Histological Parameters and “In Vitro” Cultures Identify Different Subsets of Myelofibrosis with Myeloid Metaplasia (MMM).
Authors: Servida Federica , Vener Claudia , Tubazio Viviana , Gianelli Umberto , Raviele Paola Rafaniello , Moro Alessia , Anastasia Antonella , Deliliers Giorgio Lambertenghi , Soligo Davide 
Published: April 2020

Myelofibrosis with myeloid metaplasia (MMM) is a chronic myeloproliferative disorder (CMPD) characterized by clonal hematopoietic proliferation, reactive marrow fibrosis and extramedullary hematopoiesis. Twenty CMPDs, previously diagnosed as chronic idiopathic myelofibrosis (CIMF), were reviewed according to the WHO morphological parameters (Vardiman et al., 2001) and classified as prefibrotic-CIMF (p-CIMF), fibrotic-CIMF (f-CIMF), polycythemia vera (PV) and post-polycythaemic myelofibrosis (PPMF). Patients median age was 67 yrs (range: 45–84), generally presenting cytogenetic abnormalities and treated with chemotherapy. Quantitation of peripheral blood (PB) CD34+ cells was carried out according to ISHAGE gating strategy. Clonogenic assays were performed both on bone marrow mononuclear cells (BM) and on cells extracted from the bone marrow biopsy (BMB) using either flushing and trypsin digestion. Myeloid progenitor cell number (CFU-GM) was evaluated in semisolid medium. Fibroblast progenitor cell number (CFU-F) was studied seeding cells in IMDM medium supplemented with 20% FBS. Microvessel density hot spots (MVD) was evaluated according to Weidner et. al., 1991. Data are summarized in Table 1. Circulating CD34+ cells were higher in all classes in comparison with normals but a clearcut decrease was observed from p-CIMF to f-CIMF and from PV to PPMF. A similar trend was observed in committed progenitor cells both in bone marrow aspirated (BM) and bone (BMB), suggesting that f-CIMF and PPMF may represent late stages of the disease in which hematopoietic cells have been progressively replaced with fibroblasts. Fibroblast precursors (CFU-F) were much lower in BM than in BMB (treated with flushing and trypsinization) in all group of patients even though these findings may be related to sampling artifacts. In any case, in both BM and BMB, CFU-F increased from p-CIMF to f-CIMF, suggesting an active progression on myelofibrosis; on the contrast, BMB CFU-F significantly decrease from PV to PPMF, indicating a progressive exhaustion of the fibroblast precursors. Moreover, MVD performed in a larger group of patients (n=50) showed higher median values than normal controls, particularly evident in PPMF, where an inverse correlation between fibrosis and angiogenesis was observed. These data suggest that f-CIMF and PPMF could be the fibrotic exit of two distinct MPDs, respectively p-CIMF and PV, indicating that the ethiopatogenesis of the two diseases may be intrinsically different. In conclusion, our “in vitro” data support the WHO classification and may have diagnostic and clinical relevance.

Table 1 WHO Classification CD34+ PB cells/μl CFU-GM 5X10e5 cells (BM) CFU-GM 5X10e5 cells (BMB) CFU-F 1X10e6 cells (BM) CFU-F 1X10e6 cells (BMB) MVD (vessels number) Median (Range) p-CIMF (7pt) 110 (1.16–378) 75.5 (65–86) 12 (2–22) 1 (1–15) 67.5 (0–113) 30 (26–43) f-CIMF (3pt) 42 (15–165) 59 (40–60) nd 18 (18–32) 176 (27–325) 24 (20–44.6) PV (3pt) 393 (110–676) 41.5 (18–65) 111.5 (76–147) 12.5 (1–24) 167.5 (86–249) 18.6 (7.3–33) PPMF (7pt) 178 (0.04–376) 19 (10–28) 10 (0–81) 0 (2–4) 36 (10–214) 33.8 (15–40) Controls &lt;1 100 (60–120) nd 44 (18–78) 66 (40–108) 6.6 (4.6–16.3)