title: The Droplet Digital PCR: A New Valid Molecular Approach for the Assessment ofB-RAFV600E Mutation in Hairy Cell Leukemia.
abstract: Hairy cell leukemia (HCL) is a chronic lymphoproliferative B-cell disorder where theB-RAFV600E mutation has been recently detected, as reported for solid neoplasias but not for other B-cell lymphomas. The digital droplet PCR (dd-PCR) is a molecular technique that, without standard references, is able to accurately quantitate DNA mutations. ddPCR could be an useful instrument for the detection of theB-RAFV600E mutation in HCL, where the minimal residual disease monitoring is fundamental for planning a patients-targeted treatment in the era of new anti-CD20 and anti-RAF compounds. This retrospective study enrolled 47 patients observed at the Hematology Unit of the University of Pisa, Italy, from January 2005 to January 2014: 27 patients were affected by "classic" HCL, two by the variant HCL (vHCL), and 18 by splenic marginal zone lymphoma (SMZL). The aim of the study was to compare dd-PCR to "classic" quantitative PCR (QT-PCR) in terms of sensitivity and specificity and to demonstrate its possible use in HCL. Results showed that: (1) the sensitivity of dd-PCR is about half a logarithm superior to QT-PCR (5 × 10-5vs. 2.5 × 10-4), (2) the specificity of the dd-PCR is comparable to QT-PCR (no patient with marginal splenic lymphoma or HCL variant resulted mutated), (3) its high sensitivity would allow to use dd-PCR in the monitoring of MRD. At the end of treatment, among patients in complete remission, 33% were still MRD-positive by dd-PCR versus 28% by QT-PCR versus 11% by the evaluation of the B-cell clonality, after 12 months, dd-PCR was comparable to QT-PCR and both detected theB-RAFmutation in 15% of cases defined as MRD-negative by IgH rearrangement. Moreover, (4) the feasibility and the costs of dd-PCR are comparable to those of QT-PCR. In conclusion, our study supports the introduction of dd-PCR in the scenario of HCL, also during the follow-up.
authors: Section of Hematology, Department of Clinical and Experimental Medicine, University of Pisa Pisa, Italy.
authors: Francesca
authors: F
authors: Guerrini
authors: UO hematology, Azienda Ospedaliero-Universitaria Pisana Pisa, Italy.
authors: Matteo
authors: M
authors: Paolicchi
authors: Section of Hematology, Department of Clinical and Experimental Medicine, University of Pisa Pisa, Italy.
authors: Francesco
authors: F
authors: Ghio
authors: UO hematology, Azienda Ospedaliero-Universitaria Pisana Pisa, Italy.
authors: Elena
authors: E
authors: Ciabatti
authors: Section of Hematology, Department of Clinical and Experimental Medicine, University of PisaPisa, Italy; GeNoMEC, University of SienaSiena, Italy.
authors: Susanna
authors: S
authors: Grassi
authors: Section of Hematology, Department of Clinical and Experimental Medicine, University of Pisa Pisa, Italy.
authors: Serena
authors: S
authors: Salehzadeh
authors: UO hematology, Azienda Ospedaliero-Universitaria Pisana Pisa, Italy.
authors: Giacomo
authors: G
authors: Ercolano
authors: UO hematology, Azienda Ospedaliero-Universitaria Pisana Pisa, Italy.
authors: Maria R
authors: MR
authors: Metelli
authors: Clinical Pharmacology and Pharmacogenetic Unit, Department of Clinical and Experimental Medicine, University of Pisa Pisa, Italy.
authors: Marzia
authors: M
authors: Del Re
authors: Section of Hematology, Department of Clinical and Experimental Medicine, University of Pisa Pisa, Italy.
authors: Lorenzo
authors: L
authors: Iovino
authors: Section of Pathology, Department of Translational Medicine and New Technologies, University of Pisa Pisa, Italy.
authors: Iacopo
authors: I
authors: Petrini
authors: Section of Hematology, Department of Clinical and Experimental Medicine, University of Pisa Pisa, Italy.
authors: Giovanni
authors: G
authors: Carulli
authors: UO hematology, Azienda Ospedaliero-Universitaria Pisana Pisa, Italy.
authors: Nadia
authors: N
authors: Cecconi
authors: UO hematology, Azienda Ospedaliero-Universitaria Pisana Pisa, Italy.
authors: Martina
authors: M
authors: Rousseau
authors: UO hematology, Azienda Ospedaliero-Universitaria Pisana Pisa, Italy.
authors: Giulia
authors: G
authors: Cervetti
authors: Section of Hematology, Department of Clinical and Experimental Medicine, University of Pisa Pisa, Italy.
authors: Sara
authors: S
authors: Galimberti
keywords: B-RAF; V600E; digital droplet PCR; hairy cell leukemia; immunoglobulins rearrangement; minimal residual disease