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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要/摘要背景：尽管有大量的患者，但急性髓系白血病（AML）的死亡率仍然很高。可用的治疗方法。不同的预后、治疗反应和生存很大程度上取决于细胞遗传学和表征 AML 亚型的分子畸变。检测 AML 融合的快速且经济有效的方法作为标准的下一代测序（NGS），突变可以改善患者的临床结果方法受到一些技术和生物信息问题的限制。假设：我们开发了一种基于 CRISPR 的单分子长读测序分析 (CSRL) 快速检测 AML 中临床相关的突变基因。这种新方法与新的生物信息学相结合方法，允许当天获得测序数据结果。我们假设这种技术可以改进后可检测 AML 中常见的突变和易位，并且这种快速的周转时间将通过快速进行风险分层和治疗选择，对患者护理产生积极影响。建议：在本研究中，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，我们将询问并回答 1) CSRL 检测是否会识别靶标中的所有突变和融合 NGS 和细胞遗传学检测到的基因？ 2) 单分子读数是否可以提供更准确的评估肿瘤负荷？ 3) CSRL 数据是否允许确定突变的阶段并提供额外的信息预后或预测意义？ 4) 当天采集样本并报告结果在临床上是否可行 AML CSRL 检测以及当天获得 CSRL 结果将如何影响临床决策？ 5) 什么相比之下，当前的周转时间、成本和检测性能有望得到改善到标准NGS？为了回答问题4和5，我们将进行一项试点临床试验，以检验临床的可行性。执行。研究设计/具体目标：具体目标：SA1) 扩展我们的 CSRL 测序分析并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）。这开发将使用低成本的长核酸分子多重 CRISPR 富集文库纳米孔装置。 SA2) 验证 SA1 中开发的检测方法的灵敏度和特异性并进行评估长读取和单分子定量提供的定相数据的预后影响更多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具有通过标准 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 测序在当日白血病分子诊断中的实用性。这一具体目标的目的是通过一项试点研究证明我们当天超快速分子分析测定的临床影响我们的癌症中心有 10-12 名患者。我们将测试在现实世界中实施这些方法的可行性来自诊所的样本、当天测试和报告的便利性以及研究当天结果可能产生的影响可能会对肿瘤学家如何治疗急性白血病患者产生影响。