Fibrotic Bone Marrow Research Articles

The Diagnostic Value of Circulating Blasts in Patients with Myeloproliferative Neoplasms in Accelerated or Blast Phase

Introduction: Myeloproliferative neoplasms (MPNs) are progressive cancers with variable propensity to transform to accelerated phase and blast phase (APBP), an acute leukemia-equivalent state. MPN in APBP is associated with very poor prognosis with shortened survival and is notorious for poor response to therapy. The only curative therapy is allogeneic stem cell transplant. Blast phase is defined by 20% blasts or higher in the bone marrow biopsy (BMBx) or peripheral blood (PB), while accelerated phase is defined by blast percentage 10% or higher. The diagnosis, and response to therapy often requires serial BMBx and PB testing. There is recognition of excessive circulating PB blasts in MPNs that occasionally exceeds blast count in BMBs, especially in fibrotic bone marrow. The aim of this study is to investigate the correlation between PB and BMBx blasts, and if PB testing might be a more convenient and lest costly marker for diagnosis and response assessment of MPN in APBP. Methods: This is a cross sectional study, based on single institution retrospective chart review. Subjects included all available subjects over the age of 18, diagnosed with APBP MPN between January 1, 2017, to July 1, 2022. We collected serial blast percentages in simultaneously collected BMBx and PB samples, assessed by the same hematopathologist. We also collected baseline disease characteristics upon transformation to APBP. This is reported descriptively, and statistically analyzed for significance using a paired t-test. Results: 15 subjects and 54 paired BP and BMBx blast assessments were identified. Median age was 59 (range 30 -71) and median duration since MPN diagnosis was 48 months (1-420). Median OS of the cohort since APBP diagnosis was 10 months. BMBx fibrosis was Grade II-III in 13/15 patients. Cytogenetic and mutation profiles, and therapies delivered are listed on Table 1. Based on evaluation of the results, there was no statistical correlation between blasts in the PB and BMBx both prior to therapy, and during therapy (detailed biostatistical analysis will be provided during ASH presentation) Conclusions: Patients with APBP MPN undergo frequent diagnostic testing, including a bone marrow biopsy, to evaluate the percentage of blasts in the bone marrow. Quantification of blast count in peripheral blood should not be as a standalone diagnostic lab test for APBP MPN. BMBx remains essential for this diagnosis, response assessment, as well as cytogenetic and molecular profiling. The outcomes on APBP remain poor despite utilization of modern targeted therapeutics.

Acute Panmyelosis with Myelofibrosis in an Elderly Patient- an Uncommon Cause of Pancytopenia

Introduction: Acute panmyelosis with myelofibrosis (APMF) is rare yet a fatal hematological malignancy. It had been classified as a subtype of acute myeloid leukemia (AML) under the category "AML, not otherwise specified" in the WHO classification of hematological tumors. APMF usually presents with cytopenia without any marked splenomegaly. We report a case of an 80-year-old female who presented with generalized weakness. An extensive workup unmasked the presence of acute panmyelosis with myelofibrosis. Case Report: An 80-year-old female patient with history of epilepsy, essential hypertension, and hyperlipidemia presented to the clinic for generalized weakness and confusion for two weeks. She was also having dyspnea, initially on exertion, which progressed to even at rest for the last one week and was unable to ambulate without assistance. The patient also reported easy bruising with minor trauma and bruises on the upper extremity. The patient denied having any fever, chills, chest pain, orthopnea, dizziness, loss of consciousness, history of bleeding, or weight loss. On physical examination, the patient did not have enlarged lymph nodes or hepatosplenomegaly. Initial labs showed hemoglobin of 4.8 g/dL, platelets 5K/uL, a white blood count of 1.8 K/uL with differentials of 49% segmented neutrophils, 0.85 K/uL absolute neutrophils, 4% band cells, 44% lymphocytes, 1% myelocytes, 1% nucleated RBC's, 0.09 K/uL absolute monocytes, and 3% monocytes. Her absolute reticulocyte count was 0. Red blood cells were hypochromic with anisopoikilocytosis. The coagulation profile showed PT 16.5 seconds, INR 1.4, and aPTT 37 seconds. She also underwent a CT head for the confusion which was unremarkable. Chest x-ray revealed left mid & lower zone infiltrates consistent with pneumonia and the patient was given ceftriaxone. The patient was transfused irradiated packed red blood cells. There was a concern for bone marrow disorders such as myelodysplastic syndrome and leukemia, so the patient underwent a bone marrow biopsy. Flow cytometry of bone marrow aspirate revealed no significant blast cells; however, it showed myeloid neoplasm with myelofibrosis and proliferation of the atypical megakaryocytes with variable hypercellularity and diffusely marked reticulin fibrosis. Immunohistochemistry was positive for E-cadherin on megaloblastic erythroid, myeloperoxidase (highlighted patchy foci of maturing myeloid cells), CD34 (widespread positive in the atypical megakaryocyte, but not blast cells), and CD61 (GPIIIA). These findings along with the absence of splenomegaly were consistent with Panmyelosis with myelofibrosis. After the family discussion about chemotherapy and bone marrow transplant, a combined decision was made for hospice with comfort measures and the patient died within a week. Discussion: Acute panmyelosis with myelofibrosis is not thoroughly studied. Although WHO has classified it as a type of AML, some still consider it equivalent to AMKL or an acute kind of MDS (myelodysplastic syndrome). APMF comprises less than 1% of the cases of AML. The disease typically presents with an acute onset of pancytopenia without any splenomegaly and is rapidly fatal with mean survival of less than a year. Significant differential diagnoses include acute megakaryoblastic leukemia (AML) and acute myeloid leukemia with myelodysplasia-related changes (AML-MRC). AML and APMF share multiple features like fibrotic un-aspirable bone marrow and the presence of megakaryoblasts. However, in AML, most of the blast population comprises megakaryoblasts, while in APMF, non-megakaryocytic cells are predominant. Compared to AML-MRC, abrupt clinical onset and worse survival rate point towards APMF. In addition, multilineage dysplasia led by myelodysplastic syndrome is also characteristic of AML-MRC. Similarly, exposure to leukemogenic therapeutic agents should also be ruled out, as such cases should be categorized as MDS or AML therapy-related diseases. Due to the disease being extremely rare, there are no established management protocols. The most commonly used treatment is with 3 + 7 induction chemotherapy. A retrospective study showed a poor outcome of hematopoietic cell transplantation due to a high relapse rate. Another case showed remission by 3+7 induction with idarubicin and cytarabine followed by cytosine arabinoside. In our case, hospice care was preferred considering the age and comorbidities

Bone marrow-confined IL-6 signaling mediates the progression of myelodysplastic syndromes to acute myeloid leukemia.

