Increased Sensitivity of Minimal Residual Disease Monitoring using Peripheral Blood in Pediatric Patients with Acute Lymphoblastic Leukemia

提高使用外周血监测急性淋巴细胞白血病儿科患者微小残留病的敏感性

• 批准号: 9797477
• 负责人: Keith August
• 金额: $ 24.16万
• 依托单位: UNIVERSITY OF KANSAS LAWRENCE
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9797477
• 关键词: Acute Lymphocytic Leukemia; Acute Myelocytic Leukemia; Acute leukemia; Adult Acute Myeloblastic Leukemia; Affect; Affinity; Antibodies; Antigens; Aspirate substance; Biological Assay; Blood; Blood Cells; Bone Marrow; Bone Marrow Cytometry; Bone marrow biopsy; CD19 gene; CD34 gene; CD7 gene; Cause of Death; Cell Count; Cell Line; Cell model; Cell surface; Cells; Child; Childhood; Childhood Acute Lymphocytic Leukemia; Chromosome abnormality; Clinical; Clinical Research; Clinical Sensitivity; Consumption; DNA Markers; DNA Nucleotidylexotransferase; Data; Detection; Detection of Minimal Residual Disease; Devices; Diagnostic; Disease; Disease remission; Early Diagnosis; Electromagnetics; Flow Cytometry; Fluorescent in Situ Hybridization; Hematopoietic stem cells; Image; Immobilization; Immunophenotyping; Leukemic Cell; Liquid substance; Measurement; Measures; Methods; Microfluidic Microchips; Microfluidics; Monitor; Monoclonal Antibodies; Mutate; Normal Cell; Outcome; Output; PTPRC gene; Pain; Patient-Focused Outcomes; Patients; Pediatric Oncology; Phenotype; Plastics; Precision therapeutics; Procedures; Prognostic Marker; Pump; Reagent; Recovery; Recurrent disease; Relapse; Reporter; Residual Cancers; Residual Neoplasm; Residual Tumors; Risk Assessment; Sampling; Scanning; Secure; Sedation procedure; Stains; Surface; Surface Antigens; Technology; Testing; Therapeutic Intervention; Whole Blood; Width; aged; base; cancer cell; chemotherapy; circulating leukemia cell; experience; fluorescence microscope; high risk; improved; innovation; leukemia; novel; outcome prediction; pediatric patients; peripheral blood; relapse risk; standard of care

Abstract The primary cause of death in patients with acute lymphoblastic leukemia (ALL) is sudden disease relapse (80% of patients with minimum residual disease, MRD, will relapse). For better outcome, onset of relapse must be detected early. Therefore, frequent monitoring of MRD is needed. In pediatric ALL, monitoring MRD utilizes multi- parameter flow cytometry (MFC) or qPCR from bone marrow aspirates (BMA), which is painful and an invasive procedure, hence not performed frequently. MFC/BMA detects MRD in ~40% of patients; most patients are classified as MRD negative in spite of residual cancer cells present in their blood. qPCR detection in B-ALL is labor intensive/expensive as it requires patient-specific target identification via sequencing. In some B-ALL patients, specific DNA markers cannot be identified. Although MRD status has proven to be an effective prognostic indicator allowing for risk assessment and improving survival with treatment intensification, MFC and qPCR require a BMA due to limited ability to detect rare leukemia cells in peripheral blood. Bone marrow is not the only niche where leukemia cells are found. Circulating leukemia cells (CLCs) are present in patient’s peripheral blood. Therefore, if sensitive methods for MRD detection from blood could be used, frequent MRD monitoring could improve patient outcome. Built from a successful R21 IMAT project for MRD monitoring in adult acute myeloid leukemia (AML) patients, an innovative and highly sensitive MRD platform will be delivered herein, which will consist of a fluidic cartridge that isolates and analyzes enriched CLCs from peripheral blood in an automated fashion. The cartridge is comprised of two modules, one for the affinity selection of B- or T-type CLCs from blood and the other module used to “trap” enriched cells to allow for immunophenotyping and/or FISH analyses (FISH performed on high-risk patients). In this R33 application, the utility of this microfluidic assay will be demonstrated in pediatric B-ALL and T-ALL patients. Appropriate antibodies (anti-CD19 and anti-CD7 monoclonal antibodies for B-ALL and T-ALL, respectively) will be immobilized to a surface of the cell selection module with the aid of a photocleavable linker to affinity-select cells expressing CD19 or CD7 surface antigens. Cells will be photolytically released from the capture surface and CLCs identified by the expression of aberrant markers, such as Terminal deoxynucleotidyl Transferase (TdT), in a unique microtrap (µTRAP) module. This module allows for automated cell staining and phenotyping. The CLC microfluidic cartridge will be clinically validated by monitoring MRD status in B-ALL and T-ALL pediatric patients and CLC burden will be tracked. The cartridge will perform CLC FISH analysis for high-risk patients to gain information on chromosomal aberrations that provide information to allow precision treatment. Given the strong data generated to date in AML (100% test positivity) and the urgent diagnostic need for an improved easy-to-implement MRD assay that permits frequent sampling using peripheral blood, broad patient coverage, and early detection of relapse, the proposed cartridge will fill an unmet clinical need in pediatric oncology as well as other leukemic diseases.

