We determined the allelic frequency of the JAK2-V617F mutation in DNA and assessed the expression levels of the mutant and wild-type JAK2 mRNA in granulocytes from 60 patients with essential ...thrombocythemia (ET) and 62 patients with polycythemia vera (PV) at the time of diagnosis. Using allele-specific quantitative polymerase chain reaction (qPCR), we detected JAK2-V617F in 75% of ET and 97% of PV at diagnosis. The total JAK2 mRNA levels were elevated in ET, PV, and secondary and idiopathic erythrocytosis, suggesting that hyperactive hematopoiesis alters JAK2 expression. The expression levels of JAK2-V617F mRNA were variable but strongly correlated with the allelic ratio of JAK2-V617F determined in DNA. Thus, differences in JAK2-V617F expression, markedly lower in ET than in PV, reflected different percentages of granulocytes carrying the mutation. Moreover, allelic ratios higher than 50% JAK2-V617F, indicating the presence of granulocytes homozygous for JAK2-V617F, were found in 70% of PV at diagnosis but never in ET.
Abstract 2820
Mpl, the receptor for thrombopoietin (Tpo), activates Jak2. Mpl and Jak2 are expressed both on platelets and on hematopoietic progenitors, where they initiate critical cell survival and ...proliferation signals in response to Tpo. Thus, interfering with the “Mpl/Jak2 couple” is likely to alter myelopoiesis. Indeed mutations in JAK2 or MPL are characteristic of myeloproliferative neoplasms (MPN). However, platelet Mpl expression is frequently low in MPN, including MPN with no mutation of JAK2.
To determine whether expression of wild type Jak2 (Jak2wt) is altered in MPN, and whether changes in expression of Jak2wt affect Mpl expression.
Mpl and Jak2 protein expression was analysed by immunoblotting in platelet and cell lysates. Expression of JAK2 wt and JAK2 V617F was also studied at the mRNA level using quantitative RT-PCR. Transient transfections of HEL and UKE-1 cells were used to study the effects of Jak2wt on Mpl and of MPL on Jak2wt and Jak2V617F. We also studied the cellular localization of Jak2wt, Jak2V617F and Mpl, expressed as chimeric proteins fused to Visible Fluorescent Proteins (VFP, derivatives of GFP) or detected by immunofluorescence methods.
Following SDS-PAGE and western blotting of normal platelet proteins, Mpl was typically found in two isoforms: the long, mature 84 kDa isoform (Golgi-processed, glycosylated, functional, Tpo-binding form expressed at the cell surface) and the short, 74 kDa isoform (immature Mpl). Four subgroups of MPN patients were defined according to platelet Mpl patterns: pattern 1, mature and immature Mpl expressed at comparable levels, typical of healthy donors and found in 37% of MPN tested; pattern 2, mature Mpl only, typical of reactive states, associated with high JAK2 wt expression and observed in 15% of MPN; pattern 3: immature Mpl only; and pattern 4: no Mpl. Patterns 3 and 4 were observed in 48% of MPN and were associated with low JAK2 wt levels (<200 copies/100 ABL copies). JAK2 wt mRNA levels varied widely in MPN and Mpl pattern was correlated with levels of JAK2 wt, but not JAK2 V617F, mRNA. Blotting studies performed on cell lysates from cultured lines homozygous for JAK2V617F (HEL, UKE-1) detected mainly the 74 kDa immature form of Mpl.
Transfection of JAK2wt in HEL and UKE-1 cells, alone or together with MPL, increased the yield of mature Mpl. Fluorescence studies of HEL cells revealed that both endogenous Mpl and the Mpl-VFP chimeric proteins were largely restricted to an intracellular pool, without significant co-localization with Jak2V617F. In contrast, significant levels of transfected Mpl-VFP reach the plasma membrane in K562 cells that express only Jak2wt; co-localization of Jak2 and Mpl in these cells was significant.
JAK2 wt and Mpl levels are linked and both are frequently low in MPN, including JAK2 V617F-negative MPN. Co-localization of Mpl and Jak2V617F is poor whereas Mpl and Jak2wt are strongly co-localized, consistent with constitutive association and indicating a role for Jak2wt as chaperone along the exocytic or recycling pathways. Future work will seek to determine the intracellular location of aberrantly trafficked Mpl and establish if other mutations found in MPN are linked to defects in the expression or trafficking of Mpl.
