Measureable Residual Disease testing for acute myeloid leukemia with single-cell genotyping

摘要 复发是急、慢性白血病治愈的主要障碍。可测残差的检测 疾病(MRD)是疾病负担、治疗效果的直接衡量标准，也是复发的最强预测因子。 尽管MRD已被确立为护理程序的标准和#年结果的衡量标准 慢性粒细胞白血病和急性淋巴细胞白血病的临床试验 并在急性髓系白血病(AML)中标准化，尚未纳入试验设计。我们的目标 本提案旨在建立一种单细胞DNA分子诊断预测急性髓系白血病MRD的方法。 使命生物公司已经开发并商业推出了一种新型微流控液滴平台Tapestri， 进行高通量的单细胞DNA测序。具有用户友好、经济高效和快速的 在Tapestri的工作流程中，Tapestri能够在数百个疾病相关基因座上对每次运行的10,000个细胞进行准确的基因分型。 通过合作，我们使用Tapestri生成了克隆建筑的高分辨率地图 纵向收集AML肿瘤样本，并展示了识别存在的罕见克隆的能力 占肿瘤人口的0.1%。 在这项研究中，我们将改进Tapestri的性能，以检测存在于 0.01%，并利用该平台构建AML MRD特异性靶向测序面板。Tapestri AML MRD Panel将部署在多达100个回顾AML患者样本上，使用配对的诊断和缓解 样本，我们将使用导出的数据来开发复发风险评估，使用的模型包括单一- 细胞基因分型数据、肿瘤内异质性和MRD状态。Tapestri单元格数据将 以现有技术为基准，包括流式细胞仪和批量DNA测序。 具有超灵敏的MRD检测方法，能够描述残留的白血病克隆 突变水平将通过(1)允许更准确地预测AML复发，(2)因此将是非常有益的 提供测试MRD导向的治疗强化或降级的平台，(3)识别 残留的白血病克隆中存在可作为治疗靶点的可操作突变 临床试验，以及(4)评估MRD状态作为新药批准的替代终点。

Abstract Relapse is the primary obstacle to cure in acute and chronic leukemia. The detection of measurable residual disease (MRD) is a direct measure of disease burden, treatment efficacy and is the strongest predictor of relapse. Although MRD has been established as a standard of care procedure and as a measurement of outcomes in clinical trials for chronic myeloid (CML) and acute lymphoblastic leukemia (ALL) it is more difficult to perform and standardize in acute myeloid leukemia (AML) and has not yet been integrated into trial design. Our objective in this proposal is to develop a single-cell DNA molecular diagnostic predictive for MRD in AML. Mission Bio has developed and commercially launched a novel microfluidic droplet platform, Tapestri, that performs high-throughput single-cell DNA sequencing. With a user friendly, cost effective and rapid workflow, Tapestri is capable of accurately genotyping 10,000 cells per run at hundreds of disease relevant loci. Through collaborations, we have used Tapestri to generate high-resolution maps of clonal architecture from longitudinally collected AML tumor samples and demonstrated the capability of identifying rare clones present of 0.1% of the tumor population. In this study, we will improve the Tapestri performance to allow detection of rare subclones present at 0.01% and use this platform to build an AML MRD-specific targeted sequencing panel. The Tapestri AML MRD panel will be deployed on up to 100 retrspective AML patient samples using paired diagnostic and remission samples and we will use the derived data to develop a relapse risk assessment using models that include single- cell genotyping data, intra-tumoral heterogeneity, and MRD status. Tapestri single-cell data will be benchmarked against existing technologies, including flow cytometry and bulk DNA sequencing. Having an ultra-sensitive MRD detection method capable of describing residual leukemic clones at the mutation level would be of great benefit by (1) allowing for more accurate prediction of AML relapse, (2) thus providing a platform for testing of MRD-directed intensification or de-escalation of therapy, (3) identifying actionable mutations present in residual leukemic clones that could serve as targets for therapy in the context of clinical trials, and (4) evaluating MRD status as a surrogate end point for new drug approvals.