Georg Greiner1, Michael Gurbisz1, Franz Ratzinger1, Nadine Witzeneder1,2, Ingrid Simonitsch-Klupp3, Gerlinde Mitterbauer-Hohendanner1, Matthias Mayerhofer4, Leonhard Müllauer3, Wolfgang R Sperr2,5, Peter Valent2,5, Gregor Hoermann6,5. 1. Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria. 2. Department of Internal Medicine I, Division of Hematology and Hemostaseology, Medical University of Vienna, Vienna, Austria. 3. Department of Pathology, Medical University of Vienna, Vienna, Austria. 4. Ludwig Boltzmann Institute of Osteology, Hanusch Hospital, Vienna, Austria. 5. Ludwig Boltzmann Cluster Oncology, Medical University of Vienna, Austria. 6. Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria; gregor.hoermann@meduniwien.ac.at.
Abstract
BACKGROUND: The analytically sensitive detection of KIT D816V in blood and bone marrow is important for diagnosing systemic mastocytosis (SM). Additionally, precise quantification of the KIT D816V variant allele fraction (VAF) is relevant clinically because it helps to predict multilineage involvement and prognosis in cases of advanced SM. Digital PCR (dPCR) is a promising new method for sensitive detection and accurate quantification of somatic mutations. METHODS: We performed a validation study of dPCR for KIT D816V on 302 peripheral blood and bone marrow samples from 156 patients with mastocytosis for comparison with melting curve analysis after peptide nucleic acid-mediated PCR clamping (clamp-PCR) and allele-specific quantitative real-time PCR (qPCR). RESULTS: dPCR showed a limit of detection of 0.01% VAF with a mean CV of 8.5% and identified the mutation in 90% of patients compared with 70% for clamp-PCR (P < 0.001). Moreover, dPCR for KIT D816V was highly concordant with qPCR without systematic deviation of results, and confirmed the clinical value of KIT D816V VAF measurements. Thus, patients with advanced SM showed a significantly higher KIT D816V VAF (median, 2.43%) compared with patients with indolent SM (median, 0.14%; P < 0.001). Moreover, dPCR confirmed the prognostic significance of a high KIT D816V VAF regarding survival (P < 0.001). CONCLUSIONS: dPCR for KIT D816V provides a high degree of precision and sensitivity combined with the potential for interlaboratory standardization, which is crucial for the implementation of KIT D816V allele burden measurement. Thus, dPCR is suitable as a new method for KIT D816V testing in patients with mastocytosis.
BACKGROUND: The analytically sensitive detection of KITD816V in blood and bone marrow is important for diagnosing systemic mastocytosis (SM). Additionally, precise quantification of the KITD816V variant allele fraction (VAF) is relevant clinically because it helps to predict multilineage involvement and prognosis in cases of advanced SM. Digital PCR (dPCR) is a promising new method for sensitive detection and accurate quantification of somatic mutations. METHODS: We performed a validation study of dPCR for KITD816V on 302 peripheral blood and bone marrow samples from 156 patients with mastocytosis for comparison with melting curve analysis after peptide nucleic acid-mediated PCR clamping (clamp-PCR) and allele-specific quantitative real-time PCR (qPCR). RESULTS: dPCR showed a limit of detection of 0.01% VAF with a mean CV of 8.5% and identified the mutation in 90% of patients compared with 70% for clamp-PCR (P < 0.001). Moreover, dPCR for KITD816V was highly concordant with qPCR without systematic deviation of results, and confirmed the clinical value of KITD816V VAF measurements. Thus, patients with advanced SM showed a significantly higher KITD816V VAF (median, 2.43%) compared with patients with indolent SM (median, 0.14%; P < 0.001). Moreover, dPCR confirmed the prognostic significance of a high KITD816V VAF regarding survival (P < 0.001). CONCLUSIONS: dPCR for KITD816V provides a high degree of precision and sensitivity combined with the potential for interlaboratory standardization, which is crucial for the implementation of KITD816V allele burden measurement. Thus, dPCR is suitable as a new method for KITD816V testing in patients with mastocytosis.
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