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.