A High Sensitivity Detection Technique Reveals JAK2-V617F Mutation in Additional 20% of Patients with Essential Thrombocytemia, but Not in Patients with Primary Myelofibrosis, Considered Negative with a Conventional ASO-PCR
Authors: Bertolotti Francesca , Pozzi Sarah , Ulivi Massimo , Podestà Marina , Imperiale Davide , Rosti Vittorio , Frassoni Francesco 
Published: April 2020

Abstract
Polycytemia Vera (PV), Essential Thrombocytemia (ET) and Primary Myelofibrosis (PMF) are defined as the major Philadelphia-negative myeloproliferative disorders (MPD). The JAK2-V617F gene mutation is a common feature of MPD but it is present in a variable proportion in patients: 95% of PV, 23 to 57% of ET and 30 to 57% of PMF patients. At the present, the most important role of JAK2-V617F mutation testing, over all during the initial evaluation of MPD patients, is that it can definitively confirm the diagnosis. In fact, its specificity permits to discriminate the considerable clinical overlap between reactive cellular proliferations and clonal myeloproliferative disorders. Moreover, because V617F is an acquired mutation that can be present in a small proportion of granulocytic populations, especially for ET, a highly sensitive detection method is essential. In fact, phenotypic effects ascribed to the presence of the JAK2-V617F mutation have been reported over all in studies with ET patients. Different sensitivity of various assays methods partially accounts for the wide range of mutation frequencies reported in literature for ET and PMF. Thus, to try to increase the sensitivity of JAK2 mutational status detection, we developed a Real-Time PCR technique that enhances allele discrimination between mutant and wild-type sequence. A Locked Nucleic Acid (LNA) clamping oligomer was added to the PCR reaction solution. In this macromolecules, the ribose moiety is modified with an extra bridge connecting the 2′ and 4′ carbons. The bridge “locks” the ribose in the 3′-end structural conformation. It binds preferentially the JAK2 wild type sequence preventing from polymerase elongation. The melting curve was used to analyse amplification products, avoiding post-PCR processing and supplying the diagnostic information immediately at the end of the amplification. 236 genomic DNA samples from healthy donors, ET and PMF patients were tested for JAK2-V617F mutation detection with a conventional allelespecific PCR (ASO-PCR, sensitivity: 1–3%). All samples were re-evaluated with a seminested PCR protocol and Real-Time PCR based method in order to improve the sensitivity. The cell line HEL DNA dilutions were used to assess the semi-nested PCR and the Real-Time PCR assay sensitivity level (0,1%). The frequency of JAK2-V617F point mutation increased from 52 to 72% in 168 ET samples. All results obtained with the new technique were confirmed by the semi-nested PCR protocol. In 58 PMF samples tested, 50% of patients were positive for the mutation in ASO-PCR while 55% resulted positive with the semi-nested protocol confirmed by the Real-Time PCR test. The high sensitivity in the JAK2-V617F mutation detection obtained with the semi-nested and the Real-Time PCR revealed that 20% of patients affected by ET and considered JAK2-V617F negative with a conventional ASO-PCR, were effectively positive. The comparison between three different analytical methods revealed that in ET but not in PMF patients, the mutation can be present only in a small proportion of granulocytic populations. Thus, since the prognostic relevance of V617F allele in ET, unlike in PMF, seems to be relevant, our high sensitive detection protocol can be effective for a correct molecular characterization and a diagnostic classification. The finding that a large proportion of ET patients bears a very small amount of JAK2-V617F mutated hematopoiesis further emphasizes the problem of the role of this small clone and warrants longitudinal analysis to understand whether this proportion remains stable or expands over time.