Advancing biological and clinical applications of genomic Minimal Residual Disease detection in AML

摘要 急性髓细胞白血病(AML)每年在美国影响20,000多名成年患者，造成更大的 而不是11,000人过早死亡。标准的AML治疗包括化疗诱导，以实现 白血病清除，随后单独和/或随后进行几个周期的巩固化疗 异基因骨髓移植。这种对急性髓细胞白血病的一般治疗方法是风险适应依赖的。 主要取决于公认的预后因素，包括急性髓细胞白血病的类型、年龄、基因突变和 细胞遗传学结果。尽管尽了最大努力，AML在大多数患者中仍然是无法治愈的。一个 急性髓细胞白血病治疗的主要障碍是疾病复发，尽管达到临床完全缓解(CR)后 标准的化疗方案。因此，重要的是识别和准确 AML患者缓解期间残留AML的测量，因为此类残留疾病很可能存在 旧病复发的根源。有关AML复发的几个重要问题仅处于令人满意的早期阶段 答案，包括i)如何最好地测量AML的最小残留病(MRD)，ii)是否所有的MRD都是 实际上是由残留病引起的，或者更确切地说，正如最近的数据所表明的那样，包括白血病的混合物，前期- 白血病与年龄相关的克隆性造血，三)急性髓系白血病复发的细胞来源是什么，四)如果 使用新的治疗方法可以更好地针对这种导致复发的细胞，以及v)在什么情况下 环境是MRD预后的指标，对MRD风险适应的AML临床管理是有用的。 由于缺乏合适的技术来可靠地回答上述相关问题，我们有 优化液滴数字聚合酶链式反应(DdPCR)--一种新型超高灵敏度检测人类基因组MRD的方法 AML。在最近发表的一项详细研究中(Parkin等人，JCI 2017)，我们发现AML经常复发 来自基于突变分析的类似于AML原始细胞的缓解骨髓中的罕见细胞 诊断时检测到。我们论证了在等位基因上检测AML相关基因突变的可行性 发病率低至0.002%，也提供了重要的新的预后洞察力。 在这项应用中，我们建议对突变的MRD和异常细胞进行完整的表征 使用复杂的细胞分选和单细胞组合在AML缓解期骨髓中的簇集 转录组分析与小鼠异种移植和体外集落形成试验相互补充。预期的 这些发现将改善携带AML相关基因突变的细胞的功能特征 试图识别和更好地描述急性髓细胞白血病复发的来源(S)。使用来自两个临床的AML样本 在验证队列中，我们将定义基于ddPCR的MRD评估的预后效用。总的来说，数据将 为未来实时基因组MRD指导的AML临床试验奠定基础，旨在监测和 改善急性髓细胞白血病的巩固和维持治疗以及最终的生存结果。

ABSTRACT Acute myelogenous leukemia (AML) affects more than 20,000 adult patients in the US per year causing greater than 11,000 untimely deaths. Standard AML therapy comprises chemotherapy induction to achieve leukemia debulking, followed by a few cycles of consolidation chemotherapy alone and/or followed by allogeneic bone marrow transplantation. This general therapeutic approach to AML is risk-adapted relying principally on well-established prognostic factors, including the type of AML, age, gene mutations and cytogenetic results. Despite best efforts, AML remains incurable in the majority of afflicted patients. A principle barrier to AML cure is disease relapse despite achieving a clinical complete remission (CR) following standard chemotherapy regimens. Of substantial interest therefore, is the identification and accurate measurement of residual AML that persists during remission in AML patients, as such residual disease is likely the source of relapse. Several important questions about AML relapse are only in early stages of satisfactory answers, including i) how to best measure minimal residual disease (MRD) in AML, ii) whether all MRD is indeed caused by residual disease or rather as suggested by recent data comprises a mixture of leukemia, pre- leukemia and age-related clonal hematopoiesis, iii) what constitutes the cellular source of relapse in AML, iv) if such relapse-causing cells can be better targeted using novel therapeutic approaches, and v) under what circumstances is MRD prognostic and useful for MRD risk-adapted AML clinical management. Given lack of well-suited techniques to reliably answer the relevant questions detailed above, we have optimized droplet digital PCR (ddPCR), a novel ultra-high sensitivity assay for detecting genomic MRD in AML. In a detailed recently published study (Parkin et al, JCI 2017) we found that AML frequently relapses from rare cells residing in remission marrows that based on mutation analysis resemble AML blast cells detected at diagnosis. We demonstrated the feasibility for detecting AML-associated gene mutations at allele frequencies as low as 0.002% and have also provided important novel prognostic insights. In this application, we are proposing complete characterization of mutational MRD and aberrant cellular clusters in AML remission bone marrows using a combination of sophisticated cell sorting and single cell transcriptome analyses complemented with mouse xenografting and ex vivo colony forming assays. Anticipated findings will improve the functional characterization of cells that carry AML-associated gene mutations in an attempt to identify and better characterize the source(s) of AML relapse. Using AML samples from two clinical validation cohorts, we will define the prognostic utility of ddPCR-based MRD assessments. Overall, data will lay the foundation for future real-time genomic MRD-guided clinical AML trials aiming at monitoring and improving consolidation and maintenance therapy and ultimately survival outcome in AML.