JAK2V617F Evaluation in Healthy Individuals with a Highly Sensitive Quantitative Assay
Authors: Martinaud Christophe , Maroc Nicolas , Hermitte Fabienne , Brisou Patrick , Mozziconacci Marie-Joelle 
Published: April 2020

Abstract
The JAK2V617F point mutation has been described in patients with Philadelphia negative myeloproliferative disorders. Improvement in the sensitivity of different methods has lowered the detection threshold of the percentage of mutant allele but currently, there is no consensus on which level of sensitivity is adequate and how to deal with the lowest levels of mutant allele. Moreover, development of JAK2 inhibitors sets the question of monitoring the molecular residual disease. These reinforce the need of the availability of reliable quantitative assays with high sensitivity and of the definition of the limit of significance for a positive result. The aim of this study was thus to establish a threshold for JAK2V617F detection between normal and potential MPD samples. We used the Jak2 MutaQuant™ kit (Ipsogen, Marseille, France), a quantitative highly sensitive allele specific RQ-PCR test on 200 randomly selected healthy individuals (among 366 volunteers). The study was approved by the Institutional Review Board and informed consent was provided according to the Declaration of Helsinki. Exclusion criteria were: age under 18, medical history of deep thrombosis or blood disease, haemoglobin level over 185 g/L for men and over 165 g/L for women, hematocrit over 52% for men and over 48% for women, white blood cell count (WBC) over 12 G/L and platelet count over 400 G/L. Median age was 37 years (19–88), 69% were male, median haemoglobin level was 142 g/L (SD 17.8), median hematocrit was 41.9% (SD 4.8), median WBC was 6.82 (SD 1.8) and median platelet count was 248 (SD 58.2). In parallel, 24 patients presenting with a thrombocytosis or a polycythemia were tested. Genomic DNA was extracted from whole blood. The technology used in this assay allows a sensitive, accurate and highly reproducible detection of single-nucleotide polymorphisms. This technique is based on the use of specific forward primers, for the wild type (WT) and the V617F allele, a common reverse primer, and a double dye TaqMan probe. Only perfect match between primer and target DNA allows extension and amplification in the PCR reaction. This assay benefits from the plasmid technology which allows the precise calibration and normalisation of RQ-PCR results, by generating standard curves based on the known concentration of plasmid dilutions, and precise measurement of the JAK2 WT and V617F alleles copy number in human cell samples without the need of subsequent handling such as capillary electrophoresis or melting curve analysis. Normalized results can be compared between RQ-PCR systems and testing sites. Results are expressed as percentage of JAK2V617F among total JAK2 detected sequences. Limit of blank was determined (95th percentile) on negative samples (n=56) and is approximately equal to 0.04%. Limit of detection was determined (95th percentile) on known low positive samples (n=96) and is approximately equal to 0.2%. As expected, the sensitivity of the test varies according to the total number of JAK2 sequences detectable in 25ng of genomic DNA. In our hands the median sensitivity of the test was 0.1% of mutant alleles (range 0.28–0.038). In all our healthy donor samples the percentage of mutant alleles was lower than 0.1% (% range 0.002–0.085). Among the 24 MPD suspected patients, 11 were positive (range 0.6–62%). Our data contribute to on-going questions regarding the interpretation of JAK2V617F allele burden. Levels of mutation that are significantly above those seen in healthy individuals, even low, should be clearly of clinical interest. They should benefit of a haematological follow up as they might represent a very early stage of a myeloproliferative disorder.