Table 1. Summary of characteristics of different biomarkers used in liquid biopsy.

Biomarker	Material available	Isolation technique	Clinical applications	Disadvantage
Circulating cfDNA/ctDNA	Cell free fragments of DNA released from tumor cells directly into the bloodstream	EpCAM-based and EpCAM-independent assays available	Represents the entire genome of the primary tumor; can provide diagnosis and prognosis; allows molecular profiling for targeted therapy (i.e., EGFR mutations); used as a real-time monitor of treatment (i.e., with TKI); high sensitivity to correlate with disease burden (metastasis)	Tumor ctDNA is not stable in circulation; ctDNA has a very short half-life; requires very sensitive methods to be able to separate ctDNA from normal cells cfDNA
ctDNA methylation	DNA fragments released from tumor cells directly into the bloodstream; methylation of certain gene promoter regions in cell DNA regulate the expression of genes involve in carcinogenesis, particularly methylation in tumor suppressor genes	MSP; real-time quantitative MSP; multiplex-nested MSP; Methyl-BEAMing	DNA methylation in cancer makes the circulating DNA more stable; identification of cancer-specific methylation sites can allow early-stage lung cancer detection. Has a very high sensitivity (one of the highest) for detection of early lung cancer; presence of certain methylated genes correlates to poor prognosis	Requires very sensitive methods to be able to separate ctDNA from normal cells cfDNA
CTC CellSearch™ (Veridex LLC) is the only FDA-approved method now (for breast, colon and prostate cancer)	Circulating tumor cells that originate from detachment from the primary lung cancer; requires a complex process to reach bloodstream and invade other tissues (EMT and MET)	Initial enrichment step that increases concentration of CTC in sample, with isolation of cells based on biological and physical properties (surface proteins/markers, cellular size); CellSearch™ utilizes ferroparticles and antibodies directed to epithelial targets (EpCAM)	Provide all cellular material, including RNAs, DNA and proteins; source of tissue for diagnosis and profiling of cancer; CTC count can correlate to cancer burden (metastasis and progression); monitoring of treatment; detection of residual disease after treatment; CTC level correlates to early recurrence	CTC are very rare compared to normal circulating cells, its isolation is challenging; EMT down-regulates epithelial properties of tumor cells (expression of tumor makers); Low sensitivity and difficult reproducibility; current FDA-approved technology is EpCAM-based isolation
TEP	Platelets that were in contact with primary tumor and underwent transference of cancer-associated biomolecules	Platelets isolation with extraction of tumor biomolecules and nucleic acids (RNAs)	More abundant than CTC; also contain a large amount of genetic material (RNA and DNA); diagnosis of lung cancer; monitoring of treatment	Technologies to obtain TEP are still in development
Exosomes	Small vesicles of endocytic origin; exosomes are released by a variety of cells, including lung cancer cells and are captured by other neighboring or distant targets	Isolation based on physical or biological properties of exosomes; MACS; immune-mediated isolation; sucrose gradient method; ultra-centrifugation; after isolation, PCR can be used to separate the RNA and proteins inside the exosomes	Can provide early diagnosis; provide important information about tumor’s biologic profile, growth rate, metastatic capacity and drug resistance; can predict response to treatment; its transport capacity has potential to be a vehicle for therapies	Isolation can be time consuming; physical isolation methods can alter exosome structure; there might be co-isolation of proteins and other contaminants; small sample volumes decrease exosome yield

cfDNA, cell free DNA; ctDNA, circulating tumor DNA; EpCAM, epithelial cell adhesion molecule; EGFR, epidermal growth factor receptor; CTC, circulating tumor cells; TEP, tumor educated platelets; MACS, magnetic activated cell sorting; PCR, polymerase chain reaction; FDA, Food and Drug Administration; EMT, epithelial-mesenchymal transition; MET, mesenchymal to epithelial transition; MSP, Methylation-specific PCR; TKI, tyrosine kinase inhibitor.