pSTAT3/pSTAT5 Signaling Patterns in Molecularly Defined Subsets of Myeloproliferative Neoplasms.

BCR/ABL1-negative myeloproliferative neoplasms (MPNs) are characterized by recurrent mutations in JAK2, CALR, and MPL, each of which has been reported to alter JAK/STAT signaling pathways. This report characterizes JAK/STAT signaling patterns in molecularly defined subsets of MPN utilizing immunohistochemistry for pSTAT3 and pSTAT5. Analysis of 30 BCR/ABL1-negative, nonpolycythemia vera MPN identified 15 (50%) with JAK2 V617F, 2 with MPL mutations (7%), and 8 with CALR mutations (27%). All mutations were mutually exclusive, except for 1 case with concurrent JAK2 V617F and CALR mutations. pSTAT3 staining in megakaryocyte nuclei was found in 4 cases (13%) and was not significantly associated with mutation status. pSTAT5 staining in megakaryocyte nuclei was found in 16 cases (53%), as was significantly associated with JAK2 V617F versus CALR mutation (P=0.009). Erythroid staining for pSTAT5 was seen exclusively in "triple-negative (TN)" cases lacking JAK2 V617F, MPL, and CALR mutations (P=0.006, TN vs. other genotypes), and pSTAT5 staining in megakaryocyte nuclei was seen in 2 TN cases. pSTAT5 staining in TN MPN suggests that other unknown abnormalities in this pathway may contribute to the pathogenesis of these cases. Furthermore, the demonstration of distinct STAT staining patterns in molecularly defined MPN suggests that these mutations result in divergent signaling events that may contribute to the biological and prognostic differences in these molecular subsets of MPN.

Thrombocytosis and STAT5 activation in chronic myelogenous leukaemia are not associated with JAK2 V617F or calreticulin mutations.

AIMS: Marked thrombocytosis is uncommon in chronic myelogenous leukaemia (CML) but may be associated with mutation of JAK2 V617F, calreticulin (CALR) and/or phospho-STAT5 (p-STAT5) activation in other myeloproliferative neoplasms (MPNs), particularly essential thrombocythaemia (ET). We investigated the JAK2 V617F, CALR and STAT5 activation status in patients with CML and thrombocytosis (CML-T) that mimicked ET, trying to identify a common mechanism for thrombocytosis in MPN. METHODS: Blood and bone marrow morphological findings were reviewed from seven CML-T, four otherwise typical CML and one CML in blast phase. All cases were analysed for BCR-ABL1, JAK2 V617F and CALR exon 9 mutation and p-STAT5 expression. RESULTS: Four of seven cases of CML-T had marked thrombocytosis (>1000×10(9)/L). Eleven of 12 cases had megakaryocyte morphology typical for CML. All cases were BCR-ABL1 positive. Eleven of 12 cases were negative for JAK2 V617F, while STAT5 was activated in six of seven CML-T and in four of five CML cases. No case had a detectable CALR exon 9 mutation. One case of CML developed ET-like morphology and had JAK2 V617F detected while in molecular remission for CML. CONCLUSIONS: Detection of BCR-ABL1 is critical in the distinction of ET from CML. Thrombocytosis and STAT5 activation in CML-T are not consistently associated with CALR exon 9 or JAK2 V617F mutation.

Refractory anemia with ringed sideroblasts associated with marked thrombocytosis (RARS-T), another myeloproliferative condition characterized by JAK2 V617F mutation.

JAK2 V617F mutation recently was identified as a pathogenic factor in typical chronic myeloproliferative diseases (CMPD). Some forms of myelodysplastic syndromes (MDS) show a significant overlap with CMPD (classified as MDS/MPD), but the diagnostic assignment may be challenging. We studied blood or bone marrow from 270 patients with MDS, MDS/MPD, and CMPD for the presence of JAK2 V617F mutation using polymerase chain reaction, sequencing, and melting curve analysis. The detection rate of JAK2 V617F mutants for polycythemia vera, chronic idiopathic myelofibrosis, and essential thrombocythemia (n = 103) was similar to the previously reported results. In typical forms of MDS (n = 89) JAK2 V617F mutation was very rare (n = 2). However, a higher prevalence of this mutation was found in patients with MDS/MPD-U (9 of 35). Within this group, most of the patients harboring JAK2 V617F mutation showed features consistent with the provisional MDS/MPD-U entity refractory anemia with ringed sideroblasts and thrombocytosis (RARS-T). Among 9 RARS-T patients, 6 showed the presence of JAK2 V617F mutation, and in 1 patient without mutation, aberrant, positive phospho-STAT5 staining was seen that is typically present in association with JAK2 V617F mutation. In summary, we found that RARS-T reveals a high frequency of JAK2 V617F mutation and likely constitutes another JAK2 mutation-associated form of CMPD.

