Liquid biopsy: Biomarkers, uses and limitations

Liquid biopsy trades tissue context for a broader, repeatable view of cancer. Because that view is built from scarce, mixed biological signals, what it reveals depends on which material is targeted and how the sample is collected, prepared and analyzed.

On this page

• Liquid biopsy versus tissue biopsy
• Liquid biopsy samples: Blood and other body fluids
• Circulating material and liquid biopsy biomarkers
• What is a liquid biopsy used for in cancer research?
• How liquid biopsy samples are prepared and analyzed

 

Liquid biopsy versus tissue biopsy

Liquid biopsy is the analysis of blood or another body fluid for intact tumor cells and cancer-associated features in cell-free DNA, RNA or extracellular vesicles. Blood draws are less invasive than tissue biopsies, making it easier to collect repeated samples and follow molecular changes in the cancer over time. (1, 2)

Molecular differences can occur between tumor sites and between regions within a single tumor. A tissue biopsy samples only part of a lesion, whereas a fluid sample can receive material from different regions and sites. Liquid biopsy can therefore reveal molecular differences missed in the sampled tissue. This broader view is still incomplete: contributions are unequal, and some tumors release very little detectable material into the sampled fluid. (1, 2)

Tissue biopsies, on the other hand, add the spatial context that fluid analyses cannot provide. Examining tissue reveals cell morphology, tissue architecture and tumor invasion along with the arrangement of immune and stromal cells around a tumor. These observations connect molecular findings to their location within the sampled tissue. (1, 2)

Liquid biopsy samples: Blood and other body fluids

Common samples used for liquid biopsy include blood, urine, cerebrospinal fluid (CSF) and saliva. (3) For blood samples, the material being studied determines which fraction to use. Plasma, the liquid fraction separated from blood cells, is commonly used for cell-free DNA analysis. Serum is obtained after blood has clotted and can therefore contain extra DNA released from blood cells during preparation, adding to the nontumor background. Studies of intact circulating tumor cells begin with whole blood. Detecting and counting these cells can help researchers investigate residual disease and recurrence risk, and relate changes in cell numbers to treatment response. Examining individual cells can also reveal molecular differences that may help explain treatment resistance. (1, 4, 5)

Urine can contain DNA released directly by bladder and other urinary tract tumors, making it a useful sample for studying their molecular alterations. Saliva is similarly studied as a source of tumor DNA in head and neck cancers, particularly those in the oral cavity. (6)

Fluids close to tumors may contain a higher proportion of tumor-derived material than blood. Cerebrospinal fluid, which surrounds the brain and spinal cord, is a good example. In cancers of the central nervous system, tumor DNA can be more readily detected in CSF than in plasma because the blood–brain barrier can limit its passage into the bloodstream. However, collecting CSF by lumbar puncture is more invasive than drawing blood. (6)

Blood and fluids collected near a tumor can contain different mixtures of tumor-derived material. An alteration readily detectable in one sample may be absent or too scarce to detect in the other, even when the same assay is used. (6)

Circulating material and liquid biopsy biomarkers

Liquid biopsy samples contain a mixture of material from tumor and nontumor cells. Cell-free DNA, or cfDNA, is DNA present outside intact cells; circulating tumor DNA, or ctDNA, is the tumor-derived part of that DNA. Measuring total cfDNA therefore does not establish how much came from a tumor. To study the cancer within this mixture, researchers look for cancer-associated sequence changes or methylation patterns that can help distinguish tumor-derived DNA from the nontumor background and describe its molecular characteristics. (1)

Researchers can measure different features in the same type of circulating material.

Circulating material	Examples of features measured	What the observation represents
Cell-free DNA, including ctDNA	Sequence variants or DNA methylation patterns	Genetic or epigenetic features in DNA released into the sampled fluid. Tumor-associated features must be distinguished from the nontumor background. (1)
Extracellular vesicles	RNA or proteins carried by membrane-bound particles released by cells	Features of the selected vesicles or their cargo; isolating vesicles alone does not establish their tumor origin. (7)
Circulating miRNAs	Abundance of selected microRNAs, small RNAs that regulate gene expression	RNA profiles that may change with cancer but can also reflect contributions from blood cells and other tissues. (8)
Circulating tumor cells	Cell number, phenotype or molecular characteristics	Properties of intact tumor cells recovered from blood. (4)

 

These categories overlap. Circulating miRNAs can be carried inside vesicles or associated with proteins outside them. A measurement of vesicle-associated miRNA therefore describes that selected fraction, rather than all the miRNA in a plasma sample. (8)

What is a liquid biopsy used for in cancer research?

