Harnessing liquid biopsies: Exosomes and ctDNA as minimally invasive biomarkers for precision cancer medicine

Abstract

Liquid biopsies have emerged as groundbreaking tools for minimally invasive monitoring of cancer, encompassing the analysis of Cell-Free DNA (cfDNA), circulating tumor DNA (ctDNA) and exosomes. This paradigm shift offers an emerging approach for understanding tumor dynamics, treatment responses, and disease progression. Leveraging advancements in molecular biology and technology, liquid biopsies enable clinicians to gain intricate insights from peripheral blood, thereby transforming the landscape of cancer care. This review describes the clinical impact, technological innovations, and recent evidence surrounding the integration of ctDNA and exosome analysis in cancer monitoring. Through early detection, real-time treatment response assessment, and the tracking of minimal residual disease, liquid biopsies have redefined the standards of precision oncology. Key advancements in ctDNA analysis, such as high-throughput sequencing and digital PCR, empower the detection of actionable mutations with high sensitivity. Concurrently, the characterization of exosomal cargo, facilitated by next-generation sequencing and mass spectrometry, unveils the molecular nuances of tumors. Recent studies underscore the utility of these approaches, demonstrating their efficacy in predicting relapse, guiding therapeutic decisions, and ultimately improving patient outcomes. As the field continues to evolve, liquid biopsies hold promise not only as diagnostic tools but also as agents of personalized medicine, enabling precise navigation of the intricate landscape of cancer with minimally invasiveness.

Graphical abstract

Pictorial abstractPictorial abstract of liquid biopsies. Symbols represent analysis of circulating biomarkers ctDNA and exosomes for applications in early detection, therapeutic monitoring, and residual disease surveillance, enabled by technologies like sequencing and microfluidics. Modular components highlight multifaceted clinical capabilities and ongoing innovations in this emerging paradigm for non-invasive cancer management.

Highlights

• •Liquid biopsies enable non-invasive cancer detection and monitoring through ctDNA and exosome analysis.
• •ctDNA and exosomes facilitate early diagnosis, treatment response monitoring, and residual disease detection.
• •Liquid biopsies overcome limitations of traditional invasive biopsies for cancer management.
• •Next-generation sequencing enables sensitive characterization of rare ctDNA mutations.
• •Exosomal omics analysis provides insights into cancer progression and therapy response.

Study summary

Liquid biopsies, which analyze circulating cell-free DNA (cfDNA) and exosomes in blood samples, allow minimally invasive monitoring of cancer through simple blood tests. This overcomes limitations of traditional tissue biopsies which are more invasive and difficult to repeat. Many studies have shown that liquid biopsies can be used to detect cancer early, monitor treatment effectiveness, find recurrence, and guide therapy choices. ctDNA reflects the tumor DNA and exosomes contain proteins and genetic material from tumor cells. Advanced technologies can analyze these biomarkers to reveal insights about cancer genetics, spread, and drug sensitivity. Liquid biopsies have demonstrated clinical validity and utility across cancer screening, treatment response monitoring, and residual disease detection. Further research and integration of ctDNA and exosome analysis promises to transform personalized cancer care (see Fig. 1).

Fig. 1.

Pictorial abstract of liquid biopsies. Symbols represent analysis of circulating biomarkers ctDNA and exosomes for applications in early detection, therapeutic monitoring, and residual disease surveillance, enabled by technologies like sequencing and microfluidics. Modular components highlight multifaceted clinical capabilities and ongoing innovations in this emerging paradigm for non-invasive cancer management.

1. Introduction

The advent of liquid biopsies, encompassing analyses of circulating tumor DNA (ctDNA) and exosomes, has catalyzed a paradigm shift in minimally invasive cancer monitoring. This revolutionary approach provides a multifaceted window into tumor biology, treatment response, and disease progression trajectories [1,2]. By harnessing circulating tumor analytes from peripheral bodily fluids, clinicians can glean profound molecular insights without invasive tissue biopsies. The emergence of liquid biopsies has thus heralded a transformative new era in precision oncology [3,4].

The conceptual origins of liquid biopsies trace back to the late 1990s with the discovery of circulating tumor cells (CTCs) in the peripheral blood of cancer patients, providing the first evidence of analytically relevant tumor material in bodily fluids [5,6]. Subsequent research identified cell-free circulating tumor DNA (ctDNA) as another powerful non-invasive analyte for interrogating tumor status and evolution [1,3]. ctDNA comprises short DNA fragments released into circulation through apoptosis, necrosis and secretion of tumor cells [7,8]. The isolation and characterization of exosomes represented another critical advancement - these endosomal-derived extracellular nanovesicles (30–150 nm) mirror parental tumors by carrying cargo like proteins, nucleic acids, lipids, and metabolites [9], [10], [11].

Several circulating protein biomarkers measured from serum or plasma samples are clinically validated and approved for diagnostic, prognostic or therapeutic monitoring applications across specific cancer types [12]. These include prostate-specific antigen (PSA) for prostate cancer screening and CA-125 for monitoring ovarian cancer [13], [14]. The CellSearch assay detecting CTCs received FDA clearance for monitoring metastatic breast, prostate and colorectal cancers [3]. Overall, traditional circulating protein biomarkers and the CTC-based CellSearch platform have sufficient clinical validity to inform standards of care, despite limitations.

Novel liquid biopsy analytes like circulating cell-free DNA (cfDNA), ctDNA, and exosomal cargo have demonstrated strong analytical and clinical validity as cancer biomarkers across detection, diagnosis, prognosis, and treatment response monitoring applications [2,4,15]. Targeted or whole-genome sequencing of ctDNA enables sensitive detection of tumor-associated mutations from blood for non-invasive genotyping, residual disease tracking and early therapy response assessment [16,17]. Exosomal miRNA, lncRNA, proteins, and metabolites constitute complementary biomarkers reflecting functional activity. Substantial retrospective analytic and clinical validity evidence has paved the way for prospective trials and regulatory approvals.

Analyzing ctDNA and exosomes within liquid biopsies provides a multifaceted approach for non-invasively tracking tumor burden, detecting resistance mutations, assessing intratumoral heterogeneity, and monitoring evolutionary dynamics [2,18]. ctDNA permits quantification of tumor burden and identification of genomic alterations, while exosomal cargo offers insights into functional protein and RNA activity [15]. Liquid biopsies enable serial sampling to capture tumor heterogeneity and evolutionary mutational landscapes as they emerge over time and in response to therapeutic pressures [4].

Moreover, liquid biopsies overcome many limitations of traditional invasive biopsies, including spatial and temporal heterogeneity, infeasibility of serial sampling, and risks associated with invasive tissue extraction [3]. By providing a comprehensive snapshot of tumor status, liquid biopsies promise to guide therapeutic decision-making and improve patient outcomes within the framework of precision oncology [1].

This review explores current evidence on the disruptive clinical potential of analyzing ctDNA and exosomes through liquid biopsies. Emerging data underscores their utility for early cancer detection, evaluating treatment responses, and disease monitoring for progression or recurrence to transform the paradigm of clinical cancer management. Liquid biopsies are at the nexus of major technological innovations in molecular oncology, and recent research reinforces their promise as essential components of precision medicine workflows.

2. Clinical impact

Liquid biopsies analyzing ctDNA and exosomes have demonstrated immense clinical promise and utility across the entire continuum of cancer management, right from early detection and diagnosis to assessing minimal residual disease after primary therapy. The minimally invasive liquid biopsy approach facilitates cancer screening, aids prognostication, enables dynamic monitoring of treatment response, and empowers optimization of therapeutic strategies for improved patient outcomes [17,19,20].

