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、治疗疗效的直接衡量指标，也是复发的最强预测因子。 尽管MRD已被确立为标准护理程序和结局的衡量标准， 慢性粒细胞白血病（CML）和急性淋巴细胞白血病（ALL）的临床试验， 并在急性髓性白血病（AML）中标准化，尚未纳入试验设计。我们的目标 本研究拟建立一种单细胞DNA分子诊断模型，预测AML患者MRD的发生。 使命生物已经开发并商业化推出了一种新型的微流体液滴平台，Tapestri， 进行高通量的单细胞DNA测序。具有用户友好、成本效益和快速 Tapestri能够在数百个疾病相关基因座处每次运行准确地对10，000个细胞进行基因分型。 通过合作，我们已经使用Tapestri生成了高分辨率的克隆结构图， 纵向收集AML肿瘤样品，并证明了鉴定存在的罕见克隆的能力， 占肿瘤总数的0.1%。 在这项研究中，我们将提高Tapestri的性能，以允许检测存在于 0.01%，并使用该平台构建AML MRD特异性靶向测序组。Tapestri AML MRD 将使用配对诊断和缓解对多达100例回顾性AML患者样本进行检测 样本，我们将使用衍生的数据来开发复发风险评估，使用模型，包括单一的， 细胞基因分型数据、肿瘤内异质性和MRD状态。Tapestri单细胞数据将被 以现有技术为基准，包括流式细胞术和批量DNA测序。 拥有一种能够描述残留白血病克隆的超灵敏MRD检测方法， 突变水平将有很大的好处，（1）允许更准确地预测AML复发，（2）因此， 提供用于测试MRD指导的治疗强化或降级的平台，（3）识别 残留白血病克隆中存在的可操作突变，可作为背景下治疗的靶点 （4）评价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.