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.

Introduction Somatic mutations acquired in key signalling pathway, transcription factor, spliceosome, epigenetic and tumor suppressor genes are of central importance in the development and progression of myeloid malignancies including myeloproliferative neoplasms (MPN), myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). To date, in order to evaluate all relevant genetic alterations, multiple tests are needed, requiring large amounts of DNA. As tests are typically performed sequentially this unnecessarily extends the time between sample acquisition and mutation detection. The QIAact Myeloid DNA UMI Panel in combination with the QIAGEN GeneReader NGS System provides a single solution to simultaneously test for actionable mutations, whilst also saving sample material (only 40ng DNA input required per sample), shortening test time and enabling simplification of lab operations. The QIAact Myeloid DNA UMI Panel is a multi-gene targeted sequencing panel designed to detect complex mutations throughout the most informative genes linked to myeloid disease. This allows reliable and sensitive detection of single nucleotide variants (SNV) and large Insertion/Deletion (InDel) mutations. Methods The QIAact Myeloid DNA UMI Panel targets 25 genes known to be important in myeloid leukemia. A key feature of the panel is the addition of a unique molecular index (UMI) to tag each individual original DNA molecule prior to target enrichment by PCR. UMIs enable sequencing and PCR bias corrections, allowing sensitive detection of mutations. To assess the assay performance, reference standards and blood and bone marrow samples were used. Following target enrichment, libraries were sequenced on the GeneReader NGS System and mutations analyzed using the QIAGEN Clinical Insight (QCI) Analyze software suite. Results To confirm DNA mutation detection, Horizon Discovery and SeraCare Reference Standards containing variants typical of myeloid malignancies were used. The anticipated DNA mutations were consistently identified both within and between runs. The samples used, including blood and bone marrow samples, demonstrated the ability of the assay to detect important large indels (52 bp deletion CALR type 1 variant) and key SNVs down to a minor allele fraction (MAF) of 1% for JAK2 (e.g. exon 12, 13, 14 & 15) and KIT (exon 8, 9, 10, 11 & 17). A sensitive variant detection of allele frequency <0.5% for the KIT D816V was also achieved. For the other genes covered by the panel, including ASXL1, RUNX1, NPM1, DNMT3A, IDH1/2, results show sufficiently uniform amplification and sequencing coverage to support mutation detection with a MAF of 5%. Conclusion The QIAact Myeloid DNA UMI Panel in combination with the QIAGEN GeneReader NGS System offer a fully integrated DNA to variant detection and interpretation solution. The optimized chemistry allows superior analytical sensitivity resulting in accurate and efficient mutation detection of highly relevant genetic alterations for myeloid malignancy research. Disclosures Laloux: QIAGEN France S.A.S: Employment. Biglia:HalioDx: Employment. Bona:HalioDx: Employment. Lafi:HalioDx: Employment. Charifi:HalioDx: Employment. Larsen:QIAGEN Aarhus: Employment. Lueerssen:QIAGEN Manchester Ltd: Employment. Gupta:QIAGEN Aarhus: Employment. Lauber:QIAGEN GmbH: Employment. Hughes:QIAGEN Manchester Ltd: Employment.