2.1. Early detection and diagnosis

Analysis of ctDNA and exosomal molecules allows identification of tumors at the earliest stages, even before clinical manifestations or detectability by imaging modalities [8,15,21]. ctDNA analysis enables detection of hotspot mutations characteristic of pre-malignant or early stage lesions. Cancer-specific genomic alterations can be detectable up to 2 years prior to diagnostic imaging [19,21]. Similarly, distinctive exosomal protein and RNA profiles are detectable during early tumorigenesis and tumor progression [15,20]. Overall, liquid biopsy analysis allows earlier cancer diagnosis an average of 6–9 months earlier than conventional detection strategies, with some studies showing diagnosis 1–2 years earlier [19,22]. Earlier diagnosis has immense clinical value for improving prognosis, providing wider treatment options, reducing invasive cancer morbidity, and enhancing survival outcomes.

2.2. Treatment response monitoring

Serial ctDNA and exosomal analysis during therapy provides dynamic monitoring of biomarker alterations reflective of treatment responses and resistance evolution [2,17,[23], [24], [25], [26]]. Quantification of ctDNA levels tracks tumor burden in response to cytotoxic or targeted therapies [17]. Altered ctDNA levels and emergence of new mutations enable tracking of tumor burden in real-time, and allows selection of optimal therapies [2]. Rise in ctDNA levels precedes radiographic progression by weeks or months, allowing rapid therapeutic changes [27], more so rising ctDNA levels has been shown to precede clinical progression by 2–9 months, allowing early therapeutic modification [28]. Patients demonstrating a decline in ctDNA levels post-treatment have significantly improved outcomes compared to those with persistently elevated ctDNA [29]. Exosomal analysis reveals functional responses to treatment, such as shifting protein levels or RNA expression [26], as well as facilitate identification of therapeutic resistance markers [2,15]. Overall, integrative analyses of ctDNA and exosomes enable precise monitoring of tumor evolution during therapy with associated timely assessment of tumor response kinetics and early detection of resistance to guide appropriate systemic therapy adjustments.

2.3. Minimally invasive disease surveillance

Liquid biopsies facilitate regular post-treatment disease monitoring in a minimally invasive manner compared to repetitive tissue biopsies [30]. Longitudinal analysis of ctDNA permits surveillance of mucosal malignancies and aggressive tumors where serial tissue sampling is challenging. Rise in ctDNA levels predicts disease progression or recurrence months before clinical diagnosis, allowing earlier interventions [29]. Exosomal biomarkers also facilitate disease monitoring through peripheral blood draws instead of biopsies [20]. Thus, liquid biopsies overcome limitations in tissue accessibility and allow surveillance of tumor dynamics via non-invasive blood tests.

2.4. Detection of minimal residual disease (MRD)

Liquid biopsy analyses facilitate ultrasensitive detection of minimal residual disease (MRD) otherwise undetectable even after definitive surgery or systemic therapy, allowing assessment of response and early relapse monitoring [17], [31], [32]. Low pre-operative ctDNA levels correlate with longer disease-free survival and overall survival (OS) [32]. Persistent postoperative ctDNA detection predicts early relapse and decreased OS [32]. Exosome analysis also detects residual disease post-therapy and identifies patients at high risk of recurrence [31]. Analytic sensitivities up to 0.001 % mutant allele fraction empower detection of residual tumor clones with potential implications for recurrence or metastasis [33]. MRD detection aids prognostication and guides optimization of adjuvant therapies or follow-up strategies, especially for aggressive tumors. Early intervention guided by MRD assessment has been associated with improved outcomes [34]. Thus, liquid biopsy analysis of MRD provides early indications of recurrence, optimizes post-treatment surveillance, and proffers a veritable premise for precision cancer medicine.

In summary, as shown in Table 1, liquid biopsies analysis of ctDNA and exosomes comprehensively enhance cancer management - from screening and early diagnosis to molecular disease monitoring and post-treatment disease surveillance. Non-invasive blood tests analyzing ctDNA and exosomes provide actionable dynamic information to guide clinical decision-making and improve patient outcomes.

Table 1.

Summary of recent clinical utility of liquid biopsy in oncology.

Study	Liquid Biomarker	Cancer Type	Key Findings
[21]	ctDNA methylation	Breast cancer	Detected cancer up to 2 years before diagnosis with 100 % specificity
[35]	Exosomal miRNA	Pancreatic cancer	Distinguished early stage pancreatic cancer from controls with 97 % sensitivity
[29]	ctDNA	Colorectal cancer	Detected recurrence 5 months before radiographic relapse on average
[36]	ctDNA	Breast cancer	Identified recurrence with 87 % sensitivity vs 68 % for imaging
[37]	ctDNA	NSCLC	Predicted recurrence with 95 % accuracy vs 18 % for imaging
[38]	Exosomal miRNA	Pancreatic cancer	miR-375 elevation marked MRD and predicted relapse
[39]	Exosomal PD-L1	Melanoma & NSCLC	Early changes predicted immunotherapy response
[40]	ctDNA	Ovarian cancer	Predicted PARP inhibitor response with 100 % sensitivity vs 66 % for CA-125
[41]	ctDNA	Pancreatic cancer	Identified actionable mutations in 65 % patients progressing on therapy

3. Technological advances

Disruptive technologies like next-generation sequencing, digital PCR, microfluidics, and mass spectrometry have enabled robust characterization of circulating nucleic acids, proteins, and vesicles [4]. Ultrasensitive assays allow detection of rare mutant alleles against normal background. Future multiplexing of cfDNA, ctDNA, CTCs, and exosomal analytes in multi-platform workflows promises to derive synergistic insights. Point-of-care microfluidic devices could enable decentralized liquid biopsy analysis [42]. Machine learning integration offers opportunities to unlock latent value from high-dimensional circulating biomarker data. Rapid technological advancements continue to enhance analytic sensitivity, specificity, and clinical utility. Major advances in molecular biology, genomics, microfluidics, and bioinformatics or computational methods have catalyzed the analytical and clinical potential of liquid biopsies by enabling robust characterization of rare ctDNA mutations and exosomal cargo, thus empowering the clinical integration of liquid biopsies [25,43,44].

3.1. ctDNA analysis

3.1.1. Next-generation sequencing transforms ctDNA analysis

Massively parallel next-generation sequencing (NGS) platforms have been transformative for rare tumor mutation detection in cell-free DNA. Illumina sequencing by synthesis technology allows ultra-deep sequencing of cfDNA to detect minimal residual disease and tumor-specific mutations against the vast excess of normal wild-type background [16]. Targeted gene panels permit enrichment and very high coverage sequencing (10,000x or greater depth) across curated cancer gene sets of 50–500 genes. This facilitates longitudinal tracking of known hotspot mutations, actionable drivers, and treatment response markers [25].

Moreover, unbiased whole exome or whole genome sequencing unpack the entire landscape of coding and non-coding alterations. This includes single nucleotide variants (SNVs), insertions/deletions, copy number alterations (CNAs), chromosomal rearrangements, gene fusions, and epigenetic changes, enabling comprehensive liquid biopsy profiling [45]. Combining ctDNA sequencing with CTC transcriptomic analysis could provide further synergistic insights across analytes [46].

3.1.2. Ultra-deep sequencing unlocks minimal residual disease detection

Unique molecular identifiers (UMIs) enable tagging and tracing of individual DNA molecules, allowing error correction and ultra-sensitive rare mutation detection down to 0.001 % allele fractions [17]. By digitally counting single mutant molecules, UMIs facilitate reliable detection of minimal residual disease otherwise undetectable by traditional sequencing. This level of sensitivity promises substantial clinical utility in post-treatment disease surveillance and early relapse detection. Ultra-deep sequencing technologies continue to push ctDNA analysis to unprecedented limits of detection that redefine standards for precision oncology care.

