Polycythemia vera (PV) is a classical "myeloproliferative neoplasm" characterized by clonal proliferation of myeloid progenitor cells caused by their acquisition of a mutation in the JAK2 gene. Its common complications are thrombosis and second primary malignancies. Secreted from virtually all cell types, exosomes are extracellular vesicles that carry bioactive cargo derived from their cells of origin. When engulfed, their cargo may alter the phenotype of the recipient cells. For example, neoplastic exosomes modulate the cellular makeup of bystander cells. Therefore, since mutated JAK2 is present in virtually all PV-patients, we hypothesize that PV-derived exosomes (hereafter PV-exo) carry the mutated JAK2 oncogene and are taken up by non-clonal cells, thus contributing to the thrombotic manifestations and oncogenic potential of patients with PV. We isolated and characterized exosomes from PV-patients and from the Human erythroleukemia (HEL) cell line (hereafter HEL-exo). We exposed normal cells implicated in thrombosis and skin cells to these exosomes and examined the cells' phenotypic changes and activation after exposure. Using Sanger sequencing we discovered that both PV-exo and HEL-exo, like their parental cells, carry the mutated JAK2 transcripts, allowing clonal cells to export oncogenes to distant, non-clonal sites. Our preliminary findings suggest that these exosomes promote pro-coagulant and malignant phenotypes in multiple ways. Thrombin generation assay pointed to a significantly higher generation of thrombin in the presence of PV-exo than those derived from healthy donors. Similarly, flow cytometry analysis revealed that PV-exo increased the expression of platelet activation markers. Moreover, Trans Endothelial Electrical Resistance (TEER) assay results suggest that HEL-exo induces endothelial dysfunction. Furthermore, using the Sulforhodamine B assay, we found that exposure to HEL-exo increased the proliferation of normal keratinocytes. In summary, we developed the concept of exosomes as “mini metastasis” that might promote disease manifestations either directly or indirectly. Since unlike solid tumors, hematological malignancies do not metastasize, exosomes derived from neoplastic hematological cells may fulfill this function. Such exosomes may spread oncogenes (e.g., mutated JAK2), damage non-clonal tissues (e.g., endothelial dysfunction) or act as a miniature reflection of their cells of origin (e.g., exosomes as mini platelets) in a parallel way to "real" metastasis; this concept may affect treatment related decisions. For example, by reducing clonal burden, early treatment even in “low risk” patients may prevent exosomal spread and exosomal-dependent damage to distant tissues. The results of our study highlight PV-exo as promoters of PV-complications, thrombosis and malignant transformation. Citation Format: Orit Uziel, Adi Shacham-Abulafia, Galia Spectre, Ester Ziv, Karyn Revital Geiger, Zinab Sarsor, Neria Ron, Einat Beery, Pia Raanani, Shoshana Revel-Vilk, Mira Naamad, Uri Rozovski. Exosomes in polycythemia vera: "mini platelets" with oncogenic and thrombogenic potential [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3849.

In all patients with polycythemia-vera (PV) a point mutation in JAK2 is present in hematopoietic progenitors. Patients develop thrombotic complications, leukemic transformation, and may also develop solid tumors. Preliminarily, we identified the mutated transcripts of JAK2 in PV patients-derived exosomes. This discovery led us to explore the contribution of clonal- exosomes which carry the driver mutation away from their cell-of-origin to the systemic manifestation characterizing PV, thus contributing to the thrombotic and neoplastic complications in non-hematopoietic tissues. Endothelial dysfunction may lead to those complications, either directly by introducing pro-thrombotic factors on their surface or indirectly by inducing chronic inflammatory milieu that promotes thrombosis and oncogenic transformation. Our working hypothesis was that PV-exosomes lead to endothelial dysfunction. First, we showed that similar to their parent cells, PV-exosomes also carry mutated JAK2 transcripts in all patients and the presence of wild-type JAK2 in all healthy individuals. Next, we found that FM-1-43 labelled JAK2mutated-exosomes were taken up by endothelial cells (HUVECs, by flow cytometry) in a dose- and time- dependent manner and induced endothelial dysfunction (by measuring membrane resistance). The resistance was dramatically decreased in the presence of PV-exosomes, indicating that the tight junction between adjacent endothelial cells was impaired and in line with our working hypothesis. To study whether PV-exosomes also have direct effect, we measured