Breakthroughs and challenges in liquid biopsy technologies for cancer diagnosis and treatment monitoring

 Corresponding author: Oluwaseyi Ajibola Fagbemi Email: Copyright © 2025 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution License 4.0. Breakthroughs and challenges in liquid biopsy technologies for cancer diagnosis and treatment monitoring Fagbemi Oluwaseyi Ajibola 1, *, Toluwani Samuel Ogunmoyero 2, Taiwo Bakare-Abidola 3, Abubakar Rawayau Mannir 4, Aminat Bukola Kehinde 5, Esther Chigbaziru Nwojiji 6 and Rofiat Oyiza Abdulkareem 7 1 Department of Human Anatomy, College of Medicine, Federal University Lokoja, Kogi State, Nigeria. 2 Department of Medical Laboratory Science, Ladoke Akintola University of Technology, Ogbomosho, Nigeria. 3 Department of Environmental Science, Georgia Southern University, Georgia, USA. 4 Department of Biochemistry, Umaru Musa Yaradua University, Katsina, Nigeria. 5 Department of Biomedical Ethics, CIS, Hamad Bin Khalifa University, Doha, Qatar. 6 Department of Microbiology and Parasitology, David Umahi Federal University of Health Sciences, Uburu, Nigeria. 7 Department of Pharmacology, Bayero University Kano, Nigeria. World Journal of Biology Pharmacy and Health Sciences, 2025, 22(01), 112-138 Publication history: Received on 26 February 2025; revised on 05 April 2025; accepted on 07 April 2025 Article DOI: https://doi.org/10.30574/wjbphs.2025.22.1.0327 Abstract Cancer remains a leading global health challenge, with early detection and precise monitoring playing a crucial role in improving patient outcomes. Traditional tissue biopsies, while essential for diagnosis, are invasive, limited in scope, and often fail to capture tumor heterogeneity or track disease progression dynamically. Liquid biopsy technologies have emerged as a transformative alternative, offering a minimally invasive approach to cancer detection and management by analyzing circulating tumor DNA (ctDNA), circulating tumor cells (CTCs), extracellular vesicles, and tumor-derived exosomes in bodily fluids. This review explores the technological advancements that have enhanced the sensitivity and specificity of liquid biopsies, including next-generation sequencing (NGS), droplet digital PCR (ddPCR), and machine learning-driven bioinformatics. The clinical applications of liquid biopsies are vast, encompassing early cancer detection, monitoring of therapeutic responses, identification of minimal residual disease (MRD), and real-time tracking of resistance mutations. These capabilities support the paradigm shift toward precision oncology, allowing clinicians to tailor treatments based on a dynamic understanding of tumor evolution. Despite their promise, liquid biopsies face challenges such as low biomarker abundance, standardization issues, cost barriers, and regulatory complexities, which hinder widespread clinical implementation. However, emerging innovations, including single-cell liquid biopsies, pointof-care diagnostic devices, and AI-assisted biomarker analysis, are set to overcome these limitations. As research advances, liquid biopsies are poised to revolutionize cancer diagnostics, providing a non-invasive, comprehensive, and personalized approach to cancer management that could significantly enhance survival rates and treatment efficacy. Keywords: Liquid biopsy; Circulating tumor DNA; Cancer diagnostics; Treatment monitoring; Precision oncology; Minimal residual disease; Next-generation sequencing; Biomarker detection 1. Introduction Cancer remains a formidable challenge in global health, and according to the World Health Organization, it was responsible for around 10 million mortalities in 2020 alone [1]. The relentless pursuit of improved diagnostic and therapeutic strategies has been at the forefront of medical research. Traditional cancer diagnostics have long relied on tissue biopsies, which, while informative, present several inherent limitations [2]. Recent years have seen the emergence of liquid biopsies as a viable, minimally invasive substitute that may improve cancer surveillance and early World Journal of Biology Pharmacy and Health Sciences, 2025, 22(01), 112-138 113 diagnosis. Examining the advances and difficulties of liquid biopsy technology in cancer diagnosis and therapy monitoring is the goal of this study [1,2]. 1.1. Background on Cancer Diagnostics and the Limitations of Traditional Biopsies Conventional cancer diagnosis often necessitates the extraction of tissue samples through surgical or needle biopsies. These procedures provide valuable histopathological information, enabling pathologists to determine tumor type, grade, and molecular characteristics essential for guiding treatment decisions [3]. However, the invasive nature of these procedures poses significant challenges. Patients may experience discomfort, pain, and potential complications such as bleeding or infection. Moreover, certain anatomical locations of tumors, such as those in the brain or pancreas, render biopsies particularly risky or, in some cases, unfeasible [3]. Figure 1 Lung cancer diagnosis and the two contrasting approaches: conventional tissue biopsy vs. liquid biopsy. Reproduced with permisiion from Ref. [3] Beyond the immediate procedural risks, tissue biopsies offer a static snapshot of the tumor's genetic landscape. Tumors are inherently heterogeneous, comprising subpopulations of cells with distinct genetic mutations and phenotypic traits. A single biopsy may not capture this intratumoral heterogeneity, leading to an incomplete understanding of the disease. This limitation becomes pronounced when considering tumor evolution over time, especially under therapeutic pressure. As tumors adapt and develop resistance to treatments, their molecular profiles shift, necessitating updated information to guide subsequent therapeutic interventions. However, repeated tissue biopsies for monitoring purposes are impractical due to their invasiveness and associated complications [4,5]. To appreciate the advancements in cancer diagnostics, it is essential to compare traditional tissue biopsies with emerging liquid biopsy techniques (see Figure 1). World Journal of Biology Pharmacy and Health Sciences, 2025, 22(01), 112-138 114 Table 1 outlines the key differences between these two approaches, highlighting aspects such as invasiveness, sampling frequency, and ability to capture tumor heterogeneity. Table 1 Comparison of Traditional Biopsy and Liquid Biopsy Aspect Traditional Biopsy Liquid Biopsy Invasiveness Involves surgical or needle procedures to extract tissue samples, which can be painful and carry risks such as infection. Minimally invasive; requires a simple blood draw, reducing discomfort and associated risks. Sampling Frequency Limited due to invasiveness; repeated sampling is often impractical. Allows for frequent sampling, enabling real-time monitoring of tumor dynamics. Tumor Heterogeneity Assessment May not capture the full genetic diversity of tumors, especially if sampling is from a single site. Provides a comprehensive view by detecting circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) from multiple tumor sites, offering insights into tumor heterogeneity. Cost Generally higher due to surgical procedures and hospitalization requirements. Potentially lower costs associated with outpatient blood draws and reduced need for surgical interventions. Clinical Applicability Standard method for initial diagnosis and histopathological analysis; essential for determining tumor architecture and microenvironment. Useful for early detection, monitoring treatment response, and detecting minimal residual disease; however, it may not replace the need for tissue biopsies entirely, especially when detailed histological information is required [6]. 1.2. Definition and Significance of Liquid Biopsy in Oncology Figure 2 Mechanism of Liquid Biopsy in Cancer Detection. Reproduced with permission from Ref. [3] In order to find biomarkers linked to cancer, non-solid biological tissues—most often blood—are sampled and analysed using a technique known as liquid biopsy. These biomarkers consist of extracellular vesicles, tumor-derived exosomes, circulating tumour cells (CTCs), and circulating tumour DNA (ctDNA) (see figure 2) [7]. Unlike traditional biopsies, liquid biopsies are minimally invasive, involving simple blood draws, and can be performed repeatedly, enabling realtime monitoring of tumor dynamics. The significance of liquid biopsies in oncology is multifaceted. Firstly, they facilitate World Journal of Biology Pharmacy and Health Sciences, 2025, 22(01), 112-138 115 early detection of genetic mutations and alterations associated with cancer, potentially identifying malignancies before they become clinically apparent. This early detection is crucial, as it increases the likelihood of successful treatment outcomes. Secondly, liquid biopsies allow for the assessment of treatment efficacy by monitoring changes in ctDNA levels, providing insights into tumor response or progression. Thirdly, they enable the timely identification of resistance mechanisms, such as the emergence of new mutations that confer resistance to targeted therapies, allowing clinicians to adjust treatment strategies accordingly [8,9]. For instance, the National Health Service (NHS) in England has implemented liquid biopsies to detect specific mutations in breast cancer patients, allowing for more individualized and successful approaches of therapy. Liquid biopsies being included into clinical practice represents a significant advancement in precision oncology, offering the potential for improved patient outcomes and more tailored therapeutic interventions [10]. 