Comparative Somatic Mutational Profiling of CD34+ Hematopoietic Precursors (HSC) and Circulating Endothelial Cells (CEC) in Patients with Primary Myelofibrosis (PMF)
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 
Published: November 2019

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.
CEC were collected for 12 of 15 subjects (including the normal control). No significative differences were found between pts from whom we isolated CEC and those we did not successfully recover CEC. A median of 26 (1-122) CEC in 4 ml of peripheral blood were recovered. Notably, no mutations were found in the CEC or HSC from healthy control whereas molecular alterations were discovered both on CEC and HSC in 11 pts (Fig. 1). The previously-identified MPN driver mutation was identified in MF HSC (except for one JAK2-mutated pt and for all CALR-mutated pts, consistent with the limitations of NGS in detecting these lesions). Interestingly, PMF pts demonstrated both shared and different mutations when comparing mutational profiles of HSC and CEC. In particular, 8 of 11 pts shared at least one mutation between EC and HSC. 2 pts harbored the same driver mutation (JAK2V617F), together with ABL1, IDH1, TET2, and ASXL1, respectively. The most frequently mutated genes shared between both CEC and HSC were JAK2, ASXL1, TET2, NOTCH1, and SRSF2. All of these mutations were observed as shared clonal events in at least 2 cases. We also identified individual paired HSC/CEC with shared mutations in TP53, KIT, KMT2A, IDH1, WT1 and ABL1. One pt shared 4 mutations between HSC and CEC, while 4 and 2 pts shared 1 and 2 mutations, respectively.
CONCLUSION
HSC and EC in PMF have in common one or more gene mutations implicated in the pathogenesis of PMF. For the first time, we show that mutations other than JAK2 can be identified in EC, and in most instances (70% of pts), these mutations are shared between CEC and HSC. Moreover, we present an analysis of mature cells of endothelial lineage including to the CEC. These preliminary findings using primary pts samples support the notion that PMF-initiating cells may be derived from a common HSC/EC precursor. Further data are needed to validate these findings and provide a rationale for additional studies exploring the antecedent cell of origin in MPN.

Disclosures
D'Adda: Pfizer: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees. Rossi:Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Jazz: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Mundipharma: Honoraria; BMS: Honoraria; Sandoz: Honoraria; Daiichi-Sankyo: Consultancy; Roche: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees. Levine:Qiagen: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Loxo: Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Research Funding; Prelude Therapeutics: Research Funding; Isoplexis: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Imago Biosciences: Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy; Lilly: Honoraria; Amgen: Honoraria; Celgene: Consultancy, Research Funding.