PROJECT SUMMARY/ABSTRACT Background: Acute myeloid leukemia (AML) continues to have high mortality rates despite a plethora of available treatments. Variable prognosis, treatment response and survival are largely based on cytogenetic and molecular aberrations that characterize AML subtypes. Fast and cost-effective methods of detecting AML fusions and mutations could improve clinical outcomes for patients, as standard next generation sequencing (NGS) methods are limited by several technical and bioinformatic issues. Hypothesis: We have developed a CRISPR-based single molecule long-read sequencing assay (CSRL) to quickly detect clinically relevant mutated genes in AML. This new methodology, combined with new bioinformatic approaches, allows for same day results of sequencing data. We hypothesize that such technology can be improved to detect both mutation and translocations common in AML, and that this rapid turn-around-time will positively impact patient care by allowing swift risk stratification and treatment selection. Proposal: In this study, we expand the CSRL assay to include more relevant mutations and translocations. Specifically, we will ask and answer 1) Will the CSRL assay identify all the mutations and fusion in the target genes seen by NGS and cytogenetics? 2) Do the single molecule reads provide a more accurate assessment of tumor burden? 3) Do CSRL data allow for determination of phasing of mutations and provide additional prognostic or predictive significance? 4) Is same-day sample collection and result reporting clinically feasible for the AML CSRL assay and how will obtaining same-day CSRL results impact clinical decision making? 5) What improvements can be expected upon the current turnaround time, costs, and assay performance in comparison to standard NGS? To answer questions 4 and 5, we will conduct a pilot clinical trial to test feasibility of clinical implementation. Research Design/Specific Aims: Specific aims: SA1) To expand our CSRL sequencing assay and optimize current bioinformatics workflow to allow for same-day diagnosis (~8 hours). This development will use a multiplexed CRISPR enrichment library of long nucleic acid molecules on a low cost Nanopore device. SA2) Validate sensitivity and specificity of the assay developed in SA1 and evaluate prognostic impact of phasing data provided by long reads and single molecule quantification for more accurate tumor burden assessment. We will test the proposed method on a set of clinically annotated leukemia samples with existing molecular data obtained with standard NGS and cytogenetics. SA3) To establish clinical utility of CSRL sequencing with same-day leukemia molecular diagnosis. The purpose of this specific aim is to demonstrate the clinical impact of our same day ultrarapid molecular profiling assay through a pilot study on 10-12 patients at our cancer center. We will test real world feasibility of implementing these methods on samples from the clinic, ease of same day tes...