Assessment of the interlaboratory variability and robustness of JAK2V617F mutation assays: A study involving a consortium of 19 Italian laboratories

Abstract

To date, a plenty of techniques for the detection of JAK2V617F is used over different laboratories, with substantial differences in specificity and sensitivity. Therefore, to provide reliable and comparable results, the standardization of molecular techniques is mandatory.A network of 19 centers was established to 1) evaluate the inter- and intra-laboratory variability in JAK2V617F quantification, 2) identify the most robust assay for the standardization of the molecular test and 3) allow consistent interpretation of individual patient analysis results. The study was conceived in 3 different rounds, in which all centers had to blindly test DNA samples with different JAK2V617F allele burden (AB) using both quantitative and qualitative assays.The positivity of samples with an AB < 1% was not detected by qualitative assays. Conversely, laboratories performing the quantitative approach were able to determine the expected JAK2V617F AB. Quantitative results were reliable across all mutation loads with moderate variability at low AB (0.1 and 1%; CV = 0.46 and 0.77, respectively). Remarkably, all laboratories clearly distinguished between the 0.1 and 1% mutated samples.In conclusion, a qualitative approach is not sensitive enough to detect the JAK2V617F mutation, especially at low AB. On the contrary, the ipsogen JAK2 MutaQuant CE-IVD kit resulted in a high, efficient and sensitive quantification detection of all mutation loads. This study sets the basis for the standardization of molecular techniques for JAK2V617F determination, which will require the employment of approved operating procedures and the use of certificated standards, such as the recent WHO 1st International Reference Panel for Genomic JAK2V617F.

Keywords: JAK2 V617F mutation; molecular diagnosis; myeloproliferative neoplasms; qPCR standardization.

Figure 1. Design of the study

A network of 19 Italian Centers was established and the study was conceived in 3 different rounds, in which seven, ten and nineteen laboratories participated, respectively. Each laboratory had to blindly test DNA samples with different JAK2^V617F allele burden (AB). Overall, one quantitative and four qualitative assays were evaluated.

Figure 2. II Round

ipsogen JAK2 MutaQuant kit Copy number (CN) boxplot of JAK2 wild-type (WT), JAK2 mutated (V617F) and JAK2 total (A) and JAK2^V617F mutation percentage boxplot (B) for each of the 8 DNA samples. Red dots are expected values.

Figure 3. III Round

ipsogen JAK2 MutaQuant kit Copy number (CN) boxplot of JAK2 wild-type (WT), JAK2 mutated (V617F) and JAK2^V617F mutation percentage boxplot for each of the 4 DNA samples. Red dots are expected values.