LOXL2 Highly-Expressed Induce the Transition of Stromal Cells into Cancer-Associated Fibroblasts Which Maybe Involve in Myeloproliferative Neoplasms Progession

Abstract Backgroud and objective: Myeloproliferative neoplasms (MPNs) are malignant disorders by proliferation of one of the myeloid lineages and characteristically show an increase in bone marrow reticulin reticulin-fibrosis.Lysyl oxidases like-2(LOXL2) is copper-dependent amine oxidases that play a critical role in the biogenesis of connective tissue by crosslinking extracellular matrix proteins, collagen and elastin,and Cancer associated-fibroblasts (CAFs) are major mediators in tumor microenvironment. Studies found that loxl2 stimulate CAFs grouth solid tumor,and the expression of LOXL2 is increased in MPN patients,espessionly in PMF patients.Here, we want to evaluate whether the expression of higher LOXL2 associated to CAFs during various MPN progression. Patients and methods: We compared normal bone marrows and those from patients with chronic myeloid leukemia(CML)(include CML-CP n=20,CML-BC n=13),polycythemia vera(PV)(n=18), essential thrombocythemia(ET) (n=23), and primary myelofibrosis (PMF) (n=8). We detected α-smooth actin and reticulin protein by immunohistochemical staining, examined LOXL2 expression by western blot in bone marrow and ELIZA kit in serum. Results: LOXL2 was not detected in normal bone marrows and serum.The level of LOXL2 gene is over expressed in PMF (p<0.01) and CML-BC (p=0.02). In other MPNs a differential pattern of expression were statistically significant (P< 0.010).The level of LOXL2 expression associated with reticulin protein expression in bone marrow, especially if reticulin protein expressed higher than 2+(p=0.01). We detected α-smooth actin positive stromal cells in CML-BC and PMF patients,and the level of LOXL2 expression is related to α-smooth actin positive stromal cells(p<0.05).we also detected α-smooth actin after co-cultured mesenchymal stem cell(MSCs) with sLOXL2 for 96 hour. Conclusion: Higher level of LOXL2 could be promote MPN progression by modulating several functions of surrounding stromal cells which acquire features of cancer-associated fibroblasts involved in the pathogenesis of MPN. These findings maybe used as the basis for future targeted therapy directed against MPN progression. Disclosures No relevant conflicts of interest to declare.

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Bone Marrow Stromal Cell Remodeling Is a Common Feature of Diverse Fibrotic Myeloproliferative Neoplasm Models

Blood ◽

10.1182/blood.v128.22.25.25 ◽

2016 ◽

Vol 128 (22) ◽

pp. 25-25

Author(s):

Timothy B Campbell ◽

Si Yi Zhang ◽

Alexander Valencia ◽

Emmanuelle Passegue

Keyword(s):

Bone Marrow ◽

Stromal Cells ◽

Stromal Cell ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Critical Role ◽

Myeloproliferative Neoplasm ◽

Myelogenous Leukemia ◽

Hematopoietic Stem ◽

Cell Numbers

Abstract Myeloproliferative neoplasms (MPN) are blood cancers initiated by driver mutations that transform hematopoietic stem cells. MPN exhibit gross pathologic bone marrow (BM) stromal remodeling, including damaging myelofibrotic change that leads to dependence on extramedullary hematopoiesis and more severe clinical diseases. Therapies targeting fibrotic change would have broad appeal in the treatment of these diseases. We previously demonstrated a critical role for malignant myeloid cells in remodeling endosteal mesenchymal stromal cells (MSC) into myelofibrotic osteoblast-lineage cells (OBC) in a model of chronic myelogenous leukemia (CML) driven by BCR/ABL (Schepers et al., Cell Stem Cell, 2013). In a separate study in a fibrotic MPN model driven by Jak2V617F, neuropathy and nestin-positive MSC cell death were found critical to disease progression but their involvement in myelofibrosis was not investigated (Arranz et al. Nature. 2014). Our goal is to characterize the type of BM stromal remodeling occurring in non-CML MPN models driven by various mutations and representing a spectrum of disease severity and fibrosis. This includes a minimally fibrotic transgenic Jak2V617F alone model (Jak2V617F model, Xing et al., Blood, 2008) and more advanced fibrotic models driven by MPLW515L expression (MPLW515L model, Pikman et al., PLoS Med, 2006) or combined transgenic Jak2V617F expression with conditional deletion of the polycomb gene EZH2 (Jak2V617F/EZH2-/- model, Sashida et al., JEM, 2016). We found common blood and BM hematopoietic changes in all three models, including thrombocytosis and expansion of myeloid-biased multipotent progenitor BM cells and confirmed the degree of fibrosis using picrosirius red staining of bone sections. Both MPLW515L and Jak2V617F/EZH2-/- heavily fibrotic models demonstrated inhibition of total endosteal MSC, OBC and endothelial cell (EC) numbers during disease development - in most cohorts a greater than 50% decrease in absolute stromal cell numbers was found. In addition, we observed that whole BM cells from Jak2V617F/EZH2-/-mice contained a significantly lower number of totalfibroblast colony forming cells (CFU-F). In co-culture experiments designed to measure direct MSC remodeling induced by malignant cells, both MPLW515L and Jak2V617F/EZH2-/- BM cells inhibited healthy endosteal MSC colony formation over time. In contrast, we found no inhibition of stromal cell numbers or co-culture MSC growth in the minimal fibrotic Jak2V617F model. In initial experiments measuring rare central marrow perivascular MSC, we found reduced LepR+ MSC (Ding et al., Nature, 2012) in both MPLW515L and Jak2V617F/EZH2-/- long bone sections using immunofluorescence. Our results show that fibrotic development in non-CML MPN inhibits stromal cell numbers and function likely via direct effects of malignant hematopoietic cells. This is in contrast to fibrotic CML development where myelofibrotic endosteal stromal cells are expanded. This difference could be partly explained by the type and localization of fibrosis in these various models. The CML model has focal endosteal collagen-I fibrosis which is heavily reliant on osteoblast remodeling, while the MPLW515L and Jak2V617F/EZH2-/- models have more diffuse reticulin central marrow fibrosis which may be produced through a process of stromal cell senescence or differentiation. Overall, this study underscores that a “one size fits all“ approach to understanding myelofibrosis is insufficient. To tease out these differences, we are examining qualitative and quantitative changes in additional central marrow MSC populations, including PDGFR+, Sca-1+ and Gli-1+ MSC, during MPN development as well as assaying the molecular mediators of stromal remodeling. Our long-term goal is to identify therapies that can restore a more normal BM stroma and support healthy hematopoiesis in MPN. Disclosures No relevant conflicts of interest to declare.