3.1.3. Digital PCR improves absolute mutation quantification

Complementary to sequencing, digital PCR platforms like droplet digital PCR enable exquisite sensitivity and absolute quantification of rare mutations in liquid biopsy samples independent of reaction efficiency [47]. Water-oil emulsion droplet systems partition cfDNA into millions of individual reactions for amplification and allele-specific fluorescent probe detection. This provides precise mutant allele fraction quantification down to 0.001 % ideal for serial ctDNA monitoring. Cost-effectiveness improvements will be key for broader clinical implementation.

Overall, spectacular technological advances ranging from ultra-deep sequencing to digital PCR have catalyzed the analytical capabilities of ctDNA liquid biopsy, providing powerful tools for unlocking unprecedented clinical insights into cancer genomes.

3.2. Exosome analysis

3.2.1. Optimized isolation methods enable reliable exosome extraction

Major progress has been made in standardized isolation and purification of high-quality exosomes from biofluids like plasma, urine or CSF. Ultracentrifugation at 100,000×g remains the gold standard isolation technique, leveraging differential buoyant densities to pellet exosomes away from soluble proteins and protein aggregates [43,44]. Density gradients allow fractionation of exosome sub-populations. Alternatively, immunoaffinity capture via microfluidic chips coated with anti-CD63/CD81/CD9 antibodies enables rapid isolation of exosome subsets [48]. Optimization of pre-analytical variables is critical to prevent artifacts and ensure downstream analytic reliability. Overall, advances in isolation workflows now permit efficient, consistent extraction of exosomes for multi-platform characterization.

3.2.2. Next-generation sequencing reveals exosomal genetic landscapes

RNA sequencing has enabled high-resolution profiling of exosomal transcriptomic contents, including tumor-specific mutations, gene fusions, splicing alterations, long non-coding RNAs and microRNA signatures reflective of parental tumor cells [49]. Such analyses provide functional insights into expressed cancer drivers, dysregulated pathways, and therapeutic response biomarkers missed by ctDNA genotyping alone. Longitudinal exoRNA sequencing could elucidate tumor evolution and subclonal architecture over time. Future multi-omics integration with exosomal proteome, lipidome and metabolome data will provide comprehensive insight.

3.2.3. Mass spectrometry defines the exosomal protein cargo

Shotgun proteomics via high-resolution mass spectrometry now facilitates in-depth quantification of exosomal proteome to extract diagnostic protein biomarkers like Glypican-1 while also unraveling prognostic predictors and networks dysregulated in tumorigenesis [15]. Further integration with tissue and serum proteomics enhances the biological insights from exosomal protein signatures ([39]. Surface proteins may also be leveraged as targets for therapeutic delivery or immunotherapy modulation [43,44]. Technological refinements continue to enhance the resolution and dimensionality of exosomal protein mapping through ultra-high throughput, multiplexing, and systems-wide analysis.

3.2.4. Microfluidics and computational analytics streamline exosome analysis

Microfluidic lab-on-chip devices like ExoChip enable rapid, low volume isolation and on-chip characterization of exosomal protein and nucleic acid biomarkers, facilitating integration into decentralized point-of-care testing workflows [42]. Downstream machine learning algorithms integrate multi-omics exosomal data to derive biological insights and predictive models guiding clinical decisions [43,44]. Computational analysis will be imperative to fully extrapolate clinically meaningful information from high-dimensional exosomal profiling.

Thus, major strides in isolation, mass spectrometry, sequencing, microfluidics, and analytics have enhanced exosome analysis to unlock immense biomarker potential and provide indispensable complements to ctDNA characterization for precision medicine.

As summarized in Table 2, transformative genomic, microfluidic, and computational approaches have enabled in-depth molecular analysis and comprehensive characterization of ctDNA and exosomes to uncover the intricate molecular landscape of tumors. Technological innovations continue to improve analytic validity and clinical utility, driving adoption of liquid biopsies in precision oncology workflows and clinical cancer management.

Table 2.

Key technological advances enabling ctDNA and exosomal analysis from liquid biopsies.

Technology	Description	Applications	Study
Next-Generation Sequencing	Massively parallel sequencing platforms (e.g. Illumina)	Targeted gene panels, whole exome/genome sequencing for variant detection	[16,25]
Digital PCR	Absolute quantification of rare mutations (e.g. ddPCR)	Sensitive detection of minimal residual disease	[47]
Ultracentrifugation	High-speed centrifugation for exosome isolation	Extraction of exosomes from biofluids	[50]
Density gradients	Separation of exosomes based on buoyant densities	Isolation of high-purity exosomal fractions	[50]
Immunoaffinity capture	Microfluidic isolation using antibodies against exosomal proteins	Efficient exosome capture from minute volumes	[50]
RNA sequencing	Profiling of exosomal transcriptomic content	Identification of expressed mutations, fusions, expression patterns	[51]
Proteomics	Mass spectrometry profiling of exosomal proteins	Mapping of exosomal proteome, surface proteins as biomarkers	[15]
Microfluidic chips	Miniaturized platforms for integrated exosomal processing	Rapid, low-volume exosome isolation and characterization	[42]
Computational analytics	Algorithms and machine learning for multi-omics data	Derivation of biological insights from exosomal analysis	[50]

4. Summarized timeline of milestones in liquid biopsies for minimally invasive monitoring of cancer

4.1. The origins of liquid biopsy: circulating tumor cells

The conceptual origins of liquid biopsies trace back to the pivotal discovery of circulating tumor cells (CTCs) in the 1990s, providing the first evidence that analytically relevant biomarkers could be isolated from the peripheral blood of cancer patients [5]. Using immunomagnetic enrichment and immunofluorescence microscopy, pioneering studies by Allard et al. demonstrated viable CTCs in the bloodstream of metastatic cancer patients across solid tumor types including prostate, lung and breast cancers [6]. This finding opened the door to minimally invasive "liquid biopsies", overcoming the limitations of traditional invasive tumor sampling. Subsequent research built upon CTC analysis, spurring technological innovations to isolate and molecularly characterize other circulating analytes.

4.2. Cell-free tumor DNA analysis takes center stage

In the early 2000s, the identification of cell-free circulating tumor DNA (ctDNA) fragments in plasma represented a major breakthrough in liquid biopsy analysis [1]. Anker et al. first reported the detection of tumor-associated mutations in cancer patient blood, laying the groundwork for development of "liquid biopsy" approaches [52]. This revelation led to considerable research on ctDNA over the next decade. Heitzer et al. provided key mechanistic insights, demonstrating the apoptotic and necrotic origins of truncated, double-stranded ctDNA released into circulation by tumors [7]. Soon after, advances in next-generation sequencing and digital PCR enabled the sensitive detection and molecular analysis of ctDNA, unlocking utility for non-invasive monitoring of tumor burden and guiding targeted therapy selection through identification of clinically actionable genomic alterations [25], [53], [54].

4.3. The discovery of exosomes completes the picture

In parallel to ctDNA, another major breakthrough was the discovery of exosomes – nanovesicles released by cells that encapsulated molecular cargo like proteins, lipids and nucleic acids that mirrored parental cells, including tumor cells [9], [11]. Profiling exosomes via proteomics or transcriptomics could provide insights into functional protein and RNA activity. This revelation identified exosomes as yet another circulating biomarker analyte to complete the composite picture of liquid biopsies [15]. Subsequent optimization of exosome isolation and molecular characterization further revealed the immense clinical potential of analyzing exosomal contents to elucidate tumor biology and responses [43,44].

