Rapid Acute Leukemia Genomic Profiling with CRISPR enrichment and Real-time long-read sequencing

项目摘要/摘要 背景：急性髓系白血病(AML)的死亡率仍然很高，尽管有大量的 可用的治疗方法。不同的预后、治疗反应和存活率在很大程度上是基于细胞遗传学和 AML亚型特有的分子像差。快速、经济有效的检测AML融合的方法 突变可以改善患者的临床结果，作为标准的下一代测序(NGS) 方法受到几个技术和生物信息学问题的限制。 假设：我们已经开发了一种基于CRISPR的单分子长读测序分析(CSRL)来 快速检测AML中与临床相关的突变基因。这种新的方法，与新的生物信息学相结合 方法，允许测序数据的当天结果。我们假设这样的技术可以是 改进以检测AML中常见的突变和易位，并且这种快速的周转时间将 通过允许快速风险分层和治疗选择，对患者护理产生积极影响。 建议：在这项研究中，我们扩展了CSRL分析，以包括更多相关的突变和易位。 具体地说，我们将问并回答1)CSRL检测是否会识别靶标中的所有突变和融合 NGS和细胞遗传学看到的基因？2)单分子读数是否提供了更准确的评估 肿瘤负担？3)CSRL数据是否允许确定突变的分期并提供额外的 预后或预测意义？4)当天采集样本和报告结果在临床上可行吗？ AML CSRL检测以及获得当天CSRL结果将如何影响临床决策？5)什么 与之相比，目前的周转时间、成本和分析性能都有望得到改进 到标准的NGS？为了回答问题4和5，我们将进行一项试点临床试验，以测试临床试验的可行性。 实施。研究设计/具体目标：具体目标：SA1)扩展我们的CSRL测序 分析和优化当前的生物信息学工作流程，以实现当天诊断(约8小时)。这 开发将以低成本使用长核酸分子的多路CRISPR富集库 纳米孔装置。SA2)验证在SA1中开发的检测的敏感性和特异性，并评估 长读数和单分子定量提供的分期数据对更多患者预后的影响 准确的肿瘤负荷评估。我们将在一组临床注释的白血病上测试所提出的方法。 用标准NGS和细胞遗传学获得的现有分子数据的样本。SA3)建立临床 CSRL测序在白血病当日分子诊断中的应用这一特定目标的目的 是通过一项初步研究来展示我们当天的超快分子图谱分析的临床影响 在我们癌症中心的10-12名病人身上。我们将在现实世界中测试实施这些方法的可行性 来自诊所的样本，简化了当天的测试和报告，并研究了当天结果的影响 可能对肿瘤学家如何治疗他们的急性白血病患者产生影响。

PROJECT SUMMARY/ABSTRACT Background: Acute myeloid leukemia (AML) continues to have high mortality rates despite a plethora of available treatments. Variable prognosis, treatment response and survival are largely based on cytogenetic and molecular aberrations that characterize AML subtypes. Fast and cost-effective methods of detecting AML fusions and mutations could improve clinical outcomes for patients, as standard next generation sequencing (NGS) methods are limited by several technical and bioinformatic issues. Hypothesis: We have developed a CRISPR-based single molecule long-read sequencing assay (CSRL) to quickly detect clinically relevant mutated genes in AML. This new methodology, combined with new bioinformatic approaches, allows for same day results of sequencing data. We hypothesize that such technology can be improved to detect both mutation and translocations common in AML, and that this rapid turn-around-time will positively impact patient care by allowing swift risk stratification and treatment selection. Proposal: In this study, we expand the CSRL assay to include more relevant mutations and translocations. Specifically, we will ask and answer 1) Will the CSRL assay identify all the mutations and fusion in the target genes seen by NGS and cytogenetics? 2) Do the single molecule reads provide a more accurate assessment of tumor burden? 3) Do CSRL data allow for determination of phasing of mutations and provide additional prognostic or predictive significance? 4) Is same-day sample collection and result reporting clinically feasible for the AML CSRL assay and how will obtaining same-day CSRL results impact clinical decision making? 5) What improvements can be expected upon the current turnaround time, costs, and assay performance in comparison to standard NGS? To answer questions 4 and 5, we will conduct a pilot clinical trial to test feasibility of clinical implementation. Research Design/Specific Aims: Specific aims: SA1) To expand our CSRL sequencing assay and optimize current bioinformatics workflow to allow for same-day diagnosis (~8 hours). This development will use a multiplexed CRISPR enrichment library of long nucleic acid molecules on a low cost Nanopore device. SA2) Validate sensitivity and specificity of the assay developed in SA1 and evaluate prognostic impact of phasing data provided by long reads and single molecule quantification for more accurate tumor burden assessment. We will test the proposed method on a set of clinically annotated leukemia samples with existing molecular data obtained with standard NGS and cytogenetics. SA3) To establish clinical utility of CSRL sequencing with same-day leukemia molecular diagnosis. The purpose of this specific aim is to demonstrate the clinical impact of our same day ultrarapid molecular profiling assay through a pilot study on 10-12 patients at our cancer center. We will test real world feasibility of implementing these methods on samples from the clinic, ease of same day testing and reporting, and study the impact same day results could potentially have on how oncologists treat their acute leukemia patients.