New Detection Method of V617F JAK2 Mutation; Its Use in a Clinical Setting.

Background: A single point mutation in the Janus 2 tyrosine kinase gene leading to a V617F substitution has been described in a large group of hematological pathologies such as Polycythemia Vera (PV), Essential Thrombocytaemia (ET), Idiopathic Myelofibrosis (IM) and unclassified Myeloproliferative disorders (MPDs). Mutated JAK2 is an essential biomarker which improves the understanding and classification of MPDs and offers a new target for specific therapeutics.

Methods: We adapted the V617F genotyping by amplification mutation system (ARMS) PCR described by AmyV Jones and Nicholas C.P Cross and combined it with a capillary electrophoresis performed on the Agilent Bioanalyseur 2100. DNA quantification was determined by βglobin gene amplification by quantitative PCR.100ng of samples and controls were genotyped by a DNA ARMS assay, using a primer pair that specifically amplifies the mutant sequence. We chose the homozygous HEL cell line as a positive control systematically tested in each run.

We investigated the sensitivity of the method by testing serial dilutions of 100% V617F homozygous HEL DNA into a non mutated DNA of PBL. We compared the sensitivity of simplex ARMS PCR method using a primer pair that only amplifies the mutant sequence versus a multiplex ARMS PCR method using a two primer pairs to specifically amplify the normal and mutant sequence plus a positive control band in a single reaction.

We genotyped over 6 months, 70 patients who had been screened for MPDs, BCR-ABL fusion negative, PV, ET, and isolated polycythaemia. Furthermore, we intend to collaborate with IPSOGEN SAS to compare our results with their new licensed JAK2 mutation test based on the pioneering work of Dr.Vainchenker.

Results: Sensitivity of the mutliplex and simplex techniques respectively ranged from 100% to 1% and 100% to 0.1%. We found evidence that the sensitivity of the technique was imrpoved by using a simplex ARMS PCR followed by a capillary electrophoresis than a multiplex one.

Of the 70 samples with a known or suspected diagnosis of MPD, 43(61%) were negative for V617F JAK2 mutation and 27(39%) were positive. The V617F JAK2 mutation was detected in 43% (3/7) PV, 42% (22/53) MPDs, BCR-ABL fusion gene negative, 25% (2/8) ET and none of isolated polycythaemia.

Conclusions: The poor frequency of positive V617F V617F JAK2 mutation in patients screened for an evocated PV might be due to the inclusion in PV group of patients who do not meet diagnostic criteria for PV.

The V617F JAK2 mutation in MPDs can be detected by various methods. ARMS associated with a capillary electrophoresis seems to be easy to use and reproductible for detection of single base G → T substitution in JAK2 mutation. In addition, the simplex approach improves the sensitivity of the technique leading to V617F JAK2 mutation.

This qualitative test leads to the conclusions: presence of the V617F JAK2 mutation “or absence of detection of the V617F JAK2 mutation and is essential in diagnosis and classification of MPDs whereas quantitative approach will improve the management of therapeutic response when targeted therapy against V617F JAK2 mutation will be defined.