Introduction: Liquid biopsy is a major breakthrough in cancer care. It allows for a non-invasive way to examine tumor materials found in body fluids. Unlike traditional tissue biopsies, which involve intrusive procedures, liquid biopsy isolates circulating tumor components like ctDNA, miRNAs, CTCs, and exosomes. This method enables real-time evaluation of tumor biology with less discomfort for patients. These biomarkers offer detailed genetic, epigenetic, and transcriptomic information about how tumors start, develop, and respond to treatment.
The key benefit of early cancer detection is its potential to greatly increase patient survival. This happens by allowing treatment to begin before metastasis or a heavy tumor load sets in. Traditional diagnostic methods, such as imaging and serum markers, usually identify cancer at later stages. They can also be invasive or lack precision. Liquid biopsy addresses these issues by identifying tumor-derived biomarkers even when they are present in small amounts, especially in early stages of the disease.
ctDNA carries specific mutations and methylation patterns that make it a reliable marker for cancer detection and tracking. miRNAs are stable in body fluids and help regulate cancer-related pathways, providing additional diagnostic and prognostic insights. Together, these elements support ongoing monitoring of the disease's progression, assist in personalizing treatment choices, and increase the accuracy of prognoses. With improvements in molecular tests, sequencing technologies, and data analysis using AI, liquid biopsy is becoming a key part of precision cancer care.
Methods: This review summarizes recent advances in liquid biopsy applications for early cancer detection and disease monitoring, focusing on ctDNA and miRNAs. The analysis draws from peer-reviewed clinical and translational research studies, chosen for their relevance to technological innovation, biomarker sensitivity, and clinical impact across different cancers.
ctDNA Analysis:
Blood plasma or other biofluid samples are collected and processed to isolate cell-free DNA.
ctDNA is distinguished from normal cfDNA by tumor-specific mutations, copy number changes, and methylation patterns.
Analytical platforms include digital droplet PCR (ddPCR) for ultra-sensitive measurement, quantitative PCR (qPCR) for targeted detection, and next-generation sequencing (NGS) for comprehensive mutation profiling.
Methylation-sensitive tests are used for early-stage detection when ctDNA levels are low.
miRNA Profiling:
miRNAs are extracted from body fluids like plasma, serum, saliva, and urine, often protected within extracellular vesicles or attached to proteins to prevent degradation.
Quantification and expression profiling use qPCR, ddPCR, or NGS to identify disease-specific miRNA patterns.
Panels of multiple miRNAs are analyzed to improve diagnostic accuracy and consider tumor diversity.
Biomarker Enrichment and Analysis:
Microfluidic devices and immunoaffinity capture methods are used to isolate CTCs, exosomes, and nucleic acids with high purity.
Size-based filters and antibody-coated surfaces target tumor-specific antigens (e.g., EpCAM) to enrich circulating biomarkers.
Bioinformatic pipelines combine ctDNA and miRNA data, using AI and machine learning algorithms to enhance detection accuracy, identify residual disease, and forecast treatment outcomes.
The methodology also includes comparative evaluation of ctDNA and miRNA tests for sensitivity, specificity, reproducibility, and clinical usefulness, highlighting multi-analyte integration.
Results: Liquid biopsy shows wide usefulness in cancer diagnosis, prognosis, and monitoring.
Lung Cancer: Detecting ctDNA for EGFR, ALK, and KRAS mutations helps select targeted therapies and predict outcomes. FDA-approved ctDNA tests are now available for EGFR mutation analysis. Circulating miRNAs increase sensitivity when combined with ctDNA, especially for early detection and monitoring resistance.
Breast Cancer: Measuring ctDNA levels can predict metastatic relapse and find minimal residual disease earlier than imaging or blood markers. MiRNAs like miR-21 and miR-195 are linked to tumor growth and prognosis, aiding in assessing risk and planning treatment.
Colorectal Cancer: Identifying ctDNA for KRAS, NRAS, and BRAF mutations supports decisions on targeted therapies and predicts relapse.
Exosome Analysis: Exosomal miRNAs and ctDNA fragments are more stable than cell-free versions, which improves diagnostic accuracy. They relate to disease stage, response to treatment, and recurrence risk.
Comparative Performance:
MiRNAs are more stable and abundant, which enhances sensitivity for early detection.
CtDNA provides better specificity due to unique mutations and methylation patterns found in tumors.
Combining both types of biomarkers in multi-panel tests boosts diagnostic strength, addresses tumor diversity, and improves detection of minimal residual disease.
Technological advancements in ddPCR, NGS, microfluidics, and AI tools have raised detection limits to below 0.01% mutant allele frequency, allowing for earlier intervention.
Conclusion: Liquid biopsy is changing how we diagnose cancer. It can detect, monitor, and profile cancers without invasive procedures, using ctDNA and miRNAs. This method allows for real-time monitoring of tumor changes, which helps with early detection, tailoring treatments, and tracking minimal residual disease (MRD). There are still challenges like standardization, biological differences, and cost. However, combining various omics profiles, AI-driven analysis, and single-cell techniques is likely to improve sensitivity, specificity, and clinical usefulness. With ongoing improvements and testing, liquid biopsy is set to become a standard part of precise cancer care. This, in turn, could lead to better survival rates and enhanced quality of life for patients around the world.
Keywords: Liquid biopsy; circulating tumor DNA (ctDNA); microRNAs; early cancer detection; minimal residual di