Non-invasive detection of tumor NTRK gene fusions via rapid, efficient and low-cost extracellular vesicle isolation method

快速、高效、低成本的细胞外囊泡分离方法无创检测肿瘤NTRK基因融合体

• 批准号: 10707684
• 负责人: Hongzhang He
• 金额: $ 40万
• 依托单位: CAPTIS DIAGNOSTICS
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10707684
• 关键词: ABL1 gene; Adult; Area; Biological Assay; Biopsy; Blood; Breast; Cancer Patient; Cell Line; Cells; Central Nervous System; Clinical; Clinical Laboratory Improvement Amendments; Colon; DNA; Detection; Development; Devices; Diagnostic; Diameter; Disease; Disease Progression; Encapsulated; Enzyme-Linked Immunosorbent Assay; Excision; Fibroblast Growth Factor Receptors; Gene Frequency; Gene Fusion; Genes; Glioma; Goals; Guidelines; High Density Lipoproteins; Introns; Knowledge; Label; Laboratories; Length; Lipids; Low-Density Lipoproteins; Lung; Magnetism; Malignant Neoplasms; Malignant neoplasm of lung; Malignant neoplasm of ovary; Malignant neoplasm of pancreas; Malignant neoplasm of thyroid; Membrane Lipids; Messenger RNA; Methods; Modality; Modeling; Molecular Profiling; Molecular Weight; NRG1 gene; Nanosphere; Nanotechnology; National Comprehensive Cancer Network; Non-Invasive Detection; Oncologist; Operative Surgical Procedures; Pathologic; Patient-Focused Outcomes; Patients; Performance; Phase; Plasma; Plasma Proteins; Point Mutation; Positioning Attribute; Proteins; RNA; RNA Degradation; ROS1 gene; Reading; Receptor Protein-Tyrosine Kinases; Recommendation; Ribonucleases; Sampling; Solid Neoplasm; Surface; Technology; Therapeutic; Time; Tissue Sample; Tissues; Transcript; Tropomyosin; Tumor Weights; Tumor-Derived; Ultracentrifugation; Validation; Very low density lipoprotein; Western Blotting; clinically actionable; cohort; compliance behavior; cost; cost effective; detection limit; detection sensitivity; diagnostic assay; extracellular vesicles; fusion gene; genetic variant; hepatobiliary cancer; hybrid gene; inhibitor therapy; innovation; kinase inhibitor; liquid biopsy; malignant stomach neoplasm; minimally invasive; molecular diagnostics; nanopore; nanoprobe; operation; particle; pediatric patients; precision medicine; preservation; receptor; response; screening; screening services; sequencing platform; single molecule; targeted treatment; technology platform; testing services; transcriptome; tumor; variant detection

SUMMARY/Abstract Cancer patients with neurotrophic tyrosine receptor kinase (NTRK) gene fusions who took Larotrectinib (Vitrakvi) and Entrectinib (Rozlytrek) lived significantly longer without disease progression, regardless of the disease origin and its gene fusion partners. Currently, detection of NTRK fusions is recommended by multiple National Comprehensive Cancer Network (NCCN) guidelines, including those for colon, non- small cell lung, breast, central nervous system, pancreatic, thyroid, gastric, hepatobiliary, and ovarian cancers. However, current diagnostic assays for NTRK gene fusions require highly invasive tissue samples, which are often limited by tumor position, patient compliance, sample amount, and tumor inaccessibility in cancers such as lung cancer, gliomas, and pancreatic cancers. Liquid biopsy allows for minimally invasive molecular profiling when tissue biopsies are scarce or unreachable. This means that patients’ tumors can be molecularly profiled for precision medicine with a simple blood draw instead of a surgical procedure. The significant clinical benefit of targeted, tropomyosin receptor kinase (TRKs) inhibitor therapies towards cancer patients bearing NTRK fusions bolsters the urgent unmet need for a liquid biopsy method to screen pathological NTRK fusions to identify the TRK inhibitor responders. Extracellular vesicles (EVs) in the blood plasma of cancer patients encapsulate the entire cancer transcriptome via long and high-quality mRNA fragments up to 20 kbp. EVs harbor cancer-associated mRNA transcripts, therefore perfectly for NTRK fusion detection. To meet the urgent, unmet, clinical challenge of detecting NTRK fusions, we propose to vertically integrate our proprietary, one-component lipid nanoprobe (1C-LNP) technology for isolating total EV from plasma, a new wild-type blocker amplification (wtBA) technology to NTRK fusions in EV RNA from a high background of wildtype RNA, and the Oxford Nanopore Technologies’ long-read sequencing platform for reading NTRK fusions. We will develop a minimally invasive NTRK fusion detection technology with two Aims. In Aim 1, we will optimize the 1C-LNP technology for total EV isolation in plasma. In Aim 2, we will develop and integrate 1C-LNP and wtBA to isolate high-molecular weight tumor mRNA from EVs, enrich mRNA sequence with NTRK fusions, perform single-molecule long-reads sequencing analysis using the Oxford Nanopore Flongle device, and further validate the feasibility of NTRK fusion variants detection using samples isolated from tissue and plasma EV. We anticipate significant impacts in two areas of molecular diagnostics and liquid biopsy: (1) We will create the most sensitive, state-of-the-art method to identify NTRK fusions at low variant allele frequencies; (2) We will create a minimally invasive NTRK fusion screening assay for plasma, which identifies NTRK inhibitors responders and can have an incredible impact on the patient outcomes. Phase II activities will focus on expanding this 1C-LNP integrated wtBA technology platform to other clinical actionable gene fusions such as ROS1, ALK, NRG1, RET, ETS, EWS, FGFR, ABL1 and subsequently perform the clinical validation of the gene fusion panel. Upon completion of clinical validation, we will launch this minimally invasive NTRK fusion screening test service through the Clinical Laboratory Improvement Amendments (CLIA) laboratory for oncologists.