No relevant conflicts of interest to declare.
The diagnosis of polycythemia vera (PV) is based on clinical and biological criteria defined by either the Polycythemia Vera Study Group (PVSG) or the World Health Organization (WHO). Both the PVSG ...and WHO PV criteria have proved helpful and are extensively used, yet diagnostic strategies and scheduling of biological investigations vary. We assessed the value of measuring serum erythropoietin (Epo) as a first intention diagnostic test in patients with absolute erythrocytosis (AE).
Serum and bone marrow (BM) samples of 241 patients with a suspicion of erythrocytosis were collected in 8 hospital centers. One hundred and ninety had an absolute erythrocytosis (116 had PV, 66 had secondary erythrocytosis and 4 had idiopathic erythrocytosis). Serum Epo was assayed (ELISA) in 186. Statistical analysis (ROC curves) was used to define serum Epo thresholds that were specific for PV and secondary erythrocytosis and to analyze the diagnostic value of a low or high serum Epo level.
A large majority of PV patients (87% or 101/116) had a serum Epo level below the normal range in healthy patients (3.3 IU/L), giving this value a specificity of 97% with a 97.8% positive predictive value for the diagnosis of PV. Statistical analysis (ROC curves) defined two thresholds allowing a specific and direct diagnosis of 65.6% (65/99) of untreated PV (Epo < 1.4 IU/L) and 19.7% (13/66) of those with secondary erythrocytosis (Epo > 13.7 IU/L).
Based on these data, we propose that measurement of serum Epo level, a simple, reliable and inexpensive test, should be considered as a first intention diagnostic test for patients with absolute erythrocytosis.
The predictive values of common biological criteria for the diagnosis of polycythemia vera were studied in a cohort of patients with high hematocrit. We found JAK2V617F and erythropoietin assays were ...the most relevant first tests. Classification of patients according to their JAK2V617F status and erythropoietin levels facilitated the choice of further diagnostic investigations.
A high number of circulating CD34+ cells has been advocated to distinguish primary myelofibrosis from other Philadelphia-negative myeloproliferative neoplasms. We re-evaluated the diagnostic interest ...of measuring circulating CD34+ cells in 26 healthy volunteers and 256 consecutive patients at diagnosis for whom a myeloproliferative neoplasm was suspected. The ROC curve analysis showed that a number of CD34+ <10/μl excludes the diagnosis of primary myelofibrosis with a sensitivity of 97 % and a specificity of 90 % (area under the curve: 0.93 0.89–0.98;
p
< 0.001). Patients with PMF harboring a CALR mutation had more circulating CD34+ cells than patients with either a JAK 2 or MPL mutation (
p
= 0.02 and
p
< 0.01, respectively). These results suggest that this fast, simple, non-invasive, and standardized test is of particular interest to exclude the diagnosis of primary myelofibrosis.
Objective Previous studies have suggested that two subtypes of essential thrombocythemia (ET) could be separated on the basis of their JAK2 status (V617F–positive or V617F–negative), with a continuum ...between V617F–positive ET and polycythemia vera (PV). Nevertheless, increasingly contradictory data on the impact of JAK2 –V617F (presence and load) on ET phenotype highlight the need for further investigations. Materials and Methods We investigated the influence of JAK2– V617F on ET phenotype using mass spectrometry−based analysis of serum protein profiles of ET patients, comparatively with PV patients. Results V617F–positive ET and PV displayed significant differences in their serum protein profiles. Furthermore, JAK2 –V617F presence did not impact significantly the serum proteome of ET patients: we observed very few differences in serum protein profiles of V617F–positive and –negative ET. Reciprocally, clustering of ET patients on the basis of their serum protein profiles did not correlate with JAK2 –V617F presence. Finally, the JAK2 –V617F load did not influence serum apolipoprotein A–1 levels in ET, a previously validated marker of JAK2 –V617F allele burden in PV. Conclusion Serum proteome of ET patients was not influenced by the presence of JAK2 –V617F or by high V617F allelic ratio (up to 50%) suggesting that ET phenotype is, at best, only partially influenced by the JAK2 –V617F mutation.
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