Liquid biopsy of cancer: a multimodal diagnostic tool in clinical oncology

Abstract

Over the last decades, the concept of precision medicine has dramatically renewed the field of medical oncology; the introduction of patient-tailored therapies has significantly improved all measurable outcomes. Liquid biopsy is a revolutionary technique that is opening previously unexpected perspectives. It consists of the detection and isolation of circulating tumor cells, circulating tumor DNA and exosomes, as a source of genomic and proteomic information in patients with cancer. Many technical hurdles have been resolved thanks to newly developed techniques and next-generation sequencing analyses, allowing a broad application of liquid biopsy in a wide range of settings. Initially correlated to prognosis, liquid biopsy data are now being studied for cancer diagnosis, hopefully including screenings, and most importantly for the prediction of response or resistance to given treatments. In particular, the identification of specific mutations in target genes can aid in therapeutic decisions, both in the appropriateness of treatment and in the advanced identification of secondary resistance, aiming to early diagnose disease progression. Still application is far from reality but ongoing research is leading the way to a new era in oncology. This review summarizes the main techniques and applications of liquid biopsy in cancer.

Keywords: cancer; circulating tumor DNA; circulating tumor cells; exosomes; liquid biopsy; targeted therapy.

Figure 1.

Molecular applications of circulating tumor cells (CTCs), circulating tumor DNA (ctDNA) and exosomes as liquid biopsy for personalized medicine.

Figure 2.

Flow chart of combined methods to isolate circulating tumor cells (CTCs). (a) Peripheral blood samples are subjected to density gradient stratification and leukocyte depletion is assessed by an immunomagnetic method using anti-CD45 and anti-glycophorin conjugated microbeads (AUTOMACS (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany)). (b) The CTC-enriched fraction is stained by specific fluorochrome conjugated antibodies and loaded in a dedicated cartridge which then is subjected to dielectric forces (DEPArray (Menarini Silicon Biosystems, Firenze, Italy)). (c) CTCs are visualized by dedicated software and selected by positive fluorescence for tumor-specific markers and negativity for CD45 leukocyte marker. 4’,6-diaminidino-2-phenylindole (DAPI) is used to counterstain nuclei. The CTCs are moved into a parking area and recovered as single or grouped cells in a buffer drop.

Figure 3.

The DEPArray technology is based on the use of a dielectrophoretic field (DEP) generated by electrodes in a matrix underlying a liquid layer of cells. (a) The DEPArray constellation for the creation of DEP is determined to be a determinant of the entire intrapolarization. (b) The whole cell is isolated from the individual software, and (c) after computational imaging, individual cells or groups of cells are moved and recovered by a drop of buffer in a specific tube.

Figure 4.

Mutational analysis performed on circulating tumor cells (CTCs) isolated by DEPArray using next-generation sequencing, Sanger sequencing or digital polymerase chain reaction (PCR). DAPI , 4’,6-diaminidino-2-phenylindole.

Figure 5.

Exosomes biogenesis and content. Exosomes are end products of the recycling endosomal pathway and originate from inward budding of the plasma membrane, leading to the formation of multi vesicular bodies (MVBs). Following active packaging of signaling molecules into MVBs, they fuse with the plasma membrane and release their contents into the extracellular space in the form of exosomes. Exosomes consist of a lipid bilayer which contains both transmembrane and nonmembrane proteins, as well as noncoding RNAs, mRNAs and either single-stranded or double-stranded DNA. They also express a conserved set of proteins independently by cellular origin, including CD63, CD81, and CD9 tetraspanins, while those from cancer cells are rich in tumor-associated antigens. MHC, major histocompatibility complex.