Does increasing the JAK2V617F assay sensitivity allow to identify more patients with MPN?

The detection of the JAK2V617F mutation has become an essential tool in BCR-ABL1-negative myeloproliferative neoplasms (MPNs) diagnosis, as it is present in 95% of polycythemia vera (PV) patients and 60% of essential thrombocytemia (ET) or myelofibrosis patients. JAK2V617F-positive MPNs are different from other hematological malignant disease in that, although the JAK2V617F mutation is considered as the causative origin of the disease, the tumor burden at the time of diagnosis as assessed by the % of JAK2V617F alleles in peripheral blood or bone marrow can vary from 100% to very low levels with no correlation to blood cell counts. It is still undefined which lowest level might be detected to allow the diagnosis of every MPN patients. Using the Mustascreen kit (Ipsogen, Marseilles, France), the detection limit of which was close to 2%, most of the patients were easily classified as positive or negative. However, some rare patients were considered as doubtful because the level of fluorescence detected from the mutant probe was between the negative control and the 2% reference positive kit control. Recently, we decided to move toward a more sensitive method, the Mutaquant kit (Ipsogen), characterized by a detection limit close to 0.1%. DNA was extracted from whole blood using the QIA-AMP DNA blood mini kit (Qiagen, Hilden, Germany). In the few months after we adopted a more sensitive method, we identified positive patients among patients previously tested negative. These results raised two questions: who are the patients with very low levels of JAK2V617F mutation and consequently what level of sensitivity a method should have in a routine diagnosis setting. All patients included have given informed consent for this study, which has been approved by the local ethics committee. To assess the specificity of the Mutaquant method, 49 samples taken from blood donors were tested and no sample was detected as positive using a cut-off of 0.1%. Next, we diluted one 100% mutated patient DNA sample in a negative one. The 10 2 and 10 3 dilutions were measured at 1.2% and 0.1%, respectively, whereas the 10 4 dilution was found negative. These results confirmed the lower detection limit of this method to be 0.1% mutant allele burden. Within 6 months after introducing Mutaquant in a routine setting, we prospectively investigated 688 patients. Overall, 497 were tested for the first time, whereas 191 had already been analyzed previously using Mutascreen method. In 98 cases, the previous sample was positive and the Mutaquant result confirmed the positivity. In 93 cases, the previous result was negative, but in 7 of them the Mutaquant method detected a low level of JAK2V617F mutant allele (Table 1). When reanalyzed using the novel method, a low level of positivity was also detected on a frozen aliquot of the previous sample for these seven patients. In three cases several DNA samples were available, which were all found positive with Mutaquant, even samples taken as far as 5 years before, with a constant level of positivity throughout time. Of these seven patients, three were suspected of having ET, one presented with an isolated elevated hematocrit and one with a myelofibrosis. Patient 6 presented with a high WBC count, normal