1.3. Objectives and Scope of the Review This review attempts to offer a thorough analysis of the current state of liquid biopsy technologies in oncology. It will delve into the biological components detectable through liquid biopsies, such as ctDNA and CTCs, and discuss the technological advancements that have enhanced their detection and analysis. The review will also explore the therapeutic uses of liquid biopsies, including early cancer identification, therapy monitoring, and detection of minimum residual illness. Furthermore, it will address the current limitations and challenges hindering widespread adoption, such as concerns of standardization, sensitivity, and specificity. Finally, the review will highlight future directions and emerging innovations in the field, offering insights into how liquid biopsies may continue to revolutionize cancer diagnostics and treatment monitoring. By examining these aspects, this review seeks to elucidate the revolutionary possibilities of liquid biopsy technologies in oncology, while also acknowledging the obstacles that need to be eliminated in order to properly utilize them in clinical settings. 2. Biological Components of Liquid Biopsies The use of liquid biopsy technologies is revolutionizing the detection and tracking of cancer by analyzing tumor-derived materials found in bodily fluids. These biological components provide crucial molecular insights into tumor behavior, allowing clinicians to track disease progression and treatment responses in real time. Among these components, circulating tumor DNA (ctDNA) carries genetic mutations, epigenetic modifications, and methylation patterns that reflect the evolving nature of cancer [11]. Circulating tumour cells (CTCs), which are released into the circulation from primary or metastatic tumours, are essential for metastasis and can provide important information about the phenotypic features of tumours. Extracellular vesicles and exosomes, once thought to be mere cellular debris, have recently been identified as essential mediators in communication between cells, carrying biomarkers that can aid in cancer detection. Additionally, microRNAs (miRNAs) and tumor-derived proteins are emerging as powerful tools for cancer classification and diagnosis (See Figure 3) [11,12]. This section explores the biological significance of these components, their role in cancer pathology, and their potential to enhance precision oncology. Liquid biopsies analyze various biomarkers to detect and monitor cancer. Table 2 summarizes the primary biomarkers, their sources, clinical significance, and current applications. Table 2 Key Biomarkers Detected in Liquid Biopsy Biomarker Type Source Clinical Significance Current Applications Circulating Tumor DNA (ctDNA) Blood Reflects the genetic alterations of tumors; used to identify mutations, monitor treatment response, and detect resistance mechanisms. Detection of specific mutations (e.g., EGFR in non-small cell lung cancer) to guide targeted therapy decisions [13]. Circulating Tumor Cells (CTCs) Blood Presence indicates tumor shedding into the bloodstream; associated with prognosis and metastatic potential. Prognostic indicator in cancers such as breast, prostate, and colorectal cancers; monitoring disease progression [6]. Exosomes Blood, Urine Nano-sized vesicles containing proteins, RNA, and DNA; involved in cell communication and may reflect tumor status. Investigational use in cancer diagnostics and monitoring; potential for early detection and understanding tumor behavior. World Journal of Biology Pharmacy and Health Sciences, 2025, 22(01), 112-138 116 MicroRNAs (miRNAs) Blood, Urine Small non-coding RNAs that regulate gene expression; aberrant expression patterns are linked to cancer development and progression. Research-stage biomarker for cancer diagnosis, prognosis, and therapeutic targets; not yet widely implemented in clinical practice. Figure 3 A schematic view of liquid biopsy. Blood collected from cancer patients contains circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), exosomes, and circulating tumor microRNA (ct-miRNA). Reproduced with perssion from Ref. [12] 2.1. Circulating Tumor DNA (ctDNA): Mutation Profiling, Epigenetic Alterations, Methylation Patterns The term "circulating tumour DNA" (ctDNA) describes tiny DNA fragments released into the circulation by tumour cells. These fragments, typically less than 200 base pairs in length, carry genetic and epigenetic information that mirrors the tumor's molecular landscape, offering a non-invasive window into cancer biology [14]. The analysis of ctDNA encompasses various aspects, including mutation profiling, epigenetic alterations, and methylation patterns, each providing unique insights into tumor behavior and potential therapeutic strategies. 2.1.1. Mutation Profiling Mutation profiling of ctDNA involves identifying genetic changes like point mutations, copy number variations, insertions, and deletions that drive cancer development and progression. Although useful, traditional tissue biopsies are invasive and could miss the variety of metastatic tumours. In contrast, ctDNA analysis enables the detection of mutations across all tumor sites through a simple blood draw, facilitating real-time monitoring of tumor dynamics [15]. The therapeutic usefulness of ctDNA mutation profiling has been shown in several research. For instance, Leary et al. [16] employed whole-genome sequencing of ctDNA to detect chromosomal alterations in patients with colorectal and breast cancers. Their findings revealed that ctDNA could identify structural variations and copy number changes concordant with those found in primary tumors, underscoring its potential for non-invasive genomic profiling. Moreover, ctDNA has been used to identify minimum residual diseases and track therapy results. In a study by Olsson et al. [17], ctDNA serial measures in individuals with breast cancer that has spread correlated with changes in tumor burden and provided early indications of treatment efficacy, often preceding radiographic assessments. These observations highlight the role of ctDNA as a dynamic biomarker for personalized cancer management. World Journal of Biology Pharmacy and Health Sciences, 2025, 22(01), 112-138 117 2.1.2. Epigenetic Alterations and Methylation Patterns ctDNA contains epigenetic changes that affect gene expression without changing the DNA sequence, in addition to genetic mutations [18]. One important epigenetic process controlling gene activity is DNA methylation, which involves the addition of methyl groups to cytosine residues. Cancer is characterized by aberrant methylation patterns, which can act as biomarkers for diagnosis and prognosis [18,19]. Examples of these patterns include hypermethylation of tumour suppressor genes and hypomethylation of oncogenes. The stability of methylation marks in ctDNA makes them attractive targets for liquid biopsy assays. Sun et al. [20] investigated 5-hydroxymethylcytosine (5hmC) signatures in ctDNA across various cancer types. Their study demonstrated that 5hmC profiles could distinguish between tumor and normal tissues and differentiate among cancer types, suggesting their potential for early detection and tumor classification. Advancements in sequencing technologies have enabled comprehensive methylation analysis of ctDNA. Techniques such as whole-genome bisulfite sequencing allow for the detection of methylation changes across the genome, providing insights into the epigenetic landscape of tumors. These approaches have been applied to identify cancer-specific methylation patterns, offering opportunities for developing non-invasive screening tests [19]. 2.1.3. Technological Advances and Clinical Applications The sensitivity and specificity of ctDNA analysis have been enhanced by technological innovations. Next-generation sequencing (NGS) technologies and digital PCR enable the detection of low-frequency mutations and subtle methylation changes in ctDNA, even when present at low concentrations [21]. These advancements have paved the way for integrating ctDNA assays into clinical practice. One notable application is the identification of resistance mutations in metastatic cancer. In non-small cell lung cancer (NSCLC), for example, the emergence of the EGFR T790M mutation confers resistance to first-line tyrosine kinase inhibitors [22]. Monitoring ctDNA allows for the timely identification of such mutations, guiding the selection of subsequent therapies. Desai et al. [23] demonstrated that ctDNA analysis could detect EGFR mutations with high concordance to tissue biopsies, facilitating rapid and non-invasive assessment of resistance mechanisms. Furthermore, ctDNA methylation patterns have been explored for early cancer detection. A study by Wang et al. [24] developed a blood-based assay targeting methylation markers across multiple cancer types. The assay achieved high sensitivity and specificity in detecting cancers at various stages, highlighting the promise of ctDNA methylation analysis as a screening tool. 2.1.4. Challenges and Future Directions Despite the promising applications, several challenges hinder the widespread adoption of ctDNA analysis. The low abundance of ctDNA, especially in early-stage cancers, necessitates highly sensitive detection methods. Pre-analytical variables, such as blood collection, processing, and storage conditions, can impact ctDNA yield and integrity [25]. Standardization of protocols is necessary to guarantee dependability and repeatability across labs. Additionally, distinguishing ctDNA from normal cell-free DNA requires precise analytical techniques. Bioinformatics tools capable of differentiating tumor-specific alterations from benign variations are crucial for accurate interpretation. Integrating ctDNA analysis with other biomarkers, such as circulating tumor cells and protein markers, may enhance diagnostic performance and provide a more comprehensive view of tumor biology [25,26]. To determine the therapeutic value of ctDNA biomarkers, future studies should concentrate on confirming them in sizable, prospective clinical trials. Exploring the combination of ctDNA analysis with imaging modalities and other diagnostic tools could lead to more robust cancer detection and monitoring strategies [25]. As technologies evolve and our understanding of ctDNA biology deepens, liquid biopsies have the potential to transform cancer therapy by allowing individualized and flexible therapeutic strategies. 