Myelodysplastic syndromes (MDS) are age-related myeloid neoplasms with increased risk of progression to acute myeloid leukemia (AML). The mechanisms of transformation of MDS to AML are poorly understood, especially in relation to the aging microenvironment. We previously established an mDia1/miR-146a double knockout (DKO) mouse model phenocopying MDS. These mice develop age-related pancytopenia with oversecretion of proinflammatory cytokines. Here, we found that most of the DKO mice underwent leukemic transformation at 12–14 months of age. These mice showed myeloblast replacement of fibrotic bone marrow and widespread leukemic infiltration. Strikingly, depletion of IL-6 in these mice largely rescued the leukemic transformation and markedly extended survival. Single-cell RNA sequencing analyses revealed that DKO leukemic mice had increased monocytic blasts that were reduced with IL-6 knockout. We further revealed that the levels of surface and soluble IL-6 receptor (IL-6R) in the bone marrow were significantly increased in high-risk MDS patients. Similarly, IL-6R was also highly expressed in older DKO mice. Blocking of IL-6 signaling significantly ameliorated AML progression in the DKO model and clonogenicity of CD34-positive cells from MDS patients. Our study establishes a mouse model of progression of age-related MDS to AML and indicates the clinical significance of targeting IL-6 signaling in treating high-risk MDS.

Mechanical checkpoint regulates monocyte differentiation in fibrotic niches.

Myelofibrosis is a progressive bone marrow malignancy associated with monocytosis, and is believed to promote the pathological remodelling of the extracellular matrix. Here we show that the mechanical properties of myelofibrosis, namely the liquid-to-solid properties (viscoelasticity) of the bone marrow, contribute to aberrant differentiation of monocytes. Human monocytes cultured in stiff, elastic hydrogels show proinflammatory polarization and differentiation towards dendritic cells, as opposed to those cultured in a viscoelastic matrix. This mechanically induced cell differentiation is blocked by inhibiting a myeloid-specific isoform of phosphoinositide 3-kinase, PI3K-γ. We further show that murine bone marrow with myelofibrosis has a significantly increased stiffness and unveil a positive correlation between myelofibrosis grading and viscoelasticity. Treatment with a PI3K-γ inhibitor in vivo reduced frequencies of monocyte and dendritic cell populations in murine bone marrow with myelofibrosis. Moreover, transcriptional changes driven by viscoelasticity are consistent with transcriptional profiles of myeloid cells in other human fibrotic diseases. These results demonstrate that a fibrotic bone marrow niche can physically promote a proinflammatory microenvironment.

Circulating CD34+ cells of primary myelofibrosis patients contribute to myeloid-dominant hematopoiesis and bone marrow fibrosis in immunodeficient mice.

Primary myelofibrosis (PMF) is a clonal stem cell disorder characterized by myeloid dominant hematopoiesis and dysregulated proliferation of fibroblasts in the bone marrow. However, how these aberrant myeloid cells and fibroblasts are produced remains unclear. In this study, we examined in vivo engraftment kinetics of PMF patient-derived CD34+ cells in immunecompromised NOD/SCID/IL2rgKO (NSG) mice. Engrafted human cells were analyzed with flow cytometry, and proliferation of fibroblastic cells and bone marrow fibrosis were assessed with the histo-pathological examination. Transplantation of PMF patient-derived circulating CD34+ fractions into NSG newborns recapitulates clinical features of human PMF. Engraftment of human CD45+ leukocytes resulted in anemia and myeloid hyperplasia accompanied by bone marrow fibrosis by six months post-transplantation. Fibrotic bone marrow contained CD45-vimentin+ cells of both human and mouse origin, suggesting that circulating malignant CD34+ subsets contribute to myelofibrotic changes in PMF through direct and indirect mechanisms. A patient-derived xenotransplantation (PDX) model of PMF allows in vivo examination of disease onset and propagation originating from immature CD34+ cells and will support the investigation of pathogenesis and development of therapeutic modalities for the disorder.

