Hyperplasia of morphologically abnormal megakaryocytes (MKs) is a hallmark of primary myelofibrosis (PMF) but the molecular events leading to MK abnormalities are still unclear. We previously demonstrated that, in thrombopoietin (TPO)-stimulated cultures, PMF CD34+ cells showed enhanced in vitro expansion capacity and impaired megakaryocytic differentiation compared to CD34+ cells from healthy individuals, and that the over-expression of the proto-oncogene protein kinase C epsilon (PKC) contributes to these abnormalities (Masselli et al. ASH 2013 abstr.114). Here we investigated whether clinical (included in the IPSS or DIPSS risk category) or biological (JAK2 mutational status) variables might impact the in vitro behavior of TPO-stimulated PMF CD34+ cells. We stratified 8 PMF patients according to the IPSS or DIPSS category (low/intermediate vs high risk) and JAK2 mutational status and evaluated: (1) Fold increase (FI) at day 14 of culture, (2) MK differentiation (% of CD41+ and CD42b+ cells and% of proplatelet-forming MKs) and (3) PKC protein levels (by western blot). CD34+ cells from high risk PMFs displayed increased proliferative capacity as compared to low/intermediate risk (FI: 44±0.2 vs 26.5±4.3, p=0.012), while no difference could be observed between JAK2V617F+ and JAK2V617F- PMFs (FI: 37.2±11.8 vs 27.9±5, p=0.39). Additionally, high risk PMFs revealed impaired MK differentiation potential, as indicated by the lower% of CD41+ and CD42b+ cells (respectively: 26.3±9.4 vs 54.2.6, p=0.008 and 16.1±8 vs 38.2±3, p=0.011) and proplatelet-forming MKs (0.67±0.21 vs 1.8±0.25, p=0.035). By contrast, no statistical difference was observed according to the JAK2 mutation. Finally, we found that high risk patients- derived MKs are characterized by higher expression of PKC as compared to low/intermediate risk ones (relative PKC /GAPDH OD values: 1.75±0.52 in high risk vs 0.82±0.29 in low risk, p=0.023). Conversely, PKC levels were comparable among JAK2V617F+ and JAK2V617F- PMFs (1.11±0.34 vs 1.3±0.8, p=0.72). These data indicate that the degree of in vitro growth and megakaryocytic commitment of PMF CD34+ cells is correlated to the aggressiveness of the disease (indicated by IPSS/DIPSS risk category) and not to the JAK2V617F mutation. Similarly, we found that PKC levels are significantly greater in high vs low/intermediate risk patients, leading us to speculate that PKC can be utilized as a marker of high disease burden and a more aggressive disease.
IN VITRO CHARACTERISTICS OF HEMATOPOIETIC PROGENITORS FROM PRIMARY MYELOFIBROSIS PATIENTS CORRELATE WITH IPSS/DIPSS RISK CATEGORY / Masselli, Elena; Carubbi, Cecilia; Gobbi, Giuliana; Mirandola, Prisco; Galli, Daniela; Martini, Silvia; Pozzi, Giulia; Gildone, Maria; Albanese, Cristina; Bonomini, Sabrina; Crugnola, Monica; Craviotto, Luisa; Aversa, Franco; Vitale, Marco. - In: HAEMATOLOGICA. - ISSN 0390-6078. - 100 (s3):(2015), pp. 167-167. (Intervento presentato al convegno 45° Congresso della Società Italiana di Ematologia tenutosi a Firenze nel 4-7 Ottobre 2015).
IN VITRO CHARACTERISTICS OF HEMATOPOIETIC PROGENITORS FROM PRIMARY MYELOFIBROSIS PATIENTS CORRELATE WITH IPSS/DIPSS RISK CATEGORY
VITALE, Marco
2015-01-01
Abstract
Hyperplasia of morphologically abnormal megakaryocytes (MKs) is a hallmark of primary myelofibrosis (PMF) but the molecular events leading to MK abnormalities are still unclear. We previously demonstrated that, in thrombopoietin (TPO)-stimulated cultures, PMF CD34+ cells showed enhanced in vitro expansion capacity and impaired megakaryocytic differentiation compared to CD34+ cells from healthy individuals, and that the over-expression of the proto-oncogene protein kinase C epsilon (PKC) contributes to these abnormalities (Masselli et al. ASH 2013 abstr.114). Here we investigated whether clinical (included in the IPSS or DIPSS risk category) or biological (JAK2 mutational status) variables might impact the in vitro behavior of TPO-stimulated PMF CD34+ cells. We stratified 8 PMF patients according to the IPSS or DIPSS category (low/intermediate vs high risk) and JAK2 mutational status and evaluated: (1) Fold increase (FI) at day 14 of culture, (2) MK differentiation (% of CD41+ and CD42b+ cells and% of proplatelet-forming MKs) and (3) PKC protein levels (by western blot). CD34+ cells from high risk PMFs displayed increased proliferative capacity as compared to low/intermediate risk (FI: 44±0.2 vs 26.5±4.3, p=0.012), while no difference could be observed between JAK2V617F+ and JAK2V617F- PMFs (FI: 37.2±11.8 vs 27.9±5, p=0.39). Additionally, high risk PMFs revealed impaired MK differentiation potential, as indicated by the lower% of CD41+ and CD42b+ cells (respectively: 26.3±9.4 vs 54.2.6, p=0.008 and 16.1±8 vs 38.2±3, p=0.011) and proplatelet-forming MKs (0.67±0.21 vs 1.8±0.25, p=0.035). By contrast, no statistical difference was observed according to the JAK2 mutation. Finally, we found that high risk patients- derived MKs are characterized by higher expression of PKC as compared to low/intermediate risk ones (relative PKC /GAPDH OD values: 1.75±0.52 in high risk vs 0.82±0.29 in low risk, p=0.023). Conversely, PKC levels were comparable among JAK2V617F+ and JAK2V617F- PMFs (1.11±0.34 vs 1.3±0.8, p=0.72). These data indicate that the degree of in vitro growth and megakaryocytic commitment of PMF CD34+ cells is correlated to the aggressiveness of the disease (indicated by IPSS/DIPSS risk category) and not to the JAK2V617F mutation. Similarly, we found that PKC levels are significantly greater in high vs low/intermediate risk patients, leading us to speculate that PKC can be utilized as a marker of high disease burden and a more aggressive disease.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
