V617FJAK2-Positive Endothelial Cells Are Present in Bone Marrow Neovessels of Patients with Myelofibrosis and Could Derive from the Transdifferentiation of Mutated Hematopoietic Cells

Background. Extensive neoangiogenesis is a distinctive feature of both the spleen and the bone marrow (BM) of patients with myelofibrosis (MF). We and others have previously reported that mature endothelial cells obtained from microvessels of the spleen, either by laser microdissection or by cell sorting, harbor the V617FJAK2 mutation, whereas nothing is known about the existence of such mutated endothelial cells in the BM. The origin of the mutated endothelium has not been clarified up to now, although it has been proposed that it could derive from the transdifferentiation of a mutated CD34+ hematopoietic progenitor cells into an endothelial cell, an event similar to what already observed in solid tumors. Aim. To assess the presence of the V617FJAK2 mutation in mature endothelial cells from the BM of patients with MF and to evaluate the presence of transitional cells expressing both myeloid and endothelial cell markers in the BM of MF patients. Patients and Methods. Paraffin-embedded BM sections from 5 patients with MF, harboring a V617FJAK2 mutation in their granulocytes, were used both for isolating mature endothelial cells by laser microdissection and for immunostaining with antibodies directed against CD34, CD33, and CD144 (VE-cadherin) antigens. Sections from patients with lymphomas who underwent a BM biopsy for staging of the disease were used as controls (n=2). The images were obtained by confocal laser scanning microscopy (Olympus Fluoview FV10i, 60x objective) and processed by IMAGE J software. DNA was extracted from 50 microdissected endothelial cells by a commercial kit and the presence of V617FJAK2 mutation assessed by an allele-specific real-time quantitative PCR. Results. The V617FJAK2 mutation was detected in at least 1 sample of endothelial cells of 5/5 patients, indicating that a mutated endothelium, in addition to the spleen, is also detectable in the BM of patients with MF. The allelic burden ranged from 51.4% to 84.1% of mutated alleles, and was similar to the allelic burden of hematopoietic cells in 1 case, whereas it was lower in 4. Staining of BM sections from 4 patients with antibodies specific for hematopoiesis (CD33) and endothelium (VE-cadherin), as well as common to the 2 lineages (CD34), while confirming the presence of increased neoangiogenetic processes in samples from MF patients, allowed the detection of cells, usually located close to neovessels, co-expressing both hematopoietic (CD33, CD34) and endothelial (VE-cadherin, CD34) markers. These "triple positive" cells were found in all the slides of all patients whereas they were never observed in the BM sections of controls. Conclusions. V617FJAK2 positive endothelial cells can be detected in the BM of patients with MF, similarly to what observed in the spleen. These mutated endothelial cells could derive from the transdifferentiation of hematopoietic mutated cells as suggested by the detection of cells co-expressing both myeloid and endothelial markers. These observations support the hypothesis that the endothelial lineage is involved in the process of malignant transformation of MF, providing new perspectives in our understanding of the phenotype, evolution, and therapy of the disease.