Mechanical Checkpoint Regulates Monocyte Differentiation in Fibrotic Matrix

Myelofibrosis (MF) is a progressive, myeloid malignancy characterized by deposition of collagen and reticulin fibers in the bone marrow (BM). Previous studies have shown that monocytosis is associated with poor prognosis in MF, highlighting a potential pathogenic role for monocytes in MF. Although many studies have addressed the role of cell-intrinsic and soluble extracellular factors in MF development, it is currently unknown if mechanical properties of fibrotic BM contribute to aberrant differentiation of myeloid cells and of monocytes in particular.We first defined the stiffness and viscoelastic properties of healthy and fibrotic BM. Stiffness is defined as the resistance of a matrix to deformation, while viscoelasticity is the rate of dissipation of an applied stress over time. Independent of stiffness, an applied stress relaxes rapidly in a more viscous, liquid-like matrix, whereas in a more elastic, solid-like material, stress relaxes slowly.We next generated a cohort of fibrotic and non-fibrotic mice by transplanting retrovirally transduced JAK2V617F or empty vector (EV) control hematopoietic stem and progenitor cells (HSPCs) into lethally irradiated recipients. Femurs from these mice were harvested seven months post-transplant, as well as from age- and sex-matched healthy primary mice. Nanoindentation was performed to measure BM stiffness and viscoelasticity. Fibrotic BM showed higher stiffness, as well as trending higher elastic, solid-like properties, compared to BM of control mice.We then aimed to study the effect of matrix stiffness and viscoelasticity on monocytes. Human BM-derived monocytes were encapsulated in stiff, viscous or stiff, elastic hydrogels and cultured in the presence of GM-CSF, IL-4, and PGE2 for 3 days, followed by nanoString and flow cytometry analyses. Cells in elastic gels upregulated gene sets associated with co-stimulatory molecules and cytokine receptor signaling, MHC class II antigen presentation, and regulation of extracellular matrix (ECM), compared to cells in viscous gels of the same stiffness. The fraction of dendritic cells (DCs) was significantly upregulated, as indicated by double-positive CD11c+CD1c+ (40.9% viscous vs 69.5% elastic of CD11b+HLA-DR+ cells) and CD80+ cells (20.9% viscous vs 62.7% elastic of CD11b+HLA-DR+ cells), and surface expression of HLA-DR (gMFI 2587 viscous vs 6334 elastic). Consistent with these findings, the fraction of pro-fibrotic SLAMF7+ cells (4.2% viscous vs 17.3% elastic) were also significantly higher in elastic gels. Together, these data suggest that stiff, elastic ECM drives pro-inflammatory polarization and differentiation of monocytes into antigen-presenting cells.Next, we examined the role of the cytoskeleton on human monocyte differentiation. Cortical F-actin was significantly upregulated in cells in stiff, elastic gels compared to viscous gels. Cells were exposed to a highly selective small molecular inhibitor of the γ-isoform of PI3K. Treatment with the PI3Ky inhibitor significantly reduced F-actin staining of cells in elastic gels, upregulated immature monocyte markers, reduced surface expression of HLA-DR, and downregulated the cytokines IL6, IL8, CCL4, which have previously been associated with disease progression in myelofibrosis.In line with the above human ex vivo data, BM isolated from fibrotic mice (described above) showed skewing towards Ly6G-Ly6C+ monocytes (a population enriched for inflammatory monocytes) within the CD11b myeloid compartment compared to control transplanted mice or to non-fibrotic mice that were transplanted with endogenously expressing Jak2V617F cells. Additionally, the percentage of conventional DCs (cDCs) was increased in fibrotic Jak2V617F mice compared to control mice. Importantly, 16 day in vivo treatment with the PI3Ky inhibitor significantly reduced the fraction of Ly6G-Ly6C+ monocytes within the CD11b compartment as well as the fraction of cDCs, compared to vehicle-treated Jak2V617F mice.In summary, fibrotic BM is stiffer and more elastic than normal BM. Our studies show that a stiff, elastic BM environment drives monocytes towards a more pro-inflammatory state which can in part be suppressed by PI3K-γ inhibition. Our results have relevance for human MF by demonstrating that a fibrotic BM niche is not just a consequence of chronic inflammation but is also inflammation-promoting.KHV and AEM contributed equally to this work. DisclosuresPozdnyakova: Scopio Labs: Consultancy. Mullally: Janssen, PharmaEssentia, Constellation and Relay Therapeutics: Consultancy. Wucherpfennig: Novartis: Research Funding; Nextechinvest: Membership on an entity's Board of Directors or advisory committees; Immunitas Therapeutics: Current holder of individual stocks in a privately-held company; TScan Therapeutics: Membership on an entity's Board of Directors or advisory committees; TCR2 Therapeutics: Membership on an entity's Board of Directors or advisory committees; SQZ Biotech: Membership on an entity's Board of Directors or advisory committees. Mooney: Novartis: Patents & Royalties: Licensed IP, Research Funding; Sirenex: Patents & Royalties: Licensed IP; Samyang Corp: Membership on an entity's Board of Directors or advisory committees; IVIVA: Membership on an entity's Board of Directors or advisory committees; Attivare: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Revela: Membership on an entity's Board of Directors or advisory committees; Amend Surgical: Patents & Royalties: Licensed IP; Lyell: Current equity holder in publicly-traded company, Patents & Royalties.

Pro-fibrotic phenotype of bone marrow stromal cells in Modic type 1 changes.

Modic type 1 changes (MC1) are painful vertebral bone marrow lesions frequently found in patients suffering from chronic low-back pain. Marrow fibrosis is a hallmark of MC1. Bone marrow stromal cells (BMSCs) are key players in other fibrotic bone marrow pathologies, yet their role in MC1 is unknown. The present study aimed to characterise MC1 BMSCs and hypothesised a pro-fibrotic role of BMSCs in MC1. BMSCs were isolated from patients undergoing lumbar spinal fusion from MC1 and adjacent control vertebrae. Frequency of colony-forming unit fibroblast (CFU-F), expression of stem cell surface markers, differentiation capacity, transcriptome, matrix adhesion, cell contractility as well as expression of pro-collagen type I alpha 1, α-smooth muscle actin, integrins and focal adhesion kinase (FAK) were compared. More CFU-F and increased expression of C-X-C-motif-chemokine 12 were found in MC1 BMSCs, possibly indicating overrepresentation of a perisinusoidal BMSC population. RNA sequencing analysis showed enrichment in extracellular matrix proteins and fibrosis-related signalling genes. Increases in pro-collagen type I alpha 1 expression, cell adhesion, cell contractility and phosphorylation of FAK provided further evidence for their pro-fibrotic phenotype. Moreover, a leptin receptor high expressing (LEPRhigh) BMSC population was identified that differentiated under transforming growth factor beta 1 stimulation into myofibroblasts in MC1 but not in control BMSCs. In conclusion, pro-fibrotic changes in MC1 BMSCs and a LEPRhigh MC1 BMSC subpopulation susceptible to myofibroblast differentiation were found. Fibrosis is a hallmark of MC1 and a potential therapeutic target. A causal link between the pro-fibrotic phenotype and clinical characteristics needs to be demonstrated.

Bone marrow microenvironment of MPN cells.

In this chapter, we will discuss the current knowledge concerning the alterations of the cellular components in the bone marrow niche in Myeloproliferative Neoplasms (MPNs), highlighting the central role of the megakaryocytes in MPN progression, and the extracellular matrix components characterizing the fibrotic bone marrow.

