Temporal Dynamics of Bone Marrow Fibrocytes in the Progression of Primary Myelofibrosis

Background: The mechanisms driving bone marrow (BM) fibrosis progression in primary myelofibrosis (PMF) remain poorly understood. Our previous studies identified neoplastic, monocyte-derived fibrocytes in the BM and spleen of PMF patients (Verstovsek, J Exp Med 2016; Manshouri, Cell Death Dis 2022). Recent research indicates that fibrocyte density correlates with high-risk molecular genotypes in myelofibrosis (Shi, Haematologica 2023), suggesting clonally-derived fibrocytes play a critical role in fibrosis development. However, the temporal kinetics of fibrocyte proliferation during PMF progression and their association with clinical and biological disease features have not been fully elucidated.

Methods: We conducted sequential multimodal imaging, including H&E, reticulin, trichrome, and Opal fluorescence immunohistochemistry (IHC) on serial sections from 85 treatment-naïve PMF patients, encompassing early/pre-fibrotic (MF-1, N=25) and overt PMF (MF-2-3, N=60). The Opal IHC panel included 7 markers. Fibrosis levels were quantified using an AI-assisted workflow developed in Visiopharm and correlated with traditional clinicopathologic scoring systems (Thiele, Haematologica 2005). Fibrocytes (CD45+/CD68+/Procollagen type I+ cells) were quantified using a Visiopharm-based cell segmentation and phenotyping algorithm. Clinical targeted next-generation sequencing (NGS) data (81- or 28-gene panels) correlated tumor mutational signatures with IHC findings. Cytokine analysis, performed using a 37-plex magnetic bead-based immunoassay, correlated cytokine levels with histological observations. BM biopsies (N=7) and plasma samples (N=15) from age-matched healthy donors served as controls.

Results: BM fibrocyte densities, calculated as cell counts per area, were significantly elevated in PMF patients compared to controls across all three disease stages (MF1-3; P<0.001). Our AI-assisted quantification method matched closely with the clinical pathology assessment of fibrosis severity (P<0.001). Intriguingly, despite overall increased densities compared to controls, fibrocyte density declined in advanced stages of PMF, negatively correlating with both reticulin (R²=-0.26, P=0.017) and collagen fibrosis levels (R²=-0.38, P<0.001). Additionally, BM fibrocyte densities were significantly higher in patients with splenomegaly (P=0.024), higher hemoglobin concentration (P=0.019), and platelet counts (P=0.031), indicative of an early, proliferative phenotype. Conversely, fibrocyte density negatively correlated with BM blasts (P=0.014), high LDH levels (P=0.015), weight loss (P=0.028), chromosomal abnormalities (P=0.022), and a high-risk MIPSS70+ score (P=0.009). Moreover, lower fibrocyte density was linked to worse overall survival (log-rank, P=0.013). These findings suggest a significant shift in proliferative capacity of these cells during disease progression, with selective pressures possibly favoring the survival of more resilient clonal subsets.

To further delineate these dynamics, we performed clinical NGS and plasma cytokine profiling. NGS revealed that fibrocyte numbers were not significantly influenced by the JAK2 mutation or mutant allele burden. However, 21 patients with the CALR mutation (25%) exhibited higher fibrocyte densities than others (P=0.014). In contrast, the ASXL1 mutation, detected in 25 patients (29%), was associated with relative fibrocyte depletion (P=0.006). Plasma cytokine profiling from 58 of the 85 patients showed that this depletion correlated with increased levels of several cytokines signaling through the JAK-STAT pathway, including IL-2, IL-29, and IFN-α2. Additionally, high levels of IL-32, sTNF-R1, and sTNF-R2 indicated inflammatory conditions.

Conclusion: Our analysis demonstrates for the first time that BM fibrocyte populations in PMF exhibit a biphasic pattern of temporal kinetics, characterized by expansion during the prefibrotic phase and contraction in the overt phase of the disease. The selective pressures driving this dynamic - whether induced by cell-intrinsic factors, resource depletion, microenvironmental stress, extracellular matrix changes, or immune dysregulation - require further investigation. These findings delineate the role of fibrocytes throughout BM fibrosis progression and identify potential targets for developing anti-fibrotic therapies.

Veletic:Aviceda Therapeutics: Research Funding; Avilect Biosciences: Research Funding. Masarova:MorphoSys: Other: Advisory Board Participant; Cogent: Other: Advisory Board Participant; GSK: Consultancy, Other: Travel support; PharmaEssentia: Other: Advisory Board Participant. Pemmaraju:Daiichi Sankyo: Research Funding; Cellectis: Research Funding; Affymetrix/Thermo Fisher Scientific: Research Funding; Triptych Health Partners: Consultancy; Stemline Therapeutics: Honoraria, Other: Travel Expenses, Research Funding; Springer Science + Business Media: Honoraria; Roche Molecular Diagnostics: Honoraria; Mustang Bio: Honoraria, Other: Travel Expenses, Research Funding; LFB Biotechnologies: Honoraria; Incyte: Honoraria; Protagonist Therapeutics: Consultancy; Neopharm: Honoraria; ClearView Healthcare Partners: Consultancy; Celgene: Honoraria, Other: Travel Expenses; Bristol-Myers Squibb: Consultancy; Aptitude Health: Honoraria; Novartis: Honoraria, Research Funding; DAVA Oncology: Honoraria, Other: Travel Expenses; Pacylex: Consultancy; CareDx: Honoraria; Blueprint Medicines: Consultancy, Honoraria; Immunogen: Consultancy; CTI BioPharma: Consultancy; Astellas: Consultancy; AbbVie: Honoraria, Other: Travel Expenses, Research Funding; Plexxikon: Research Funding; Samus Therapeutics: Research Funding; Blueprint Medicines OncLive PeerView Institute for Medical Education: Consultancy, Other: advisory board; ASH Committee on Communications ASCO Cancer.NET Editorial Board: Other: Leadership; Karger Publishers: Other: Licenses; National Institute of Health/National Cancer Institute (NIH/NCI): Research Funding; HemOnc Times/Oncology Times: Other: uncompensated. Bose:AbbVie: Honoraria; BMS: Honoraria, Research Funding; Incyte: Honoraria, Research Funding; Karyopharm: Honoraria; Kartos: Honoraria, Research Funding; Cogent: Honoraria, Research Funding; PharmaEssentia: Honoraria; MorphSys: Honoraria, Research Funding; Disc Medicine: Research Funding; Blueprint: Honoraria, Research Funding; Ionis Pharmaceuticals: Research Funding; Novartis: Honoraria; Telios: Research Funding; Astellas: Research Funding; Pfizer: Research Funding; GSK: Honoraria; CTI Biopharma Corp: Honoraria, Research Funding; NS Pharma: Research Funding; Promedior: Research Funding. Kantarjian:AbbVie, Amgen, Ascentage, Ipsen Biopharmaceuticals, KAHR Medical, Novartis, Pfizer, Shenzhen Target Rx, Stemline,Takeda: Consultancy, Honoraria. Dunbar:Morphosys: Consultancy. Abbas:Alamar Biosciences: Honoraria; Molecular Partners: Consultancy; Illumina: Honoraria, Other: Inkind Support, Research Funding; Ascentage: Research Funding; Blueprint Medicines Corporation: Research Funding; GlaxoSmithKline: Research Funding; Genentech: Research Funding; Enzyme By Design: Research Funding.