12th Congress of the European Hematology ... - Haematologica

Aims. Aim of this study was to predict the presence of the JAK2V617F
mutation by a simple flow cytometry analysis of LAP expression. Methods.
Patients with Polycythemia Vera (PV, n=146), Essential Thrombocythemia
(ET, n=329) and Idiopathic Erythrocytosis (IE, n= 56) were
selected among those regularly followed up in our out patient clinic.
Healthy volunteers were selected from those referred to our institutional
blood bank for blood donation (n=40). The JAK2V617F mutation
screening was performed by allele specific Polymerase Chain Reaction.
The surface expression of Leukocyte Alkaline Phosphatase was quantified
as arbitrary units of Mean Fluorescence Intensity (MFI) by flow
cytometry using the lB12.1 (IgG1) antibody and a FACS analyzer. PRV-
1 gene expression was performed by Real Time Quantitative PCR (RQ-
PCR) using RNA from purified granulocytes and a 7700 ABI platform.
The discriminant ability of LAP and PRV-1 expression in identifying the
JAK2V617F mutation, was performed by a Receiving Operating Characteristic
(ROC) analysis, using each value of the rating variable (LAP and
PRV-1) as a possible discriminant cut-point. Results. The mutational
analysis for JAK2V617F showed that 132/146 PV patients (90%) and
174/329 ET patients (53%) were mutated, while none of the IE patients
proved positive. The LAP expression detected on granulocytes isolated
from ET patients lacking the JAK2V617F mutation was not different
from that of normal donors and IE patients. On the contrary, a progressive
and highly significant increase of the LAP Mean Fluorescence Intensity
was registered in ET and PV patients carrying a heterozygous or
homozygous JAK2V617F mutation (Figure1 A). A ROC curve analysis
allowed to identify a LAP MFI value of 90 (AUC=0.8295; 95% confidence
interval: 0.7965-0.8625) as the optimal cut-off to discriminate,
with a good score of sensitivity (76.87%) and specificity (75.76%),
patients carrying a JAK2 wild type gene as compared to those with a
JAK2V617F mutation (Figure 1 B). The presence of a homozygous
JAK2V617F mutation strongly correlates with the highest values of LAP
expression (Figure 1 C). The simultaneous RQ-PCR evaluation of PRV-
1 gene expression in peripheral blood granulocytes provided evidence of
a remarkable linear correlation of results obtained by these assays (βcoefficient:
399.85, p