IN VITRO CHARACTERISTICS OF HEMATOPOIETIC PROGENITORS FROM PRIMARY MYELOFIBROSIS PATIENTS CORRELATE WITH IPSS/DIPSS RISK CATEGORY

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.