Introduction: Myelofibrosis (MF) is characterized by clonal hemopoiesis, inflammatory microenvironment and mutations in JAK2, MPL, or CALR genes. Around 10% of patients (pts) does not carry the 3 driver mutations (Triple negative (TN)) displaying, along with lower haemoglobin level and platelet number, significantly worse survival-rates. The malignant hemopoietic stem/progenitor cells compartment of TN MF pts has never been characterized. Here, we compared the in vitro effects of crucial factors of the inflammatory microenvironment on the functional behaviour of TN circulating CD34+ cells vs the JAK2(V617F) mutated counterparts. Methods: Peripheral blood was collected from 9 MF pts (at diagnosis or out of cytotoxic treatment for at least 3 months) and 10 age/sex-matched healthy donors (HD). MF pts were JAK2(V617F) (n=5) mutated and TN (n=4). Circulating CD34+ cells from pts were enumerated by flow cytometry. Immunomagnetically isolated CD34+ cells were in vitro treated with selected inflammatory factors (Interleukin (IL)-1β, Tumor Necrosis Factor (TNF)-α, Interleukin-6) for 12/24 hours and migration/survival ability was investigated. The plasma concentration of these cytokines was evaluated by ELISA. Gene expression profiling analysis (GEP) was performed in CD34+ cells from 3 TN vs 3 JAK2(V617F) mutated pts (Human Transcriptome array 2.0, Affymetrix) and gene set enrichment analysis was conducted by GSEA. Results: GEP analysis showed a total of 165 genes differentially expressed (121 downmodulated and 44 upregulated in CD34+ cells from TN- vs JAK2(V617F) mutated pts). Specifically, we found that the expression of selected anti-apoptotic (TSPYL5, GFI-1 and FCMR) and pro-apototic (TNFSF10, TP53INP1) genes was significantly down- and up-regulated, respectively, in TN-CD34+ cells. Consistent with the GEP data, the in vitro survival of untreated TN-CD34+ cells was significantly decreased as compared with the JAK2(V617F) mutated counterparts. GEP analysis identified also a network of genes involved in cell adhesion, proliferation and inflammation which were mainly down-regulated in TN-CD34+ cells. Specifically, GSEA highlighted an enrichment of cell adhesion, migration and chemotaxis signatures in TN-CD34+ cells. Accordingly, we found that the absolute number of circulating CD34+ and CD34+ CD184+ cells was significantly increased in TN pts as compared with the JAK2(V617F) mutated counterparts (p<0.05). Plasma concentration of IL-1β, IL-6 and TNF-α was significantly increased (p<0.05) in MF pts with no differences between the two groups. However, at variance with the CD34+ cells from the JAK2(V617F) mutated pts, the survival and migration of TN-CD34+ cells was not significantly affected by the combined IL-1β, IL-6 and TNF-α. Conclusions: Altogether these findings suggest that TN-CD34+ cells show distinct in vitro functional features which may contribute to explain, at least in part, the defective hemopoiesis of TN-pts.

CIRCULATING CD34+ STEM/PROGENITOR CELLS FROM TRIPLE NEGATIVE PATIENTS WITH MYELOFIBROSIS SHOW DIFFERENT NUMBER, GENE EXPRESSION PROFILE AND IN VITRO RESPONSE TO INFLAMMATORY STIMULI AS COMPARED WITH THE JAK2(V617F) MUTATED COUNTERPARTS

D. Sollazzo;D. Forte;G. Simonetti;S. Bruno;M. Barone;G. Auteri;M. Romano;G. Martinelli;N. Vianelli;M. Cavo;F. Palandri;L. Catani
2018

Abstract

Introduction: Myelofibrosis (MF) is characterized by clonal hemopoiesis, inflammatory microenvironment and mutations in JAK2, MPL, or CALR genes. Around 10% of patients (pts) does not carry the 3 driver mutations (Triple negative (TN)) displaying, along with lower haemoglobin level and platelet number, significantly worse survival-rates. The malignant hemopoietic stem/progenitor cells compartment of TN MF pts has never been characterized. Here, we compared the in vitro effects of crucial factors of the inflammatory microenvironment on the functional behaviour of TN circulating CD34+ cells vs the JAK2(V617F) mutated counterparts. Methods: Peripheral blood was collected from 9 MF pts (at diagnosis or out of cytotoxic treatment for at least 3 months) and 10 age/sex-matched healthy donors (HD). MF pts were JAK2(V617F) (n=5) mutated and TN (n=4). Circulating CD34+ cells from pts were enumerated by flow cytometry. Immunomagnetically isolated CD34+ cells were in vitro treated with selected inflammatory factors (Interleukin (IL)-1β, Tumor Necrosis Factor (TNF)-α, Interleukin-6) for 12/24 hours and migration/survival ability was investigated. The plasma concentration of these cytokines was evaluated by ELISA. Gene expression profiling analysis (GEP) was performed in CD34+ cells from 3 TN vs 3 JAK2(V617F) mutated pts (Human Transcriptome array 2.0, Affymetrix) and gene set enrichment analysis was conducted by GSEA. Results: GEP analysis showed a total of 165 genes differentially expressed (121 downmodulated and 44 upregulated in CD34+ cells from TN- vs JAK2(V617F) mutated pts). Specifically, we found that the expression of selected anti-apoptotic (TSPYL5, GFI-1 and FCMR) and pro-apototic (TNFSF10, TP53INP1) genes was significantly down- and up-regulated, respectively, in TN-CD34+ cells. Consistent with the GEP data, the in vitro survival of untreated TN-CD34+ cells was significantly decreased as compared with the JAK2(V617F) mutated counterparts. GEP analysis identified also a network of genes involved in cell adhesion, proliferation and inflammation which were mainly down-regulated in TN-CD34+ cells. Specifically, GSEA highlighted an enrichment of cell adhesion, migration and chemotaxis signatures in TN-CD34+ cells. Accordingly, we found that the absolute number of circulating CD34+ and CD34+ CD184+ cells was significantly increased in TN pts as compared with the JAK2(V617F) mutated counterparts (p<0.05). Plasma concentration of IL-1β, IL-6 and TNF-α was significantly increased (p<0.05) in MF pts with no differences between the two groups. However, at variance with the CD34+ cells from the JAK2(V617F) mutated pts, the survival and migration of TN-CD34+ cells was not significantly affected by the combined IL-1β, IL-6 and TNF-α. Conclusions: Altogether these findings suggest that TN-CD34+ cells show distinct in vitro functional features which may contribute to explain, at least in part, the defective hemopoiesis of TN-pts.
Haematologica XV Congress of the Italian Society of Experimental Hematology Rimini, Italy, October 18-20, 2018 ABSTRACT BOOK
D. Sollazzo, D. Forte, G. Simonetti, S. Bruno, M. Barone, G. Auteri, E. Ottaviani, M. Romano, G. Martinelli, N. Vianelli, M. Cavo, F. Palandri, L. Catani
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11585/662924
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