4.4. Translating liquid biopsies into clinical practice

By the late 2010s, substantial evidence on the disruptive potential of ctDNA and exosomal analysis as circulating biomarkers catalyzed efforts to translate liquid biopsies into clinical practice [8,55]. Liquid biopsy tests began integration into clinical trials across cancer applications such as early screening, monitoring therapeutic response, detecting minimal residual disease, and selecting targeted therapy [17,19,41]. In 2018, the FDA granted the first tissue-agnostic drug approval for larotrectinib, a TRK inhibitor, on the basis of NTRK gene fusions detected via liquid biopsy [56]. The stage was set for liquid biopsies to transition from academic curiosity to clinical standard-of-care.

5. Emerging microfluidic and nanotechnology approaches for enhanced liquid biopsy analysis

Ongoing technological innovations continue to unlock the immense potential of liquid biopsies by enhancing the analytical capabilities for ctDNA and exosome characterization. Recent advances in microfluidic techniques have enabled rapid and ultrasensitive analysis of ctDNA and exosomes from small volume liquid biopsies. For instance, a magnetofluidic immuno-PCR platform allowed highly accurate COVID-19 antibody testing from just 1 μL saliva samples, demonstrating clinical readiness for decentralized diagnostics [48,57]. Additionally, novel microfluidic devices integrating one-step RNA extraction and digital PCR facilitated sensitive detection of EGFR mutations in ctDNA for non-invasive lung cancer screening [58].

For ovarian cancer screening, analysis of ctDNA mutations have been shown to outperform conventional protein biomarkers like CA-125, with the potential to achieve greater diagnostic accuracy and sensitivity even at early stages [59]. Integration of ctDNA analysis with CA-125 could enhance ovarian cancer detection from liquid biopsies. Further, emerging microfluidic platforms enable epigenetic profiling of ctDNA through digital melt analysis and methylation-specific PCR, providing additional layers of molecular information [60,61].

Nanotechnology advancements have also augmented exosome isolation and analysis. For instance, an integrated rotating micromagnet array has been developed to enable rapid, label-free exosome capture, while iron oxide nanoparticle probes have been used to facilitate exosome quantification via magnetic susceptibility changes [62]. In other studies, plasmonic gold nanoparticles have enabled surface-enhanced Raman spectroscopy analysis of exosomal contents [63].

In summary, disruptive microfluidic, nanotechnology and analytical approaches continue to expand the frontiers of liquid biopsy analysis. By enabling comprehensive and ultrasensitive ctDNA and exosomal characterization from minuscule volumes, these advances promise to augment the clinical utility of liquid biopsies for early cancer detection, diagnosis, minimal residual disease monitoring, and guiding therapeutic decisions. Continued innovation in this domain will be integral for establishing liquid biopsies as standard-of-care in clinical oncology.

6. Accruing evidence supporting liquid biopsies as clinical standard-of-care in oncology

Extensive research over the past decade continues to support the clinical validity and utility of liquid biopsy analysis of ctDNA and exosomes across the spectrum of cancer management.

6.1. Cancer screening and early detection

ctDNA methylation biomarkers detects breast cancer up to 2 years prior to clinical diagnosis with 100 % specificity [21]. Analysis of serum exosomes via microRNA panels distinguishes patients with early-stage pancreatic cancer from healthy controls with 97 % sensitivity [35]. Exosomal lncRNAs shows the ability to detect early-stage colorectal cancer with 87 % sensitivity and 93 % specificity [64].

6.2. Disease monitoring and recurrence detection

In stage II/III lung cancer patients post-surgery, ctDNA-based detection of relapse was shown to precede radiographic relapse by an average of 5 months, enabling timely interventions [65]. ctDNA analysis detected recurrence with a sensitivity of 87 % vs 68 % for imaging in breast cancer, allowing earlier treatment initiation [36]. In NSCLC patients, elevated ctDNA levels post-resection predicted recurrence with 95 % accuracy vs 18 % for imaging [37]. Elevated ctDNA after curative lung cancer resection was associated with >90 % recurrence rate within 1 year [37]. Exosomal miRNA models could detect pancreatic cancer with 97 % sensitivity and differentiate malignant from benign disease [35]. Plasma exosomal miRNAs distinguished localized and metastatic prostate cancer with 95 % accuracy [66]. In advanced ovarian cancer, longitudinal ctDNA tracking detected recurrence an average of 7.9 months before CA-125 or imaging [19].

6.3. Detecting minimal residual disease

Ultrasensitive ctDNA analysis detected residual disease in >50 % breast cancer patients deemed disease-free by conventional methods [36]. In stage II colon cancer patients, postoperative ctDNA detection was prognostic of recurrence within 1 year [29]. Exosome analysis identified miR-375 elevation as a marker of minimal residual disease in pancreatic cancer [67].

6.4. Therapy response monitoring

In EGFR-mutant NSCLC patients on erlotinib, emergence of resistance mutations in ctDNA preceded progression by 2.2–5.2 months compared to CT scans [68]. Early changes in exosomal PD-L1 levels predicted treatment response to anti-PD-1 immunotherapy in melanoma [69]. Exosomal PD-L1 dynamics on immunotherapy correlated with radiographic response in NSCLC patients [70]. CtDNA analysis predicted response to PARP inhibition in ovarian cancer patients with 100 % sensitivity vs 66 % for CA-125 [40]. In metastatic breast cancer patients on palbociclib, changes in ctDNA predicted progression ∼2 months before CT scans [71]. In metastatic castration-resistant prostate cancer (mCRPC) patients, declines in AR-V7 splice variant in CTCs/ctDNA predicted better responses to taxane therapy [72].

6.5. Guiding targeted therapy

ctDNA sequencing identified actionable mutations in 65 % mCRPC patients progressing on standard therapy, enabling personalized recommendations [41]. ctDNA analysis detected emerged resistance mutations informing therapeutic decisions in 32 % of EGFR NSCLC patients progressing on osimertinib [73]. Exosomal RNA analysis revealed activation of PI3K/AKT/mTOR pathway in refractory disease guiding treatment decisions [74]. Plasma exosomal RNA analysis identified actionable fusions in ALK/ROS1/NTRK negative NSCLC patients [75]. Exosomal lncRNAs revealed activation of immunomodulatory pathways in melanoma guiding use of anti-PD-1 therapy [76].

A recent review highlighted the potential of ctDNA and exosomal analysis in precision oncology and tracking tumor evolution over time [1]. A rapidly growing body of evidence underscores the clinical utility of liquid biopsy analysis of ctDNA and exosomes to transform cancer management and enable personalized/precision medicine approaches.

7. Contemporary liquid biopsy-based precision oncology approaches

7.1. Multi-analyte approaches for comprehensive liquid biopsy profiling

While ctDNA and exosomes have substantial clinical utility, combining multiple circulating biomarkers may enable more comprehensive liquid biopsy profiling than any single analyte [77]. CTCs provide complementary information on intact tumor cells, while emerging analytes like tumor-educated platelets uniquely reveal insights into tumor microenvironment interactions [58,78]. Intelligent integration of ctDNA, exosomes, CTCs and platelets could harness synergistic molecular information towards holistic characterization of multifaceted tumor complexity. For instance, concordant TP53 mutations between ctDNA and CTCs improves detection specificity in metastatic breast cancer [79]. Future adoption of multi-analyte workflows promises to advance liquid biopsies beyond piecemeal insights.