2.2. Circulating Tumor Cells (CTCs): Role in Metastasis and Phenotypic Analysis Cancer cells that separate from the main or metastasized tumours and enter the bloodstream are known as circulating tumour cells, or CTCs. Their presence is a critical factor in the metastatic cascade, leading to the spread of cancer to distant organs. Understanding the role of CTCs in metastasis and their phenotypic characteristics offers valuable insights into cancer progression and potential therapeutic interventions [27]. World Journal of Biology Pharmacy and Health Sciences, 2025, 22(01), 112-138 118 2.2.1. Role in Metastasis The phases involved in the metastatic process include local invasion, bloodstream intravasation, extravasation into distant tissues, survival in circulation, and colonization to produce new tumours [28]. CTCs are essential to this procedure, particularly during intravasation and dissemination. Recent studies have challenged the traditional view that CTCs travel as single cells [27,29]. The "cancer exodus hypothesis" posits that CTC clusters—aggregates of two or more tumor cells—maintain their multicellular structure throughout metastasis. These clusters intravasate, circulate, and extravasate as cohesive units, significantly enhancing their metastatic potential compared to single CTCs [30,31]. This multicellularity provides advantages such as increased survival, proliferation, and resistance to apoptosis. For instance, research has shown that patients with prostate cancer exhibiting CTC clusters have a shorter mean survival rate compared to those with only single CTCs, underscoring the aggressive nature of clustered CTCs [30]. Moreover, because of their distinct gene expression patterns, CTC clusters are more resistant than individual tumour cells and can avoid several cancer treatments. This resistance further complicates treatment strategies and highlights the need for targeted therapies addressing the specific characteristics of CTC clusters [32]. 2.2.2. Phenotypic Analysis Phenotypic characterization of CTCs involves assessing their morphological and molecular attributes, which can provide insights into their origin, metastatic potential, and resistance mechanisms. CTCs can be categorized based on the expression of epithelial markers, size, and apoptotic status: • Traditional CTCs: These cells exhibit an intact, viable nucleus; express epithelial markers such as EpCAM and cytokeratins; lack hematopoietic markers like CD45; and are typically larger with irregular shapes [33]. • Cytokeratin-negative CTCs: These lack epithelial markers, possibly indicating an undifferentiated state or a mesenchymal phenotype due to epithelial-mesenchymal transition (EMT). Such cells may be more resistant to therapies and possess higher metastatic potential [34]. • Apoptotic CTCs: These are CTCs undergoing programmed cell death, identifiable by nuclear fragmentation or cytoplasmic blebbing. Monitoring the ratio of viable to apoptotic CTCs can provide clues to treatment efficacy [35]. • Small CTCs: These are cytokeratin-positive and CD45-negative but similar in size and shape to white blood cells. They have been implicated in aggressive disease progression and may differentiate into small cell carcinomas, requiring distinct therapeutic approaches [36]. CTC clusters can be homotypic, consisting solely of tumor cells, or heterotypic, incorporating other cell types such as white blood cells, fibroblasts, endothelial cells, and platelets. Heterotypic clusters, also known as microemboli, might enhance metastatic potential by facilitating immune evasion and promoting survival in circulation [36,37]. 2.2.3. Clinical Implications Identifying and evaluating CTCs, especially clusters, using liquid biopsies provides useful prognostic data. The presence of CTC clusters is associated with increased metastatic potential and poorer prognosis. For example, research has indicated that individuals with prostate cancer who have only single CTCs exhibit an eight-fold longer mean survival rate compared to those with CTC clusters [38]. Understanding the phenotypic diversity of CTCs can inform treatment strategies. Identifying EMT markers or stem celllike properties in CTCs may indicate resistance to conventional therapies, suggesting the need for alternative or combination treatments. Additionally, monitoring apoptotic CTCs can serve as a real-time indicator of therapeutic efficacy, allowing for timely adjustments to treatment plans [39]. 2.3. Extracellular Vesicles & Exosomes: Biomarker Potential and Intercellular Communication Cells release particles into the extracellular environment called extracellular vesicles (EVs), which include exosomes and microvesicles and are encased in a lipid bilayer. These vesicles carry bioactive substances including nucleic acids, proteins, and lipids between cells, facilitating intercellular communication. In oncology, EVs have garnered interest due to their potential as biomarkers and their role in mediating tumor progression [40]. World Journal of Biology Pharmacy and Health Sciences, 2025, 22(01), 112-138 119 2.3.1. Biogenesis and Composition of Exosomes Exosomes, a subset of EVs, originate from the endosomal pathway. Their formation starts with the endosomal membranes budding inward, which produces multivesicular bodies (MVBs) containing intraluminal vesicles. These intraluminal vesicles are discharged as exosomes into the extracellular area when MVBs fuse with the plasma membrane. Exosomes usually have a diameter of 30 to 150 nanometres and contain molecular cargo that is representative of the cell from which they originated. This load includes various proteins, lipids, mRNAs, and microRNAs, enabling exosomes to influence recipient cell behavior [41]. Biomarker Potential of Exosomes The molecular composition of exosomes provides a snapshot of the parent cells' physiological or pathological condition, making them valuable as non-invasive biomarkers for the detection and tracking of cancer. Tumor-derived exosomes have been found to contain specific proteins and nucleic acids associated with malignancy. For instance, exosomes from glioblastoma multiforme (GBM) patients have been shown to carry amplified oncogene sequences and retrotransposon elements, which can be detected in the circulation, offering a potential diagnostic avenue. Moreover, exosomal integrins have been implicated in organ-specific metastasis. Hoshino et al. [42] demonstrated that tumor exosome integrins determine organotropic metastasis, suggesting that the integrin profiles of circulating exosomes could predict metastatic sites, thereby aiding in prognosis and personalized treatment strategies. Intercellular Communication Mediated by Exosomes By delivering their chemical payload to recipient cells, exosomes play a crucial part in intercellular communication and influence a number of physiological and pathological processes. This transfer can influence immune responses, angiogenesis, and tumor progression. For example, bone marrow progenitor cells can be taught to adopt a prometastatic phenotype by tumor-derived exosomes via the MET receptor tyrosine kinase pathway, facilitating the establishment of pre-metastatic niches [43]. Additionally, exosomes have been shown to mediate the horizontal movement of genetic material across cells, including microRNAs and mRNAs. This mechanism allows for the modulation of gene expression in recipient cells, contributing to tumorigenesis and the tumor microenvironment's dynamic nature. For instance, exosomes derived from melanoma cells can transfer microRNAs to recipient cells, promoting tumor growth and metastasis [43,44]. 2.3.2. Clinical Applications and Future Directions The unique properties of exosomes have spurred interest in their clinical applications. Their stability in bodily fluids and capacity to mirror their parent cells' molecular traits make them attractive candidates for liquid biopsy approaches in cancer diagnostics. Standardized techniques for isolating and characterizing exosomes are being developed to harness their full potential as biomarkers. Furthermore, exosomes are being explored as therapeutic vehicles due to their natural biocompatibility and ability to deliver cargo to specific cells. Modifying exosomes to transport antimicrobial agents like short interfering RNAs or chemotherapeutic drugs, offers a promising strategy for targeted cancer therapy [45]. 2.4. MicroRNAs (miRNAs) and Proteins: Emerging Roles in Cancer Detection and Classification With respect to liquid biopsies, microRNAs (miRNAs) and proteins have emerged as pivotal biomarkers, offering profound insights into cancer detection and classification. These molecular entities, detectable in various body fluids, provide a non-invasive window into the oncogenic processes, facilitating early diagnosis and personalized treatment strategies [46]. 