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TGF-Beta Signaling in Mesenchymal Stromal Cells Contributes to Myelofibrosis but Not Hematopoietic Phenotypes in Myeloproliferative Neoplasms

Blood ◽

10.1182/blood-2018-99-113745 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3053-3053

Author(s):

Juo-Chin Yao ◽

Grazia Abou Ezzi ◽

Joseph R. Krambs ◽

Eric J. Duncavage ◽

Daniel C. Link

Keyword(s):

Bone Marrow ◽

Growth Factor ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Transforming Growth Factor ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Hematopoietic Cells ◽

Dismal Prognosis ◽

Collagen Iii

Abstract The development of myelofibrosis in patients with myeloproliferative neoplasms (MPNs) is associated with a dismal prognosis. The mechanisms responsible for the progression to myelofibrosis are unclear, limiting the development of therapies to treat or prevent it. The cell of origin responsible for the increased collagen deposition is controversial, with recent studies implicating Gli1+ or leptin receptor+ mesenchymal stromal cells, monocytes, or even endothelial cells. Moreover, the signals generated by malignant hematopoietic cells in MPN that induce increased collagen expression are uncertain. There is some evidence that elevated expression of cytokines/chemokines in the bone marrow microenvironment of patients with MPN may contribute. In particular, recent studies have implicated transforming growth factor-β (TGF-β), platelet-derived growth factor and CXCL4 in the development of myelofibrosis. Here, we test the specific hypothesis that TGF-β signaling in mesenchymal stromal cells is required for the development of myelofibrosis. Moreover, we hypothesize that TGF-β signaling, by altering the expression of key niche factors by mesenchymal stromal cells, contributes to the myeloid expansion in MPN. To test this hypothesis, we abrogated TGF-β signaling in mesenchymal stem/progenitor cells (MSPCs) by deleting Tgfbr2 using a doxycycline-repressible Sp7 (osterix)-Cre transgene (Osx-Cre), which targets all mesenchymal stromal cells in the bone marrow, including CXCL12-abundant reticular (CAR) cells, osteoblasts, adipocytes, or arteriolar pericytes. We previously showed that TGF-β signaling plays a key role in the lineage specification of MSPCs during development (2017 ASH abstract #2438). In contrast, we show that post-natal deletion of Tgfbr2, by removing doxycycline at birth, is not associated with significant changes in mesenchymal stromal cells in the bone marrow. Moreover, expression of key niche factors, including Cxcl12 and stem cell factor, and basal hematopoiesis were normal in these mice. Thus, we used the post-natal Osx-Cre; Tgfbr2-deleted mice as recipients to assess the role of TGF-β signaling in mesenchymal stromal cells on the hematopoietic and myelofibrosis phenotype in Jak2V617For MPLW515Lmodels of MPN. Specifically, we transplanted hematopoietic cells from Mx1-Cre; Jak2V617Fmice (4 weeks after pIpC treatment) or hematopoietic cells transduced with MPLW515Lretrovirus into irradiated wildtype or post-natal Osx-Cre; Tgfbr2-deleted mice. Both MPN models have elevated Tgfb1 expression in the bone marrow. As reported previously, transplantation of MPLW515Ltransduced hematopoietic cells into wildtype recipients produced a rapidly fatal MPN characterized by neutrophilia, erythrocytosis, thrombocytosis, splenomegaly, and reticulin fibrosis in the bone marrow. A similar hematopoietic phenotype was observed in Osx-Cre; Tgfbr2fl/flrecipients. However, a trend to decreased reticulin fibrosis was observed in Osx-Cre; Tgfbr2fl/flcompared to wildtype recipients (reticulin histology score: 0.5 versus 1.1, respectively, n=5, p=0.23). Likewise, the degree of neutrophilia, erythrocytosis, thrombocytosis, and splenomegaly in wildtype and Osx-Cre; Tgfbr2fl/flrecipients of Jak2V617Fcells was similar. As reported previously, we did not observe overt myelofibrosis in this model (as measured by reticulin staining). However, we were able to detect increased collagen III deposition using immunofluorescence staining in 4 of 5 wildtype recipients compared to 1 of 4 Osx-Cre Tgfbr2fl/flrecipients of Jak2V617Fcells (p=0.21). In conclusion, our data suggest that TGF-β signaling in mesenchymal stromal cells contributes, but is not absolutely required, for the development of myelofibrosis. Alterations in mesenchymal stromal cells induced by increased TGF-β signaling do not appear to be a major driver of the myeloid expansion in MPN. The contribution of increased TGF-β signaling in hematopoietic cells or other bone marrow stromal cell populations to the MPN phenotype is under investigation. Disclosures No relevant conflicts of interest to declare.

