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

Abstract

Reliable detection of JAK2-V617F is critical for accurate diagnosis of myeloproliferative neoplasms (MPNs); in addition, sensitive mutation-specific assays can be applied to monitor disease response. However, there has been no consistent approach to JAK2-V617F detection, with assays varying markedly in performance, affecting clinical utility. Therefore, we established a network of 12 laboratories from seven countries to systematically evaluate nine different DNA-based quantitative PCR (qPCR) assays, including those in widespread clinical use. Seven quality control rounds involving over 21,500 qPCR reactions were undertaken using centrally distributed cell line dilutions and plasmid controls. The two best-performing assays were tested on normal blood samples (n=100) to evaluate assay specificity, followed by analysis of serial samples from 28 patients transplanted for JAK2-V617F-positive disease. The most sensitive assay, which performed consistently across a range of qPCR platforms, predicted outcome following transplant, with the mutant allele detected a median of 22 weeks (range 6-85 weeks) before relapse. Four of seven patients achieved molecular remission following donor lymphocyte infusion, indicative of a graft vs MPN effect. This study has established a robust, reliable assay for sensitive JAK2-V617F detection, suitable for assessing response in clinical trials, predicting outcome and guiding management of patients undergoing allogeneic transplant.

Figure 1

Summary of QC rounds used to identify JAK2-V617F assays with the best performance profile. Seven successive QC rounds, designated QC1–7, were conducted involving the assessment of plasmid standards and DNA derived from dilutions of cell lines harboring JAK2-V617F (HEL or UKE-1) in cells with wild-type JAK2 (K562 or PB mononuclear cells), abbreviated: HEL/K562, UKE-1/PBMNC, as indicated. Assays found to exhibit consistently poorer performance in multiple test laboratories were not taken forward to subsequent QC rounds—reasons for assay exclusion are provided. Details of the JAK2-V617F assays (designated Assays 1–9) and control gene assays (Albumin, Cyclophilin) are provided in Supplementary Table 1.

Figure 2

Plasmid standards. In QC rounds QC3–7, in addition to the assessment on cell line dilutions, JAK2 and control gene assays were tested on serial dilutions of plasmid standards. The efficiencies of JAK2-V617F Assays 2, 5 and 7 were found to be superior to that of Assay 6, with slope values much closer to −3.32 (the slope associated with a PCR reaction with maximal efficiency). y-axis: Mean cycle threshold (Ct) values; x-axis: log dilution.

Figure 3

Interlaboratory concordance of JAK2-V617F quantification. Left panel: shows the design of Assays 2, 5 and 7, which were found to afford the greatest sensitivity for the detection of JAK2-V617F. Right panel: graphs show mean Ct value for the respective JAK2-V617F assays reported by each laboratory obtained on the analysis of DNA from serial dilutions of HEL in K562 cells in the third QC round (QC3). Plots of the dilution series were observed to lie largely in parallel, with all laboratories successfully detecting the 0.5% JAK2-V617F dilution (with Ct values <40 cycles recorded by all laboratories with each assay). The Ct values observed with neat K562 cells (labeled as 0% JAK2-V617F) generally exceeded 40 cycles (with the exception of laboratory 2 and laboratory 4 for Assay 2 and Assay 7, respectively), consistent with specificity of the JAK2-V617F assays for the mutant allele with the test conditions applied in the majority of the laboratories.

Figure 4

Investigation of best-performing JAK2-V617F qPCR assays to predict outcome following allogeneic transplantation. Stored PB samples from patients with JAK2-V617F-positive myeloid disorders were tested in parallel using JAK2-V617F Assays 5 and 7, in conjunction with ALB as an independent control gene. Samples were determined to be positive or negative for JAK2-V617F according to the ΔCt between the JAK2-V617F and ALB amplification plots, taking into account the cut offs defined in control PB samples for each mutant assay (see Supplementary Figure 3), with PCR-positive and -negative samples denoted by filled and empty data points, respectively. JAK2-V617F percentage for PCR-positive samples was calculated using the matching wild-type and total JAK2 assays for the respective assays. PCR profiles are shown from four (UPN1–4) of the 28 patients evaluated, who underwent allogeneic transplant with reduced intensity conditioning with an unrelated (UPN1) or sibling donor (UPN2–4) for PMF (UPN2, UPN3) or JAK2-V617F-positive secondary acute myeloid leukemia arising on a background of MPN (UPN1, UPN4). In each case, the clinical relapse post-transplant was predicted by JAK2-V617F positivity with a rise in mutant level (UPN1, UPN3 and UPN4) or persistently high levels (UPN2). JAK2-V617F detection by qPCR preceded loss of donor chimerism in UPN1, UPN3 and UPN4. Three patients received DLI, denoted by blue arrows, inducing molecular remission. Chimerism was assessed by microsatellite marker analysis in UPN1, UPN3 and UPN4 and by fluorescence in situ hybridization analysis of X and Y chromosomes in UPN2.