P556: a new multiplex pcr tool enables timely identification of actionable targets for molecular-guided therapy in patients with acute myeloid leukemia

Background: The initial diagnostic work-up of patients with newly diagnosed acute myeloid leukemia (AML) requires the evaluation of various genetic abnormalities such as mutations and gene fusions. The results of these analyses are crucial for risk stratification and molecular-guided therapy. Traditionally, a combination of molecular analyses and time-consuming cytogenetic methods with a turnover time often exceeding three to five days are used. Aims: We aimed to develop multiplex PCR assays simultaneously detecting gene fusions and mutations relevant to the ELN guideline resulting in significantly faster results compared to NGS or conventional Sanger sequencing. Methods: Two multiplex PCR assays for the MODAPLEX platform (Biotype GmbH) were developed. The Modaplex System merges PCR and capillary electrophoresis into one automated workflow to enable high degree multiplexing. Each Modaplex PCR reaction consists of multiple fluorescent-labeled primers and probes, unique for assay targets. After a PCR cycle, the PCR products are injected into the capillary gel. Here they will get separated based on their physical properties and subsequently will be detected after passing two lasers. This process is repeated over several PCR cycles during a Modaplex run, thus generating real-time data for each well and each target. Prototypes were tested on cell lines as well as clinical samples from AML patients included in the prospective Study Alliance Leukemia (SAL) registry to obtain performance data. The study was approved by the ethics committee of the TU Dresden (BO-EK-8012021). Results: Due to the simple workflow, 11 gene fusions covering up to 54 different variants and six mutation hotspots in IDH1, IDH2, NPM1, FTL3-TKD, and FLT3-ITD were performed and analyzed in only 4 hours with minimal hands-on time. Through optimization of the process, the analysis of all specified targets was performed using only two reaction wells per patient. This enables the simultaneous testing of parameters on genomic (DNA) as well as on transcriptomic level (fusion genes) in one MODAPLEX run. During the development of the assays artificial material was used to also cover rare genetic aberrations. For initial determination of sensitivity and specificity a total number of 19 pre-characterized and well-established cell lines were analyzed with the two assays. Together the results confirm that all tested mutations and gene fusion variants are detectable with the new assay. Preliminary data showed a limit of detection (LoD) between 1 to 5% mutation content for both assays. In addition, comparable sensitivity was achieved to the Mentype® AMLplexQS CE IVD assay on for example the Kasumi (RUNX1::RUNX1T1) and FKH-1 (DEK::NUP214) cell lines. Although not recommended recently in the ELN recommendations, the assay also calculates allelic ratio for FLT3-ITD, which was comparable to the pre-characterized samples. In a beta-testing regime with 100 clinical samples from AML patients at diagnosis, the prototype of the mutation assay achieved a sensitivity of 94%, a specificity of 100% and an accuracy of 97%. The gene fusion prototype assay achieved a sensitivity, specificity, and accuracy of 96%, respectively. Summary/Conclusion: In this proof-of-principle study, we demonstrate for the first time the successful combination of the analysis of AML-relevant gene mutations, duplications and translocations using two multiplex PCR assays in parallel, thus enabling ELN-concordant analyses in a timely manner.Keywords: AML, Molecular markers, PCR