7.2. Machine learning-assisted extraction of insights from multi-omics data

Machine learning offers enormous potential for integrating ctDNA, exosomal, radiomic, and other omics data to inform clinical decision-making [63]. Deep learning models enable heterogeneous data fusion for enhanced diagnostic, prognostic and predictive modeling. Federated learning allows collaborative multi-site model development without data sharing, accelerating real-world evidence generation [80]. Reinforcement learning agents could continually optimize models and adapt treatment recommendations as new liquid biopsy data accrues over the clinical course. Overall, machine learning promises to unlock the immense latent value in multi-dimensional liquid biopsy data.

7.3. Microfluidic and paper-based platforms for decentralized testing

Point-of-care microfluidic devices allow decentralized liquid biopsy analysis and personalized therapy selection at the bedside [48,57]. Paper-based assays further facilitate accessible community-based testing. For instance, a nanowire microfluidic platform could detect EGFR mutations in 15 min from 1 ml plasma [81]. Wearable sensors also enable continuous monitoring through non-invasive sweat-based liquid biopsy [82]. Democratizing liquid biopsy analytics through decentralized, low-cost technologies can help expand access and real-time precision guidance.

7.4. Hybrid capture approaches for mutation detection

Hybrid capture combining targeted gene panels (100–200 genes) with whole exome sequencing enables focused interrogation of known actionable drivers alongside discovery of novel mutations from scarce liquid biopsy specimens [83]. This balances assay sensitivity for established biomarkers with unbiased genome-wide analysis. Highly multiplexed panels using barcoded probes facilitate enrichment for improved variant calling [84]. Hybrid targeted-whole exome NGS promises to maximize information recovery from limited ctDNA.

7.5. Epigenetic profiling of ctDNA for precision medicine

Novel microfluidic and nanotechnology platforms facilitate epigenetic profiling of methylation, nucleosome positioning, and histone modifications in plasma ctDNA [60,85]. For example, quantum dot barcodes enable digital melt analysis for quantitative hypermethylation detection [60]. Epigenetic ctDNA signatures provide complementary information to mutations for early diagnosis, therapeutic decisions and relapse monitoring [86]. Integrating genetic and epigenetic profiling will provide more comprehensive ctDNA liquid biopsy characterization.

7.6. Exploring functional roles beyond diagnostic biomarkers

Prospective functional roles of ctDNA and exosomes beyond non-invasive biomarkers warrant greater exploration [43,44,87]. Exosomes could deliver encapsulated drugs or RNA therapeutics, while native tumor-derived exosomes influence immunotherapy response [26,48,57]. ctDNA could trigger anti-tumor immunity or serve as an immunotherapeutic target [88]. Elucidating such mechanistic roles through rigorous in vivo studies is key to translating ctDNA and exosomes into actionable clinical realities as therapeutic agents.

8. Major clinical studies evaluating liquid biopsies

Liquid biopsy represents an exciting frontier in cancer management, with circulating biomarkers offering invaluable insights into tumor genetics, residual disease, molecular evolution, and early relapse. Extensive clinical studies are underway to evaluate circulating biomarkers including cfDNA, ctDNA, exosomes and multi-analyte liquid biopsies across cancer screening, diagnosis, and management applications [8]. As shown in Table 3, ongoing clinical trials continue to define optimal integration of liquid biopsy into clinical practice.

Table 3.

Summary of major liquid biopsy clinical trials.

Trial	BioFluid	Circulating Biomarker	Cancer Type	Treatment/Intervention	Endpoints	ClinicalTrial.gov ID
GRETeL	Plasma	ctDNA/cfDNA	Glioblastoma	Standard chemo/radiation	Outcomes association	NCT05695976
SIBYL	Plasma	ctDNA	Breast, Colorectal, NSCLC	Systemic therapy	Response assessment	NCT05935384
PERCEIVE-I	Plasma	cfDNA/ctDNA/miRNA	Gynecologic	None	Early detection	NCT04903665
HCCGenePanel	Plasma	ctDNA	HCC	Immunotherapy/targeted therapy	Genomic profiling, therapy selection	NCT04111029
ExoLuminate	Plasma	Exosome/Extracellular vesicles	Pancreatic	None	Early detection	NCT05625529
PEDALB	CSF	cfDNA	Pediatric brain tumors	None	Diagnosis, monitoring, genetics	NCT05934630
LAP-GC	Plasma	CTCs/ctDNA/cfDNA	Gastric	Neoadjuvant chemotherapy	Response monitoring	NCT03957564
GEDI	Plasma	Proteins/cfDNA/ctDNA/RNA	Gastric	HER2-targeted therapy	Response to targeted therapy, Prognosis	NCT02610218
CCGA	Plasma	cfDNA	Multi-cancer	None	Prognosis for early detection	NCT02889978
IMPROVE	Plasma	ctDNA	Colorectal	Post-surgical standard care	Treatment optimization	NCT03637686
MEDOCC-CrEATE	Plasma	ctDNA	Colon	Adjuvant chemotherapy	Treatment decisions, outcomes	NCT05288897
B-FAST	Plasma	ctDNA	NSCLC	Targeted therapy	Mutation detection, therapy selection	NCT03178552
TIGER-X	Plasma	ctDNA	NSCLC	EGFR inhibitor	Early relapse detection	NCT01526928
BESPOKE CRC	Plasma	ctDNA	Colorectal	Adjuvant chemotherapy	Optimization, early relapse detection	NCT04264702

∗ClinicalTrials.gov. (n.d.). Retrieved from https://clinicaltrials.gov/.

The GRETeL trial (NCT05695976) evaluates whether ctDNA or cfDNA dynamics correlate with glioblastoma outcomes and immune suppression. The SIBYL trial (NCT05935384) generates validity data on using Guardant360 ctDNA assays to measure therapeutic response of unresectable advanced solid tumors. A gynecological cancer study (NCT04903665) aims to validate combined methylation, mutation, and miRNA assays for early detection. Another trial (NCT04111029) examines ctDNA for monitoring hepatocellular carcinoma and selecting targeted therapy. Researchers are also evaluating integrated assays to enable pancreatic cancer screening (NCT05625529). Detecting cerebrospinal fluid cfDNA could facilitate non-invasive monitoring in pediatric brain tumors (NCT05934630). Studies are also examining liquid biopsy for gastric cancer management (NCT03957564, NCT02610218).

The CCGA trial (NCT02889978) revealed a cfDNA methylation sequencing assay for multi-cancer early detection [89]. Cancer cases not detected by the cfDNA test had significantly better 3-year overall survival compared to those detected, even after adjusting for clinical stage and diagnosis method. CfDNA test-negative cancers also exceeded expected survival from SEER data. Additionally, the test differentiated more aggressive from less aggressive malignancies within screen-detectable types like breast and colorectal cancer. The IMPROVE trial (NCT03637686) investigates the benefit of ctDNA-guided postoperative surveillance in patients with Union for International Cancer Control (UICC) stage II or III CRC, with the aim of optimizing treatment in early-stage CRC [90]. The MEDOCC-CrEATE trial (N = 1320) examines willingness for ctDNA-directed adjuvant chemotherapy in patients with stage II colon cancer with the ultimate aim of reducing the risk of relapse in a high-risk population [91]. The B-FAST trial (NCT03178552) showed blood-based NGS accurately identified actionable mutations in unresectable, advanced NSCLC, achieving high response rates to Alectinib; Alectinib treatment based on blood ctDNA identification of ALK rearrangements achieved an impressive durable response rate of 87.4 %, with 75.9 % 12-month response duration [92]. The TIGER-X trial (NCT01526928) of rociletinib in previously-treated patients with EGFR mutant-positive advanced NSCLC, is evaluating longitudinal tracking of EGFR activating and resistance mutations in ctDNA via digital PCR during treatment to assess molecular residual disease and early relapse in NSCLC [93]. Reduced EGFR variant allele fractions in ctDNA predict treatment response, while increased levels indicate progression 5–6 months prior to imaging.

