Quantative Analysis of Somatic Mosaicism

Multiple prospective clinical trials have demonstrated that the speed in which a child with leukemia is put into remission is a critical prognostic indicator of the child’s ultimate outcome. Historically, remission was determined subjectively by manual review of bone marrow, which was insufficient to detect persistent, but minimal, remaining leukemia. In recent years, the implementation of “minimal residual disease” (MRD) has greatly improved upon therapeutic and outcome stratification by using a combination of flow cytometry for unique cell surface markers and in-situ hybridization for leukemia-specific VDJ recombination. These methods have a sensitivity of roughly 1:1000 and detect remaining leukemia that would likely be missed by manual review. However, there remain a subset of patients who still suffer poor outcomes despite being MRD-negative, suggesting that MRD analysis is not sensitive enough to detect all cases of residual leukemia. Thus, we have designed a novel strategy for detecting leukemia-specific DNA via next-generation sequencing by labeling every single DNA molecule with a unique index. “Double-indexing” of source DNA also enables us to computationally exceed the inherent error rate of 1:1000 on most high-throughput sequencing platforms. Preliminary experiments suggest that we can detect a unique sequence variant at a frequency of 1:10,000, and perhaps lower. This would enable an order of magnitude increase in our ability to detect residual leukemia and further improve outcomes for children or adults with incompletely-treated leukemia.