Quantitative Analysis of Wild Type and V617F JAK-2 Expression in Neutrophils of Polycythemia Vera and Essential Thrombocythemia Patients at Diagnosis.
Authors: Lippert Eric , Girodon Francois , Kralovics Robert , Boissinot Marjorie , Dobo Irene , Praloran Vincent , Boiret Nathalie , Skoda Radek C. , Hermouet Sylvie 
Published: April 2020

Abstract
The reported frequency of the V617F mutation of JAK-2 appears to vary depending on diagnosis, techniques and cohorts of patients, often already treated. Our aim was to determine the frequency of V617F JAK-2 in polycythemia vera (PV) and essential thrombocythemia (ET) at diagnosis and to quantify its expression at the mRNA level. Neutrophil cDNA was available for 113 MPD patients at diagnosis (56 PV, 57 ET) and 47 controls (26 secondary erythrocytosis (SE), 21 reactive thrombocytosis (RT)) from 5 centers; diagnosis was made following WHO criteria. All patients had a test of endogenous colony formation and a PRV-1 mRNA assay; all ET patients had a bone marrow biopsy in favour of ET. Reverse transcription followed by quantitative real-time PCRs (qPCRs) specific for wild type (WT) JAK-2, V617F JAK-2 and ABL were performed in duplicate; results were expressed as number of copies of WT or V617F JAK-2/100 copies of ABL. V617F JAK-2, never found in SE and RT, was expressed in 96.6% of PV and 70.2% of ET at diagnosis; in this series, the frequency of endogenous colony formation was 84.3% in PV and 78.6% in ET. All but 2 PV patients had elevated PRV-1 levels; one of the 2 PV with normal PRV-1 expression did not express V617F JAK-2. In the ET series, only 15/57 patients (26.3%) had elevated expression of PRV-1; all but one expressed V617F JAK-2. In both PV and V617F JAK-2-positive ET, PRV-1 and mutant JAK-2 expression levels were correlated (PV: r = 0.450, p = 0.0006; ET: r = 0.540, p = 0.0005). Levels of expression of both WT and V617F JAK-2 were significantly different in PV and in ET. ET neutrophils expressed an average of 144 V617F JAK-2 and 462 WT JAK-2 /100 ABL vs. 496 WT JAK-2/100 ABL for RT patients; the V617F/WT ratio for ET was always &lt; 0.7. In PV, two groups of patients were distinguished. The first group (the majority, 60.8%) expressed an average of 975 WT JAK-2 and 733 V617F JAK-2/100 ABL, hereby doubling the total expression of JAK-2 compared to SE (average: 821 WT JAK-2/100 ABL); the V617F/WT ratio of these patients was always &lt; 3. The second group (the minority, 39.2%) expressed low levels of WT JAK-2 (average: 247 copies/100 ABL) and high levels of V617F JAK-2 (average: 1515 copies/100 ABL), hence also doubling the amount of JAK-2 mRNA but with a V617F/WT ratio ≥ 3. Analysis of the cDNA of patients with a V617F/WT ratio &lt; 3 by sequencing showed both WT and mutated (G/T) sequences, whereas patients with a V617F/WT ratio ≥ 3 showed only the mutated (T/T) sequence. In conclusion, 96.6% of PV and 70.2% of ET at diagnosis expressed V617F JAK-2 as measured by allele-specific RT-qPCR. PV was characterized by moderate or high levels of V617F JAK-2 and global over-expression of JAK-2; in contrast, both WT and V617F-JAK-2 were expressed at low levels in ET. Such differences in V617F JAK-2 expression would be expected if clonal neutrophils harbouring the mutation represented a higher percentage of total neutrophils in PV than in ET; in addition, transcription of WT JAK-2 in neutrophils appears to be regulated differently in PV and in ET.