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Endogenous TGF-beta1 Mediated Osteogenic Impairment of Medullar Mesenchymal Stromal Cells in Primary Myelofibrosis

Blood ◽

10.1182/blood.v124.21.1873.1873 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1873-1873

Author(s):

Christophe Martinaud ◽

Christophe Desterke ◽

Johanna Konopacki ◽

Lisa Pieri ◽

Rachel Golub ◽

...

Keyword(s):

Bone Marrow ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasm ◽

Hematopoietic Cells ◽

Primary Myelofibrosis ◽

Osteogenic Potential ◽

Healthy Donors

Abstract Primary myelofibrosis (PMF) is myeloproliferative neoplasm characterized by clonal myeloproliferation, dysmegakaryopoiesis, extramedullary hematopoiesis associated with myelofibrosis and altered stroma in bone marrow and spleen. Mesenchymal stromal cells (MSCs) are reported to play a pivotal role in fibrosis and stromal changes are considered as a reactive counterpart of the cytokine production by clonal hematopoietic cells. The present study shows that MSCs from patients demonstrate functional abnormalities that are unexpectedly maintained ex-vivo, in culture. Material and Methods: we studied MSCs and bone marrow sections from PMF patients (n=12) as compared to healthy donors (HDs) (n=6). We tested their proliferation, immunophenotype, hematopoiesis supporting capacities, differentiation abilities, in-vivo osteogenic assays, and performed secretome and transcriptome analysis. Results: We found that PMF-MSCs exhibit similar proliferative capacity and long-term hematopoiesis supporting abilities as compare to healthy donors. They overproduce interleukin 6, VEGF, RANTES, PDGF, BMP-2 and surprisingly TGF-beta1. MSCs from fibrotic PMF patients express high levels of glycosaminoglycans. Adipocytes and chondrocytes differentiation abilities were not different as compared to HDs but PMF-MSCs exhibit an increased in vitro potential. Implementation on scaffold in nude mice confirmed, in vivo, this increased osteogenic potential. We then looked into gene expression and discovered that PMF-MSCs show an original transcriptome signature related to osteogenic lineage and TGF-beta1. Indeed, osteogenic genes such as Runx2, Dlx5, Twist1, Noggin, Sclerostin, GDF5 and Serpine1 are deregulated and suggest a potential osteoprogenitor priming of PMF-MSCs. These molecular results also advocated for a TGF-beta1 impregnation that prompted us to study its impact on PMF-MSCs osteogenic differentiation. First, we then showed that Smad2 is intrinsically over-activated in PMF-MSC and that stimulation by TGF-beta1 is associated with an increase phospho-Smad2 level and an enhancement of bone master gene regulator Runx2 expression. Then, we inhibited TGF-beta1 pathway by by SB-431542 and evidenced a specific behavior of osteogenic MSCs differentiation in patients, suggesting involvement of TGF-beta1 in osteogenic impairment. Conclusion: Altogether, our results identify a signature of PMF-MSCs and suggest that they participate in PMF osteogenic dysregulation independently from in vivo local stimulation by clonal hematopoietic cells Disclosures No relevant conflicts of interest to declare.

