Design and Application of a Novel PNA Probe for the Detection At Single Cell Level of JAK2^V617F Mutation

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 JAK2^V617F), 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 JAK2^V617F 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 (JAK2^V617F/PNA). Slides were analyzed by fluorescence confocal microscopy.

The analysis revealed that among JAK2^V617F PV patients the distribution pattern is fairly similar to that reported by Scott and colleagues in 2006 analyzing JAK2^V617F 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 JAK2^V617F positive PV are capable of generating JAK2^V617F negative colonies. In addition these data indicate that fluorescinated JAK2^V617F/PNA probe displays a very high specificity towards a single base-pair mismatch. Interestingly, when evaluating the presence of JAK2^V617Fpositive cells collected from 3 JAK2 wild type subjects defined by sequencing and by Q-PCR, we identified a small percentage of cells positive for the JAK2^V617F/PNA staining. However, this apply only to patients with PV but not to PMF and ET patients. Quantitatively, this percentage did not exceed 3% of the CD34+ cell population indicating a high level of sensitivity of the procedure since the PNA/FISH technique may detect JAK2^V617F-positive cells within a CD34+ population isolated from patients reported as JAK2^V617Fnegative by standard approaches. Importantly, the lack of positivity detected in CD34 positive cells from 20 healthy subjects demonstrates a high specificity of this method.

JAK2V617F/PNA-FISH method displays high specificity and reliability in discriminating cell subpopulations harbouring the JAK2^V617F mutation. In addition, it allows to analyze the CD34+ population at the single cell level, avoiding the time consuming analysis of hematopoietic colonies. In addition, this approach allows to monitor longitudinally the evolution of a defined cell population over time in MPNs and the characterization of the CD34+ compartment in patients with MPNs which still represents a challenging issue.

No relevant conflicts of interest to declare.