The BESPOKE trial (NCT04264702) assesses integrating postoperative ctDNA analysis to guide CRC adjuvant treatment and enable early relapse detection [94]. Deep learning models also show promise to leverage multi-analyte ctDNA, exosomal, protein and metabolite patterns for screening and early detection [95]. Overall, ongoing studies continue integrating cutting-edge technical capabilities continue to evaluate circulating biomarkers across the entirety of precision oncology pathways, and to optimize integration of liquid biopsy into clinical practice to guide treatment and improve cancer outcomes through sensitive monitoring of molecular tumor dynamics. Standardization of isolation protocols and analytic interpretation is still needed. Multi-platform liquid biopsy analysis promises to unlock synergistic insights, provided rigorous validation and standardization establishes firm clinical validity and utility.

9. Critical appraisal of key controversies and challenges surrounding exosomes and ctDNA for liquid biopsies in precision cancer medicine

Liquid biopsies analyzing circulating tumor DNA (ctDNA) and exosomes have shown immense potential for enabling non-invasive cancer monitoring and guiding precision therapy [4,15]. However, several controversies remain regarding their clinical validity, utility, and adoption into widespread oncology practice.

9.1. Analytic validity

A major ongoing challenge hampering the clinical validity of liquid biopsies is the lack of standardization and variability in pre-analytical protocols as well as isolation methods for extracting ctDNA and exosomes from biofluids. Differences in sample collection tubes, processing intervals, storage conditions, and isolation techniques can substantially impact the quantity and quality of extracted biomarkers [8]. This introduces biases and discrepancies in downstream molecular characterization. For instance, lengthy processing delays can induce artifactual mutations through DNA damage while variability in exosome isolation platforms significantly alters expression profiles [43,44,96]. Ultracentrifugation, density gradients, and immunoaffinity capture each enrich different exosome sub-populations. Such technical factors confound analysis, interpretation, and cross-validation of biomarker findings across different laboratories.

Moreover, assay sensitivity remains a key concern surrounding rare tumor-derived ctDNA and exosomes that typically represent less than 1 % of total cell-free DNA and extracellular vesicles in biofluids [16,43,44]. Detecting minority mutations against the vast excess of normal background requires highly optimized assays. While ultra-deep next generation sequencing has aimed to improve detection down to 0.01 % allele fractions, reproducibility and concordance of such findings across labs and techniques remains poor [17]. Significant heterogeneity still exists between analyzer platforms, bioinformatics pipelines, coverage depth, and variant calling algorithms leading to discrepant results [97]. Lack of integration with standardized controls and spike-in references further impedes reliable detection of minimal residual disease. Overall, overcoming fundamental challenges in pre-analytical and analytical optimization will be critical to establish the clinical validity of liquid biopsy tests.

9.2. Clinical utility

Despite encouraging evidence, the clinical utility of integrating ctDNA or exosomal analysis into precision oncology workflows remains controversial. While studies have demonstrated ability to detect mutations or expression changes, large-scale randomized controlled trials firmly establishing improved clinical outcomes through guidance of therapeutic decisions are severely limited [8,20]. Most research has focused on analytic and clinical validity, while impact on overall survival, quality of life, and health economics remains sparsely explored. Additionally, utility has been examined in narrow subpopulations and academic centers, while real-world evidence across diverse settings is lacking [8,98].

Another key controversy is that upfront infrastructure requirements, costs of biomarker testing, and ensuing clinical actions may outweigh benefits [19]. For example, while detecting actionable mutations earlier through ctDNA sequencing could enable timely therapy adjustments, expenses of serial testing must be weighed against survival gains. Similarly, utility of spared radiographic imaging must account for liquid biopsy costs. Scalable health economic models and data across the clinical continuum in heterogeneous populations are imperative prior to widespread adoption in standard oncology practice. Beyond clinical validity, demonstrating real-world clinical utility and value will be essential to drive payer coverage and clinician buy-in for disruptive integration of liquid biopsy approaches.

9.3. Therapeutic utility

The utility of ctDNA and exosomes extends beyond diagnostic and prognostic biomarker applications. However, their potential role as therapeutic targets or vectors for drug delivery remains fraught with controversies surrounding lack of putative preclinical and clinical evidence.

Most ctDNA and exosomal research has focused on elucidating their biology and demonstrating biomarker validity for non-invasive monitoring [15,16]. In contrast, exploration of therapeutic functions has lagged significantly behind. Early in vitro studies suggest exosomes could deliver encapsulated drugs or siRNAs to tumors [48,57]. However, translation to in vivo efficacy remains elementary with huge challenges in targeting specificity and cargo optimization [43]. While native tumor-derived exosomes could theoretically influence immunotherapy response, manipulation for therapeutic modulation has solely been hypothetical so far [57].

Similarly, prospective mechanistic roles of ctDNA include triggering anti-tumor immunity or serving as an immunotherapeutic target [47]. But beyond correlative immune associations, direct in vivo evidence is lacking. Demonstrating safety and efficacy in preclinical models will be imperative before clinical advancement. Compared to the well-established merits as biomarkers, the therapeutic utility of ctDNA and exosomes remains nebulous and controversial warranting extensive investigation before becoming actionable realities. Overall, realizing clinical applications beyond liquid biopsy diagnostics hinges on explicating functional roles of ctDNA and exosomes through rigorous in vivo experimentation in the future.

9.4. Analyte limitations

Both ctDNA and exosomes suffer from intrinsic limitations in comprehensively capturing the multifaceted complexity of tumors. Neither analyte fully represents the extensive heterogeneity, evolutionary dynamics, and microenvironment interactions within tumors [99,100]. ctDNA provides a snapshot of a subset of mutations, while exosomes reflect the biology of cells releasing them. Spatial subclonal variations, epigenetic dysregulation, and stromal components are poorly profiled.

Emerging evidence suggests combining ctDNA, CTCs, and exosomes could enable more holistic profiling by integrating complementary molecular information ([77];[57], [87]. For example, concordant TP53 mutations between ctDNA and CTCs improves detection specificity in metastatic breast cancer [79]. Additionally, tumor-educated platelets expressing cancer-associated RNA and proteins may reveal insights into the tumor microenvironment [78]. Intelligent machine learning frameworks integrating ctDNA, exosomal data, and radiomic features from medical imaging could further enhance diagnostic, predictive, and prognostic modeling [43,44,59].

Overall, while ctDNA and exosomes have substantial utility as liquid biopsy analytes, combining multiple circulating biomarkers and leveraging computational integration promises more comprehensive liquid profiling than either single analyte. A multi-platform approach harnessing synergistic strengths of diverse analytes is imperative to capture the multifaceted interconnected complexities of cancer biology.

9.5. Regulatory hurdles

The lack of liquid biopsy tests approved by regulatory agencies like the FDA reinforces existing gaps in validating clinical utility beyond analytic validity [8]. Thus far, the FDA has only granted one tissue-agnostic indication for larotrectinib in 2018 based on presence of NTRK fusions detected via liquid biopsy [56]. This highlights the extensive regulatory hurdles still facing commercial development of ctDNA and exosome tests, which face challenges through lengthy, expensive approval pathways.

Manufacturers have to demonstrate robust analytic validity, definitive clinical validity linking biomarker findings to health outcomes, and sufficient real-world clinical utility changing patient management to justify clinical adoption [101]. Ambiguities exist around validating mutations with unclear pathological or therapeutic significance. Regulators recommend co-development of targeted therapies alongside companion diagnostics to accelerate liquid biopsy approvals. However, personalized assay platforms are difficult to standardize into mass-market products. Post-market surveillance studies are also required to continually gather ongoing evidence.

