PROJECT SUMMARY
Solitary pulmonary nodules (SPNs) are hazy opacities less than or equal to 3 cm in diameter that are
surrounded by aerated lung. The incidence of SPN in the United States is approximately 1.5 million per year. It
can be observed in both benign and malignant etiology, with differential diagnosis proving to be a challenge.
Currently, imaging-based diagnosis takes months or even years of surveillance to assess benignity/malignancy
with a moderate degree of accuracy. The periodic imaging examinations also increase the burden of
healthcare costs, delay relevant treatment, and may increase the risk of metastasis. In addition, there is no
consensus on optimal size threshold or methods for tissue sampling of SPN in clinical care due to multiplicity,
heterogeneity, spatial limitations, etc. In brief, molecular diagnostics for prompt, definitive detection of
malignant SPN is absent but highly desired. Liquid biopsy offers a method of precision diagnosis, yet it has not
been explored in SPN. Extracellular vesicles (EVs) are cell released vesicles of sub-micrometer diameter. EVs
contain a tissue-specific signature wherein a variety of proteins and nucleic acids are selectively packaged.
Growing evidence has shown important biological roles and clinical relevance of EVs in cancers. Recently, our
preliminary study demonstrated that malignant SPNs can be identified by interrogating the molecules
contained within EVs, and it is probable that a definitive diagnosis can be made with a five-day turnaround time
after the initial visit. Consequently, in the pursuit of clinical translation, we propose the development of an
assay and mutation panel for liquid biopsy of SPN with EVs. This ultimate goal will be achieved by fulfilling
three major milestones. First, we will develop and validate a microdevice for efficient isolation of pure EVs from
up to 5 ml plasma for reliable downstream molecular analyses. The disposable device also features ready-to-
use, low-cost, automated, and high-throughput processing of various body fluids. Second, to strengthen the
coverage and depth of sequencing, we customized a mutation panel that consists of 565 genes that are
involved in lung adenocarcinoma. Meanwhile, the sequencing library preparation that uses EV DNA and
associated targeted sequencing will be optimized and validated. Third, we will analyze mutation concordance
between malignant SPN tissue DNA and plasma EV DNA. Based on identified mutations, mutation patterns in
relation to smoking history and sex in malignant SPN will be investigated. Genomic evolution of malignant
SPNs will be studied among malignant SPN tissue DNA, normal tissue DNA, and plasma EV DNA. In parallel,
we will customize a minimized gene panel for SPN differential diagnosis without compromising detection
sensitivity and specificity. Altogether, we aim to develop an assay for diagnosis of SPNs in clinical settings.
Moreover, mutations and genomic evolution of malignant SPNs will be studied. While designed to specifically
target SPNs as proof-of-concept, the new device in combination with other molecular detection modalities will
provide a transformative platform for basic and clinical investigation of a wide range of diseases.