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TP53 Mutation Is Rare In Primary Myelofibrosis

Blood ◽

10.1182/blood.v118.21.5241.5241 ◽

2011 ◽

Vol 118 (21) ◽

pp. 5241-5241

Author(s):

Wesley O. Greaves ◽

Shalini Verma ◽

Tigist Bisrat ◽

Hamed Rahimi ◽

Abhaya Paladugu ◽

...

Keyword(s):

Bone Marrow ◽

Acute Leukemia ◽

Sanger Sequencing ◽

Tp53 Mutation ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Melting Curve ◽

Primary Myelofibrosis ◽

Hrm Analysis ◽

Exon 4

Abstract Abstract 5241 Introduction TP53 is the most frequently mutated tumor suppressor gene in human cancers and is usually associated with an aggressive disease course. TP53 mutation has been described in a variety of hematopoietic neoplasms, and has been suggested to play a role in leukemic transformation of myeloproliferative neoplasms (MPN). However, the incidence as well as the clinical and pathogenetic implications of TP53 mutation in each sub-category of MPN, including primary myelofibrosis, have not been described. In this study, we investigated the presence and potential clinical significance of TP53 mutations in a large series of primary myelofibrosis cases. Patients and Methods We retrieved archival bone marrow DNA from 51 consecutive patients diagnosed with primary myelofibrosis at The University of Texas MD Anderson Cancer Center between the years 2005 and 2007. Diagnosis was based on morphologic, immunophenotypic, cytogenetic and molecular evaluation of bone marrow in conjunction with clinical data. Only 2 patients had blast counts >10% (11 and 14%). Twenty nine of 50 (58%) patients showed JAK2 p.V617F mutation and all patients were negative for BCR-ABL1 translocation on routine clinical testing. DNA samples were assessed for sequence variation in exons 4 through 9 of TP53 by both high resolution melting curve (HRM) analysis using LightCycler® 480 System (Roche, Indiana IN) and bidirectional Sanger sequencing using 3730XL DNA Analyzer (Life technologies, Carlsbad CA). Results The mean overall survival was 5.7 years. Five patients developed acute leukemia, all of whom died of disease. By Sanger sequencing, only one (1.9%) case showed an amino acid-altering mutation in TP53: c.707A>G (TAC to TGC) in codon 236 (p.Y236C) of exon 7. In addition, 8 cases showed silent mutations/single nucleotide polymorphisms of unknown significance - c.36G>A (CCG to CCA) in exon 4 (n=3) and c.213A>G (CGA to CGG) in exon 6 (n=6). The p.R72P polymorphism in exon 4 which has been described in other hematopoietic neoplasms was present in 1 patient. All cases with a mutant sequence by Sanger sequencing also showed a variant melting curve pattern by HRM analysis. The patient with TP53 mutation died 2 years after presentation from progressive myelofibrosis without developing acute leukemia. Conclusion TP53 mutation is very rare in primary myelofibrosis. Disclosures: No relevant conflicts of interest to declare.

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Macrophage frequency in the bone marrow correlates with morphologic subtype of myeloproliferative neoplasm

Annals of Hematology ◽

10.1007/s00277-020-04304-y ◽

2020 ◽

Vol 100 (1) ◽

pp. 97-104

Author(s):

David C. A. Molitor ◽

Peter Boor ◽

Andreas Buness ◽

Rebekka K. Schneider ◽

Lino L. Teichmann ◽

...

Keyword(s):

Bone Marrow ◽

Stromal Cells ◽

Myeloid Leukemia ◽

Clinical Course ◽

Myeloproliferative Neoplasms ◽

Philadelphia Chromosome ◽

Myeloproliferative Neoplasm ◽

Primary Myelofibrosis ◽

Neoplastic Processes ◽

And Control

AbstractBone marrow (BM) fibrosis in myeloproliferative neoplasms (MPNs) is associated with a poor prognosis. The development of myelofibrosis and differentiation of mesenchymal stromal cells to profibrotic myofibroblasts depends on macrophages. Here, we compared macrophage frequencies in BM biopsies of MPN patients and controls (patients with non-neoplastic processes), including primary myelofibrosis (PMF, n = 18), essential thrombocythemia (ET, n = 14), polycythemia vera (PV, n = 12), and Philadelphia chromosome–positive chronic myeloid leukemia (CML, n = 9). In PMF, CD68-positive macrophages were greatly increased compared to CML (p = 0.017) and control BM (p < 0.001). Similar findings were observed by CD163 staining (PMF vs. CML: p = 0.017; PMF vs. control: p < 0.001). Moreover, CD68-positive macrophages were increased in PV compared with ET (p = 0.009) and reactive cases (p < 0.001). PMF had higher frequencies of macrophages than PV (CD68: p < 0.001; CD163: p < 0.001) and ET (CD68: p < 0.001; CD163: p < 0.001). CD163 and CD68 were often co-expressed in macrophages with stellate morphology in Philadelphia chromosome–negative MPN, resulting in a sponge-like reticular network that may be a key regulator of unbalanced hematopoiesis in the BM space and may explain differences in cellularity and clinical course.

