International external quality assurance of JAK2 V617F quantification

The discovery of the c.1849G>T mutation leading to the p.Val617Phe (V617F) substitution in JAK2 [1‐4] has been a landmark in molecular diagnosis of the myeloproliferative neoplasms (MPN) polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). Quantification of the mutation has shown that mutation burden also could reflect different subtypes of MPN. The majority of patients with PV or fibrotic PMF have been reported to have more than 50% JAK2 V617F while the opposite has been seen in ET patients [5, 6]. In addition, quantification of the allelic burden in JAK2 V617F-positive patients is increasingly used to monitor treatment response of new targeted therapies as well as in transplanted patients [7‐9].

For molecular diagnosis, it has been recommended that the assay should be sensitive enough to detect a mutant burden around 1% [10]. The combination of a sensitive detection and reproducible quantification of JAK2 V617F challenges the methodology used in a routine setting. Conventional Sanger sequencing does not show the required sensitivity in cases with low mutation burden, and methodologies involving next generation sequencing are unnecessarily labor intensive and expensive for mutation detection of a single nucleotide substitution. Instead, the use of quantitative polymerase chain reaction (qPCR) has been shown to be a both sensitive and cost-effective method [11] and superior in sensitivity compared to qualitative methods [12]. As a step towards standardization of reliable molecular diagnostics, the European Leukemia Net (ELN) and MPN&MPNr-EuroNet have evaluated performance of different allele-specific (AS)-qPCR assays [8]. This work, involving 12 laboratories from seven countries recommended a JAK2 V617F qPCR assay which showed consistent performance across different qPCR platforms [13]. Even so, variation between laboratories and different instrumental setups can be substantial despite the use of the same experimental protocol. To ensure high quality and standardized quantitative results, external quality assurance (EQA) programs are vital. A program dedicated to JAK2 V617F detection by qPCR is advantageous since no additional bias on quantification would be introduced by comparison to a different methodology. MPN&MPNr-EuroNet has performed two rounds of EQA based on qPCR assays. In addition to providing an EQA to participating laboratories in the network, the aim was to identify parameters critical for the quantification of JAK2 V617F. Such factors would have a substantial impact also on an EQA result, and thus need to be identified in order to design a beneficial EQA program which would be useful in clinical routine.

Bias altering qPCR results may occur at several steps of JAK2 V617F assays, even when laboratories use the same methodology. Starting material for the analysis as well as technical platform, assay design and batch variations can influence the results. Even among laboratories using the same qPCR protocol for quantitative assays, considerable variation has been reported [14]. Standardized results are vital not only to aid in diagnosis of patients but also in clinical, multicenter studies. One way to test how well individual laboratories align to predicted results is through participating in EQA. Moreover, EQA are central tools for the accreditation and assessment of laboratory performance. To design a useful EQA for quantitative analysis, it is important to take into account the variation of the methodology in focus. If expectations of consistency in results are set too high, beyond the limits of the technology used, there is a risk that a well-performing laboratory will get poor or inadequate results just because of natural variation in the method, or because of the influence of a particular parameter which has not been identified as important for outcome. Therefore, it is essential to recognize factors which would cause substantial outliers in the tests, as well as which variation could be expected from different qPCR technical platforms.

A previous study has shown that the results obtained for the detection of the JAK2 mutation were comparable in whole blood and in purified granulocytes, and that no false negative was reported in whole blood if the qPCR assay used was able to detect < 1% JAK2 V617F [15]. However, in this study, the allelic ratio was reported to be on average 15% lower in whole blood than in purified granulocytes; the low-average JAK2 V617F values was due to a minority of the whole blood samples. The choice of the starting material could thus be of importance in individual cases depending on the question asked. In the present study, the starting material used for the analysis did not affect the performance in EQA for the majority of laboratories.

In both QA1 and QA2, samples with low mutation burden (< 2% JAK2 V617F) were included, and a greater variation was seen for these samples. This reflects the sensitivity of the assay and the qPCR setup in each laboratory. In addition, when dealing with low JAK2 V617F copy numbers stochastic variation will add to the overall variation. However, for low mutation burden, specificity of the assay is an equally important issue. The background level where cross-reaction with the wild-type allele could occur must be clearly defined by each laboratory to avoid false positive results [10].

To be able to compare results, over time as well as between laboratories, there is a need to standardize the results with respect to the quantitative level of mutation burden. In chronic myeloid leukemia, where the level of expression of the fusion gene BCR-ABL1 is correlated to prognosis, a conversion factor has been established to correct for differences across laboratories. This factor is used to align results to an international scale which is anchored to clinical results [16, 17]. However, the original conversion factor was based on the sample exchange with a reference laboratory and this procedure is both time-consuming and expensive and a risk for inborn errors due to bias cannot be ruled out. To overcome this, primary references intended for the calibration of a secondary reference material have been established [18]. In addition, a certified reference plasmid for the calibration of BCR-ABL1 quantification has been manufactured [19]. As reported in a previous international study [11], a common reference material remains a useful tool for laboratories also for JAK2 V617F, as it allows decreasing or suppressing differences in copy numbers in certain laboratories. In addition, it also allows adjustment for batch variations, e.g., due to differences in quality of primer oligonucleotides. A first WHO reference panel for JAK2 V617F has recently been established and is now available [20]. This holds promise to further improve assay standardization. With increasing clinical demands for molecular monitoring, both EQA programs and standardized JAK2 V617F reference material are needed to identify and maintain validated laboratories.