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