2.4.1. MicroRNAs (miRNAs) in Cancer Detection Small, non-coding RNA molecules called miRNAs control post-transcriptional expression of genes. Their dysregulation has been linked to the development and spread of a number of cancers. Notably, miRNAs exhibit remarkable stability in body fluids, making them ideal candidates for liquid biopsy-based diagnostics. New developments have resulted in the development of miRNA biosensors, which enable the detection of specific miRNA signatures associated with different cancer types. These biosensors have been integrated into point-of-care (POC) testing devices, allowing for sensitive and quick miRNA analysis in clinical settings. For instance, urine samples containing miR-21 can be detected using electrochemical biosensors, achieving detection limits as low as 2 nanomolar within a two-hour timeframe. Such innovations underscore the potential of miRNA-based diagnostics in facilitating early cancer detection and monitoring disease progression [47]. World Journal of Biology Pharmacy and Health Sciences, 2025, 22(01), 112-138 120 2.4.2. Proteins as Biomarkers in Liquid Biopsies Proteins, being the functional executors of cellular processes, reflect the physiological and pathological states of an organism. In the context of cancer, aberrant protein expression, post-translational modifications, and the presence of tumor-specific isoforms serve as valuable biomarkers for disease detection and classification [48]. A notable advancement in this arena is the development of a urine-based test for early lung cancer detection. Researchers have identified proteins released by senescent cells—often referred to as "zombie" cells—that can reprogram their environment to support cancer cell emergence. By utilizing an injectable sensor that interacts with these proteins, a detectable compound is released into the urine, signaling potential early-stage lung cancer. This innovative approach, currently progressing towards human trials, exemplifies the utility of protein biomarkers in non-invasive cancer diagnostics [48,49]. 2.4.3. Integration of miRNA and Protein Biomarkers The convergence of miRNA and protein biomarker analysis holds promise for enhancing the liquid biopsies' sensitivity and specificity. Combining these molecular signatures can give a thorough rundown of tumour biology, enabling more accurate cancer detection and classification [46,50]. As research advances, the integration of multi-omic approaches in liquid biopsy platforms is anticipated to revolutionize personalized oncology, leading to improved patient outcomes through tailored therapeutic interventions. 3. Technological Advancements in Liquid Biopsy The evolution of liquid biopsy technology has transformed how cancer is detected, monitored, and managed. With continuous improvements in sensitivity, specificity, and throughput, novel platforms now enable the identification of minute traces of tumor-derived material circulating in body fluids. These advancements (see figure 4) are redefining early diagnosis, allowing clinicians to capture critical molecular changes in real time without relying on invasive tissue biopsies. From next-generation sequencing (NGS) to digital PCR and microfluidic-based enrichment techniques, the growing arsenal of cutting-edge tools is enhancing the precision of cancer diagnostics and treatment monitoring [51]. This section delves into the key breakthroughs that are influencing liquid biopsy's future, highlighting the technologies that are improving the identification of extracellular vesicles, circulating tumour cells (CTCs), circulating tumour DNA (ctDNA), and other critical biomarkers. Various technologies have been developed to detect circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) in liquid biopsies. Table 3 compares major detection techniques, focusing on sensitivity, specificity, cost, and their respective advantages and limitations. Table 3 Comparison of Detection Techniques for ctDNA and CTCs Technology Target Sensitivity Specificity Cost Major Advantages Limitations NextGeneration Sequencing (NGS) ctDNA High High High Comprehensive genomic profiling; detects multiple mutations simultaneously. Requires complex data analysis; higher cost; longer turnaround time. Droplet Digital PCR (ddPCR) ctDNA Very High Very High Moderate High sensitivity and specificity; quantifies rare mutations; faster results. Limited to known mutations; not suitable for broad mutation discovery. BEAMing (Beads, Emulsion, Amplification, Magnetics) ctDNA Very High Very High High Combines digital PCR and flow cytometry; highly sensitive; allows for rare mutation detection. Complex and laborintensive; higher cost; limited availability. CellSearch System CTCs Moderate High High FDA-approved for certain cancers; standardized method; provides prognostic information. Limited sensitivity; may miss CTC subpopulations; expensive equipment. World Journal of Biology Pharmacy and Health Sciences, 2025, 22(01), 112-138 127 malignancy, reaching 51.5% overall: First degree - 16.8%; second degree - 40.4%; third degree - 77.0%; and fourth degree - 90.1%. For 12 pre-specified cancer categories that cause almost two-thirds of all cancer-related fatalities in the United States each year, the sensitivity from first degree to third degree was 67.6%. Additionally, in 88.7% of truepositive instances, the test correctly identified the target tissue for the malignancy [93]. The Galleri test is intended to complement existing cancer screenings, such as mammography and colonoscopy, and is recommended for adults at a higher risk of developing cancer, particularly those aged 50 or older. It requires a prescription from a licensed healthcare provider [93]. The NHS in England is conducting a randomized controlled trial, known as NHS-Galleri, to evaluate how well the test works to lower the incidence of late-stage cancer. Over 140,000 individuals aged 50 to 77 without a cancer diagnosis have been enrolled, with results expected in the summer of 2026 [94]. While the Galleri test shows promise in detecting multiple cancer types early, it is essential to consider its limitations, such as varying sensitivity across different cancer stages and types. Ongoing studies aim to further validate its clinical efficacy and cost-effectiveness before widespread implementation. 4.2. Real-Time Treatment Monitoring: Tracking Tumor Evolution and Therapeutic Response The dynamic nature of cancer necessitates continuous monitoring to effectively assess therapeutic efficacy and adapt treatment strategies accordingly. Traditional imaging modalities and tissue biopsies, while informative, often fall short in recording the tumours' temporal variability due to their invasive nature and limited sampling frequency. In this regard, liquid biopsy has become a game-changing technique that allows for the study of circulating biomarkers including circulating tumour DNA (ctDNA) and circulating tumour cells (CTCs), allowing for real-time monitoring of tumour progression and therapy response [95]. 4.2.1. Monitoring Therapeutic Response The quantification of ctDNA levels in plasma serves as a non-invasive biomarker for evaluating tumor burden and therapeutic response. A decline in ctDNA concentrations post-treatment initiation often correlates with a favorable response, whereas stable or rising levels could be a sign of disease progression or resistance. For instance, the Guardant360 test, a comprehensive liquid biopsy assay, has been utilized to monitor ctDNA dynamics in patients undergoing targeted therapies, facilitating timely adjustments to treatment regimens [96,97]. 4.2.2. Detecting Minimal Residual Disease (MRD) Assays for liquid biopsies have been useful in identifying minimal residual disease (MRD), which is the existence of cancer cells that may recur. The Guardant Reveal test, for example, is a blood-only liquid biopsy designed to detect recurring and persistent disease in colorectal cancer by identifying ctDNA. This enables oncologists to detect recurrence earlier than using conventional approaches and to identify individuals with residual disease who might benefit from extra therapy [97]. 4.2.3. Assessing Treatment Resistance In oncology, the formation of clones that are resistant to treatment presents serious difficulties. The identification of such mutations linked to resistance is made possible via liquid biopsy, allowing for the timely modification of therapeutic strategies. In breast cancer management within the NHS, liquid biopsies have been implemented to identify mutations such as ESR1, which can develop after hormone treatment and promote cancer growth. Patients testing positive for the ESR1 mutation can now access elacestrant, a targeted therapy, thereby personalizing treatment and potentially improving outcomes [8]. 4.2.4. Advantages Over Traditional Monitoring Compared to conventional tissue biopsies, liquid biopsies offer several advantages in monitoring tumor evolution and therapeutic response. One of the primary benefits is their non-invasiveness, as they require only a simple blood draw, minimizing patient discomfort and the risks associated with surgical or needle biopsies. Additionally, liquid biopsies enable real-time monitoring of tumor dynamics through serial sampling, allowing clinicians to track disease progression and detect relapse at an earlier stage. This continuous assessment is particularly valuable in guiding timely therapeutic adjustments [8]. Furthermore, liquid biopsies provide a more comprehensive tumor profile by capturing circulating tumor DNA (ctDNA) from multiple tumor sites, thereby reflecting the heterogeneity of the disease more accurately than World Journal of Biology Pharmacy and Health Sciences, 2025, 22(01), 112-138 128 a single-site tissue biopsy. This holistic approach enhances precision medicine strategies, improving patient outcomes by tailoring treatments based on a more complete understanding of tumor evolution [69]. 