Liquid biopsy can identify molecular alterations relevant to treatment-selection research and track changes associated with response, resistance or recurrence. A single sample can reveal a mutation that points to a potential therapeutic target, including when tumor tissue is difficult to obtain. Repeated samples extend the question from which alterations are present to how the cancer changes during and after treatment. (1, 9)

The observation sought depends on the study:

• Treatment response: changes in a tumor-associated signal across samples can be compared with response to treatment. For example, in colorectal cancer research, serial ctDNA measurements tracked tumor dynamics during surgery and chemotherapy. (10)
• Acquired resistance: newly detected resistance-associated mutations can help explain progression during targeted therapy. In a study of metastatic colorectal cancer, ctDNA revealed mutations in EGFR-pathway genes in patients who had initially responded to EGFR blockade and then progressed. (9)
• Minimal residual disease (MRD) and recurrence: cancer cells may remain after treatment even when no tumor is visible on imaging. Detecting ctDNA in follow-up samples can provide evidence of this residual disease and help researchers assess the risk of recurrence. (1, 10)

Liquid biopsy is less established for screening people without a known cancer than for molecular profiling of diagnosed disease. (11) When screening people without a known cancer, researchers must detect a cancer-associated signal and distinguish it from nontumor signals without knowing in advance which tumor alterations to look for. Small or localized cancers may also release little detectable ctDNA. Performance in patients with established cancer therefore does not by itself establish performance in a screening population. (1, 3, 9)

How liquid biopsy samples are prepared and analyzed

Preserve the mixture collected

Sample preservation must limit changes after collection that could alter the molecular signals being studied. Normal blood cells that break down after collection release DNA into plasma, lowering the proportion of tumor-derived DNA in the sample. Prompt processing or appropriate cell stabilization helps limit this release. (1) PAXgene Blood ccfDNA Tubes stabilize blood cells and help prevent release of intracellular DNA into plasma during storage and transport.

Recover the material the question requires

Recovery methods must match the physical material being studied. Extracting DNA, capturing vesicles and enriching rare cells require different approaches. Vesicle studies may require intact particles or the RNA carried inside them, so the intended analysis determines what must be recovered.

The recovered material must also have sufficient yield and purity for the intended analysis. For cfDNA, extraction methods differ in both yield and the fragment sizes recovered; losses can reduce the material available for downstream detection. (1) Contaminants carried through purification can also inhibit downstream reactions.

Research need	Product or products	Relevant capability
Recover and concentrate cfDNA	QIAamp MinElute ccfDNA Kits	Purify cfDNA from 1–10 mL of human plasma or serum and elute it in as little as 20 µL, concentrating scarce DNA for downstream analysis.
Prepare cfDNA for sequencing	QIAseq cfDNA All-in-One Kits	Combine extraction from plasma with sequencing-library preparation, allowing extracted DNA to enter library preparation without a separate quantification step.
Recover intact extracellular vesicles	exoEasy Maxi Kit	Isolate intact vesicles by membrane affinity for physical or biochemical analysis.
Recover RNA from extracellular vesicles	exoRNeasy Midi and Maxi Kits	Capture vesicles by membrane affinity and purify their RNA, including miRNA.
Recover mRNA from circulating tumor cells	EZ2 AdnaTest CTC Select Kit	Combine immunomagnetic CTC enrichment from human whole blood with mRNA isolation on EZ2 Connect.

 

Match the measurement to the intended finding

A targeted sequencing panel examines a defined set of genes or regions, allowing researchers to look for multiple possible variants in the same sample. The analysis can identify which alterations are present within the regions covered by the panel.

When a study focuses on a known mutation, digital PCR (dPCR) can quantify the mutant sequence against a background of wild-type DNA. Repeating that measurement across samples allows researchers to follow changes in its abundance over time.