Depletion of Neoplastic CD11b Positive Cells in Jak2V617F Mutant Mice Reduced Fibrocytes in Bone Marrow and Improved Myelofibrosis

Myelofibrosis (MF) associated with myeloproliferative neoplasms (MPN) has been considered to be a reactive phenomenon caused by mesenchymal stromal cells (MSCs) stimulated by cytokines such as TGFb-1 overproduced by neoplastic megakaryocytes (MKs) and platelets. TGFb-1 stimulates non-neoplastic mesenchymal cells to produce collagen and fibronectin and to induces bone marrow (BM) fibrosis. However, the involvement of neoplastic fibrocyte in MF has recently been reported (Verstovsek et al. JEM 2016), and among blood cells, monocytes in particular are considered to be the main source of neoplastic fibrocytes. In this study, we assesed the role of neoplastic fibrocytes using a mouse model of MPN induced by Jak2V617F (Shide et al. Leukemia 2008). First, the distribution of neoplastic fibrocyte in the BM of Jak2V617F transgenic (TG) mice was examined. We transplanted wild-type (WT) or Jak2V617F TG cells (B6-CD45.2), together with WT BM cells (B6-CD45.1) into irradiated WT recipient mice (B6-CD45.1). Only recipient mice transplanted with a mixture of Jak2V617F cells and WT cells developed BM fibrosis. In immunofluorescent staining of fibrotic BM, cells expressing the fibrocyte marker CD45/Collagen-1(Col-1) were observed much more than cells expressing the fibroblast marker CD90(usually positive for MSCs)/Col-1. As for CD45/Col-1 positive cells, cells expressing CD45.2/Col-1 were much more than cells expressing CD45.1/Col-1, clearly indicating that these cells were derived from Jak2V617F mutant blood cells. On the other hand, in the BM of recipient mice transplanted with control WT cells, few cells expressing CD45/Col-1 or CD90/Col-1 were present. To examine the differentiation ability of Jak2V617F blood cells to fibrocytes directly, peripheral blood (PB) mononuclear cells (MNC) of Jak2V617F mice or WT mice were cultured in vitro. After 5 days of culture, PB MNCs from Jak2V617F mice differentiated into mature fibrocytes exhibiting a long spindle shape with Col-1 expression. On the other hand, there were very few fibrocytes differentiated from PB MNC from WT mice. Next, we depleted monocytes, the main source of fibrocytes, and observed its effects on BM fibrosis in vivo. Jak2V617F TG mice were mated with CD11b-diphtheria toxin receptor (DTR) TG mice (Duffield et al. JCI 2005) to obtain Jak2V617F/CD11b-DTR double TG mice. Mice transplanted with BM cells from Jak2V617F/CD11b-DTR double TG mice (hereinafter called Jak2V617F/CD11b-DTR mice) exhibit leukocytosis, thrombocytosis, anemia, splenomegaly, and BM fibrosis with increased megakaryocytes. Jak2V617F/CD11b-DTR mice was administered diphtheria toxin (DT) intraperitoneally to deplete monocytes. One day after DT administration, the number of PB monocytes (CD11b+/F4/80+) drastically decreased in Jak2V617F/CD11b-DTR mice, and the reduction of monocyte was maintained by every-other-day DT administration. After 8 weeks DT treatment, mice were sacrificed and analyzed. As a control group, Jak2V617F/CD11b-DTR mice treated with PBS were examined. DT treatment drastically decreased the number of neoplastic fibrocytes expressing CD45.2/Col-1 in BM and spleen of Jak2V617F/CD11b-DTR mice compared with control mice treated with PBS. Consistently, reticulin fibers were eliminated almost completely and collagen fibers almost fully disappeared in BM, which led to a reversal of the decrease in BM cellularity, although the number of MKs was not affected. Similar findings were observed in the spleen, although not completely. Plasma TGF-b1 level were about 2-fold higher in Jak2V617F/CD11b-DTR mice than in WT mice. Neoplastic monocyte depletion significantly decreased TGF-b1 level. Since MK numbers did not change, this indicates that fibrocytes are one of the main sources of TGF-b1. In other features of MF in Jak2V617F/CD11b-DTR mice, splenomegaly was ameliorated by DT treatment. Microscopic analysis revealed an improvement in the damaged spleen architecture and the disappearance of splenic fibrosis. In summary, most collagen-producing cells in BM were neoplastic fibrocytes in Jak2V617F-induced MPN, indicating that neoplastic fibrocytes played an essential role and mesenchymal fibroblasts had a minor contribution in fibrosis in MPN. Depletion of neoplastic monocytes also improved splenomegaly as well as BM fibrosis in mice, and this cell fraction could be a promising therapeutic target. Disclosures No relevant conflicts of interest to declare.

Platelet-derived growth factor receptor β activation and regulation in murine myelofibrosis.

There is prevailing evidence to suggest a decisive role for platelet-derived growth factors (PDGF) and their receptors in primary myelofibrosis. While PDGF receptor β (PDGFRβ) expression is increased in bone marrow stromal cells of patients correlating with the grade of myelofibrosis, knowledge on the precise role of PDGFRβ signaling in myelofibrosis is sparse. Using the Gata-1low mouse model for myelofibrosis, we applied RNA sequencing, protein expression analyses, multispectral imaging and, as a novel approach in bone marrow tissue, an in situ proximity ligation assay to provide a detailed characterization of PDGFRβ signaling and regulation during development of myelofibrosis. We observed an increase in PDGFRβ and PDGF-B protein expression in overt fibrotic bone marrow, along with an increase in PDGFRβ–PDGF-B interaction, analyzed by proximity ligation assay. However, PDGFRβ tyrosine phosphorylation levels were not increased. We therefore focused on regulation of PDGFRβ by protein tyrosine phosphatases as endogenous PDGFRβ antagonists. Gene expression analyses showed distinct expression dynamics among PDGFRβ-targeting phosphatases. In particular, we observed enhanced T-cell protein tyrosine phosphatase protein expression and PDGFRβ–T-cell protein tyrosine phosphatase interaction in early and overt fibrotic bone marrow of Gata-1low mice. In vitro, T-cell protein tyrosine phosphatase (Ptpn2) knockdown increased PDGFRβ phosphorylation at Y751 and Y1021, leading to enhanced downstream signaling in fibroblasts. Furthermore, Ptpn2 knockdown cells showed increased growth rates when exposed to low-serum growth medium. Taken together, PDGF signaling is differentially regulated during myelofibrosis. Protein tyrosine phosphatases, which have so far not been examined during disease progression, are novel and hitherto unrecognized components in myelofibrosis.

