Feature Review
Liquid Biopsies in Cancer Diagnosis, Monitoring, and Prognosis

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Highlights

Liquid biopsies are emerging as a favorable alternative to conventional tissue biopsies, providing a noninvasive approach for the detection and monitoring of cancer biomarkers.

The FDA approval of a companion diagnostic test for lung cancer and a screening test for colorectal cancer based on the analysis of ctDNA were important milestones in the clinical implementation of liquid biopsies.

CTC enumeration using the CellSearch® CTC test is FDA approved for the prognosis of breast, colorectal, and prostate cancers.

Circulating EVs, RNAs, and TEPs are promising new constituents of liquid biopsies.

Recent technological developments in the field have the potential to address current limitations of liquid biopsies and enhance the sensitivity of cancer biomarker detection.

Liquid biopsies, comprising the noninvasive analysis of circulating tumor-derived material (the ‘tumor circulome’), represent an innovative tool in precision oncology to overcome current limitations associated with tissue biopsies. Within the tumor circulome, circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) are the only components the clinical application of which is approved by the US Food and Drug Administration (FDA). Extracellular vesicles (EVs), circulating tumor RNA (ctRNA), and tumor-educated platelets (TEPs) are relatively new tumor circulome constituents with promising potential at each stage of cancer management. Here, we discuss the clinical applications of each element of the tumor circulome and the prevailing factors that currently limit their implementation in clinical practice. We also detail the most recent technological developments in the field, which demonstrate potential in improving the clinical value of liquid biopsies.

Section snippets

Liquid Biopsies: Investigating the ‘Tumor Circulome’

Cancer is one of the leading causes of death worldwide, with 9.6 million cancer deaths estimated in 2018i. In the USA, more than 1 735 350 diagnoses of cancer were estimated in 2018, causing more than 609 640 deathsii. The development of ‘omics’ technologies has led to the field of precision oncology, which comprises tailoring treatment regimens to the molecular characteristics of each patient’s tumor [1]. The current gold standard for genetic profiling of tumors typically involves the use of

Circulating Tumor-Derived Proteins

The measurement of circulating protein markers has historically been the gold standard approach used for noninvasive diagnosis, screening, and postoperative follow-up in cancer management. Notable examples of circulating tumor-derived protein markers include the prostate-specific antigen (PSA) for prostate cancer screening [9] and cancer antigen (CA) 15-3 for postoperative follow-up of breast cancer recurrence [10]. These are compromised by high false positive rates, which can lead to

Circulating Tumor DNA

ctDNA comprises the fraction of circulating cell-free DNA (cfDNA) originating from cancer cells. This includes short nucleosome-associated fragments (80–200 bp) [13] and longer fragments (>10 kb) encapsulated within EVs [14]. The mechanisms of ctDNA release into circulation include apoptosis, necrosis, lysis of CTCs, and active secretion from the tumor [15]. The proof of the suitability of ctDNA as a cancer biomarker came with the identification of KRAS gene mutations in ctDNA from the blood of

Circulating Tumor Cells

CTCs are a population of tumor cells that have detached from the primary tumor and can be found in the peripheral blood of patients. Their presence is thought to be fundamental to the development of metastasis [31]. CTCs present systemically through active intravasation, with epithelial-to-mesenchymal transition (EMT) as a fundamental step [32], or through passive shedding from the primary tumor. This latter mechanism is supported by the presence of CTC aggregates or circulating tumor

Extracellular Vesicles

EVs are membranous particles released from all cell types under physiological and pathological conditions, as well as following different types of stimuli, including proteases, ADP, thrombin, inflammatory cytokines, growth factors, biomechanical shear and stress inducers, and apoptotic signals [40]. They can be found in almost every bodily fluid, especially blood [41]. Once considered a simple means to eliminate unneeded cellular components from the cytoplasm of cells, over the past decade, EVs

Circulating Tumor RNA

The fraction of circulating cell-free RNA originating from cancer cells is referred to as ctRNA. The existence of extracellular RNA was first documented in 1978 [66] and the first report of its potential as a cancer biomarker was published several years later [67]. Compared with DNA, RNA is a relatively unstable molecule, the naked half-life of which in plasma is ∼15 s [68]. Its stability is enhanced by its association with proteins [69], proteolipid complexes [67], and EVs [44].

Tumor-Educated Platelets

TEPs are perhaps the latest components of the tumor circulome to be considered for biomarker analysis. The concept of ‘platelet education’ by cancer refers to the presence of specific RNA signatures in platelets from patients with cancer. This was first reported in 2010 and 2011 with the observations that: (i) in patients with metastatic lung cancer, 197 platelet genes were downregulated and several genes were differentially spliced compared with controls [88]; and (ii) in glioma,

Controversies over the Use of Liquid Biopsies in Cancer Management

Despite reports demonstrating the potential of liquid biopsies in addressing current needs in cancer management, numerous controversies remain on their utility. This is particularly true for ctDNA and CTCs, which have already found application in clinical management. In a recent report, Torga and Pienta [92] compared the performance of two commercially available NGS-based ctDNA tests for metastatic prostate cancer, finding an astonishingly low concordance (7.5% patients studied) between the two

Concluding Remarks and Future Directions

Current standards for patient stratification and treatment selection include the analysis of tumor genetic alterations from tissue biopsies. Despite their undoubted value, tissue biopsies have important limitations, being highly invasive procedures that fail to capture tumor clonal heterogeneity. Liquid biopsies, comprising the analysis of circulating tumor-derived factors (the tumor circulome), are gaining exceptional attention as a valuable alternative. The tumor circulome is a source of

Glossary

4′,6-diamidino-2-phenylindole (DAPI)
a fluorescent dye that binds to AT-rich regions on DNA and is used to stain nuclei.
Apoptosis
programmed cell death; can be induced by external stimuli and is a tightly regulated process.
Beads, emulsion, amplification, magnetics (BEAMing)
highly sensitive dPCR method that combines emulsion PCR and flow cytometry to identify and quantify DNA mutations.
Cancer antigen 19-9 (CA 19-9)
a portion of the Sialyl-Lewis A antigen. Its presence is highly correlated with

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