Problem: With a 5-year survival of ~30%, AML is the 6th deadliest cancer, and >20,000 new patients are diagnosed each year in the United States. Although most patients achieve Complete Remission (CR) with aggressive therapy, most will eventually relapse. The criteria for CR, however, is based on historical diagnostic methods, and patients in CR may carry up to 1010 residual leukemia blasts. Significant effort has gone into developing tools to detect Minimal amounts of Residual Disease (MRD), including multi-parametric flow cytometry (MFC) and polymerase chain reaction (PCR). MRD is the strongest predictor of relapse, and several AML trials have demonstrated that survival was significantly better when MRDpos patients were subjected to intensified therapy. Yet, lack of sensitivity is a clear problem with MFC and PCR. For example, relapses occur in ~40% of patients who are MRDneg by MFC after chemotherapy. Next Generation Sequencing (NGS) holds the promise to identify MRD by detecting mutations associated with residual AML cells. Yet, the sensitivity of conventional NGS is limited by a relatively high error rate, which makes it difficult to differentiate sequencing errors from true low-frequency mutations. Solution: Duplex Sequencing (DS) is an ultra-sensitive NGS technology which uses specialized biochemistry and informatics to improve the accuracy of standard DNA sequencers by more than ten-thousand-fold. In our prior Phase I SBIR study, we developed a broadly-applicable DS-based AML MRD assay that overcomes the above limitations. The DS assay targets many MRD-relevant genes simultaneously, with sensitivity and specificity rivaling or exceeding single-gene PCR assays. In Phase II, we demonstrated excellent reproducibility across labs, applied the optimized assay to retrospectively banked cohorts, and showed superior clinical performance vs. MFC. However, based on many customer interviews, the most substantial barrier to widespread commercial adoption we face is the lack of large-scale prospective clinical trial data. Specific Aims: In the present Phase 2b application we propose generating this comprehensive clinical utility data set through collaboration with the world-class US National Marrow Donor Program (NMDP) and other top AML MRD key opinion leaders. The primary goals of our proposed study include: Aim 1: Prospectively validate the prognostic value of pre-transplantation DS AML MRD testing for predicting post-transplant relapse and survival; Aim 2: Prospectively generate data supporting the ability of post-transplantation DS AML MRD testing to assess relapse risk for patients treated with different transplant conditioning intensities; and Aim 3: Demonstrate the potential of using tumor-informed DS AML MRD testing for patients without a driver gene variant targeted by our fixed gene panel. Impact: This combined approach may bring NGS MRD testing up to 100% patient applicability. Blood MRD comparison may present an equally sensitive but less invasive alternative to marrow. Our anticipated data will also support the use of the technology in assigning high-risk patients to intensified therapies and as an early marker of anti-leukemic efficacy for novel drug development efforts.

PROJECT NARRATIVE Despite the fact that most patients with acute myeloid leukemia receiving intensive chemotherapy are able to achieve a remission, many have residual disease below the level of morphological detection (measurable residual disease, MRD), which eventually leads to relapse. Current methods of MRD detection are limited in sensitivity, are not applicable to all AML patients, and are challenging to perform and interpret. In this Phase 2b project we propose utilizing our Duplex Sequencing AML-29 MRD assay to generate a comprehensive clinical utility data set through a collaboration with the world-class US National Marrow Donor Program (NMDP). The currently accruing MEASURE (“Molecular Evaluation of AML patients after Stem cell transplant to Understand Relapse Events”) protocol offers a historical opportunity to generate definitive evidence of the clinical utility of our commercial DuplexSeq™ AML MRD testing product before and after bone transplant in a 500 patient cohort.