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Constitutive Heterozygous Expression of JAK2V617F Mutated Kinase Triggers Severe Myeloproliferative Disorder in Knock-in Mice.

Blood ◽

10.1182/blood.v114.22.2902.2902 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2902-2902

Author(s):

Caroline Marty ◽

Catherine Lacout ◽

Linda Fong ◽

Antoine Martin ◽

William Vainchenker ◽

...

Keyword(s):

Bone Marrow ◽

Germ Line ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Disease Onset ◽

Primary Myelofibrosis ◽

Selective Advantage ◽

Advanced Degree ◽

Major Drawback ◽

Hydroxyurea Treatment

Abstract Abstract 2902 Poster Board II-878 Myeloproliferative neoplasms (MPNs) are frequent malignant hematopoietic pathologies. The acquired Jak2V617F mutation is found in the majority of polycythemia vera (PV), and in approximately half of essential thrombocytosis (ET) and primary myelofibrosis (PMF). Animal models using transgenic or retroviral bone marrow transplant approaches allowing for JAK2V617F expression in hematopoietic cells have demonstrated that this sole mutation is sufficient to induce a MPN in mice. They have also shown that the level of JAK2V617F expression is crucial for MPN phenotypes. Therefore, these over-expression models appeared not suited to study human MPNs. In order to circumvent this problem, we developed a Jak2V617F constitutive and a conditional Knock-in (KI) mouse models. Jak2V617F allele was introduced into the endogenous Jak2 locus, allowing physiological expression of the mutated kinase. The Jak2V617F heterozygocity status was confirmed by allele-specific fluorescent competitive probes in quantitative real-time polymerase chain reaction. Constitutively activated JAKV617F kinase activity was assessed by analyzing STAT5a phosphorylation level in bone marrow and spleen cells by Western blotting. Expression of endogenous JAK2V617F in constitutive KI mice induced a severe PV- to PMF-like phenotype disease, at 5 months of age, characterized in blood by marked polycythemia (Hct 71% ± 3.6%), granulocytosis (WBC counts 79 ± 11 × 106 cells/mL) and thrombocytosis (4.4 ± 0.7 × 109 platelets/mL). Mice displayed enlarged spleens (1.6 ± 0.3 g compared to 0.12 ± 0.01 g for WT mice). Histological examinations of the spleens and femurs revealed advanced degree of fibrosis and trilineage hyperplasia. Flow cytometry analysis showed high levels of TER-119-positive (erythroid) cells in bone marrow and spleen and a marked decrease in lymphocyte percents. The number of CFU-E increased in spleen and included an extremely high percentage of endogenous erythroid colonies (EECs). EECs were also detected in bone marrow. The numbers of BFU-E and CFU-GM were only slightly increased in the spleen but there were no significantly changed in the bone marrow. A major drawback of this constitutive and ubiquitous KI mouse model resides in the inability of female to reproduce even after recovery of normal blood parameters induced by hydroxyurea treatment. Progeny was only obtained through KI male breeding. Embryonic expression of endogenous JAK2V617F was also obtained by crossing Jak2V617F conditional KI mice with CMV-Cre transgenic mice. The offspring presented a similar phenotype than the constitutive KI mice. We performed serial competitive bone marrow (BM) transplantations (BMTs) in irradiated WT recipient mice using from 10% to 100% CMV-Cre Jak2V617F KI mouse BM cells diluted with WT mouse BM cells. Time of disease onset and survival were shorter in mice grafted with high compared to low percentages of KI-derived cells. Reduced occurrences of disease were observed along with successive BMTs or limiting dilution of Jak2V617F-positive clone, suggesting that Jak2V617F provides little or no selective advantage to HSC. This study demonstrates that embryonic endogenous JAK2V617F heterozygous expression results in a severe lethal MPN. This result may explain why no germ line transmission of the Jak2V617F-positive human disease has been reported. Disclosures: No relevant conflicts of interest to declare.

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Bone Marrow Biopsy Revision According to WHO Criteria in 272 Patients of the Registro Italiano Trombocitemia (RIT): Preliminary Report On Clinical and Histopathological Implications.

Blood ◽

10.1182/blood.v114.22.4974.4974 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4974-4974

Author(s):

Luigi Gugliotta ◽

Stefano Ascani ◽

Silvia Asioli ◽

Emanuela Boveri ◽

G. Fraternali Orcioni ◽

...