For DNA methylation, targeted sequencing can compare methylation across selected CpG sites. Pyrosequencing offers focused quantification of methylation at individual CpG sites. For miRNAs, qPCR after reverse transcription measures targets chosen in advance, whereas sequencing supports broader miRNA profiling.

Research need	Product or products	Relevant capability
Examine variants across selected genes or regions	QIAseq Targeted cfDNA Ultra Panels	Enrich selected genes and regions in human cfDNA to prepare libraries for targeted sequencing.
Quantify a specified mutation	dPCR LNA Mutation Assays with QIAcuity and QIAcuity MasterMix	Detect selected mutant and wild-type sequences and quantify the mutation by dPCR.
Examine methylation at selected CpG sites	QIAseq Targeted Methyl Panels	Prepare targeted sequencing libraries from bisulfite-converted DNA, including cfDNA, for methylation analysis.
Quantify methylation at individual CpG sites	PyroMark CpG assays with PyroMark Q48 Autoprep	Quantify methylation at the CpG sites covered by the assay by pyrosequencing PCR-amplified, bisulfite-converted DNA; assay design and DNA input must be suitable for the sample.
Measure selected miRNAs	miRCURY LNA miRNA PCR Assays	Measure individual miRNAs by qPCR after reverse transcription.
Profile miRNAs more broadly	QIAseq miRNA Library Kit	Prepare miRNA sequencing libraries from RNA recovered from biofluids, including plasma and serum.

 

The limitations of liquid biopsy extend beyond assay sensitivity. A tumor-associated signal can be missed if too little tumor material reaches the sample, if it is obscured by abundant normal background or if material is lost during preparation. Even when the material is present, the assay may not cover the relevant molecular feature. A negative result therefore does not exclude cancer. (1, 9)

References

1. Bronkhorst AJ, Ungerer V, Holdenrieder S. The emerging role of cell-free DNA as a molecular marker for cancer management. Biomol Detect Quantif. 2019;17:100087. doi.org/10.1016/j.bdq.2019.100087
2. Podder PS, Bhadra D, Martinelli C, Cortellino S, Giordano A, Cinti S. Liquid biopsy in breast cancer: current biomarker platforms and clinical applications. Explor Target Antitumor Ther. 2026;7:1002394. doi.org/10.37349/etat.2026.1002394
3. Rendek T, et al. Current challenges of methylation-based liquid biopsies in cancer diagnostics. Cancers (Basel). 2024;16(11):2001. doi.org/10.3390/cancers16112001
4. Lin D, et al. Circulating tumor cells: biology and clinical significance. Signal Transduct Target Ther. 2021;6(1):404. doi.org/10.1038/s41392-021-00817-8
5. Kustanovich A, Schwartz R, Peretz T, Grinshpun A. Life and death of circulating cell-free DNA. Cancer Biol Ther. 2019;20(8):1057–1067. doi.org/10.1080/15384047.2019.1598759
6. Tivey A, Church M, Rothwell D, Dive C, Cook N. Circulating tumour DNA – looking beyond the blood. Nat Rev Clin Oncol. 2022;19(9):600–612. doi.org/10.1038/s41571-022-00660-y
7. Doyle LM, Wang MZ. Overview of extracellular vesicles, their origin, composition, purpose, and methods for exosome isolation and analysis. Cells. 2019;8(7):727. doi.org/10.3390/cells8070727
8. Mo MH, Chen L, Fu Y, Wang W, Fu SW. Cell-free circulating miRNA biomarkers in cancer. J Cancer. 2012;3:432–448. doi.org/10.7150/jca.4919
9. Bettegowda C, et al. Detection of circulating tumor DNA in early- and late-stage human malignancies. Sci Transl Med. 2014;6(224):224ra24. doi.org/10.1126/scitranslmed.3007094
10. Diehl F, et al. Circulating mutant DNA to assess tumor dynamics. Nat Med. 2008;14(9):985–990. doi.org/10.1038/nm.1789
11. National Cancer Institute. Tumor Markers. Reviewed December 7, 2023. NCI fact sheet