Design and Application of a Novel PNA Probe for the Detection At Single Cell Level of JAK2V617F Mutation

• Published in: Blood 2012-11, Vol.120 (21), p.1761-1761
• Main Authors: Cilloni, Daniela, Rosso, Valentina, Torti, Davide, Carnuccio, Francesca, Serra, Anna, Nicoli, Paolo, Gaidano, Valentina, Campia, Valentina, Signorino, Elisabetta, Calabrese, Chiara, Carturan, Sonia, Favole, Alessandra, Saglio, Giuseppe, Frassoni, Francesco, Bracco, Enrico
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V120.21.1761.1761

Abstract 1761 Mutation(s) of the JAK2 gene (V617F) has been described in a significant proportion of Philadelphia negative MPN patients and its detection is now a cornerstone in the diagnostic algorithm. The method most frequently used for measuring the distribution of cell populations is based on JAK2 sequencing and Q-PCR. Therefore the chance of distinguishing the JAK2 wild type or mutated population at the single-cell level still represents a challenge. The aim of the study was to developed a novel assay based on peptide nucleic acid (PNA) technology coupled to immuno-fluorescence microscopy (PNA-FISH) for the specific detection at a single cell level of JAK2-mutation thus improving both the diagnostic resolution and the study of clonal prevalence. We designed a fluorescently-labelled PNA probe, coupled to FISH technology, which allows to distinguish with a high degree of specificity between CD34+ progenitor stem cells harbouring the mutated (V617F) or the wild type form of JAK2. CD34+ cells were enriched from 24 PV patients (5 of them were selected for the absence of JAK2V617F), 13 PMF (10 with the mutation and 3 JAK2 wild type) and 6 ET patients (2 of them were wild type). In addition 20 BM samples were collected from healthy donors and used as control. Patients were a priori found to be either positive or negative for the JAK2V617F mutation by standard sequencing and by Q-PCR. CD34+ progenitors cells were enriched by MACS and then cytospun onto slides and hybridized with human species-specific fluorescinated 15 base pairs (bp)-long oligo-PNA, specifically recognizing the human JAK2 sequence surrounding the nucleotide at position 1849, which is responsible for the V617F substitution (JAK2V617F/PNA). Slides were analyzed by fluorescence confocal microscopy. The analysis revealed that among JAK2V617F PV patients the distribution pattern is fairly similar to that reported by Scott and colleagues in 2006 analyzing JAK2V617F in colonies. We found, with a rather wide variability occurring among patients, a percentage of mutated CD34+ cells ranging from 40% to 100% in PV patients, from 15% to 80% in ET and from 25% to 100% in PMF. These findings are in agreement with previous data reporting that a variable proportion of progenitors from patients affected by JAK2V617F positive PV are capable of generating JAK2V617F negative colonies. In addition these data indicate that fluorescinated JAK2V617F/PNA probe displays a very high specificity towards a single base-