4.2.5. Real Case Studies with Guardant360 Guardant360 is a liquid biopsy test that looks for genetic changes in cancer patients by analysing circulating tumour DNA (ctDNA) in blood samples. This non-invasive method offers a comprehensive genomic profile, aiding in the selection of targeted therapies, particularly for metastatic non-small cell lung cancer (NSCLC) patients. Clinical Validation and Impact on Survival Rates A prospective study involving 193 advanced cancer patients, including those with NSCLC, demonstrated the clinical utility of the Guardant360 assay. In the NSCLC cohort, patients matched to targeted therapy based on Guardant360 results exhibited an objective response rate of 87% and a disease control rate of 100%. Notably, median overall survival more than doubled for these patients (31.8 months) compared to those receiving non-targeted cytotoxic therapy (12.7 months) [98]. Additionally, a study published in JCO Precision Oncology assessed the use of Guardant360 to monitor molecular response in metastatic NSCLC patients undergoing pembrolizumab-based therapy. Individuals who showed a molecular response—which is characterised by a reduction of at least 50% in the mean variant allele fraction—had better median progression-free survival (14.1 months vs. 4.4 months) and overall survival (22.1 months vs. 12.0 months) than those who did not [99]. Advantages Over Traditional Tissue Biopsy A head-to-head study comparing Guardant360 liquid biopsy to standard tissue biopsy in advanced NSCLC patients revealed that Guardant360 detected 23.6% more actionable mutations when used as a first-line test. This suggests that liquid biopsy can uncover additional therapeutic targets, potentially leading to improved patient outcomes [100]. 4.3. Minimal Residual Disease (MRD) Detection The term "minimum residual disease" (MRD) describes the little quantity of cancer cells that might still be present in a patient's body following therapy, which may cause recurrence. Traditional imaging methods often lack the sensitivity to detect these residual cells, making early intervention challenging. Liquid biopsies have emerged as a transformative approach in this context, enabling the detection of circulating tumor DNA (ctDNA) in blood samples to monitor MRD with high precision [101,102]. Guardant Health's Guardant Reveal test exemplifies this advancement. This blood-only liquid biopsy test is intended to identify ctDNA after surgery, which helps identify individuals who may benefit from further treatment. It is used to detect residual and recurrent disease in colorectal cancer (CRC). Notably, Medicare's recent decision to cover Guardant Reveal for colon cancer patients underscores its clinical utility and potential to enhance patient outcomes [103]. By facilitating early detection of MRD, liquid biopsies empower clinicians to make informed decisions regarding adjuvant therapies, thereby reducing recurrence risk and improving survival rates. 4.3.1. Real Case Studies with Signatera for MRD Test Signatera, developed by Natera, is a personalized molecular residual disease (MRD) test designed to detect circulating tumor DNA (ctDNA) in the bloodstream of colorectal cancer patients. By identifying ctDNA, Signatera can detect minimal residual disease that may not be visible through conventional imaging, thereby predicting cancer recurrence earlier and informing treatment decisions [104,105]. Clinical Evidence Supporting Signatera's Efficacy A European study involving 265 patients with stage I-III colorectal cancer utilized the Signatera test shortly after surgery and periodically over several months. The findings revealed that among the 20 patients with detectable ctDNA post-surgery, 75% experienced relapse, compared to only 13.6% of those who tested negative. Moreover, serial ctDNA analysis predicted recurrence before imaging scans by a median of eight months, demonstrating greater accuracy than the carcinoembryonic antigen (CEA) blood test [106]. According to a different study, with a mean lead time of 8.7 months, serial ctDNA analyses could predict disease recurrence up to 16.5 months before radiologic imaging [105]. 4.4. Predicting Drug Resistance: Identifying Resistance Mutations for Adaptive Therapy Strategies One major obstacle to cancer treatment is the establishment of medication resistance, which frequently results in therapeutic failure. Real-time, non-invasive detection of resistance mutations by liquid biopsies enables prompt modification of treatment approaches. In breast cancer management, the integration of liquid biopsies into clinical practice has marked a significant advancement. The NHS in England, for instance, has adopted ultra-sensitive blood World Journal of Biology Pharmacy and Health Sciences, 2025, 22(01), 112-138 129 tests to detect tumor DNA, enabling the identification and monitoring of mutations such as ESR1. This mutation can develop after hormone treatment and promote cancer progression. Patients testing positive for the ESR1 mutation can now access elacestrant, a targeted therapy that offers a more personalized treatment approach [107-109]. Furthermore, the work of researchers like Alberto Bardelli has shed light on the molecular mechanisms underlying resistance to targeted therapies. By analyzing liquid biopsies, Bardelli's team has uncovered how drug-resistant clones emerge and evolve, providing insights that inform the development of adaptive therapy strategies [110,111]. By making it possible to identify resistance mutations early, liquid biopsies facilitate the implementation of adaptive therapy strategies. This proactive approach allows clinicians to modify treatment plans in response to evolving tumor profiles, thereby enhancing the effectiveness of cancer therapies and improving patient outcomes. 4.4.1. Clinical Examples The introduction of the T790M mutation frequently results in resistance to firstand second-generation tyrosine kinase inhibitors (TKIs) in the treatment of non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) mutations [112]. Finding this mutation is essential for directing further treatment. Role of Liquid Biopsy in Detecting T790M Mutation To find genetic changes like the T790M mutation, circulating tumour DNA (ctDNA) in the bloodstream is analysed using liquid biopsy, which is a non-invasive technique. When tumour tissue is inaccessible, this method can be very helpful since it provides a less intrusive substitute for conventional tissue biopsies. Research has demonstrated that liquid biopsies can successfully identify the T790M mutation, enabling timely therapeutic interventions [113,114]. Osimertinib: Targeted Therapy for T790M-Positive NSCLC Osimertinib is a third-generation EGFR-TKI that is specifically intended to target the T790M resistant mutation as well as EGFR-sensitizing mutations. Clinical trials have demonstrated its efficacy in patients with T790M-positive NSCLC, leading to its approval for this indication [115,116]. Implementing liquid biopsy to detect the T790M mutation allows for the early identification of resistance, facilitating a timely switch to osimertinib. This strategy has been associated with improved progression-free survival and overall outcomes in patients with EGFR-mutant NSCLC [115]. 5. Current Limitations and Challenges Despite the ability of liquid biopsies to transform in oncology, several obstacles continue to hinder its widespread clinical adoption. Sensitivity and specificity remain critical concerns, as distinguishing tumor-derived signals from normal cell-free DNA can be challenging, particularly in early-stage cancers. Standardization of protocols across different platforms and laboratories is another pressing issue, with variations in sample collection, processing, and data interpretation affecting reproducibility. Moreover, the high cost of some advanced techniques limits accessibility, making integration into routine clinical practice difficult in resource-limited settings. This section delves into these challenges, examining the technological, biological, and regulatory barriers that must be addressed to fully realize the promise of liquid biopsy in precision oncology [117]. 5.1. Sensitivity and Specificity Issues Liquid biopsies hold promise for non-invasive cancer diagnostics, yet challenges persist in ensuring high sensitivity and specificity. Detecting low levels of circulating tumor DNA (ctDNA) amidst abundant normal cell-free DNA can lead to false negatives, particularly in early-stage cancers [78]. Conversely, benign mutations or clonal hematopoiesis may result in false positives. Advanced techniques like Cancer Personalized Profiling by Deep Sequencing (CAPP-Seq) have enhanced detection capabilities, achieving sensitivity to detect one mutant DNA molecule among 10,000 healthy ones [118]. However, issues such as sample cross-contamination, allelic bias, and PCR or sequencing errors can still affect accuracy. Moreover, the lack of standardized protocols across laboratories contributes to variability in results, underscoring the need for consistent methodologies to ensure reliable clinical applications [78]. 5.2. Cost and Accessibility: Economic Barriers to Widespread Clinical Adoption The implementation of liquid biopsy technologies is often hindered by economic factors. The development and execution of sophisticated assays require substantial financial investment, leading to high costs that can limit accessibility, especially in resource-constrained settings. This economic barrier poses challenges to the widespread World Journal of Biology Pharmacy and Health Sciences, 2025, 22(01), 112-138 130 clinical adoption of liquid biopsies, potentially exacerbating healthcare disparities. Efforts to streamline technologies and reduce costs are essential to make these advanced diagnostics more affordable and universally accessible [79,119]. 