Importance of JAK2 V617F Allele Burden in the Diagnosis of Myeloproliferative Diseases and Its Association to Age.

The aim of this study is to compare the diagnostic value of several methods, with different sensitivities, to detect JAK2 V617F mutation. Three new methods based on Real Time PCR to detect JAK2 V617F mutation were employed. The first two, based on hybridization probes and Peptide Nucleic Acid Probe (PNA) specific for wild allele; and the third employing Allele Specific Oligonucleotide (ASO) primers for JAK2 V617F mutant allele and MGB TaqMan probe. Healthy subjects (n 149) and patients with Essential Thrombocythemia (ET) (n 63), Polycythemia Vera (PV) (n 31), Secondary Thrombocythosis (ST) (n 38), Secondary Erytrocythosis (SE) (n 35) and other haematological malignancies (n 72) were included in the study. Results. Validity test study for JAK2 617 using HP PCR showed: A-. For PV patients: Sensitivity (Se) was 87.1%, Specificity (Sp) 100%, Positive Predictive value (PPV) 100%, and Negative Predictive Value (NPV) 97.9%. B-. For ET patients: Se was 61.3%, Sp 100%, PPV 100%, and NPV 93.9%. With JAK2 617 PNA PCR: A.- For PV patients: Se was 93.6%, Sp 97.8%, PPV 87.9%, and NPV 98.9%. B. - For ET patients: Se was 71%, Sp 95.7%, PPV 73.3%, and NPV 95.2%. With JAK2 V671F ASO quantitative PCR (JAK2 617 ASO qPCR): A.- For PV patients: Se was 93.5%, Sp 98.5%, PPV 90.6%, and NPV 98.9%. B.-For ET patients: Se was 80.6%, Sp 95.9%, PPV 75.8%, and NPV 96.7%. Cutoff point of 1% was established by ROC curves as mutation burden to distinguish PV or ET versus secondary causes. Three healthy subjects were positive (2%, 3/149) by JAK2 617 PNA PCR method. With JAK2 617 ASO qPCR, 16 healthy subjects would be positive (10%) if cutoff burden established in 0.1%. Significant differences were obtained when comparing Myeloproliferative Disease (MPD) patients over 40 years (n 64) to patients under 40 (n 15). JAK2 V617F median allele burden was 18.41% and 7.26% respectivily (p=0.016).

Conclusions: JAK2 617 HP and ASO qPCR are the best tests in differential diagnosis of Polyglobulias and Thrombocytosis. When JAK2 V617F allele burden is low, the best test for MPD diagnosis is JAK2 617 ASO qPCR. The number of JAK2 V617F mutated cells is higher in patients over 40, which leads to think that age may influence in tumour burden.