Quantitative PCR Assessment of JAK2 Mutational Status in Philadelphia-Chromosome Negative Chronic Myeloproliferative Disorders - The JAK2 V617F Mutation Is an Event in an Early Stem Cell and the Clonal Involvement of Different Haematopoietic Cells Varies between Individual Patients.
Authors: Larsen Thomas S. , Hasselbalch Hans C. , Pallisgaard Niels 
Published: October 2019

Abstract
Introduction: The JAK2 V617F mutation is believed to occur in an early stem cell. Analysis of the JAK2 mutational status of patients suspected of a chronic myeloproliferative disorder has already been widely introduced into the diagnostic procedure. The ability to determine the size of the mutated clone and to monitor its evolution over time would be of great interest. Most methods are based on sequencing and qualitative or semi-quantitative PCR methods with limited sensitivity We present a highly sensitive real-time quantitative PCR assay, and have determined the percentage of mutated alleles in granulocytes, monocytes, T-lymphocytes and B-lymphocytes.
Methods: Peripheral blood from 13 patients (PV=10, IMF=2, ET=1) with a known JAK2 mutation was collected. CD66-FITC, CD14-PE-Cy7, CD19-PE and CD3-APC were used to label granulocytes, monocytes, B-lymphocytes and T-lymphocytes, respectively. The cells were collected by a four-way FACS sort with a specificity of 99%. DNA was extracted by standard procedure. A real-time quantitative assay with a common forward primer, a Taqman probe and a wildtype specific reverse primer or a mutation specific reverse primer, was used to determine the ratio of mutated vs. wildtype alleles. The sensitivity of the PCR assay was determined by dilution series to be better than 1:10000. In the cell fractions we defined a minimum of 5 % mutated alleles to be significant.
Results: All 13 patients had JAK2 V617F clonal granulocytes with a median proportion of mutated alleles of 60%, ranging from 8–96%. Eight patients (PV= 6, IMF=2) had more than 50 % mutated alleles in their granulocytes indicating homozygocity. Twelve (PV=10, IMF=2) out of 13 patients had clonal monocytes, median proportion of mutated alleles 35%, range (2–84%). Five patients had homozygote monocytes. Median proportion of mutated alleles in T-lymphocytes was 3%, range (0.24–83%), and four patients (PV=2, IMF=1, ET=1) had T-lymphocytes with more than 5% mutated alleles, 2 of them being homozygote. In B-lymphocytes only 2 patients had more than 5 % mutated alleles, none were homozygote. Median proportion of mutated alleles 3.5 %, range (1–46%).
Conclusions: By a highly sensitive realtime quantitative PCR assay we have demonstrated that the JAK2 V617F mutation is an event in an early stem cell with the potential of both myeloid and lymphoid differentiation. The fraction of mutated alleles varies not only within the different cell types, but also to a great extend between individual patients. As expected all 13 patients harboured the mutation in their granulocytes Twelve, five and two patients had clonal JAK2 mutated monocytes, T-cells and B-cells respectively. The ratio of mutated vs. wt alleles allowed us to determine homozygocity in 8 cases within a specific cell type, mostly granulocytes, but homozygocity was also detected in monocytes and T-cells. In conclusion this highly sensitive quantitative assay may detect more JAK2 V617F patients than previously reported assays and may be useful in monitoring the size of the mutated clone during cytoreductive therapy.