Keyword(s):

Bone Marrow ◽

Preliminary Report ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Primary Myelofibrosis ◽

Treatment Rate ◽

Who Criteria ◽

Bone Marrow Trephine Biopsy ◽

Hemorrhagic Events ◽

Wbc Count

Abstract Abstract 4974 Background The bone marrow trephine biopsy (BMB) has a crucial role for the diagnosis of essential thrombocythemia (ET), both according to the PVSG and the WHO criteria. The WHO 2001 criteria enhanced the role of BMB also by distinguishing the true-ET (ET) from the prefibrotic and the early fibrotic chronic idiopathic myelofibrosis. The WHO 2008 criteria, in the JAK2 era, confirmed the diagnostic and prognostic relevance of the histopathological features in ET as well as in the other Ph-neg myeloproliferative neoplasms (MPN). Otherwise, only few validated data are presently available, and the reproducibility in the evaluation of some morphological details is still controversy. Objective To validate ET diagnosis in a large registry-based series of patients by revising the BMB specimens according to the WHO criteria and to evaluate the potential relationship between the histopathological and the clinical parameters at presentation. Methods The hematological centers of the Registro Italiano Trombocitemia (RIT) were invited to participate to this study by sending the BMB specimens (hematoxylin-eosin, Giemsa and silver stains) obtained at diagnosis. The clinic-pathological panel of the RIT (with three hematopathologists as permanent members coordinated by a chairman) performed a centralized revision of the BMB specimens, concomitantly (multiheaded microscopy) and blindly (only patient sex and age were known). The panel described for each case the morphological features and gave a diagnostic conclusion according to WHO criteria as follows: true-ET (ET); or initial primary myelofibrosis (i-PMF) distinguished in prefibrotic PMF (pf-PMF/MF-0) and early fibrotic PMF (ef-PMF/MF-1); or advanced PMF (MF-2 and MF-3); or early polycythemia vera (e-PV); or MPN unclassifiable (MPN-U); or diagnosis other than MPN (No MPN). Results Thirteen centers sent the specimen of BMB at diagnosis of 272 patients registered into the RIT and diagnosed in the years 1986-2002 (group A, cases 66, 24.3%), 2003-2005 (group B, cases 95, 34.9%) and 2006-2008 (group C, cases 111, 40.8%). The patients, 104 (38%) males and 168 (62%) females, had at diagnosis: age over 50 in 64% of cases (median 58); PLT count (109/L) >1000 in16% and <= 600 in 24% of cases (median 789); Hgb level (g/dL) >17 in 1% and <=9 in 5% of cases (median 14.3); WBC count (109/L) >9 in 44% of cases (median 8.6); splenomegaly in 18% of cases. Disease symptoms, thrombotic and hemorrhagic events were reported in 40%, 21%, and 7% of cases, respectively. During the follow-up (median 2.7 years) 65% of patients received cytoreductive drugs (Hydroxyurea 59%, Anagrelide 20%, Interferon 14%, Pipobroman 6.5%, Busulfan 0.5%). The revision of the 272 BMB specimens allowed to the following diagnosis: ET 142 cases, 52.2%; i-PMF 72 cases, 26.5% (distinguished in pf-PMF 19 cases, 7% and ef-PMF 53 cases, 19.5%); e-PV 13 cases, 4.8%; MPN-U 16 cases, 14.3%; No MPN 8 cases, 2.9%. In the patients of groups A, B, and C the rate of ET was increasing (from 44%, to 50% and to 59%, respectively) while the rate of i-PMF was decreasing (from 34%, to 31% and to 18%, respectively); moreover, the rate of e-PV was 0%, 6%, and 6%, respectively. The ET patients compared with the i-PMF patients showed at diagnosis a lower rate of splenomegaly (16/142, 11.3% vs 24/72, 33.3%, p<0.0001), of age >50 years ( 84/142, 59.2% vs 53/72, 73.6%, p<0.03), and a higher rate of PLT count (109/L) <=600 (39/142, 27.5% vs 11/72, 15,3%, p<0.04 ); no significant differences were found between the two groups for sex, Hgb level, WBC count, symptoms, thrombotic and hemorrhagic events. The treatment rate was lower in ET than in i-PMF (ratio 0.7). Conclusion This preliminary report on the revision of the BMB at diagnosis in 272 patients of the Registro Italiano Trombocitemia (RIT) shows that: the rate of true ET is continously increasing (from 44% before 2003 to 59% in the period 2006-2008), with i-PMF rate concomitantly decreasing (from 34% to 18%); the ET patients, compared with the i-PMF ones, were younger, with lower PLT count and with lower rate of splenomegaly, and received a less intensive cytoreductive treatment. Disclosures No relevant conflicts of interest to declare.

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Cytokine Consistency Between Bone Marrow and Peripheral Blood in Patients With Philadelphia-Negative Myeloproliferative Neoplasms

Frontiers in Medicine ◽

10.3389/fmed.2021.598182 ◽

2021 ◽

Vol 8 ◽

Author(s):

Pu Chen ◽

Boting Wu ◽

Lili Ji ◽

Yanxia Zhan ◽

Feng Li ◽

...