5.3. Regulatory Hurdles: FDA Approvals and Global Implementation Challenges Navigating the regulatory landscape presents significant challenges for the integration of liquid biopsy technologies into clinical practice. In the United States, obtaining FDA approval necessitates rigorous validation to demonstrate safety and efficacy, a process that can be time-consuming and costly [120]. Globally, disparate regulatory standards and approval processes further complicate the implementation of these technologies. Harmonizing regulatory frameworks and establishing clear guidelines are crucial steps toward facilitating the global adoption of liquid biopsies in standard clinical care [25]. 5.4. Ethical Considerations The utilization of liquid biopsies raises important ethical considerations, particularly concerning data privacy and the implications of genetic risk information. Safeguarding patient genetic data is paramount to prevent misuse or discrimination. Additionally, the interpretation of genetic risk requires careful consideration to avoid unnecessary anxiety or interventions. Establishing robust ethical guidelines and engaging in transparent communication with patients are essential to address these challenges responsibly [121,122]. 6. Future Directions and Emerging Innovations The rapid evolution of liquid biopsy technologies is paving the way for breakthroughs that could redefine cancer diagnostics and treatment monitoring. Researchers are refining analytical methods to improve sensitivity, enhance multi-analyte detection, and integrate artificial intelligence for more precise data interpretation. Innovations in microfluidics and single-molecule sequencing are pushing the boundaries of early cancer detection, while novel biomarkers, such as tumor-educated platelets and circulating mitochondrial DNA, are expanding the scope of liquid biopsy applications [123]. Table 6 highlights key emerging technologies and their potential impact on the field. As these advancements progress, the focus is shifting toward making liquid biopsy not only a complementary tool but a primary diagnostic strategy in precision oncology. This section explores the promising developments shaping the future of this field and the challenges that must be addressed to bring these innovations into routine clinical practice. Table 6 Emerging Technologies and Their Potential Impact on Liquid Biopsy Technology Development Stage Potential Advantages Current Challenges Single-Cell Liquid Biopsy Experimental Allows analysis of individual tumor cells, offering insights into heterogeneity and resistance mechanisms. Expensive and complex; requires advanced microfluidics. Wearable Biosensors for Liquid Biopsy Early research Real-time monitoring; non-invasive detection of biomarkers from sweat, saliva, or interstitial fluids. Limited biomarker range; requires validation for accuracy. AI-Integrated Liquid Biopsy Analysis Pilot trials Improves accuracy by analyzing large datasets; enhances early cancer detection. Data privacy concerns; regulatory approval challenges. Microfluidics-Based ctDNA Isolation Experimental High efficiency in capturing rare ctDNA fragments; minimal sample requirement. Requires standardization for clinical use. 6.1. Single-Cell Liquid Biopsies: Advancing Precision in CTC Analysis Single-cell liquid biopsies are enhancing the precision of circulating tumor cell (CTC) analysis by allowing for the examination of individual tumor cells isolated from blood samples. This approach provides detailed insights into tumor heterogeneity, metastatic potential, and treatment resistance mechanisms. Technologies such as the NanoVelcro Chip have been developed to capture and analyze single CTCs, facilitating personalized treatment strategies and real-time monitoring of tumor dynamics [124-126]. World Journal of Biology Pharmacy and Health Sciences, 2025, 22(01), 112-138 131 6.2. Wearable and Point-of-Care Liquid Biopsy Devices The development of wearable and point-of-care liquid biopsy devices is revolutionizing real-time patient monitoring. These innovations enable continuous tracking of biomarkers, allowing for early detection of cancer recurrence and timely therapeutic interventions. Integrating microfluidic technologies into portable devices facilitates rapid, on-site analysis of bodily fluids, enhancing patient convenience and enabling more responsive healthcare delivery [127]. 6.3. Combination with AI and Blockchain for Secure Data Management Integrating artificial intelligence (AI) and blockchain technology with liquid biopsy data management systems enhances diagnostic accuracy and ensures secure handling of sensitive patient information. AI algorithms can analyze complex datasets to identify patterns indicative of cancer, while blockchain provides a decentralized, immutable ledger for secure data storage and sharing, addressing concerns related to data privacy and integrity. However, regulatory scrutiny is essential to validate the efficacy and safety of these AI-driven diagnostic tools, as highlighted by recent evaluations of health tech firms' claims [128]. 6.4. Potential for Universal Cancer Screening The potential of liquid biopsies for universal cancer screening lies in their ability to detect multiple cancer types through a simple blood test, facilitating large-scale, population-based applications. Implementing such screening programs could lead to early detection and improved survival rates across diverse populations. However, challenges such as ensuring test accuracy, managing healthcare infrastructure, and addressing ethical considerations related to widespread genetic testing must be carefully navigated to realize this potential fully [129,130]. 7. Conclusion Liquid biopsies have transformed cancer diagnostics and monitoring by enabling the detection of circulating tumor DNA (ctDNA) in the bloodstream, providing a non-invasive alternative to traditional tissue biopsies.This advancement allows for real-time insights into tumor dynamics, facilitating early detection of mutations and personalized treatment strategies. The integration of artificial intelligence (AI) and genetic sequencing has further enhanced the precision of these diagnostics, offering tailored therapies based on individual cancer profiles. Despite these advancements, challenges persist. Economic factors, such as the high costs associated with advanced diagnostics, limit accessibility, particularly in resource-constrained settings. Additionally, disparities in research funding and pharmaceutical interest have slowed progress in treating less common but lethal cancers, highlighting the need for equitable investment across all cancer types. Looking ahead, the continued evolution of liquid biopsy technologies, coupled with AI integration, holds the potential to revolutionize precision oncology. These innovations could lead to earlier detection, more accurate monitoring, and personalized treatment plans, ultimately improving patient outcomes and survival rates. Addressing current challenges through collaborative efforts and equitable resource allocation will be crucial in realizing the full potential of these advancements in cancer care. Compliance with ethical standards Acknowledgments The authors wish to acknowledge the collaborative effort of all contributing scholars and colleagues who jointly authored and edited this review paper. This work was conducted entirely through the intellectual and academic contributions of the authoring team, without external funding or assistance from any individual, institution, or organization. Disclosure of conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. World Journal of Biology Pharmacy and Health Sciences, 2025, 22(01), 112-138 132 References [1] Horgan, D., Mia, R., Erhabor, T., Hamdi, Y., Dandara, C., Lal, J. A., ... & Barrera-Saldana, H. A. (2022, October). Fighting cancer around the world: A framework for action. In Healthcare (Vol. 10, No. 11, p. 2125). MDPI. [2] Pulumati, A., Pulumati, A., Dwarakanath, B. S., Verma, A., & Papineni, R. V. (2023). Technological advancements in cancer diagnostics: Improvements and limitations. Cancer Reports, 6(2), e1764. [3] Khan, A., Raza, F., & He, N. (2024). Nanoscale Extracellular Vesicle-Enabled Liquid Biopsy: Advances and Challenges for Lung Cancer Detection. Micromachines, 15(10), 1181. [4] Turajlic, S., Sottoriva, A., Graham, T., & Swanton, C. (2019). Resolving genetic heterogeneity in cancer. Nature Reviews Genetics, 20(7), 404-416. [5] Deshmukh, S., & Saini, S. (2020). Phenotypic heterogeneity in tumor progression, and its possible role in the onset of cancer. Frontiers in Genetics, 11, 604528. [6] Cleveland Clinic. (n.d.). Liquid biopsy: What it is & procedure details. Cleveland Clinic. Retrieved March 19, 2025, from https://my.clevelandclinic.org/health/diagnostics/23992-liquid-biopsy [7] Marrugo-Ramírez, J., Mir, M., & Samitier, J. (2018). Blood-based cancer biomarkers in liquid biopsy: a promising non-invasive alternative to tissue biopsy. International journal of molecular sciences, 19(10), 2877. [8] Nikanjam, M., Kato, S., & Kurzrock, R. (2022). Liquid biopsy: current technology and clinical applications. Journal of hematology & oncology, 15(1), 131. [9] Lone, S. N., Nisar, S., Masoodi, T., Singh, M., Rizwan, A., Hashem, S., ... & Macha, M. A. (2022). Liquid biopsy: a step closer to transform diagnosis, prognosis and future of cancer treatments. Molecular cancer, 21(1), 79. [10] Connal, S., Cameron, J. M., Sala, A., Brennan, P. M., Palmer, D. S., Palmer, J. D., ... & Baker, M. J. (2023). Liquid biopsies: the future of cancer early detection. Journal of translational medicine, 21(1), 118. [11] Zhang, L., Liang, Y., Li, S., Zeng, F., Meng, Y., Chen, Z., ... & Yu, F. (2019). The interplay of circulating tumor DNA and chromatin modification, therapeutic resistance, and metastasis. Molecular cancer, 18, 1-20. [12] Ricciardi, E., Giordani, E., Ziccheddu, G., Falcone, I., Giacomini, P., Fanciulli, M., ... & Valenti, F. (2023). Metastatic melanoma: liquid biopsy as a new precision medicine approach. International journal of molecular sciences, 24(4), 4014. [13] U.S. Food and Drug