Liquid biopsy: current technology and clinical applications

Abstract

Liquid biopsies are increasingly used for cancer molecular profiling that enables a precision oncology approach. Circulating extracellular nucleic acids (cell-free DNA; cfDNA), circulating tumor DNA (ctDNA), and circulating tumor cells (CTCs) can be isolated from the blood and other body fluids. This review will focus on current technologies and clinical applications for liquid biopsies. ctDNA/cfDNA has been isolated and analyzed using many techniques, e.g., droplet digital polymerase chain reaction, beads, emulsion, amplification, and magnetics (BEAMing), tagged-amplicon deep sequencing (TAm-Seq), cancer personalized profiling by deep sequencing (CAPP-Seq), whole genome bisulfite sequencing (WGBS-Seq), whole exome sequencing (WES), and whole genome sequencing (WGS). CTCs have been isolated using biomarker-based cell capture, and positive or negative enrichment based on biophysical and other properties. ctDNA/cfDNA and CTCs are being exploited in a variety of clinical applications: differentiating unique immune checkpoint blockade response patterns using serial samples; predicting immune checkpoint blockade response based on baseline liquid biopsy characteristics; predicting response and resistance to targeted therapy and chemotherapy as well as immunotherapy, including CAR-T cells, based on serial sampling; assessing shed DNA from multiple metastatic sites; assessing potentially actionable alterations; analyzing prognosis and tumor burden, including after surgery; interrogating difficult-to biopsy tumors; and detecting cancer at early stages. The latter can be limited by the small amounts of tumor-derived components shed into the circulation; furthermore, cfDNA assessment in all cancers can be confounded by clonal hematopoeisis of indeterminate potential, especially in the elderly. CTCs can be technically more difficult to isolate that cfDNA, but permit functional assays, as well as evaluation of CTC-derived DNA, RNA and proteins, including single-cell analysis. Blood biopsies are less invasive than tissue biopsies and hence amenable to serial collection, which can provide critical molecular information in real time. In conclusion, liquid biopsy is a powerful tool, and remarkable advances in this technology have impacted multiple aspects of precision oncology, from early diagnosis to management of refractory metastatic disease. Future research may focus on fluids beyond blood, such as ascites, effusions, urine, and cerebrospinal fluid, as well as methylation patterns and elements such as exosomes.

Keywords: CTC; Liquid biopsy; Precision medicine; cfDNA; ctDNA.

Fig. 1

A Types of liquid biopsies. Liquid biopsy is most commonly obtained via blood sampling, but can also be derived from urine, CSF, ascites fluid, and pleural fluid. cfDNA/ctDNA, CTCs, RNA, and extracellular vesicles can be isolated from these fluids. Protein expression and methylation patterns can also be assessed with liquid biopsy. Boxes represent examples of fluids biopsied and circles represent examples of materials isolated/evaluated. B Clinical applications of liquid biopsies. Liquid biopsies (cfDNA/ctDNA and CTCs) have been utilized for a variety of purposes as noted. C Examples of types of liquid biopsies, material isolated/analyzed, and clinical applications. Liquid biopsy is most commonly obtained via blood sampling, but can also be derived from a variety of other fluids. cfDNA/ctDNA and CTCs are the most commonly isolated and analyzed materials. cfDNA: cell-free DNA, ctDNA: circulating tumor DNA, CTCs: circulating tumor cells

Fig. 2

Advantages and disadvantages of liquid versus tissue biopsy. Liquid biopsies are non-invasive, less expensive, can assess multiple tumor sites, and can be obtained serially. Low tumor DNA shed, CHIP, and reproducibility issues may limit usage. CTC isolation can also be technically challenging. Image created with help of Biorender.com. CHIP: clonal hematopoiesis of indeterminate potential, CTCs: circulating tumor cells