Development of a Universal Assay for Minimal Residual Disease in Acute Myeloid Leukemia using Duplex Sequencing

问题：AML的5年存活率约为30%，是第六大致死性癌症，新增加的20,000名患者 在美国每年都会被确诊。尽管大多数患者通过治疗获得完全缓解(CR) 积极治疗，大多数人最终会复发。然而，CR的标准是基于历史诊断 方法，CR患者可携带多达1010个残留白血病原始细胞。已经在以下方面做出了重大努力 开发工具以检测最少量的残留疾病(MRD)，包括多参数血流 细胞计数法(MFC)和聚合酶链式反应(PCR)。MRD是复发的最强预测因子，还有几个 急性髓细胞白血病试验表明，当MRDpos患者接受 强化治疗。然而，缺乏敏感性是MFC和PCR的一个明显问题。例如，复发会发生 在化疗后MFC检测到MRDneg的患者中，约有40%。下一代测序(NGS)掌握着 承诺通过检测与残留AML细胞相关的突变来识别MRD。然而，它的敏感性 传统的NGS受错误率较高的限制，这使得排序难以区分 来自真正的低频突变的错误。解决方案：双链测序(DS)是一种超敏感的NGS 利用专门的生物化学和信息学来提高标准DNA准确性的技术 测序器增加了一万多倍。在我们之前的第一阶段SBIR研究中，我们开发了一种广泛适用的 基于DS的AML MRD检测克服了上述限制。DS检测针对许多与MRD相关的 基因同时检测，灵敏度和特异度可与单基因聚合酶链式反应相媲美或超过。在第二阶段， 我们在实验室中表现出了极好的重复性，将优化后的检测方法应用于回溯性银行 队列，并显示出优于MFC的临床表现。然而，根据许多客户采访， 我们面临的最大障碍是缺乏大规模的前瞻性临床试验 试验数据。具体目标：在目前的2b期应用中，我们建议生成此综合临床 通过与世界一流的美国国家骨髓捐赠者计划(NMDP)和其他机构合作建立的实用数据集 顶级反洗钱MRD关键舆论领袖。我们提议的研究的主要目标包括：目标1：前瞻性 验证移植前DS-AML-MRD检测预测移植后复发的预后价值 和存活率；目标2：前瞻性地生成支持移植后DS AML MRD能力的数据 测试以评估接受不同移植条件强度治疗的患者的复发风险；以及目标3： 证明在没有驱动基因变异的患者中使用肿瘤信息DS AML MRD检测的潜力 被我们固定的基因小组锁定。影响：这种组合方法可能会使NGS MRD测试达到100% 患者适用性。血液MRD比对可能提供一种同样敏感但侵入性更小的替代方案 骨髓。我们的预期数据也将支持使用该技术将高危患者分配给 加强治疗，并作为新药开发工作的抗白血病疗效的早期标志。

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.