Pb1701: replacing the current methods in leukemia diagnostics with optical genome mapping and cas9-directed nanopore sequencing.

Topic: 1. Acute lymphoblastic leukemia - Biology & Translational Research Background: Leukemias are clinically and genetically heterogeneous and the type of genetic aberration(s) is important for progression prognosis and choice of treatment. Consequently, a high-throughput and comprehensive genetic diagnosis is needed. Due to the different types of aberrations, a combination of techniques (SNP-array, karyotyping and FISH) is needed for diagnosis and even then relevant abnormalities may go undetected because of resolution and other limitations of the different techniques. Aims: The current techniques are time-consuming, labor-intensive and as mentioned, may not detect all aberrations. Therefore, we want to test whether we can replace the current diagnostic workflow with optical genome mapping (OGM). Methods: Here we studied an unselected cohort of 18 bone marrow aspirates using routine diagnostics alongside OGM and tested if we were able to identify all previously reported aberrations. For additional aberrations, not verified using current methods, Cas9-directed guide RNAs were designed to perform long read (Nanopore) sequencing 1) to confirm these aberrations and 2) to define the precise breaking points of suspected deletions and duplications between 2 OGM labels. Results: All reported aberrations were identified with OGM and 12 additional aberrations with putative clinical relevance were detected. We could confirm 4/12 extra findings with current methods, whereas 8/12 could not be assessed due to a lack of resolution of these methods. We confirmed the remaining variants with Nanopore sequencing. Moreover, we showed the added value of redefining the breaking points, e.g., in 3 patients with suspected deletions in a 42 Kb region between 2 OGM labels on chromosome 17, including TP53. Using nanopore sequencing, the deletions were redefined to be 1.2 Kb deletions localized beside (and not including) TP53 and consequently had no clinical relevance. Summary/Conclusion: We showed that our diagnostic workflow can be replaced by OGM. Nanopore sequencing can be used for confirmation of variants and to redefine the breaking points when needed. Guide RNAs can be designed in advance because the low resolution OGM regions with clinical relevance are known, which allows confirmation within to 1 to 2 days. Keywords: Chromosomal abnormality, Cytogenetics