Identification of the JAK2 V617F mutation in chronic myeloproliferative disorders using FRET probes and melting curve analysis.

We developed and validated a real-time polymerase chain reaction assay using fluorescent hybridization probes and melting curve analysis to identify the JAK2 V617F mutation, which is implicated in a substantial proportion of chronic myeloproliferative disorders (CMPDs). DNA from 161 samples was isolated from peripheral blood granulocytes and formalin-fixed bone marrow clot sections in patients with CMPDs and without myeloproliferative disorders previously genotyped for the JAK2 V617F (G-->T) mutation, which included 114 wild types (GG) and 47 mutants (GT and TT). Melting curve analysis of these samples yielded 114 wild types, 42 heterozygotes, and 5 homozygotes showing 100% concordance. Analytic sensitivity of the assay for mutant DNA was 5% for the LightTyper (Roche Applied Sciences, Indianapolis, IN) and 10% for the LightCycler (Roche Applied Sciences). Consistent with earlier reports, 78% of the non-chronic myelogenous leukemia CMPD patients and 8% of non-CMPD patients displayed this mutation. This study demonstrates that clinical genotyping of the JAK2 V617F mutation can be performed by melting analysis using both freshly isolated and formalin-fixed tissues.

Lysophosphatidylcholine regulates human microvascular endothelial cell expression of chemokines.

The role of lysophosphatidylcholine (LPC) in the induction of MCP-1, IL-8 and RANTES, which are chemotactic factors to monocytes, neutrophils and lymphocytes, respectively, by human vascular endothelial cells (EC), was examined. LPC induced the expression of MCP-1 and IL-8 in a concentration- and time-dependent manner in microvascular EC (MVEC) and in large vessel EC from aorta, pulmonary artery and umbilical vein. LPC also induced RANTES in MVEC but not in large vessel EC. Signaling pathways responsible for LPC induction of chemokines were examined in MVEC. LPC and TNFalpha, a cytokine secreted in sites of inflammation, additively stimulated RANTES expression. LPC did not augment TNFalpha induction of MCP-1 or IL-8. A platelet-activating factor receptor antagonist (BN52021) failed to block LPC induction of MVEC chemokines, but the G(i)-protein inhibitor pertussis toxin partially blocked LPC induction of RANTES and IL-8. LPC activated multiple kinases in MVEC; it increased the phosphorylation of ERK1/2, AKT and p38 MAP kinase in a time-dependent manner. An inhibitor of the MAPK/ERK pathway, PD98059, blocked the phosphorylation of ERK1/2 and RANTES induction by LPC, but augmented IL-8 induction. LY294002, a specific inhibitor of phosphoinositide 3 kinase (PI3 kinase), blunted the phosphorylation of AKT and inhibited LPC induction of RANTES more strongly than IL-8. Inhibition of p38 MAP kinase pathway by SB202190 also blocked LPC-induced expression of IL-8 and RANTES. Our results suggest that LPC induction of chemokines in MVEC is distinct from that in large vessel EC, and required the activities of MAP kinases and PI3 kinase for the induction of RANTES and IL-8. We speculate that the presence of LPC, a bioactive lipid product of phospholipase A(2) (PLA(2)) and a constituent of oxidized low-density lipoprotein, can differentially influence the chemotaxis of particular leukocyte subpopulations during inflammation.

Membrane microviscosity regulates endothelial cell motility.

Endothelial cell (EC) movement is an initiating and rate-limiting event in the neogenesis and repair of blood vessels. Here, we explore the hypothesis that microviscosity of the plasma membrane (PM) is a key physiological regulator of cell movement. Aortic ECs treated with membrane-active agents, such as alpha-tocopherol, cholesterol and lysophospholipids, exhibited a biphasic dependency on membrane microviscosity, in which moderate increases enhanced EC migration, but increases beyond a threshold markedly inhibited migration. Surprisingly, angiogenic growth factors, that is, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), also increased membrane microviscosity, as measured in live cells by fluorescence recovery after photobleaching (FRAP). The localization of Rac to the PM was modified in cells treated with membrane-active agents or growth factors, suggesting a molecular mechanism for how membrane microviscosity influences cell movement. Our data show that angiogenic growth factors, as well as certain lipophilic molecules, regulate cell motility through alterations in membrane properties and the consequent relocalization of critical signalling molecules to membranes.