the rate of thrombin generation in platelets-poor-plasma to assess the thrombogenic potential of PV-exosomes. An increase in all parameters of the thrombin concentration in the presence of PV-exosomes compared with exosomes from healthy individuals was obtained. We also detected an upregulation of three markers of platelet aggregation in response to PV- exosomes compared with their healthy-derived counterparts. Finally, we found that PV-exosomes have the capacity to increase the proliferation of normal cells from various tissues. We conclude that like their parental cells, PV-derived exosomes carry mutant JAK2 transcripts. These exosomes are pro-thrombotic and have transforming potential both directly and indirectly through endothelial dysfunction. In all patients with polycythemia-vera (PV) a point mutation in JAK2 is present in hematopoietic progenitors. Patients develop thrombotic complications, leukemic transformation, and may also develop solid tumors. Preliminarily, we identified the mutated transcripts of JAK2 in PV patients-derived exosomes. This discovery led us to explore the contribution of clonal- exosomes which carry the driver mutation away from their cell-of-origin to the systemic manifestation characterizing PV, thus contributing to the thrombotic and neoplastic complications in non-hematopoietic tissues. Endothelial dysfunction may lead to those complications, either directly by introducing pro-thrombotic factors on their surface or indirectly by inducing chronic inflammatory milieu that promotes thrombosis and oncogenic transformation. Our working hypothesis was that PV-exosomes lead to endothelial dysfunction. First, we showed that similar to their parent cells, PV-exosomes also carry mutated JAK2 transcripts in all patients and the presence of wild-type JAK2 in all healthy individuals. Next, we found that FM-1-43 labelled JAK2mutated-exosomes were taken up by endothelial cells (HUVECs, by flow cytometry) in a dose- and time- dependent manner and induced endothelial dysfunction (by measuring membrane resistance). The resistance was dramatically decreased in the presence of PV-exosomes, indicating that the tight junction between adjacent endothelial cells was impaired and in line with our working hypothesis. To study whether PV-exosomes also have direct effect, we measured the rate of thrombin generation in platelets-poor-plasma to assess the thrombogenic potential of PV-exosomes. An increase in all parameters of the thrombin concentration in the presence of PV-exosomes compared with exosomes from healthy individuals was obtained. We also detected an upregulation of three markers of platelet aggregation in response to PV- exosomes compared with their healthy-derived counterparts. Finally, we found that PV-exosomes have the capacity to increase the proliferation of normal cells from various tissues. We conclude that like their parental cells, PV-derived exosomes carry mutant JAK2 transcripts. These exosomes are pro-thrombotic and have transforming potential both directly and indirectly through endothelial dysfunction.

Introduction The safety of neuro-axial anaesthesia (epidural/spinal) at labour of women with partial factor XI (FXI) deficiency is uncertain. Although FXI deficiency is frequent in Ashkenazi Jews, it is not routinely measured before labour. Our institute serves a large Ashkenazi population. We assumed that 10% of them have undiagnosed FXI deficiency. Aim Assess the incidence, bleeding tendency and coagulation status among Jewish Ashkenazi women with FXI deficiency that underwent neuro-axial anaesthesia at delivery. Methods Jewish Ashkenazi women who underwent neuro-axial anaesthesia at labour completed the SSC ISTH bleeding assessment tool (BAT) and had blood drawn for coagulation tests, FXI and thrombin generation after labour. Estimation for 10 years was calculated from the 1-year sample. Results We recruited 261 women during 12 months. Among them, 39 (15%) had FXI deficiency (<70%) with median FXI levels of 63% (range: 33%-70%). Around 50% of them underwent amniocentesis in the current pregnancy and prior neuro-axial anaesthesia with no bleeding complications. BAT score and thrombin generation did not differ between women regardless of FXI status. aPTT was longer in women with partial FXI deficiency (median - 28.6 sec vs 26.3 sec, P < .001, Table 2), although within the normal range in all women. No bleeding complications after neuro-axial anaesthesia at delivery were reported in our centre in the last decade though, and according to our estimation,at least2150 women had partial FXI deficiency. Conclusions A significant number of Jewish Ashkenazi women with undiagnosed partial FXI deficiency undergo neuro-axial anaesthesia at labour without bleeding complications.