Standard chemotherapy for acute myeloid leukemia (AML) targets proliferative cells and efficiently induces complete remission; however, many patients relapse and die of their disease. Relapse is caused by leukemia stem cells (LSC), the cells with selfrenewal capacity. Self-renewal and proliferation are separate functions in normal hematopoietic stem cells (HSC) in steadystate conditions. If these functions are also separate functions in LSCs, then antiproliferative therapies may fail to target selfrenewal, allowing for relapse. We investigated whether proliferation and self-renewal are separate functions in LSCs as they often are in HSCs. Distinct transcriptional profiles within LSCs of Mll-AF9/NRAS G12V murine AML were identified using single-cell RNA sequencing. Single-cell qPCR revealed that these genes were also differentially expressed in primary human LSCs and normal human HSPCs. A smaller subset of these genes was upregulated in LSCs relative to HSPCs; this subset of genes constitutes "LSCspecific" genes in human AML. To assess the differences between these profiles, we identified cell surface markers, CD69 and CD36, whose genes were differentially expressed between these profiles. In vivo mouse reconstitution assays resealed that only CD69 High LSCs were capable of self-renewal and were poorly proliferative. In contrast, CD36 High LSCs were unable to transplant leukemia but were highly proliferative. These data dem-onstrate that the transcriptional foundations of self-renewal and proliferation are distinct in LSCs as they often are in normal stem cells and suggest that therapeutic strategies that target selfrenewal, in addition to proliferation, are critical to prevent relapse and improve survival in AML.

Clinical laboratories have adopted next generation sequencing (NGS) as a gold standard for the diagnosis of hereditary disorders because of its analytic accuracy, high throughput, and potential for cost-effectiveness. We describe the implementation of a single broad-based NGS sequencing assay to meet the genetic testing needs at the University of Minnesota. A single hybrid capture library preparation was used for each test ordered, data was informatically blinded to clinically-ordered genes, and identified variants were reviewed and classified by genetic counselors and molecular pathologists. We performed 2509 sequencing tests from August 2012 till December 2017. The diagnostic yield has remained steady at 25%, but the number of variants of uncertain significance (VUS) included in a patient report decreased over time with 50% of the patient reports including at least one VUS in 2012 and only 22% of the patient reports reporting a VUS in 2017 (p = .002). Among the various clinical specialties, the diagnostic yield was highest in dermatology (60% diagnostic yield) and ophthalmology (42% diagnostic yield) while the diagnostic yield was lowest in gastrointestinal diseases and pulmonary diseases (10% detection yield in both specialties). Deletion/duplication analysis was also implemented in a subset of panels ordered, with 9% of samples having a diagnostic finding using the deletion/duplication analysis. We have demonstrated the feasibility of this broad-based NGS platform to meet the needs of our academic institution by aggregating a sufficient sample volume from many individually rare tests and providing a flexible ordering for custom, patient-specific panels.

We have successfully demonstrated the feasibility of using the NGS platform to multiplex genetic tests for several rare diseases and the use of cloud computing for bioinformatics analysis as a relatively low-cost solution for implementing NGS in clinical laboratories.