摘要/摘要 神经营养性酪氨酸受体激酶（NTRK）基因融合的癌症患者 （vitrakvi页 疾病起源及其基因融合伙伴的伴侣。目前，建议检测NTRK融合 由多个国家综合癌症网络（NCCN）指南，包括结肠，非 - 小细胞肺，乳房，中枢神经系统，胰腺，甲状腺，胃，肝胆和卵巢癌。 但是，当前对NTRK基因融合的诊断测定需要高度侵入性的组织样本， 通常受肿瘤位置，患者依从性，样本量和肿瘤在这种癌症中的限制 作为肺癌，神经胶质瘤和胰腺癌。液体活检允许微创分子分析 当组织活检稀缺或无法到达时。这意味着患者的肿瘤可以分子 用简单的血液抽血而不是外科手术进行了精确药物的介绍。重要的 针对癌症患者的靶向肌球蛋白受体激酶（TRK）抑制剂疗法的临床益处 轴承ntrk融合体巩固了对筛查病理学的液体活检方法的紧急需求 NTRK融合以识别TRK抑制剂响应者。 癌症患者血浆中的细胞外蔬菜（EV）封装了整个癌症转录组 通过长达20 kbp的长长和高质量mRNA碎片。 EV携带癌症相关的mRNA转录物， 因此，完美地用于NTRK融合检测。满足检测NTRK的紧急，未满足，临床的挑战 融合，我们建议垂直整合我们的专有的单组脂质纳米探针（1C-LNP）技术 为了将总电动汽车隔离为等离子体，这是一种新的野生型阻滞剂扩增（WTBA）技术 来自WildType RNA的高背景的EV RNA和牛津纳米孔技术的长阅读测序 读取NTRK融合的平台。我们将与 两个目标。在AIM 1中，我们将优化1C-LNP技术，用于血浆中的总电动汽车隔离。在AIM 2中，我们将 开发和整合1C-LNP和WTBA，以分离出EVS的高分子重量肿瘤mRNA，富集mRNA 使用NTRK融合序列，使用牛津进行单分子长阅读测序分析 纳米孔inthe设备，并进一步验证使用样品检测NTRK融合变体的可行性 从组织和血浆EV中分离出来。我们预计分子诊断和 液体活检：（1）我们将创建最敏感，最先进的方法，以识别低变体的NTRK融合 等位基因频率； （2）我们将为血浆创建一个微创的NTRK融合筛选测定法，该测定法 识别NT​​RK抑制剂响应者，并可能对患者结局产生不可思议的影响。第二阶段 活动将着重于将此1C-LNP集成的WTBA技术平台扩展到其他临床可行 诸如ROS1，ALK，NRG1，RET，ETS，EWS，FGFR，ABL1之类的基因融合，然后进行临床 基因融合面板的验证。完成临床验证后，我们将启动此最低侵入性 NTRK融合筛查测试服务通过临床实验室改进修订（CLIA）实验室 对于肿瘤学家。