Keyword(s):

Bone Marrow ◽

Inflammatory Cytokines ◽

Peripheral Blood ◽

Myeloproliferative Neoplasms ◽

Critical Role ◽

Primary Myelofibrosis ◽

Diagnostic Value ◽

Inflammatory Status ◽

Bm Niche ◽

Cytokine Levels

Background: Inflammation might play a critical role in the pathogenesis and progression of Philadelphia-negative myeloproliferative neoplasms (Ph−MPNs) with elevated inflammatory cytokines in peripheral blood (PB). However, the inflammatory status inside the bone marrow (BM), which is the place of malignancy origin and important microenvironment of neoplasm evolution, has not yet been elucidated.Methods: Inflammatory cytokine profiles in PB and BM of 24 Ph-MPNs patients were measured by a multiplex quantitative inflammation array. Cytokines that correlated between PB and BM were selected and then validated by ELISA in a separate cohort of 52 MPN patients. Furthermore, a panel of cytokines was identified and examined for potential application as non-invasive markers for the diagnosis and prediction of fibrosis progress of MPN subtypes.Results: The levels of G-CSF, I-309, IL-1β, IL-1ra, IL-12p40, IL-15, IL-16, M-CSF, MIG, PDGF-BB, and TIMP-1 in BM supernatants were significantly higher than those in PB (all p < 0.05). Linear correlations between BM and PB levels were found in 13 cytokines, including BLC, Eotaxin-2, I-309, sICAM-1, IL-15, M-CSF, MIP-1α, MIP-1δ, RANTES, TIMP-1, TIMP-2, sTNFRI, and sTNFRII (all R > 0.4 and p < 0.05). Levels of BLC, Eotaxin-2, M-CSF, and TIMP-1 in PB were significantly different from those in health controls (all p < 0.05). In PB, levels of TIMP-1 and Eotaxin-2 in essential thrombocythemia (ET) group were significantly lower than those in groups of prefibrotic primary myelofibrosis (pre-PMF) [TIMP-1: 685.2 (322.2–1,229) ng/ml vs. 1,369 (1,175–1,497) ng/ml, p = 0.0221; Eotaxin-2: 531.4 (317.9–756.6) pg/ml vs. 942.4 (699.3–1,474) pg/ml, p = 0.0393] and primary myelofibrosis (PMF) [TIMP-1: 685.2 (322.2–1229) ng/ml vs. 1,365 (1,115–1,681) ng/ml, p = 0.0043; Eotaxin-2: 531.4 (317.9–756.6) pg/ml vs. 1,010 (818–1,556) pg/ml, p = 0.0030]. The level of TIMP-1 in myelofibrosis (MF) >1 group was significantly higher than that in MF ≤ 1 group.Conclusion: Abnormal inflammatory status is present in MPN, especially in its BM microenvironment. Consistency between PB and BM levels was found in multiple inflammatory cytokines. Circulating cytokine levels of BLC, M-CSF, Eotaxin-2, and TIMP-1 reflected inflammation inside BM niche, suggesting potential diagnostic value for MPN subtypes and prognostic value for fibrosis progression.

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A Novel Immunohistochemical Sequential Multi-Labelling and Erasing Technique Enables Epitope Characterization of Bone Marrow Pericytes In Primary Myelofibrosis

Blood ◽

10.1182/blood.v116.21.4108.4108 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4108-4108

Author(s):

Ann Brinch Madelung ◽

Michael Bzorek ◽

Henrik Bondo ◽

Eva M.K. Zetterberg ◽

Ole Weis Bjerrum ◽

...

Keyword(s):

Bone Marrow ◽

Light Microscopy ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Primary Myelofibrosis ◽

