LIQUID BIOPSY

Liquid biopsy is a non-invasive method for collecting samples from blood or other body fluids to detect molecular changes, tumor cells, and metabolites [45, 46]. In asymptomatic individuals, it is invaluable for early cancer detection and intervention. Compared to tissue biopsies, liquid biopsy plays an important role in early screening. Blood and urine are the most commonly used specimens. Since liquid biopsy is minimally invasive and easier to perform, it offers the potential for continuous monitoring of tumor progression [47, 48]. Various molecular markers can be detected through liquid biopsy, including circulating tumor DNA (ctDNA), Circulating Tumor Cells (CTCs), Tumor-Educated Platelets (TEPs), Tumor-derived Extracellular Vesicles (TEVs), and circulating free RNA (cfRNA) [49]. Current research primarily focuses on detecting exosomes, ctDNA, and CTCs.

Circulating Tumor Cells

CTCs are cells shed from primary and metastatic tumors into the bloodstream or lymphatic vessels of cancer patients, circulating in peripheral blood [50]. Although their proportion in blood is low (approximately one CTC per million leukocytes), most CTCs do not survive beyond 1 to 2.5 hours [48, 51]. Despite this, CTCs hold promise as a valuable tool for cancer diagnosis, aiding clinical decision-making and research (Fig. 4) [52]. Despite this, CTCs hold promise as a valuable tool for cancer diagnosis, aiding clinical decision-making and research. Detection methods rely on specific marker expressions, including epithelial cell adhesion molecule (EpCAM), vimentin, and N-cadherin, using techniques such as EpCAM enrichment, immunomagnetic separation, and microfluidic devices [53]. Among these, the CellSearch® method remains the only FDA-approved technique for monitoring CTC levels in blood samples [54].

Exosomes

Exosomes are considered miniature versions of their parental cells due to their complex composition, which includes uniquely sorted proteins, lipids, nucleic acids, and other components [55]. Secreted by nearly every cell type, exosomes are characterized by their endosomal origin and small size, typically ranging from 30 to 100 nm in diameter. These exosomes can be released through the trans-Golgi network or via inducible release (Fig. 5) [56]. The trans-Golgi network serves as a primary sorting station for secretory pathways, processing extracellular materials and recycled molecules from endocytic compartments before directing them to various subcellular destinations. Additionally, proteins from the Rab family of small GTPases are crucial for regulating intracellular vesicular trafficking, including cytoskeletal transport, vesicle budding, and docking/fusion activities [57].

Circulating Tumor DNA

ctDNA is a type of extracellular nucleic acid found in circulation [58]. It can be extracted from the bloodstream and is derived from the tumor. However, employing ctDNA sequencing for cancer screening encounters substantial challenges [59, 60]. In asymptomatic individuals, the concentration of ctDNA is relatively low, ranging between 1 and 15 ng/mL. To achieve optimal sensitivity, especially in breast cancer screening, large blood samples, typically between 150 and 300 mL, are needed [61]. Currently, ctDNA assays exhibit higher efficacy compared to traditional cancer-derived antigens, such as prostate-specific antigens and carcinoembryonic antigens [62]. Several studies support the potential use of ctDNA for early lung cancer diagnosis, detecting mutations like Kirsten Rat Sarcoma Virus (KRAS) and TP53 as much as 2 years before cancer diagnosis. Beyond DNA mutations, combining ctDNA level quantification and DNA methylation enhances the robustness of results. Notably, the detection of SEPT9 gene methylation is FDA-approved for colorectal cancer screening, surpassing protein markers in terms of sensitivity and specificity [63-65].

In the realm of treatment selection and prognosis, ctDNA sequencing provides a real-time snapshot of tumor-specific molecular profiles, guiding precision medicine [66]. Its short half-life allows for dynamic monitoring of treatment effects, aiding in treatment adjustment and prognosis assessment [67]. Regulatory bodies have approved ctDNA-based EGFR mutation testing for therapy guidance in NSCLC patients [68, 69]. For those undergoing immunotherapies, ctDNA emerges as an early marker of therapeutic efficacy, predicting survival outcomes more accurately than conventional methods. The most commonly used technologies for mutation detection in ctDNA are Scorpion Amplified Refractory Mutation System (ARMS) PCR, droplet digital PCR (ddPCR), and NGS [70].

Despite these advancements, ctDNA biological challenges include its highly fragmented nature and low concentration in the blood. Isolating ctDNA for quantification is challenging due to the potential loss or degradation of small fragments. Biological knowledge gaps, such as clearance rates and the quantitative relationships between circulating tumor DNA (ctDNA) and early cancer development, hinder further clinical applications (Fig. 6) [71]. ctDNA also faces challenges in identifying low-frequency mutations due to limited sensitivity. To overcome this, Wang et al. (2024) present ‘HiCASE,’ a highly sensitive and specific method to detect ctDNA mutations, utilizing a PCR-based CRISPR approach coupled with restriction enzymes [72]. Kamps et al. (2017) described the use of NGS in liquid biopsies across a range of cancers, including lung, HCC, gynecologic, pediatric, gastrointestinal tract tumors, and urothelial cancers [73]. The NGS-based analysis of liquid biopsy samples does not require an invasive biopsy of the tumor tissue and provides significant advantages in early cancer detection. It also supports monitoring of treatment response, identification of potential therapy resistance, and helps guide decisions for alternative treatment options (Table 3) [74, 75].