Modic type 1 change is an autoimmune response that requires a proinflammatory milieu provided by the ‘Modic disc’

Background ContextModic changes (MCs) are magnetic resonance imaging (MRI) evidence of inflammatory and fibrotic vertebral bone marrow lesions that associate with adjacent disc degeneration and end plate damage. Although MC etiology is uncertain, historical data suggest a linkage to an autoimmune response of bone marrow triggered by the nucleus pulposus (NP). PurposeThe aim of this study was to test whether bone marrow has an autoimmune response to NP cells that is amplified by an inflammatory milieu and ultimately leads to MC development in vivo. We hypothesized that an inflammatory co-stimulus is required for bone marrow/NP crosstalk to stimulate MC. Study DesignThis is an in-vitro cell co-culture study plus in-vivo experiments in rat caudal vertebrae. MethodsIn in-vitro study, bone marrow mononuclear cells (BMNCs) and NP cells (NPCs) from rats were co-cultured with and without interleukin (IL)-1α stimulation. Cell viability (n=3) of BMNCs and NPCs and gene expression (n=7) were analyzed. In in-vivo study, proinflammatory lipopolysaccharide (LPS) and control disc nucleus surrogates (NP micromass pellets) were generated in vitro from rat NPCs and implanted into rat tail vertebrae, and the response was compared with sham surgery (n=12 each). Tissue changes were investigated with T1w and T2w MRI (7T), histology, and immunohistochemistry (tumor necrosis factor, CD3) 1 (n=6) and 2 weeks (n=6) after implantation. ResultsBMNC/NPC co-culture significantly increased lymphocyte viability (42%–69%, p<.05) and reduced NPC viability (96%–88%, p<.001), indicating immunogenicity of NPC. However, IL-1α was required to cause significant transcriptional upregulation of IL-1, IL-6, IL-10, and tropomyosin receptor kinase A. Therefore, an inflammatory activation is required to amplify the immune response. Immunogenicity of the NP was corroborated in vivo by CD3 cell accumulation around LPS and control disc surrogates at Day 7. However, only the LPS disc surrogate group demonstrated infiltration of CD3 cells at Day 14. Furthermore, end plate defects (p<.05, LPS: n=4/6, Ctrl: n=0/6, sham: n=0/6) and MC1-like MRI changes (T2w hyperintensity, p<.05) were only seen with LPS disc surrogates. ConclusionsNPCs are immunogenic but cannot trigger MC without an additional proinflammatory stimulus. Our data suggest that MC requires end plate defects that allow marrow/NPC co-mingling plus an adjacent inflammatory “MC disc” that can amplify the immune response.

Targeting the fibrotic bone marrow niche for treatment of myeloproliferative neoplasms

Targeting the fibrotic bone marrow niche for treatment of myeloproliferative neoplasms

Histopathological examination of spine tumors after treatment with radiosurgery.

OBJECTIVE Increased survival time after diagnosis of neoplastic disease has resulted in a gradual increase in spine tumor incidence. Radiosurgery is frequently a viable alternative to operative management in a population with severe medical comorbidities. The authors sought to assess the histopathological consequences of radiosurgery in the subset of patients progressing to operative intervention. METHODS Eighteen patients who underwent radiosurgery for spine tumors between 2008 and 2014 subsequently progressed to surgical treatment. A histopathological examination of these cases was performed. Indications for surgery included symptomatic compression fractures, radiographic instability, and symptoms of cord or cauda equina compression. Biopsy samples were obtained from the tumor within the radiosurgical zone in all cases and were permanently fixated. Viable tumor samples were stained for Ki 67. RESULTS Fifteen patients had metastatic lesions and 3 patients had neurofibromas. The mean patient age was 57 years. The operative indication was symptomatic compression in 10 cases (67%). The most frequent metastatic lesions were breast cancer (4 cases), renal cell carcinoma (3), prostate cancer (2), and endometrial cancer (2). In 9 (60%) of the 15 metastatic cases, histological examination of the lesions showed minimal evidence of inflammation. Viable tumor at the margins of the radiosurgery was seen in 9 (60%) of the metastatic cases. Necrosis in the tumor bed was frequent, as was fibrotic bone marrow. Vascular ectasia was seen in 2 of 15 metastatic cases, but sclerosis with ectasia was frequent. No evidence of malignant conversion was seen in the periphery of the lesions in the 3 neurofibroma cases. In 1 case of neurofibroma, the lesion demonstrated some small areas of remnant tumor in the radiosurgical target zone. CONCLUSIONS This case series demonstrates important histopathological characteristics of spinal lesions treated by SRS. Regions with the highest exposure to radiation appear to be densely necrotic and show little evidence of tumor growth, whereas peripheral regions distant from the radiation dosage are more likely to demonstrate viable tumor in malignant and benign neoplasms. Physiological tissue appears to be similarly affected. With additional investigation, a more homogenized field of hypofractionated radiation exposure may allow for tumor obliteration with relative preservation of critical anatomical structures.

