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

利用 CRISPR 富集和实时长读长测序进行快速急性白血病基因组分析

• 批准号: 10651543
• 负责人: CECILIA C YEUNG
• 金额: $ 25.58万
• 依托单位: FRED HUTCHINSON CANCER CENTER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10651543
• 关键词: Acute Myelocytic Leukemia; Acute leukemia; Allogenic; Bioinformatics; Biological Assay; Cancer Center; Characteristics; Chemistry; Chromosome abnormality; Clinic; Clinical; Clinical Services; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Computing Methodologies; Cytogenetics; DNA; DNA sequencing; Data; Data Analyses; Detection; Development; Devices; Diagnosis; Disease; Drug Targeting; Ensure; FLT3 gene; Flow Cytometry; Gene Mutation; Genes; Genomics; Goals; Guide RNA; Guidelines; Hematopoietic Neoplasms; Hour; Individual; JAK2 gene; Karyotype; Laboratories; Libraries; Methodology; Methods; Molecular; Molecular Abnormality; Molecular Diagnosis; Molecular Profiling; Morphology; Mutate; Mutation; NPM1 gene; National Comprehensive Cancer Network; Newly Diagnosed; Nucleic Acids; Oncologist; Outcome; Pathology; Patient Care; Patient-Focused Outcomes; Patients; Performance; Phase; Physicians; Pilot Projects; Polymerase Chain Reaction; Prediction of Response to Therapy; Preparation; Prognosis; Prognostic Marker; Protocols documentation; RUNX1 gene; Reagent; Recommendation; Reporting; Reproducibility; Research Design; Running; Sampling; Selection for Treatments; Sensitivity and Specificity; Software Tools; Specimen; Surveys; System; TP53 gene; Technology; Testing; Time; Training; Tumor Burden; Variant; Visualization software; clinical decision-making; clinical implementation; clinically actionable; clinically relevant; cost; cost effective; cost efficient; design; detection method; feasibility testing; fusion gene; hematopoietic cell transplantation; improved; instrument; leukemia; mortality; multiplex assay; nanopore; next generation sequencing; novel; pilot trial; predictive marker; prognostic; prognostication; response; risk stratification; sample collection; single molecule; skills; standard of care; t(8; 21)(q22; q22); targeted treatment; treatment response

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.

项目摘要/摘要 背景：急性髓性白血病（AML）尽管有大量的化疗，但死亡率仍然很高。 可用的治疗。可变的预后、治疗反应和存活率主要基于细胞遗传学和 AML亚型的分子畸变特征。检测AML融合的快速且具有成本效益的方法 和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：我们已经开发了基于CRISPR的单分子长读段测序测定（CSRL）， 快速检测AML中的临床相关突变基因。这种新的方法，结合新的生物信息学 方法，允许测序数据的同一天结果。我们假设这种技术可以 改进以检测AML中常见的突变和易位，这种快速的周转时间将 通过允许快速的风险分层和治疗选择来积极影响患者护理。 建议：在这项研究中，我们扩展了CSRL检测，以包括更多相关的突变和易位。 具体来说，我们将提问和回答1）CSRL检测是否会识别靶标中的所有突变和融合 NGS和细胞遗传学发现的基因2)单分子读数是否能更准确地评估 肿瘤负荷？3)CSRL数据是否允许确定突变的定相并提供额外的 预后或预测意义？4)同一天的样本采集和结果报告在临床上是否可行， AML CSRL检测试剂盒以及获得当天CSRL结果将如何影响临床决策？5)什么 相比之下，根据目前的周转时间、成本和分析性能， 标准NGS？为了回答问题4和5，我们将进行一项试点临床试验，以测试临床治疗的可行性。 实施.研究设计/具体目标：具体目标：SA 1）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重CRISPR富集文库 纳米孔装置。SA 2）验证SA 1中开发的检测试剂盒的灵敏度和特异性，并评估 通过长读数和单分子定量提供的定相数据的预后影响， 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。 使用标准NGS和细胞遗传学获得的具有现有分子数据的样本。SA 3）建立临床 CSRL测序与当天白血病分子诊断的实用性。这一具体目标的目的 是通过一项初步研究来证明我们当天超快速分子分析测定的临床影响， 在我们癌症中心的10-12个病人身上。我们将测试在真实的世界中实现这些方法的可行性。 从诊所获得的样本，当天检测和报告的便利性，以及研究当天结果的影响， 对肿瘤学家如何治疗急性白血病患者有潜在的影响。