Superiority of Droplet Digital PCR Over Real-Time Quantitative PCR for JAK2 V617F Allele Mutational Burden Assessment in Myeloproliferative Neoplasms: A Retrospective Study.

Francesco La Rocca,Simona Laurino,Vitina Grieco,Vitalba Ruggieri,Oreste Villani,Pietro Zoppoli,Giovanni Calice,Emanuela Zifarone,Anna Marinaccio,Sabino Russi,Maria Iole Natalicchio,Pellegrino Musto,Francesco Albano,Geppino Falco

doi:10.3390/diagnostics10030143

Abstract

JAK2V617F mutational status is an essential diagnostic index in myeloproliferative neoplasms (MPNs). Although widely used for detection of JAK2 V617F mutation in peripheral blood (PB), sensitive real-time quantitative PCR (qPCR) presents some methodological limitations. Recently, emerging alternative technologies, like digital droplet PCR (ddPCR), have been reported to overcome some of qPCR’s technical drawbacks. The purpose of this study was to compare the diagnostic utility of ddPCR to qPCR for JAK2 V617F detection and quantification in samples from MPNs patients. Sensitivity and specificity of qPCR and ddPCR in the detection of the mutation were assessed by using a calibrator panel of mutated DNA on 195 JAK2 positive MPN samples. Based on our results, ddPCR proved to be a suitable, precise, and sensitive method for detection and quantification of the JAK2 V617F mutation.

Highlights

Detection of the JAK2 V617F variant in patients with Philadelphia-negative myeloproliferative neoplasms (MPNs), including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) has changed the MPN molecular diagnostics routine over recent years [1,2].Diagnostics 2020, 10, 143; doi:10.3390/diagnostics10030143 www.mdpi.com/journal/diagnosticsThe mutation occurs in more than 90% of PV cases and in about 50–60% of those with ET or PMF.Interestingly, several studies showed that different MPNs phenotypes correlate with the mutant allelic burden [3,4]
A JAK2 V617F allele burden may have prognostic significance as well, since it correlates with clinical endpoints in MPN patients [10]
Peripheral blood samples were collected from 195 JAK2-positive MPN patients at time of diagnosis and from 50 healthy controls at the Department of Hematology, IRCCS-CROB (Referral Cancer Center of Basilicata, Rionero in Vulture, Italy)

Summary

Introduction

Detection of the JAK2 V617F variant in patients with Philadelphia-negative myeloproliferative neoplasms (MPNs), including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) has changed the MPN molecular diagnostics routine over recent years [1,2]. Several studies showed that different MPNs phenotypes correlate with the mutant allelic burden [3,4]. Patients with ET, for instance, have the lowest allele burden (90%) [5,6]. The presence of an allele burden greater than 50% increases the probability of an overt PV or myelofibrotic evolution [7,8,9]. A JAK2 V617F allele burden may have prognostic significance as well, since it correlates with clinical endpoints in MPN patients [10]

Objectives

Methods

Conclusion