Collagen I and collagen III expression in fibrotic bone marrow

Objective: The aim of this study was to assess the expression of collagen I and III in fibrotic bone marrow (BM) in different pathologies. Background: A wide variety of benign conditions and malignant disorders are associated with BM fibrosis. BM fibrosis can be demonstrated by histochemical staining using silver impregnation or by trichrome stains. Immunohistochemical stains, especially anticollagen I and III, are also used for more accurate assessment of BM fibrosis. This study demonstrates the expression of collagen I and III in fibrotic marrow in different pathologies. No clinical data, prognosis , survival were mentioned in this study and so the impact was not investigated. Materials and methods: In this study 50 formalin-fixed paraffin-embedded BM biopsy samples were selected from archived cases at the Clinical Pathology Department, Cairo University. These 50 cases included seven cases with myeloproliferative neoplasms, 14 with lymphoproliferative disorders, three with acute leukemia, four with metastasis, and 22 with reactive BM changes. Cases were divided into two groups according to the Masson's trichrome staining pattern: the fibrotic group, which included 13 cases, and the nonfibrotic group, which included 37 cases. The included cases were subjected to reticulin and trichrome stains, in addition to assessment of collagen I and III expression by immunohistochemistry. Results: As regards collagen I and III staining grades, grade 4 was significantly higher in the fibrotic group, whereas grade 0 was significantly higher in the nonfibrotic group. Conclusion: Collagen I and III are markedly expressed in fibrotic marrow and marrow fibrosis; in particular, collagen fibrosis appears to be more evident in myeloproliferative neoplasms and in tumor metastasis to the BM.

Expression of HMGA2 Collaborates with JAK2V617F to Progress Myeloproliferative Neoplasms

Myeloproliferative neoplasms (MPN) are characterized by chronic proliferation of myeloid cells, extramedullary hematopoiesis and occasional leukemic transformation. Mutations in JAK2, CALR and MPL have been established as drivers of myeloproliferative phenotype, but their roles in disease progression with clonal expansion remain unclear. In addition, studies have shown mutations in epigenetic modifiers including TET2, DNMT3A, ASXL1 and EZH2, and aberrant expressions of microRNAs in MPN, but downstream of these changes is also largely unknown. Recently, we showed high expression of HMGA2 mRNA partly correlated with reduced microRNA let-7 in granulocytes of patients with MPN, including 100% patients with primary myelofibrosis (MF) and 20% polycythemia vera and essential thrombocythemia (Harada-Shirado et al, Brit J Haematol, 2015). In mice, loss of epigenetic modifiers such as BMI1 and EZH2, along with the Arf/Ink4a knockout (Oguro et al, J Exp Med, 2012) or the JAK2 V617F (Sashida et al, ASH, 2013), leads to overexpression of HMGA2 with accelerating MPN. We have generated transgenic (Tg) mice of Hmga2 cDNA with truncated 3'UTR (ΔHmga2) lacking binding sites of let-7 thatrepresses expression of HMGA2 (Ikeda et al, Blood, 2011). Δ Hmga2 mice overexpress HMGA2 and develop MPN-like disease, and represent a clonal advantage in competitive repopulations with serial bone marrow (BM) transplants (BMT). Here, to clarify if HMGA2 affect JAK2 V617F+ hematopoiesis, we crossed Δ Hmga2+/- mice with JAK2 V617F+/- Tg mice (Shide et al, Leukemia, 2008). Δ Hmga2-/-JAK2 V617F-/- wild type (WT), Δ Hmga2+/-JAK2 V617F-/- (Δ Hmga2 -Tg), Δ Hmga2-/-JAK2 V617F+/- (JAK2 V617F-Tg) and Δ Hmga2+/-JAK2 V617F+/- (double-Tg) mice were born at expected Mendelian ratios and we could analyze 5 - 6 of each. At 3 months old, leukocytosis, thrombocytosis, anemia and splenomegaly were most severe in double-Tg compared with JAK2 V617F-Tg or Δ Hmga2 -Tg mice. Relative to WT, peripheral leukocyte and platelet counts were nearly 16- and 4-fold higher in double-Tg, while 3- and 2-fold higher in JAK2 V617F-Tg mice, respectively. Mean spleen weights were 0.067, 0.10, 0.83 and 2.8 g in WT, Δ Hmga2 -Tg, JAK2 V617F-Tg and double-Tg mice, while BM cell counts were 2.4, 2.8, 0.4 and 1.2 x 107/femur, respectively. However, JAK2 V617F-Tg and double-Tg equally showed MF whereas no MF was detected in WT and DHmga2-Tg, suggesting that HMGA2 partly recovers cellularity in fibrotic BM. In the absence and presence of JAK2 V617F, HMGA2 augments lineage- Sca1+ Kit+ cells (WT: Δ Hmga2-Tg: JAK2 V617F-Tg: double-Tg= 0.17%: 0.19%: 0.17%: 0.27% in BM cells), endogenous erythroid colonies (1: 11: 13: 21 CFU-E/104 BM cells) and CD71+ Ter119+ erythroblasts (23%: 29%: 5.7%: 10% in BM and 2.0%: 4.4%: 7.9%: 16% in spleen cells), indicating HMGA2 contributes to expansion of hematopoietic stem/progenitor cells (HSPC) and erythroid commitment in JAK2 V617F+ hematopoiesis. Most Δ Hmga2-Tg and JAK2 V617F-Tg survived for over one year, but all double-Tg mice died within 4 months after birth due to severe splenomegaly and MF with no acute leukemia. To study the effect of HMGA2 on JAK2 V617F+ HSPC activity, we performed BMT with 0.25 x 106 Ly5.2+Δ Hmga2-Tg, JAK2 V617F-Tg or double-Tg cells with 0.75 x 106 Ly5.1+ competitor WT cells to lethally irradiated Ly5.1+ WT mice. Proportions of Ly5.2+ cells were higher in recipients of Δ Hmga2 -Tg than double-Tg cells, while JAK2 V617F-Tg cells were almost rejected at 8 weeks after BMT. To confirm role of HMGA2 without let-7 repression in JAK2 V617F+ hematopoiesis, we performed another BMT with 1 x 104 KIT+ cells of JAK2 V617F-Tg mice transduced with retroviral vector of Hmga2 with each let-7 -site-mutated full-length 3'UTR (Hmga2-m7) to sublethally irradiated WT mice. Recipients of JAK2 V617F-Tg cells with Hmga2-m7 developed MPN-like disease, whereas donor cells were rejected in recipients of JAK2 V617F cells with empty vector. In conclusion, HMGA2 may play a crucial role in hematopoiesis harboring JAK2 V617F by expanding HSPC, leading to disease progression. DisclosuresNo relevant conflicts of interest to declare.

