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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. [ABSTRACT FROM AUTHOR]

Quantitative PCR (qPCR) for detection of fusion transcripts and overexpressed genes is a promising tool for following minimal residual disease (MRD) in patients with hematological malignancies. Its widespread clinical use has to some extent been hampered by differences in data analysis and presentation that complicate multicenter clinical trials. To address these issues, we designed a highly flexible MRD-reporting software program, in which data from various qPCR platforms can be imported, processed, and presented in a uniform manner to generate intuitively understandable reports. The software was tested in a two-step quality control (QC) study; the first step involved eight centers, whose previous experience with the software ranged from none to extensive. The participants received cDNA from consecutive samples from a BCR-ABL+ chronic myeloid leukemia (CML) patient and an acute myeloid leukemia (AML) patient with both CBFβ-MYH11 and WT1 target genes, they conducted qPCR on their respective hardware platforms and generated a series of reports with pre-defined features. In step two, five centers used the software to report BCR-ABL+ MRD in a harmonized manner, applying their recently obtained CML international scale conversion factors. The QC study demonstrated that this MRD-reporting software is suitable for efficient handling of qPCR data, generation of MRD reports and harmonization of MRD data. [ABSTRACT FROM AUTHOR]