Cell Types ◽

Normal Donor ◽

Bone Marrow Biopsies ◽

Color Palette ◽

Morphological Studies

Abstract Abstract 4108 Background: In Philadelphia-negative chronic myeloproliferative neoplasms (MPN) increased microvascular density, bizarre vessel architecture and increased number of pericytes are distinct histopathological features; apart from the characteristic proliferation of myeloid cell lines and the progressive accumulation of connective tissue in the bone marrow. Pericytes express several markers such as CD146, CD271, Smooth Muscle Actin (SMA), Desmin, Platelet-derived growth factor receptor beta and Neuron-glial 2. However, these markers are also expressed in other cell types of which some are related to vascular structures. Immunofluorescence labelling is the golden standard for detection of co-expressed cellular antigens, but due to the crowded cellular environment in bone marrow and excessive autofluorescence, identification of cell types by light microscopy is preferred. Aim: This is a methodological study aiming to identify pericyte marker profiles by light microscopy in bone marrow biopsies, contributing to our understanding of the pathogenesis of MPN. Method and results: Formalin fixed, decalcified and paraffin-embedded blocks of bone marrow trephine specimens from normal donor (n=1) and patients with primary myelofibrosis (PMF) (n=3) were included. Specimens were subjected to an immunohistochemical sequential multi-labelling and erasing technique (SE-technique), inspired by the work of Glass et al. 2009 (J Histochem Cytochem). Briefly, antigens of interest in the first and/or second layer were detected with an immunoperoxidase system and visualised with Amino-Ethyl-Carbazole (AEC). After imaging, erasing of AEC with 96% alcohol and blocking of immunoreagents, the slides were stained with a traditional double immuno-labelling procedure. We successfully applied up to four layers of antibodies using CD146, SMA, CD34, CD271, and Ki67 in different combinations; either displayed as single or single followed by traditional double sequential staining runs (figure 1). In addition to the conventional light microscopy analysis we applied a Photoshop color palette, where the different immunohistochemical reactions in the staining sequence were assigned to the different color channels creating a single composite image. The SE-technique significantly improves morphological studies especially in bone marrow trephines with the cells of interest intermingled with other cells. Additionally, the SE-technique makes it possible to detect more than two antigens regardless of immunoglobulin type or animal host. Conclusion: To our knowledge, the SE-technique described in this study, is the first to multi-label antigens, identifying vessel and pericyte architecture in bone marrow trephines at light microscopic level. This technique may unravel novel aspects of the composition of the microvessel structures in patients with PMF and related neoplasms. The SE-technique displayed as single staining images and Photoshop color palette combined image. Panel A-D shows an identical area in the different steps of the method. A) CD146. Positive pericytes (arrow). B) SMA. Positive pericytes (arrow). C) CD34. Negative pericytes (arrow). D) Combined image with CD146 (green channel), CD34 (red channel), and SMA (blue channel). Coexpression of CD146/CD34 is seen as yellow reaction deposit, and coexpression of CD146/SMA as cyan reaction deposit (arrow). Note, as a negative control, the CD146 positive fat cell in A (arrowhead) - negative in B and C, and SMA positive pericyte in B (arrow) – negative in C. Disclosures: No relevant conflicts of interest to declare.

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MSCs From Patients with Myelofibrosis Display a Fibrotic Phenotype

Blood ◽

10.1182/blood.v118.21.5160.5160 ◽

2011 ◽

Vol 118 (21) ◽

pp. 5160-5160 ◽

Cited By ~ 1

Author(s):

Rebekka K Schneider ◽

Isabelle Leisten ◽

Susanne Ziegler ◽

Anne Schumacher ◽

Tim H Brümmendorf ◽

...

Keyword(s):

Bone Marrow ◽

Extracellular Matrix ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Primary Myelofibrosis ◽

Collagen I ◽

Rt Pcr ◽

Healthy Donors ◽

Collagenous Matrix ◽

Significant Difference

Abstract Abstract 5160 Myeloproliferative neoplasms (MPNs) are characterized by the excessive production of terminally differentiated nonlymphoid cells or platelets in the bone marrow. They represent a heterogenous group of clonal hematologic malignancies and are classified into chronic myeloid leukemia (CML), essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF) and other rarer diseases. MPNs are often associated with extramedullary hematopoiesis and progressive hepatosplenomegaly due to displacement of hematopoietic progenitor cells from the BM to spleen and liver. Progenitor mobilization follows the enhanced deposition of extracellular matrix proteins, which can be found in the bone marrow of MPN patients. Nonhematopoietic bone marrow stromal cells (BMSCs) and their precursors (mesenchymal stem cells) are believed to be conditioned by abundantly released growth factors from the pathological hematopoietic clone in MPNs and in turn contribute to a modified niche environment which participates in the maintenance of the malignant clone and in disease progression. We therefore aimed at the comparative analysis of BMSCs from MPN patients (displaying myelofibrosis 0–1) and healthy donors as well as at their matrix remodelling capacity. BMSCs from PMF patients were obtained by trephine biopsies and isolated by plastic adherence in IMDM, 20% FCS and cortisone. BMSCs from PMF patients fulfilled MSC criteria according to common consensus (Dominici et al., 2007). To compare their potential to support hemato-lymphopoiesis, we analyzed their hemato-lymphotropic cytokine transcription by RT-PCR. MSCs from MPN patients and healthy donors expressed gene transcripts for M-CSF, SDF-1, LIF, FLT3L, Oct-4, SCF, IL-6 and IL-7, suggesting no significant difference in cytokine production. However, when activated through contact with collagen I/III and embedded in three-dimensional scaffolds, MSCs from MPN-patients extensively remodelled the collagenous matrix compared to healthy donors. Under osteogenic and undifferentiated culture conditions, the extracellular matrix (ECM) production by MPN-MSC was strongly enhanced compared to MSCs from healthy controls as shown by RT-PCR and immunohistochemistry for collagen I, IV, fibronectin, laminin and osteopontin. Furthermore, MSCs from MPN patients significantly contracted and densified the collagenous matrix and the ECM deposition by MSCs from MPN patients was highly comparable to the ECM changes observed in corresponding bone punches. We therefore hypothesize that MSCs from MPN patients are primed to produce enhanced ECM proteins, whereas their capacity to support hematopoiesis seems to be unchanged. Disclosures: No relevant conflicts of interest to declare.

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