Abstract A38: Remodeling of the malignant bone marrow niche represents a therapeutic target.

Abstract Myeloproliferative neoplasms (MPNs) are malignant diseases of the hematopoietic stem cell (HSC). Targeting driver mutations in these diseases has had variable success. In chronic myelogenous leukemia (CML), targeting the BCR/ABL pathway has completely changed the way these patients are treated; however, there still remain a significant proportion of patients who have no targetable mutation for therapy and treatments centered on non-cell autonomous pathways of disease progression may have significant value. Previously, our lab showed that malignant myeloid cells from a mouse model of CML overexpressing BCR/ABL (BA mice) expand a remodeled mesenchymal stem cell (MSC) derived osteoblast population which leads to a fibrotic bone marrow (BM) niche and preserves malignant hematopoiesis (Schepers et al. Cell Stem Cell 2013; 13: 285-299). Disrupting the establishment of this fibrotic niche hopefully could delay clinical MPN progression and restore normal hematopoiesis. Bisphosphonates are known to act locally in the BM microenvironment on many cell types and improve the survival of patients with BM-involved disease, including multiple myeloma and breast cancer (Modi and Lentzsch. Leukemia 2012; 26:589-594). The focus of our current studies is to test the effect of zoledronic acid (ZA), a widely used bisphosphonate, on in vitro MSC fibrotic colony formation induced by malignant CML cells. Using whole BM from BA or control mice co-cultured with wild type MSCs, we found that ZA potently inhibited the increase in MSC colony formation induced by CML cells. The significant four-fold increase in MSC colony size (measured by cell number per colony) in BA co-cultures was completely negated by ZA at concentrations as low as 500 nM. The BA co-cultures were exquisitely sensitive to ZA and no breakthrough colonies were seen as compared to a 65 percent reduction in colony size in control co-cultures with 500 nM ZA. Our results suggest that zoledronic acid could be an interesting therapeutic compound to target the fibrotic BM niche in MPNs. Ongoing studies seek to understand the mechanism of action of ZA and to test its efficacy in preventing fibrosis and restoring a healthy BM niche in the BA model of CML. The goal is also to expand these results to other, more strongly fibrotic, mouse models of MPNs and human MPN samples. Citation Format: Timothy B. Campbell, Emmanuelle Passegué. Remodeling of the malignant bone marrow niche represents a therapeutic target. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr A38.

Microarray-based genomic profiling and in situ hybridization on fibrotic bone marrow biopsies for the identification of numerical chromosomal abnormalities in myelodysplastic syndrome.

BackgroundMyelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematological malignancies. In MDS patients with a fibrotic bone marrow the aspiration of cells often fails (dry-tap), which hampers standard karyotyping. Obtaining genetic data from these fibrotic marrows is therefore challenging, and up till now in situ hybridization applied to bone marrow biopsies is the only option. The microarray-based genomic profiling technology has already proven its value for bone marrow aspirates and peripheral blood samples, but has never been applied to the technically challenging bone marrow biopsies. We describe an approach for microarray-based genomic profiling on bone marrow biopsies and demonstrate its ability to obtain clinically relevant cytogenetic aberrations. In addition the data were compared with those obtained by in situ hybridization and karyotyping.ResultsWe have evaluated the success rate of microarray-based genomic profiling by studying twenty-one bone marrow biopsies (7 fibrotic MDS, 12 non-fibrotic MDS and 2 reactive), by microarray-based genomic profiling and in situ hybridization (12 of 21 cases). The data obtained with these techniques were compared with conventional karyotyping data on corresponding bone marrow aspirates. Of the 15 copy number aberrations that were detected by in situ hybridization, 13 were concordant with microarray-based genomic profiling and karyotyping, whereas two hybridizations were misinterpreted. In 20 of 21 patients, the data obtained by microarray-based genomic profiling and karyotyping were identical or differences could be explained by the presence of marker chromosomes, complex karyotypes, clonal heterogeneity or disease progression.ConclusionsWe demonstrate that genome wide microarray-based genomic profiling performed on bone marrow biopsies has a similar success rate compared to in situ hybridization, and prevents misinterpretation of chromosomal losses as observed by FISH. In addition, equal to even higher resolutions were obtained with genomic profiling compared to conventional karyotyping. Our findings indicate that microarray-based profiling, even on bone marrow biopsies, is a valid approach for the identification of genetic abnormalities. This is a valuable substitution in cases of fibrotic MDS lacking cytogenetic results.

Expression of JAK2V617I Does Not Result in Overt Myeloproliferative Disorder, but Results in Cytokine Hypersensitivity

