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

概要/摘要 服用Larotrectinib的神经营养性酪氨酸受体激酶（NTRK）基因融合的癌症患者 （Vitrakvi）和Entrectinib（Rozlytrek）在无疾病进展的情况下生存时间显著延长， 疾病的起源和它的基因融合伙伴。目前，推荐检测NTRK融合 根据多个国家综合癌症网络（NCCN）指南，包括结肠癌，非结肠癌， 小细胞肺癌、乳腺癌、中枢神经系统癌、胰腺癌、甲状腺癌、胃癌、肝胆癌和卵巢癌。 然而，目前NTRK基因融合的诊断测定需要高度侵入性的组织样品，这是非常困难的。 通常受到肿瘤位置、患者依从性、样本量和肿瘤不可接近性的限制， 肺癌，神经胶质瘤，胰腺癌液体活检允许微创分子分析 当组织活检很少或无法获得时。这意味着患者的肿瘤可以在分子上 通过简单的抽血而不是外科手术来进行精准医疗。的重大 靶向原肌球蛋白受体激酶（TRKs）抑制剂治疗癌症患者的临床获益 携带NTRK融合物支持了对液体活检方法的迫切需求， NTRK融合以鉴定TRK抑制剂应答者。 癌症患者血浆中的细胞外囊泡（EV）包裹整个癌症转录组 通过长达20 kbp的长而高质量的mRNA片段。EV携带癌症相关的mRNA转录物， 因此非常适合NTRK融合检测。为了应对检测NTRK的紧迫、未满足的临床挑战， 融合，我们建议垂直整合我们的专有，单组分脂质纳米探针（1C-LNP）技术 为了从血浆中分离总EV，一种新的野生型阻断剂扩增（wtBA）技术， 来自野生型RNA高背景的EV RNA，以及牛津纳米孔技术公司的长读测序 用于阅读NTRK融合的平台。我们将开发微创NTRK融合检测技术， 两个目标。在目标1中，我们将优化1C-LNP技术，以实现血浆中的完全EV隔离。在目标2中，我们将 开发并整合1C-LNP和wtBA，从EV中分离高分子量肿瘤mRNA，富集mRNA 序列与NTRK融合，使用牛津测序仪进行单分子长读段测序分析。 Nanopore Flongle装置，并进一步验证NTRK融合变异体检测的可行性，使用样品 分离自组织和血浆EV。我们预计在分子诊断和 液体活检：（1）我们将创建最敏感，最先进的方法来识别低变异的NTRK融合。 等位基因频率;（2）我们将建立一个微创NTRK融合筛查检测血浆， 识别NTRK抑制剂反应者，并对患者的结果产生令人难以置信的影响。二期 活动将侧重于将这种1C-LNP集成wtBA技术平台扩展到其他临床可行的 基因融合体，如ROS 1、ALK、NRG 1、RET、ETS、EWS、FGFR、ABL 1，并随后进行临床 基因融合组的验证。在完成临床验证后，我们将推出这种微创 通过临床实验室改进修正案（CLIA）实验室提供NTRK融合筛查测试服务 给肿瘤学家。

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.