Comparative Somatic Mutational Profiling of CD34+ Hematopoietic Precursors (HSC) and Circulating Endothelial Cells (CEC) in Patients with Primary Myelofibrosis (PMF)

• Published in: Blood 2019-11, Vol.134 (Supplement_1), p.1684-1684
• Main Authors: Farina, Mirko, Bernardi, Simona, Polverelli, Nicola, Zanaglio, Camilla, Malagola, Michele, Re, Federica, Cattina, Federica, D'Adda, Mariella, Rossi, Giuseppe, Zollner, Tatiana, Dunbar, Andrew, Levine, Ross L., Russo, Domenico
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2019-127451

INTRODUCTION Endothelial cells (EC) play an important role in thrombosis and are increased in frequency in Myeloproliferative Neoplasms (MPN). Notably, the JAK2V617F mutation has been identified in micro-laser-dissected EC in peripheral vasculature (Sozer, 2009) and is present in a subset of endothelial progenitor cells (Teofili, 2011). Primary myelofibrosis (PMF) is also characterized by increased bone marrow vascularity. These data suggest that PMF clones could derive from a common precursor shared between CD34-hematopoietic stem cells (HSC) and EC. We explored this hypothesis with a prospective study aimed at comparing the mutational profiles of paired Circulating Endothelial Cells (CEC) and HSC in patients (pts) with PMF. METHODS 14 PMF pts not previously treated with JAK2 inhibitors, along with one healthy control, were enrolled in the MyCEC0617 study beginning in 2018. The study was approved by the local Ethical Committee and all pts gave written informed consent. HSC were selected using CD34+ immunomagnetic bead-column separation. CEC were detected using the CellSearch system, which uses immunomagnetic selection incorporating ferrofluid nanoparticles and fluorophore-labelled antibodies. The markers CD146, CD105, CD45, and DAPI were used to isolate CEC. We identified cells as CEC when we observed characteristic morphological features and a surface immunophenotype of CD146+, CD105 +, DAPI+ and CD45-. Putative CEC were then sorted using the DEPArray system, which uses a combination of di-electrophoresis technology and high-quality image-based cell selection to manipulate individual cells. Sequencing data was then assessed with the MiSeq Illumina NGS platform using a 54-gene custom panel focused on genes mutated in PMF. The cutoffs to confirm the presence of the mutations were identification of mutant alleles in 30 and 50 reads both in forward and reverse, for HSC and CEC, respectively. RESULTS The characteristics of pts are reported in Figure 1. Median age of pts was 69.5 ys (35-85) and male/female ratio was 9:6. Median time from diagnosis to sample collection was 4 (1-211) months. 2/14 pts had had a previous thrombosis, 78% had splenomegaly, and 29% presented with constitutional symptoms. Considering the molecular profile, 9 pts were JAK2V617F mutated, 3 were CALR mutated, and 1 was MPLW515L positive. 2 pts were triple-negative. Most of the pts (7) presented with an Intermediate-1 DIPSS score, while 5 were intermediate-2, and 2 were high risk DIPSS. C