Additionally, clinician education is imperative regarding appropriate interpretation and integration of liquid biopsy findings into clinical practice [8]. Misuse or overinterpretation of ctDNA and exosomal data could lead to overtreatment, inappropriate change in therapy, or worse outcomes. Multidisciplinary input alongside test results is vital to enhance decision-making. Overcoming regulatory and clinical implementation hurdles through ongoing interdisciplinary research, clinical trials, and accrual of real-world evidence will be key to unlock the potential of disruptive liquid biopsy integration into precision oncology care in the future.

9.6. Health economics

Another key barrier impeding integration of liquid biopsies into clinical practice is lack of health economic data demonstrating value to support payer coverage and clinician adoption [8]. Beyond just clinical validity, liquid biopsy tests must show real-world clinical utility that improves patient outcomes in a cost-effective manner.

Economic models must account for upfront costs of establishing predictive biomarker infrastructure, expenses associated with serial ctDNA/exosomal testing, and ensuing downstream costs like additional imaging or follow-up visits prompted by results [19]. These costs must be weighed against benefits like spared imaging from earlier response assessment, avoiding ineffective therapies, or improving survival through early intervention. Test access, adherence, clinician behavior changes, and heterogeneity across settings should be incorporated.

Cost-effectiveness data across the continuum from screening to minimal residual disease monitoring is currently sparse. Insurers like Medicare have limited reimbursement largely to single gene companion diagnostics. Scalable economic models and multicenter data are imperative to justify clinical adoption and expanded coverage. Demonstrating health economics value centering patient benefits will be key to disruptive liquid biopsy integration into oncology practice.

9.7. Critical needs for standardization

The reliability and consistency of liquid biopsy testing across laboratories remains a major concern hampering clinical validity and utility [8]. Substantial variations exist in pre-analytical protocols, analytical methods, and post-analytical interpretation practices that introduce discrepancies and confound validation efforts [34].

Standardizing and controlling pre-analytical variables like sample collection tubes, processing intervals, and storage conditions is essential to prevent artifacts and ensure isolated ctDNA/exosomes reflect true tumor-derived profiles. Consensus on analytical techniques for extraction, sequencing, mutation calling, and computational analysis is imperative to reduce technical biases and improve reproducibility. Post-analytical standardization of clinical interpretation and reporting practices is also needed to avoid misuse or misrepresentation of test results that could lead to patient harm through over-treatment or inappropriate changes in therapy.

Globally adopted standard operating procedures surrounding liquid biopsies are urgently required to address these standardization gaps and inconsistencies that currently impede validation, regulation, and widespread clinical implementation. Multi-disciplinary efforts by researchers, regulators, diagnostics companies and clinics will be key to establish robust consensus standards and guidelines to enhance the reliability of liquid biopsy testing.

Overall, while ctDNA and exosomal analysis shows immense potential to transform non-invasive cancer management, overcoming these challenges will be key to unlocking their full disruptive impact and establishing liquid biopsies as standard of care in precision oncology [98]. Extensive research already underway in analytic optimization, multi-center utility studies, and machine learning integration promises to help conquer existing barriers to clinical adoption.

10. Expert consensus and recommendations on clinical integration of liquid biopsies

Professional societies and thought leaders have issued recommendations guiding appropriate clinical adoption of liquid biopsy analysis of cfDNA, exosomes and other circulating biomarkers in oncology [8,55,102]. The joint consensus guideline by the American Society of Clinical Oncology (ASCO) and College of American Pathologists (CAP) provides a comprehensive framework encompassing analytic validity, clinical validity, clinical utility and ethical considerations [8]. Rigorous evidence demonstrating improved decision-making and patient outcomes from liquid biopsy testing is emphasized, beyond just technical accuracy. For advanced cancers, targeted Next-generation sequencing of cfDNA is endorsed to identify actionable mutations and guide targeted therapy selection when tissue re-biopsy is infeasible [101]. However, guidelines emphasize that cfDNA testing should complement rather than replace standard tissue biopsy. Multidisciplinary interpretation integrating cfDNA, imaging and clinical findings is advised to enhance sensitivity and specificity [8].

Many experts underscore the high clinical utility of longitudinal cfDNA analysis for monitoring response to systemic therapy based on serial quantification of variant allele fractions [17,65]. Rising cfDNA levels predict progression 2–6 months prior to radiological detection across cancer types, allowing early therapy switches [27]. Beyond overt progression, ultrasensitive cfDNA profiling facilitates surveillance for molecular residual disease following curative therapy, enabling adjuvant treatment intensification when indicated [103]. However, experts caution against over-interpreting isolated cfDNA elevations amidst high background noise and emphasize combining molecular and radiographic insights [8]. Leveraging advanced cfDNA sequencing for early cancer detection has elicited divergent expert opinions, with some touting potential benefits while others urging caution about analytic validity and clinical utility [19,104]. Advocates highlight 2+ year lead time from ctDNA detection till clinical diagnosis within prospective trials, enabling early interventions and better prognosis in high-risk groups [21]. However, critics argue trial populations are enriched versus real-world screening cohorts. They advocate further technological refinements to improve assay specificity before population-level implementation [105]. Overall, expert consensus supports investigational development but urges rigorous evidence generation encompassing diverse populations before endorsing early cancer screening [8].

Thought leaders strongly recommend combining orthogonal circulating biomarkers like circulating tumor cells (CTCs), exosomes, proteins alongside cfDNA to derive synergistic insights and enable holistic liquid profiling of cancers [4,106]. Each analyte provides unique and complementary information on tumor status. Consolidated analyses harness multidimensional insights into morphology, functional biology, microenvironment, and genomic landscape [107]. Computational integration platforms leverage machine learning to assimilate biomarker inputs for enhanced diagnostic and predictive modeling [108]. However, analytical and clinical validity must be demonstrated for novel integrated classifiers before clinical adoption [8].

Many experts highlight the need to establish defined contexts where liquid biopsy-guided clinical decision-making improves meaningful outcomes versus standard-of-care [8,102]. While technical and diagnostic accuracy are necessary prerequisites, sufficient evidence linking test results to improved clinical actions and patient benefits is imperative to demonstrate utility. Health economic models must weigh upfront costs of serial cfDNA testing against deferred downstream expenses from earlier response assessment and therapy adjustments [109,110]. Larger collaborative trials across the patient journey are urged to generate real-world evidence beyond demonstration projects at specialized centers. There is strong advocacy for developing and adhering to standardized pre-analytical protocols for sample acquisition, handling, processing and storage prior to cfDNA isolation and analysis [111,112]. Controlling these variables is essential to ensure derivation of high-quality representative cfDNA and analytic reproducibility. Consensus guidelines recommend immediate centrifugation and freezing of plasma, use of specialized collection tubes, nuclease inhibitors, cold chain logistics, and nucleic acid stabilization buffers [112]. Global harmonization of protocols promises to enhance result consistency and clinical validity across laboratory sites.

Many experts advise integrating analysis of clinically actionable somatic hotspot mutations rather than large gene panels with unclear significance into sequencing workflows [101,113]. Focusing on known pathogenic variants in the ∼50–300 most informative cancer genes balances analytic sensitivity and specificity. Standardizing targeted content, bioinformatics pipelines, quality control metrics and reporting practices is also recommended to reduce inter-laboratory discordance and misuse [8]. Alignment on curated gene/variant lists promises to refine clinical validity and utility of cfDNA tests. According to thought leaders, critical knowledge gaps remain regarding origins, kinetics and biological roles of circulating nucleic acids that warrant unraveling to optimize liquid biopsy implementation [11], [114]. Elucidating the varied release mechanisms underlying cfDNA and exosomal cargo across cancer types could refine isolation protocols and analytic approaches. Understanding in vivo stabilities and clearance rates will inform serial sampling timelines. Exploring functional immunomodulatory effects beyond diagnostic biomarkers may uncover therapeutic opportunities. Addressing these fundamental biological questions through rigorous experimentation will likely maximize future clinical value.

