doppler examinations (P=0.049). Although without reaching statistical significance, the AUC of Doppler measurements (AUC=0.99) was numerically greater than the AUC of scintigraphy measurements (AUC=0.857+/-0.099) (P=0.15) in differentiating proximal femura with regard to their fractional vascular areas in the late post-operative stage. In contrast, scintigraphy tended to perform better (AUC=0.984+/-0.022) than Doppler ultrasound (AUC=0.746+/-0.131) to demonstrate the vascularity intensity per area unit (P=0.07) in the late stage. Our results warrant further investigation to determine the value of different imaging modalities for assessment of pathologic changes following hip surgery. Power Doppler sonography demonstrated larger AUCs (representing higher accuracy) for the discrimination of vascular fractional areas and scintigraphy, for discrimination of the number of vessels per area unit.

Elevated soluble urokinase plasminogen activator receptor in plasma from patients with idiopathic myelofibrosis or polycythaemia vera.

Extracellular proteolytic enzymes of the urokinase-type plasminogen activator (uPA) system and the family of metalloproteases play a crucial role in the matrix degradation and tissue remodelling processes characteristic of malignant disorders. The receptor for urokinase plasminogen activator (uPAR) serves to localise and intensify the action of uPA and is expressed on the surface of malignant as well as tumour stromal cells including fibroblasts. A soluble form of uPAR (suPAR) cleaved from its glycosyl-phosphatidylinositol anchor is detected in plasma from healthy individuals and increased levels of suPAR have been found in advanced malignancy, suggesting that suPAR may be a marker of extensive tissue remodelling. In an attempt to clarify whether suPAR might be a marker for bone marrow tissue remodelling we measured plasma suPAR levels in a patient cohort comprising 17 with myelofibrosis (MF), 17 with polycythaemia vera (PV), 15 with essential thrombocythaemia (ET), one with a transitional myeloproliferative disorder evolving from PV and 30 controls. Compared with controls suPAR levels were significantly higher in the patients (P < 0.0001) (median 3.35 vs. 2.32 microg L(-1)). Moreover, in subgroup analyses including patients with MF, PV, and ET, respectively, suPAR levels differed significantly with the highest levels found in patients with MF and PV (MF vs. PV vs. ET; P = 0.0003). When comparing suPAR levels of the individual patient subgroups with controls, only suPAR levels of PV and MF patients were significantly increased (P < 0.0001). Sixty-five percent of patients with PV and MF (22/34) had suPAR plasma values that were above the mean +2 standard deviations (SD) of controls. The concentration of suPAR was significantly correlated to plasma lactate dehydrogenase, thrombomodulin, and complex of tPA:PAI-1 in the patients. There was no difference between patients and controls when comparing plasma uPA levels. Increased plasma suPAR levels in patients with chronic myeloproliferative disorders may reflect increased uPAR production in the bone marrow, leading to enhanced bone marrow remodelling.

Bone Marrow Transplantation for Infantile Malignant Osteopetrosis

Most patients diagnosed with malignant osteopetrosis die during infancy or early childhood from hemorrhage and infection due to bone marrow failure. Allogeneic bone marrow transplantation (BMT) has been reported to provide curative therapy for this disorder. We report our experience with eight patients with malignant osteopetrosis who underwent BMT. Between May 1987 and August 1992, eight children with malignant osteopetrosis underwent allogeneic BMT. Median age at BMT was 9 months (range, 2-36 months). Six patients received marrow from HLA-identical sibling donors, one from phenotypically matched father, and one from a one antigen mismatched father. BMT conditioning for all patients was busulfan 16 mg/kg and cyclophosphamide 200 mg/kg each administered over 4 days. Graft versus host disease (GVHD) prophylaxis included cyclosporin A in six patients or cyclosporin A and methotrexate in two patients. Six patients, including those who received bone marrow from their father's, engrafted as documented by bone marrow biopsy showing an increase in osteoclasts in all cases and by chromosomal analysis in four patients. Two patients died without engraftment. Three out of six patients engrafted are alive and well at the follow-up of 48, 63, and 81 months. Serum calcium, alkaline, and acid phosphatase levels normalized within 2 months. These patients have full bone marrow reconstitution. Serial radiologic studies revealed bone marrow remodelling and a new nonsclerotic bone formation. Vision improved dramatically in the youngest patient. BMT offers cure to patients with malignant osteopetrosis with reconstitution of bone marrow and correction of metabolic disturbances. In our experience, reversibility in neurosensory deficit is possible when BMT is done at an early age.