Administration. (2020, August 7). FDA approves first liquid biopsy next-generation sequencing companion diagnostic test. https://www.fda.gov/news-events/press-announcements/fda-approves-first-liquidbiopsy-next-generation-sequencing-companion-diagnostic-test [14] Shegekar, T., Vodithala, S., & Juganavar, A. (2023). The emerging role of liquid biopsies in revolutionising cancer diagnosis and therapy. Cureus, 15(8). [15] Liao, H., Zhang, J., Zheng, T., Liu, X., Zhong, J., Shao, B., ... & Li, H. (2022). Identification of mutation patterns and circulating tumour DNA-derived prognostic markers in advanced breast cancer patients. Journal of Translational Medicine, 20(1), 211. [16] Leary, R. J., Sausen, M., Kinde, I., Papadopoulos, N., Carpten, J. D., Craig, D., ... & Velculescu, V. E. (2012). Detection of chromosomal alterations in the circulation of cancer patients with whole-genome sequencing. Science translational medicine, 4(162), 162ra154-162ra154. [17] Olsson, E., Winter, C., George, A., Chen, Y., Howlin, J., Tang, M. H. E., ... & Saal, L. H. (2015). Serial monitoring of circulating tumor DNA in patients with primary breast cancer for detection of occult metastatic disease. EMBO molecular medicine, 7(8), 1034-1047. [18] Lianidou, E. (2021). Detection and relevance of epigenetic markers on ctDNA: recent advances and future outlook. Molecular Oncology, 15(6), 1683-1700. [19] Delpu, Y., Cordelier, P., Cho, W. C., & Torrisani, J. (2013). DNA methylation and cancer diagnosis. International journal of molecular sciences, 14(7), 15029-15058. [20] Sun, K., Jiang, P., Chan, K. A., Wong, J., Cheng, Y. K., Liang, R. H., ... & Lo, Y. M. D. (2015). Plasma DNA tissue mapping by genome-wide methylation sequencing for noninvasive prenatal, cancer, and transplantation assessments. Proceedings of the National Academy of Sciences, 112(40), E5503-E5512. World Journal of Biology Pharmacy and Health Sciences, 2025, 22(01), 112-138 133 [21] Bronkhorst, A. J., & Holdenrieder, S. (2023). The changing face of circulating tumor DNA (ctDNA) profiling: Factors that shape the landscape of methodologies, technologies, and commercialization. Medizinische Genetik, 35(4), 201-235. [22] Minari, R., Bordi, P., & Tiseo, M. (2016). Third-generation epidermal growth factor receptor-tyrosine kinase inhibitors in T790M-positive non-small cell lung cancer: review on emerged mechanisms of resistance. Translational lung cancer research, 5(6), 695. [23] Desai, A., Vázquez, T. A., Arce, K. M., Corassa, M., Mack, P. C., Gray, J. E., & Pellini, B. (2024). ctDNA for the evaluation and management of EGFR-mutant non-small cell lung cancer. Cancers, 16(5), 940. [24] Wang, T., Li, P., Qi, Q., Zhang, S., Xie, Y., Wang, J., ... & Wang, C. (2023). A multiplex blood-based assay targeting DNA methylation in PBMCs enables early detection of breast cancer. Nature Communications, 14(1), 4724. [25] Ntzifa, A., & Lianidou, E. (2023). Pre-analytical conditions and implementation of quality control steps in liquid biopsy analysis. Critical Reviews in Clinical Laboratory Sciences, 60(8), 573-594. [26] Han, X., Wang, J., & Sun, Y. (2017). Circulating tumor DNA as biomarkers for cancer detection. Genomics, proteomics & bioinformatics, 15(2), 59-72. [27] Majidpoor, J., & Mortezaee, K. (2021). Steps in metastasis: an updated review. Medical Oncology, 38(1), 3. [28] Li, Y., Liu, F., Cai, Q., Deng, L., Ouyang, Q., Zhang, X. H. F., & Zheng, J. (2025). Invasion and metastasis in cancer: molecular insights and therapeutic targets. Signal Transduction and Targeted Therapy, 10(1), 57. [29] Keller, L., & Pantel, K. (2019). Unravelling tumour heterogeneity by single-cell profiling of circulating tumour cells. Nature Reviews Cancer, 19(10), 553-567. [30] Allen, T. A., Asad, D., Amu, E., Hensley, M. T., Cores, J., Vandergriff, A., ... & Cheng, K. (2019). Circulating tumor cells exit circulation while maintaining multicellularity, augmenting metastatic potential. Journal of Cell Science, 132(17), jcs231563. [31] Wikipedia contributors. (2025, February 4). Cancer exodus hypothesis. In Wikipedia, The Free Encyclopedia. Retrieved 14:23, March 19, 2025, from https://en.wikipedia.org/w/index.php?title=Cancer_exodus_hypothesis&oldid=1273891976 [32] Hong, Y., Fang, F., & Zhang, Q. (2016). Circulating tumor cell clusters: What we know and what we expect. International journal of oncology, 49(6), 2206-2216. [33] Yaghoubi Naei, V., Bordhan, P., Mirakhorli, F., Khorrami, M., Shrestha, J., Nazari, H., ... & Ebrahimi Warkiani, M. (2023). Advances in novel strategies for isolation, characterization, and analysis of CTCs and ctDNA. Therapeutic Advances in Medical Oncology, 15, 17588359231192401. [34] Agnoletto, C., Corrà, F., Minotti, L., Baldassari, F., Crudele, F., Cook, W. J. J., ... & Volinia, S. (2019). Heterogeneity in circulating tumor cells: the relevance of the stem-cell subset. Cancers, 11(4), 483. [35] Yadav, D. K., Bai, X., Yadav, R. K., Singh, A., Li, G., Ma, T., ... & Liang, T. (2018). Liquid biopsy in pancreatic cancer: the beginning of a new era. Oncotarget, 9(42), 26900. [36] Lozar, T., Jesenko, T., Kloboves Prevodnik, V., Cemazar, M., Hosta, V., Jericevic, A., ... & Grasic Kuhar, C. (2020). Preclinical and clinical evaluation of magnetic-activated cell separation technology for CTC isolation in breast cancer. Frontiers in Oncology, 10, 554554. [37] Wang, G., Benasutti, H., Jones, J. F., Shi, G., Benchimol, M., Pingle, S., ... & Simberg, D. (2018). Isolation of Breast cancer CTCs with multitargeted buoyant immunomicrobubbles. Colloids and Surfaces B: Biointerfaces, 161, 200209. [38] Wikipedia contributors. (2025, March 5). Circulating tumor cell. In Wikipedia, The Free Encyclopedia. Retrieved 14:34, March 19, 2025, from https://en.wikipedia.org/w/index.php?title=Circulating_tumor_cell&oldid=1278944230 [39] Orrapin, S., Udomruk, S., Lapisatepun, W., Moonmuang, S., Phanphaisarn, A., Phinyo, P., ... & Chaiyawat, P. (2022). Clinical implication of circulating tumor cells expressing epithelial mesenchymal transition (EMT) and cancer stem cell (CSC) markers and their perspective in HCC: a systematic review. Cancers, 14(14), 3373. [40] Battistelli, M., & Falcieri, E. (2021). Apoptotic bodies: particular extracellular vesicles involved in intercellular communication. Advances in Medical Biochemistry, Genomics, Physiology, and Pathology, 473-486. World Journal of Biology Pharmacy and Health Sciences, 2025, 22(01), 112-138 134 [41] Kalluri, R., & LeBleu, V. S. (2020). The biology, function, and biomedical applications of exosomes. science, 367(6478), eaau6977. [42] Hoshino, A., Costa-Silva, B., Shen, T. L., Rodrigues, G., Hashimoto, A., Tesic Mark, M., ... & Lyden, D. (2015). Tumour exosome integrins determine organotropic metastasis. Nature, 527(7578), 329-335. [43] Maurizi, A., Ponzetti, M., & Rucci, N. (2021). How the “seed” prepares the “soil”: the bone/bone marrow premetastatic niche. Journal of Cancer Metastasis and Treatment, 7, N-A. [44] Ramachandran, S., & Palanisamy, V. (2012). Horizontal transfer of RNAs: exosomes as mediators of intercellular communication. Wiley interdisciplinary reviews: RNA, 3(2), 286-293. [45] Avgoulas, D. I., Tasioulis, K. S., Papi, R. M., & Pantazaki, A. A. (2023). Therapeutic and diagnostic potential of exosomes as drug delivery systems in brain cancer. Pharmaceutics, 15(5), 1439. [46] Fernández-Lázaro, D., García Hernández, J. L., García, A. C., Córdova Martínez, A., Mielgo-Ayuso, J., & CruzHernández, J. J. (2020). Liquid biopsy as novel tool in precision medicine: Origins, properties, identification and clinical perspective of cancer’s biomarkers. Diagnostics, 10(4), 215. [47] Wikipedia contributors. (2025, January 21). MicroRNA biosensors. In Wikipedia, The Free Encyclopedia. Retrieved 15:05, March 19, 2025, from https://en.wikipedia.org/w/index.php?title=MicroRNA_biosensors&oldid=1270866920 [48] Das, S., Dey, M. K., Devireddy, R., & Gartia, M. R. (2023). Biomarkers in cancer detection, diagnosis, and prognosis. Sensors, 24(1), 37. [49] Marsh, S. (2024, December 6). Lung cancer 'could be detected by world-first urine test'. The Guardian. https://www.theguardian.com/society/2024/dec/06/lung-cancer-could-be-detected-by-world-first-urine-test [50] Lone, S. N., Nisar, S., Masoodi, T., Singh, M., Rizwan, A., Hashem, S., ... & Macha, M. A. (2022). Liquid biopsy: a step closer to transform diagnosis, prognosis and future of cancer treatments. Molecular cancer, 21(1), 79. [51] Clack, K., Soda, N., Kasetsirikul, S., Mahmudunnabi, R. G., Nguyen, N. T., & Shiddiky, M. J. (2023). Toward personalized nanomedicine: the critical evaluation of micro and nanodevices for cancer biomarker analysis in liquid biopsy. Small, 19(15), 2205856. [52] Wikipedia contributors. (2024, December 17). CAPP-Seq. In Wikipedia, The Free Encyclopedia. Retrieved 16:05, March 19, 2025, from https://en.wikipedia.org/w/index.php?title=CAPP-Seq&oldid=1263587646 [53] Nakamura, Y., Olsen, S., Zhang, N., Liao, J., & Yoshino, T. (2022). Comprehensive genomic profiling of circulating tumor DNA in patients with previously treated metastatic colorectal cancer: analysis of a real-world healthcare claims database. Current Oncology, 29(5), 3433-3448. [54] Guardant Health. (2021, November 11). Study shows Guardant360® liquid biopsy test helps guide treatment for patients with HER2-driven metastatic colorectal cancer. https://investors.guardanthealth.com/pressreleases/press-releases/2021/Study-Shows-Guardant360-Liquid-Biopsy-Test-Helps-Guide-Treatment-forPatients-with-HER2-Driven-Metastatic-Colorectal-Cancer/default.aspx [55] Moreno-Manuel, A., Calabuig-Fariñas, S., Obrador-Hevia, A., Blasco, A., Fernández-Díaz, A., Sirera, R., ... & JantusLewintre, E. (2021). dPCR application in liquid biopsies: divide and conquer. Expert Review of Molecular Diagnostics, 21(1), 3-15. [56] Shi, J., Zhang, Y., Fan, Y., Liu, Y., & Yang, M. (2024). Recent