BACKGROUNDA germline JAK2V617I mutation has recently been identified in a family with hereditary thrombocytosis (Mead et al, NEJM 2012). Like acquired MPN, family members with JAK2V617I have thrombocytosis and megakaryocytic hyperplasia in the marrow with increased risk of thrombosis. But unlike acquired MPN, individuals with this germline mutation do not develop a fibrotic bone marrow, splenomegaly, or transform to acute leukemia. Why germline JAK2V617I recapitulates some aspects but not others of the MPN phenotype in humans is unclear. To delineate the differences between JAK2V617F and JAK2V617I we compared the phenotype of mice with hematopoietic cells expressing JAK2V617F or JAK2V617I. METHODS AND RESULTSLethally irradiated C57B/6 mice were transplanted with bone marrow cells infected with retrovirus expressing JAK2V617F, JAK2V617I, or empty MSCV-IRES-GFP (MIG) vector. As expected, mice transplanted with JAK2V617F-expressing cells developed erythrocytosis and leukocytosis, whereas mice transplanted with JAK2V617I-expressing cells had peripheral blood counts similar to empty vector mice. Humans with germline JAK2V617I do not display constitutive activation of the kinase, but they do demonstrate cytokine hyper-responsiveness as evidenced by increased phosphorylation of STATs at low concentrations of ligand. We compared phosphorylated STAT5 in peripheral blood of mice transplanted with JAK2V617I, JAK2V617F, and MIG empty vector following stimulation with increasing concentrations of GM-CSF. At all concentrations of GM-CSF tested JAK2V617I and JAK2V617F-expressing cells had exaggerated phosphorylation of STAT5 as compared to MIG empty vector mice. We also measured phospho-STAT3 and STAT5 in unstimulated bone marrow and spleen from each mouse at time of euthanasia, there was no difference between JAK2V617I and MIG empty vector mice. JAK2V617F mice did demonstrate phosphorylation of STAT3 and STAT5 in the absence of GM-CSF, confirming the ability of JAK2V617F but not JAK2V617I to constitutively activate downstream signaling pathways. Next, to evaluate for histologic evidence of MPN and assess spleen size, all mice were euthanized at 120 days post-transplant. JAK2V617F mice had splenomegaly as expected, spleens from JAK2V617I mice appeared larger than empty vector mice, but spleen weight was not statistically different (p>0.05). While JAKV617I mice had increased cellularity of their marrow with increased numbers of megakaryocytes as compared to empty vector mice, this was not nearly to the extent of JAK2V617F mice. Mild fibrosis was seen in JAK2V617I mice, JAK2V617F mice had severe reticulin fibrosis in the marrow as expected. In the spleen architecture was preserved in the JAK2V617I mice, whereas in the JAK2V617F mice splenic architecture was disrupted by invasion of myeloid cells including megakaryocytes.To identify whether JAK2V617I affects the frequency of stem and progenitor cells or expands mature myeloid lineage cells we measured the frequency of hematopoietic stem cells, myeloid progenitors, and mature myeloid populations in JAK2V617F, JAK2V617I, and MIG empty vector. The bone marrow of JAK2V617I mice contained an increased percentage of GMP and MEP populations as compared to both the MIG empty vector and the JAK2V617F mice. Mature granulocyte (Gr-1+CD11b+) and erythroid (Ter119+) populations were expanded in the bone marrow and spleen of JAK2V617F but not JAK2V617I mice. CONCLUSIONSThese data demonstrate that the JAK2V617I mouse model recapitulates the effect of germline expression of JAK2V617I seen in humans: it results in cytokine hyper-responsiveness without the ability to constitutively activate downstream signals in the absence of ligand. Why JAK2V617F is so exquisitely conserved in acquired MPN is still unknown. DisclosuresNo relevant conflicts of interest to declare.

Enhanced Expression of Stim, Orai, and TRPC Transcripts and Proteins in Endothelial Progenitor Cells Isolated from Patients with Primary Myelofibrosis

BackgroundAn increase in the frequency of circulating endothelial colony forming cells (ECFCs), the only subset of endothelial progenitor cells (EPCs) truly belonging to the endothelial phenotype, occurs in patients affected by primary myelofibrosis (PMF). Herein, they might contribute to the enhanced neovascularisation of fibrotic bone marrow and spleen. Store-operated Ca2+ entry (SOCE) activated by the depletion of the inositol-1,4,5-trisphosphate (InsP3)-sensitive Ca2+ store drives proliferation in ECFCs isolated from both healthy donors (N-ECFCs) and subjects suffering from renal cellular carcinoma (RCC-ECFCs). SOCE is up-regulated in RCC-ECFCs due to the over-expression of its underlying molecular components, namely Stim1, Orai1, and TRPC1.Methodology/Principal FindingsWe utilized Ca2+ imaging, real-time polymerase chain reaction, western blot analysis and functional assays to evaluate molecular structure and the functional role of SOCE in ECFCs derived from PMF patients (PMF-ECFCs). SOCE, induced by either pharmacological (i.e. cyclopiazonic acid or CPA) or physiological (i.e. ATP) stimulation, was significantly higher in PMF-ECFCs. ATP-induced SOCE was inhibited upon blockade of the phospholipase C/InsP3 signalling pathway with U73111 and 2-APB. The higher amplitude of SOCE was associated to the over-expression of the transcripts encoding for Stim2, Orai2–3, and TRPC1. Conversely, immunoblotting revealed that Stim2 levels remained constant as compared to N-ECFCs, while Stim1, Orai1, Orai3, TRPC1 and TRPC4 proteins were over-expressed in PMF-ECFCs. ATP-induced SOCE was inhibited by BTP-2 and low micromolar La3+ and Gd3+, while CPA-elicited SOCE was insensitive to Gd3+. Finally, BTP-2 and La3+ weakly blocked PMF-ECFC proliferation, while Gd3+ was ineffective.ConclusionsTwo distinct signalling pathways mediate SOCE in PMF-ECFCs; one is activated by passive store depletion and is Gd3+-resistant, while the other one is regulated by the InsP3-sensitive Ca2+ pool and is inhibited by Gd3+. Unlike N- and RCC-ECFCs, the InsP3-dependent SOCE does not drive PMF-ECFC proliferation.

Challenges in bone marrow pathology

Bone marrow diagnosis presents unique challenges: distinction between benign and neoplastic conditions may be subtle and the pathologist must effectively incorporate information from several morphologic modalities (trephine biopsy, aspirate smear, peripheral blood smear) as well as a myriad of ancillary testing results. In this Short Course, several difficult diagnostic scenarios will be explored using actual clinical cases that illustrate key points in the differential diagnosis of bone marrow diseases. These will include hypocellular bone marrow (in which distinction between hypoplastic myelodysplastic syndrome and aplastic anemia may be difficult), fibrotic bone marrow, and marrow lymphoid infiltrates, with discussion of both B-cell and T-cell lymphomas. Hypercellular bone marrow raises distinct differential diagnoses depending on whether the patient is cytopenic or presents with elevated counts, and can reflect a myeloid neoplasm or various reactive conditions in either situation. An accurate diagnosis relies on understanding the spectrum of diseases that can produce specific patterns in the bone marrow and awareness of clues that help distinguish among the differential diagnostic possibilities. Depending on the clinical context, the pathology may variably weigh morphology, clinical information, and ancillary test results in arriving at the final diagnosis.