Sustained Alterations In Bone Marrow Stromal Cells From Patients With Myeloproliferative Neoplasms (MPN) Contribute To Remodelling Of The Bone Marrow Microenvironment Prior To The Manifestation Of Myelofibrosis
Authors: Schneider Rebekka K. , Leisten Isabelle , Ziegler Susanne , Schumacher Anne , Rath Björn , Fahrenkamp Dirk , Mueller-Newen Gerhard , Crysandt Martina , Wilop Stefan , Jost Edgar , Knuechel Ruth , Bruemmendorf Tim H. , Ziegler Patrick 
Published: October 2019

Abstract
Myeloproliferative neoplasms (MPN) are characterized by the loss of normal hematopoiesis and the excessive production and accumulation of non-lymphoid cells and platelets in the BM. Clonal hematopoiesis in MPN is assumed to generate factors which induce profound changes in the non-clonal BM microenvironment. Alterations include massive deposition of extracellular matrix proteins (ECM), progression to bone marrow (BM)-fibrosis and osteosclerosis. We hypothesize that particularly in MPN, alterations in the cross talk between hematopoietic and bone marrow stromal cells (BMSC) play a critical role for an impaired bone marrow microenvironment long before overt signs of myelofibrosis can be detected by conventional methods.
To dissect the hematopoiesis supporting capacity and extracellular matrix remodelling of BMSC from patients with MPN, we isolated BMSC from BM of patients with essential thrombocytemia (ET, n=5), polycythemia vera (PV, n=5), chronic myeloid leukemia (CML, n=5) and control BM (n=6). BMSC isolates were taken only from pre-fibrotic MPN patients (bone marrow trephine biopsy reticulin staining graded 0 or 1) and the resulting expansion cultures fullfilled MSC criteria according to the common consensus (Dominici et al., Cytotherapy, 2006; 8(4):315-317). When subjected to myeloid colony forming unit assays, MPN-BMSC conditioned supernatants showed a significantly reduced capacity to stimulate a CFU-GM/G/M growth of non-malignant hematopoietic stem and progenitor cells as compared to control BMSC (control (n=6) vs CML (n=5): p= 0.0032; control vs PV (n=5): p=0.016, control vs ET (n=4) not significant; student«s T- test ). BMSC-dependent matrix remodelling was analysed in a previously established robust matrix remodelling assay in vitro. MPN-BMSC displayed a pronounced increased matrix remodelling capacity compared to control BMSC. Interestingly, among the different MPN subtypes, this effect was highly significant in BMSC derived from patients with ET (Control (n=6) vs. ET (n=5): p<0.001). Furthermore, in vitro ECM production by MPN-BMSC was paralleled by ECM changes observed in matched bone marrow punches as shown by fibronectin immunohistochemistry. Co-expression of the stroma marker CD271 and fibronectin -as shown by confocal microscopy- points towards stroma-mediated ECM production in vivo. As upregulation of fibronectin expression was also detected in reticulin 0 graded BM punches, we hypothesized that fibronectin staining might be a potential marker for pre-fibrotic ECM changes in –so far- reticulin-negative MPN biopsies. To validate this hypothesis, we stained fibronectin in a tissue microarray (TMA), containing primary BM biopsies from patients with ET (n=14), PV (n=14), CML (n=14), MF (n=11) and controls (Non-Hodgkin's lymphoma without bone marrow involvement, n=17). Interestingly, within the reticulin-negative subcohort of pre-fibrotic MPN (n=34), fibronectin stained positive (grade 1 or higher) in 5/7 cases with ET (71%), 6/9 cases with PV (66%) and in 14/14 cases with CML (100%) as well as in all cases with PMF (100%). Furthermore, fibronectin staining correlated significantly with patients' decreased haemoglobin levels as shown by ANOVA analysis of routine clinical parameters (F=5.71; Prob> F 0.0037).
We conclude that prior to the manifestation of fibrosis in MPN, BMSC loose their capacity to support non-malignant hematopoiesis and increase their matrix remodelling potential. As to our surprise, these effects are stably maintained in the absence of the malignant hematopoietic clone and since BMSC cultures develop over numerous population doublings in vitro from proliferating mesenchymal precursors, we hypothesize that cell intrinsic effects in BMSC from patients with MPN contribute to the development of myelofibrosis in MPN.Figure 1reticulin-negative subcohort of pre-fibrotic MPNFigure 1. reticulin-negative subcohort of pre-fibrotic MPN

Disclosures:
Bruemmendorf: Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Ariad: Consultancy, Honoraria.