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-pair mismatch. Interestingly, when evaluating the presence of JAK2V617Fpositive 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 JAK2V617F/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 JAK2V617F-positive cells within a CD34+ population isolated from patients reported as JAK2V617Fnegative 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 JAK2V617F 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.
Asterisk with author names denotes non-ASH members.