Design and Evaluation of a Real-Time PCR Assay for Quantification of JAK2 V617F and Wild-Type JAK2 Transcript Levels in the Clinical Laboratory

Abstract

The somatic mutation JAK2 V617F is associated with BCR-ABL1-negative myeloproliferative neoplasms. Detection of this mutation aids diagnosis of these neoplasms , and quantification of JAK2 V617F may provide a method to monitor response to therapy. For these reasons , we designed a clinical assay that uses allele-specific PCR and real-time detection with hydrolysis probes for the quantification of JAK2 V617F , wild-type JAK2 , and GAPDH transcripts. Mutant and wild-type JAK2 were quantified by using external plasmid standards that contain the relevant JAK2 V617F or JAK2 sequence , respectively. We tested 55 peripheral blood specimens from patients with suspected myeloproliferative neoplasms and 55 peripheral blood specimens from patients not known to have myeloproliferative neoplasms. Low-level , nonspecific amplification was detected in reactions containing a high copy number of plasmid standards and in specimens from patients not known to have myeloproliferative neoplasms , necessitating the use of a laboratory-established mutant to wild-type cutoff. The limit of detection established by using cell line dilutions is 0.1% , and this method identified three JAK2 V617F-positive patients who were not detected by a less sensitive method. The assay characteristics and our initial evaluation indicate this method can be used for the detection and quantification of JAK2 V617F, which should be useful for diagnosis of myeloproliferative neoplasms and potentially for monitoring minimal residual disease in future trials of therapies targeted to myeloproliferative neoplasms.

Establishing optimal quantitative-polymerase chain reaction assays for routine diagnosis and tracking of minimal residual disease in JAK2-V617F-associated myeloproliferative neoplasms: a joint European LeukemiaNet/MPN&MPNr-EuroNet (COST action BM0902) study

“…Importantly, it has become clear that the level of JAK2 -V617F detectable in the PB of some patients with bona fide MPNs can be as low as 1% at the time of diagnosis, 6, 7, 8, 9, 10 which is below the detection limit of some standard diagnostic methods, such as pyrosequencing. 10, 11 Since the discovery of the JAK2 -V617F mutation, numerous qPCR assays have been designed to detect this abnormality, 10, 12, 50 which we have shown vary markedly in their sensitivity and specificity. Previous studies have highlighted the potential of qPCR assessment of JAK2 -V617F allele burden to predict outcome following allogeneic transplant in PMF, 26, 27, 28, 29 with reduction of JAK2 -V617F to <1% by 1 month 30 or achievement of PCR negativity in the PB by 6 months post-transplant, distinguishing patients at markedly differing risk of subsequent disease relapse.…”

Revisiting diagnostic criteria for myelodysplastic/myeloproliferative neoplasms with ring sideroblasts and thrombocytosis: Borderline cases without anemia exist

“…The same primers were used for bidirectional Sanger sequencing. JAK2 V617F sequencing was performed as previously described …”

In situ detection of JAK2V617F within viable hematopoietic cells using gold nanoparticle technology

“…First‐strand complementary DNA (cDNA) was synthesized from total 20 ng RNA with SCRIPT cDNA Synthesis‐Kit (Jena Bioscience) according to the manufacturer's instructions. The sequences of the primers and PCR conditions were applied as previously that uses allele‐specific PCR and real‐time detection with hydrolysis probes for the quantification of JAK2 V617F and JAK2 wt transcript levels . The fold change in the expression of the JAK2 V617F and JAK2 wt relative to the control cells (HEL and K562) and housekeeping gene GAPDH were studied.…”

“…After performing titrations, the optimal concentrations were applied as following: Background Control -Cy3+Uptake-Cy5 8 µL, Beta-Actin-Cy3 32 µL, Beta-Actin- The sequences of the primers and PCR conditions were applied as previously that uses allele-specific PCR and real-time detection with hydrolysis probes for the quantification of JAK2V617F and JAK2wt transcript levels. 9 The fold change in the expression of the JAK2V617F and JAK2wt relative to the control cells (HEL and K562) and housekeeping gene GAPDH were studied. The detailed formulation of 2 -ΔΔCT calculation method is reported elsewhere.…”