advances in droplet‐based microfluidics in liquid biopsy for cancer diagnosis. Droplet, 3(1), e92. [57] Mirabile, A., Sangiorgio, G., Bonacci, P. G., Bivona, D., Nicitra, E., Bonomo, C., ... & Musso, N. (2024). Advancing Pathogen Identification: The Role of Digital PCR in Enhancing Diagnostic Power in Different Settings. Diagnostics, 14(15), 1598. [58] Wikipedia contributors. (2024, October 30). Digital polymerase chain reaction. In Wikipedia, The Free Encyclopedia. Retrieved 19:37, March 19, 2025, from https://en.wikipedia.org/w/index.php?title=Digital_polymerase_chain_reaction&oldid=1254262218 [59] Hudecova, I. (2015). Digital PCR analysis of circulating nucleic acids. Clinical biochemistry, 48(15), 948-956. [60] Naoumi, N., Michaelidou, K., Papadakis, G., Simaiaki, A. E., Fernández, R., Calero, M., ... & Gizeli, E. (2022). Acoustic array biochip combined with allele-specific PCR for multiple cancer mutation analysis in tissue and liquid biopsy. ACS sensors, 7(2), 495-503. World Journal of Biology Pharmacy and Health Sciences, 2025, 22(01), 112-138 135 [61] Wikipedia contributors. (2023, November 29). BEAMing. In Wikipedia, The Free Encyclopedia. Retrieved 19:43, March 19, 2025, from https://en.wikipedia.org/w/index.php?title=BEAMing&oldid=1187515873 [62] Forthun, R. B., Hovland, R., Schuster, C., Puntervoll, H., Brodal, H. P., Namløs, H. M., ... & Straume, O. (2019). ctDNA detected by ddPCR reveals changes in tumour load in metastatic malignant melanoma treated with bevacizumab. Scientific reports, 9(1), 17471. [63] Chabon, J. J., Hamilton, E. G., Kurtz, D. M., Esfahani, M. S., Moding, E. J., Stehr, H., ... & Diehn, M. (2020). Integrating genomic features for non-invasive early lung cancer detection. Nature, 580(7802), 245-251. [64] Wikipedia contributors. (2024, October 18). Cancer Likelihood in Plasma. In Wikipedia, The Free Encyclopedia. Retrieved 19:57, March 19, 2025, from https://en.wikipedia.org/w/index.php?title=Cancer_Likelihood_in_Plasma&oldid=1251937621 [65] Wikipedia contributors. (2025, February 20). Dxcover. In Wikipedia, The Free Encyclopedia. Retrieved 20:01, March 19, 2025, from https://en.wikipedia.org/w/index.php?title=Dxcover&oldid=1276711032 [66] Wikipedia contributors. (2024, December 30). EPIC-Seq. In Wikipedia, The Free Encyclopedia. Retrieved 20:04, March 19, 2025, from https://en.wikipedia.org/w/index.php?title=EPIC-Seq&oldid=1266172608 [67] Abian, A. I., Khan Raiaan, M. A., Karim, A., Azam, S., Fahad, N. M., Shafiabady, N., ... & De Boer, F. (2024). Automated diagnosis of respiratory diseases from lung ultrasound videos ensuring XAI: an innovative hybrid model approach. Frontiers in Computer Science, 6, 1438126. [68] Di Sario, G., Rossella, V., Famulari, E. S., Maurizio, A., Lazarevic, D., Giannese, F., & Felici, C. (2023). Enhancing clinical potential of liquid biopsy through a multi-omic approach: A systematic review. Frontiers in Genetics, 14, 1152470. [69] Gorgannezhad, L., Umer, M., Islam, M. N., Nguyen, N. T., & Shiddiky, M. J. (2018). Circulating tumor DNA and liquid biopsy: opportunities, challenges, and recent advances in detection technologies. Lab on a Chip, 18(8), 11741196. [70] Wikipedia contributors. (2024, September 6). Precision diagnostics. In Wikipedia, The Free Encyclopedia. Retrieved 20:18, March 19, 2025, from https://en.wikipedia.org/w/index.php?title=Precision_diagnostics&oldid=1244332160 [71] Macklin, A., Khan, S., & Kislinger, T. (2020). Recent advances in mass spectrometry based clinical proteomics: applications to cancer research. Clinical proteomics, 17(1), 17. [72] Birhanu, A. G. (2023). Mass spectrometry-based proteomics as an emerging tool in clinical laboratories. Clinical proteomics, 20(1), 32. [73] Danzi, F., Pacchiana, R., Mafficini, A., Scupoli, M. T., Scarpa, A., Donadelli, M., & Fiore, A. (2023). To metabolomics and beyond: a technological portfolio to investigate cancer metabolism. Signal Transduction and Targeted Therapy, 8(1), 137. [74] Kumar, A., & Misra, B. B. (2019). Challenges and opportunities in cancer metabolomics. Proteomics, 19(21-22), 1900042. [75] Wikipedia contributors. (2024, September 6). Precision diagnostics. In Wikipedia, The Free Encyclopedia. Retrieved 20:48, March 19, 2025, from https://en.wikipedia.org/w/index.php?title=Precision_diagnostics&oldid=1244332160 [76] Chakraborty, S., Sharma, G., Karmakar, S., & Banerjee, S. (2024). Multi-OMICS approaches in cancer biology: New era in cancer therapy. Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease, 1870(5), 167120. [77] Tellez-Gabriel, M., Knutsen, E., & Perander, M. (2020). Current status of circulating tumor cells, circulating tumor DNA, and exosomes in breast cancer liquid biopsies. International Journal of Molecular Sciences, 21(24), 9457. [78] Fu, S. W., Tang, C., Tan, X., & Srivastava, S. (2024). Liquid biopsy for early cancer detection: technological revolutions and clinical dilemma. Expert Review of Molecular Diagnostics, 24(10), 937-955. [79] Ignatiadis, M., Sledge, G. W., & Jeffrey, S. S. (2021). Liquid biopsy enters the clinic—implementation issues and future challenges. Nature reviews Clinical oncology, 18(5), 297-312. [80] Streck. (2023, July 13). FDA clearance brings liquid biopsy into a new era. Streck. https://www.streck.com/blog/fda-clearance-brings-liquid-biopsy-into-a-new-era/ World Journal of Biology Pharmacy and Health Sciences, 2025, 22(01), 112-138 136 [81] Cavallo, J. (2021, October 10). The evolution of liquid biopsy in cancer care. The ASCO Post. https://ascopost.com/issues/october-10-2021/the-evolution-of-liquid-biopsy-in-cancer-care/ [82] U.S. Food and Drug Administration. (2020, October 26). FDA approves liquid biopsy NGS companion diagnostic test for multiple cancers and biomarkers. https://www.fda.gov/drugs/resources-information-approved-drugs/fdaapproves-liquid-biopsy-ngs-companion-diagnostic-test-multiple-cancers-and-biomarkers [83] U.S. Food and Drug Administration. (2020, August 7). FDA approves first liquid biopsy next-generation sequencing companion diagnostic test. https://www.fda.gov/news-events/press-announcements/fda-approves-first-liquidbiopsy-next-generation-sequencing-companion-diagnostic-test [84] American Association for Cancer Research. (n.d.). Liquid biopsy approved for lung cancer. https://www.aacr.org/patients-caregivers/progress-against-cancer/liquid-biopsy-approved-lung-cancer-fda/ [85] Foundation Medicine. (n.d.). FoundationOne® Liquid CDx. Foundation Medicine. Retrieved March 19, 2025, from https://www.foundationmedicine.com/test/foundationone-liquid-cdx [86] Guardant Health. (n.d.). Guardant360® CDx: Fastest FDA-approved liquid CGP panel. https://www.guardantcomplete.com/products/guardant360-cdx [87] Wikipedia contributors. (2024, December 13). Guardant Health. In Wikipedia, The Free Encyclopedia. Retrieved 21:50, March 19, 2025, from https://en.wikipedia.org/w/index.php?title=Guardant_Health&oldid=1262911842 [88] Coronado, G. D., Jenkins, C. L., Shuster, E., Johnson, C., Amy, D., Cook, J., ... & Mummadi, R. (2024). Blood-based colorectal cancer screening in an integrated health system: a randomised trial of patient adherence. Gut, 73(4), 622-628. [89] Kong, X., Gao, P., Wang, J., Fang, Y., & Hwang, K. C. (2023). Advances of medical nanorobots for future cancer treatments. Journal of Hematology & Oncology, 16(1), 74. [90] Kishore, C., & Bhadra, P. (2021). Targeting brain cancer cells by nanorobot, a promising nanovehicle: new challenges and future perspectives. CNS & Neurological Disorders-Drug Targets-CNS & Neurological Disorders), 20(6), 531-539. [91] Montoya Mira, J. L., Quentel, A., Patel, R. K., Keith, D., Sousa, M., Minnier, J., ... & Fischer, J. M. (2025). Early detection of pancreatic cancer by a high-throughput protease-activated nanosensor assay. Science Translational Medicine, 17(785), eadq3110. [92] Neal, R. D., Johnson, P., Clarke, C. A., Hamilton, S. A., Zhang, N., Kumar, H., ... & Sasieni, P. (2022). Cell-free DNA– based multi-cancer early detection test in an asymptomatic screening population (NHS-Galleri): design of a pragmatic, prospective randomised controlled trial. Cancers, 14(19), 4818. [93] Hall, M. P., & Aravanis, A. M. (2023). The Galleri Assay. In Circulating Tumor Cells: Advances in Liquid Biopsy Technologies (pp. 633-664). Cham: Springer International Publishing. [94] Marlow, L. A., Schmeising-Barnes, N., Warwick, J., & Waller, J. (2023). Psychological Impact of the Galleri test (sIG (n) al): protocol for a longitudinal evaluation of the psychological impact of receiving a cancer signal in the NHSGalleri trial. BMJ open, 13(7), e072657. [95] Bi, W. L., Hosny, A., Schabath, M. B., Giger, M. L., Birkbak, N. J., Mehrtash, A., ... & Aerts, H. J. (2019). Artificial intelligence in cancer imaging: clinical challenges and applications. CA: a cancer journal for clinicians, 69(2), 127157. [96] Reece, M., Saluja, H., Hollington, P., Karapetis, C. S., Vatandoust, S., Young, G. P., & Symonds, E. L. (2019). The use of circulating tumor DNA to monitor and predict response to treatment in colorectal cancer. Frontiers in genetics, 10, 1118. [97] Chen, K., Shields, M. D., Chauhan, P. S., Ramirez, R. J., Harris, P. K., Reimers, M. A., ... & Chaudhuri, A. A. (2021). Commercial ctDNA assays for minimal residual disease detection of solid tumors. Molecular diagnosis & therapy, 1-18. [98] Gutierrez, M. E. (2017, November 6). Genomic profiling of advanced non–small cell lung cancer in community settings: Gaps and opportunities. Clinical Lung Cancer. https://guardanthealth.eu/clinical-lung-cancer/ [99] Sternberg, A. (2021, April 7). Molecular response by Guardant360 liquid biopsy translates to clinical outcomes in NSCLC. Cancer Network. https://www.cancernetwork.com/view/molecular-response-by-guardant360-liquidbiopsy-translates-to-clinical-outcomes-in-nsclc