抽象的 急性淋巴细胞白血病患者的死亡原因（全部）是突然的疾病缓解（80％ 在最小残留疾病的患者中，MRD将中继）。为了更好的结果，救济的发作必须是 提早检测到。因此，经常需要监测MRD。在小儿所有人中，监测MRD都使用了多种多样的 参数流式细胞仪（MFC）或骨髓抽吸物（BMA）的QPCR，这是痛苦且侵入性的 程序，因此不经常执行。 MFC/BMA检测到约40％的患者中的MRD；大多数患者是 尽管血液中存在残留的癌细胞，但仍被归类为MRD阴性。 b-all中的qPCR检测是 劳动密集型/昂贵，因为它需要通过测序进行特定于患者的目标识别。在某个B-all中 患者，无法识别特定的DNA标记。虽然MRD状态已被证明是有效的 预后指标允许通过治疗强化，MFC和MFC进行风险评估并提高生存率 由于检测到外周血中罕见的白血病细胞的能力有限，QPCR需要BMA。骨髓不是 发现白血病细胞的唯一利基市场。患者的循环白血病细胞（CLC）存在 外周血。因此，如果可以使用血液中MRD检测的敏感方法，则经常MRD 监测可以改善患者的结果。由成功的R21 IMAT项目构建，用于成人MRD监控 急性髓样白血病（AML）患者，将在这里提供创新且高度敏感的MRD平台， 它将由液体墨盒组成，该墨盒分离并分析了从外周血中富集的CLC 自动化时尚。该墨盒由两个模块组成，一个用于B-或T型CLC的亲和力选择 从血液和其他用于“捕获”富集细胞的模块，以允许免疫表型和/或鱼类 分析（对高危患者进行鱼）。在此R33应用中，该微流体测定的实用性将 在小儿B-ALL和T-ALL患者中得到证明。适当的抗体（抗CD19和抗CD7 B-all和T-All的单克隆抗体将被固定在细胞选择的表面上 借助于表达CD19或CD7表面抗原的亲和力 - 选择细胞的可光链接头模块。 细胞将从捕获表面释放，并通过异常表达识别CLC 在独特的Microtrap（µTRAP）模块中，标志物，例如末端脱氧核苷酸转移酶（TDT）。这 模块允许自动细胞染色和表型。 CLC微流体墨盒将在临床上 通过监测B-ALL和T-ALL小儿患者和CLC烧伤的MRD状态的验证。这 墨盒将对高风险患者进行CLC鱼分析，以获取有关染色体畸变的信息 提供信息以允许精确治疗。给定迄今为止在AML中生成的强数据（100％测试 阳性）以及对改进易于实施的MRD测定的紧急诊断需求，该测定法可以允许频率 使用外周血，广泛的患者覆盖范围和早期发现浮雕的早期检测 将满足儿科肿瘤学以及其他白血病疾病的未满足的临床需求。