High costs of cfDNA sequencing may impede population-level uptake absent demonstrated clinical utility and value to guide payer coverage decisions [109]. To convince payers like Medicare, robust health economic models must demonstrate superior outcomes from liquid biopsy-directed care delivery across diverse real-world populations beyond carefully curated trial cohorts [110]. Reducing economic barriers through reimbursement policies and development of low-cost decentralized point-of-care platforms promises to accelerate equitable oncology integration [115]. While cfDNA analysis shows great promise, thought leaders caution liquid biopsy results must not divert focus away from fundamental preventive, diagnostic and therapeutic interventions known to improve outcomes [104]. Health systems with resource constraints in particular should prioritize strengthening primary care, early diagnosis and access to established treatments over novel ancillary technologies like cfDNA testing that lacks extensive validation [104]. A measured evidence-based approach is urged to judiciously integrate liquid biopsy guided precision oncology without detracting from fundamental oncologic care standards.

In summary, while expert consensus recognizes the emerging potential of cfDNA analysis to advance non-invasive cancer care, recommendations emphasize carefully validating clinical utility, multidisciplinary interpretation, developing standardized protocols, focusing on actionable variants, unraveling biological underpinnings, evaluating value, and promoting equitable access to responsibly translate liquid biopsies into clinical practice.

11. Unanswered questions and future directions

Despite rapid advances, several critical questions and challenges remain unresolved regarding integration of ctDNA and exosomal analyses into widespread clinical practice. Ongoing research aims to address these gaps to further optimize and advance liquid biopsy approaches.

A major current limitation is the low analytic sensitivity of detecting rare tumor-derived ctDNA and exosomes against a high normal background in biofluids. ctDNA and exosomes typically represent less than 1 % of cell-free DNA and extracellular vesicles in samples [8,43,44]. Developing ultrasensitive and customized assays, along with improvements in isolation and enrichment techniques, will be critical for robust analysis in the presence of high background [8]. Standardization of pre-analytical variables, analytical validation protocols, and test reporting practices is also needed to ensure consistency across laboratories before full clinical adoption [8].

Additionally, large-scale studies on the clinical validity, utility, and health economics of liquid biopsies in real-world settings are still lacking. Multicenter trials across diverse patient populations are required to firmly establish the benefits of integrating liquid biopsies into clinical workflows for improved patient management and outcomes [19]. Cost-effectiveness data accounting for infrastructure, testing, and follow-up costs are also vital.

Emerging directions include combining ctDNA and exosomal analysis into a multi-analyte approach to derive synergistic molecular information about tumors [4]. Machine learning integration of ctDNA, exosomal data, and radiomic features from medical imaging could enable enhanced diagnostic, prognostic, and predictive modeling [43,44,59]. Point-of-care microfluidic platforms may allow decentralized liquid biopsy analysis at the bedside or in outpatient settings [48].

Elucidating the varied biological mechanisms behind ctDNA and exosome release across cancer types could reveal functional roles beyond diagnostic biomarkers [87]. Exploring these research frontiers through interdisciplinary efforts promises to drive the disruptive impact of liquid biopsies in precision oncology.

12. Conclusion

The advent of liquid biopsies for the analysis of circulating tumor DNA (ctDNA) and exosomes has catalyzed a paradigm shift in the field of oncology, providing clinicians with the unprecedented ability to unlock tumor insights through minimally invasive blood tests. Molecular profiling of cancer has traditionally relied on invasive tissue biopsies fraught with challenges like spatial heterogeneity, infeasibility of repeat sampling, and risks associated with procedural complications [4,116]. The analysis of ctDNA and exosomes isolated from biofluids like blood, urine, saliva, and CSF overcomes these limitations, facilitating dynamic monitoring of cancer with a simple blood draw.

Over the past decade, technological innovations have enabled the comprehensive characterization of rare tumor-derived molecules in the circulation, revealing the complex genomic and transcriptomic alterations underlying cancer initiation, progression, and therapeutic responses [15,117]. Advances in next-generation sequencing, digital PCR, microfluidics, exosome isolation, mass spectrometry, and computational analytics have been instrumental in unlocking these insights [16,43,44]. Extensive investigation has established the clinical validity of ctDNA and exosomal biomarkers as surrogates of tumor status and precision indicators of disease evolution over time and treatment [4,20].

As discussed earlier, several traditional circulating protein biomarkers are clinically validated for diagnostic and monitoring applications in specific cancers [12], [13], [14]. Novel liquid biopsy analytes like ctDNA and exosomal molecules have also demonstrated analytical and clinical validity as complementary biomarkers, with ongoing research to accelerate regulatory approvals [2,15]. Technological innovations continue to enhance analytic sensitivity and specificity to unlock the full potential of these emerging approaches.

Translating these biomarkers from bench to bedside, liquid biopsies have already demonstrated immense clinical potential across the entire continuum of cancer management. In early detection, ctDNA and exosomal analysis will likely facilitate screening and diagnosis of tumors ∼9–24 months prior to conventional strategies, allowing more conservative interventions [15,19]. For therapy monitoring, molecular changes detectable weeks or months earlier than radiographic changes can enable rapid adaptation of ineffective treatments [118,39]. Non-invasive surveillance through serial sampling detects actionable minimal residual disease otherwise undetectable and have been shown to identify recurrence prior to clinical manifestation [17,30]. Overall, liquid biopsies arm oncologists with real-time dynamic information to optimize therapeutic decisions and improve patient outcomes.

Given the promise of liquid biopsies, investments in research and clinical implementation continue to accelerate. Ongoing studies like the PATHFINDER are assessing the clinical utility of ctDNA analysis across cancer types and stages of management (NCT04241796) [119,120]. In 2020, the FDA approved the first tissue-agnostic indication for entrectinib based on NTRK gene fusions identified by liquid biopsy [121]. The CLIA-certification of laboratory-developed liquid biopsy tests signals increasing adoption in clinical practice [122]. Overall, liquid biopsies are transitioning rapidly from scientific curiosity to standard of care in oncology.

Looking ahead, realizing the full potential of liquid biopsies warrants continued research to establish clinical utility across diverse contexts, standardization of pre-analytical and analytical protocols, clinician education, and demonstrate sufficient fiscal value to expand payer coverage [4,8]. Furthermore, integrative analyses combining ctDNA, exosomes, and other emerging analytes like EVs, platelets, and tumor-educated platelets, hold promise for a more comprehensive liquid biopsy [78,100]. Overall, liquid biopsies are poised to radically advance clinical cancer management, enabling early diagnosis, real-time molecular monitoring, and surveillance to ultimately improve patient outcomes through precision medicine approaches.

Funding

The authors declare that no funds, grants or other support were received during the preparation of this manuscript.

Availability of data and materials

The data and materials used during the current study are available from the corresponding author on reasonable request.

Authors' contributions

All authors made substantial intellectual contribution to the study. Study conception and design: OAB. Literature review and analyses: OAB, C-CC, TRP. Manuscript writing: OAB, TRP. Provision of resources and administrative overseeing: OAB. All authors approved the final version of the manuscript for publication.

Consent for publication

Not applicable.

Ethics approval

This is a review of literature, and as such exempted from the need for ethical approval. Literature review was performed in compliance with applicable international guidelines and regulations.

Declaration of competing 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.