Breast cancer represents a major global health challenge.

Early identification of breast cancer can increase the likelihood of effective treatment and improve clinical outcomes.

Mammography remains an important screening modality, but its performance can be influenced by breast density, age, and other patient characteristics.

These limitations have encouraged researchers to investigate molecular approaches that could complement existing screening methods.

Molecular biomarkers are measurable biological characteristics that can indicate the presence or biological behavior of disease.

In breast cancer, biomarkers may originate from tumor cells, stromal cells, immune cells, or systemic responses to malignant transformation.

Potential biomarker sources include blood, plasma, serum, urine, and tissue samples.

Liquid biopsy-based biomarkers are particularly attractive because they can potentially be obtained through minimally invasive sampling.

Molecular biomarkers may also provide biological information that cannot be obtained from imaging alone.

For example, changes in circulating nucleic acids can reflect tumor-associated genomic and epigenetic alterations.

Similarly, changes in proteins and metabolites can reflect alterations in cellular signaling and metabolism.

The development of reliable molecular biomarkers for early breast cancer detection could therefore complement imaging and improve risk stratification.

Biological Basis of Molecular Biomarkers in Breast Cancer

Breast cancer development involves genetic mutations, epigenetic alterations, abnormal signaling, metabolic reprogramming, and interactions with the tumor microenvironment.

These changes can generate measurable molecular signals.

Tumor cells may release DNA fragments, RNA molecules, proteins, metabolites, and extracellular vesicles into the circulation.

The concentration and molecular characteristics of these components can change during tumor development.

This creates an opportunity to identify cancer-associated molecular signatures before extensive clinical progression.

Major Molecular Biomarkers Investigated in Breast Cancer

Several classes of biomarkers have been studied for early breast cancer detection.

Circulating MicroRNAs

MicroRNAs are small non-coding RNA molecules that regulate gene expression.

They can influence cellular proliferation, apoptosis, differentiation, migration, and metabolism.

Cancer-associated changes in microRNA expression have been reported in breast tumors.

Some microRNAs may also be detected in circulation.

Their relative stability in blood makes them attractive candidates for liquid biopsy applications.

However, individual microRNAs are rarely specific to breast cancer.

For this reason, panels containing several microRNAs may provide greater diagnostic value than single markers.

miR-21

miR-21 is one of the most extensively investigated oncogenic microRNAs.

Increased miR-21 expression has been reported in several malignancies, including breast cancer.

It can regulate genes involved in apoptosis, proliferation, and tumor progression.

Although miR-21 may provide useful information, its expression is not specific to breast cancer and can be altered in other pathological conditions.

miR-155

miR-155 participates in immune regulation and inflammatory signaling.

Its dysregulation has been reported in breast cancer and other malignancies.

Circulating miR-155 has therefore been investigated as a potential component of molecular biomarker panels.

Its clinical usefulness may increase when combined with other molecular markers.

miR-200 Family

The miR-200 family is closely associated with epithelial characteristics and epithelial-to-mesenchymal transition.

Changes in these microRNAs may reflect alterations in tumor-cell plasticity.

Their expression patterns have been investigated in breast cancer tissue and circulation.

However, additional validation is required before they can be routinely applied to early screening.

Cell-Free DNA

Cell-free DNA consists of extracellular DNA fragments found in biological fluids.

A proportion of circulating cell-free DNA may originate from normal tissues, while another fraction can originate from tumor cells.

Tumor-associated cell-free DNA may contain genetic and epigenetic alterations.

The analysis of cell-free DNA therefore provides a potential route for detecting molecular evidence of malignancy.

Circulating Tumor DNA

Circulating tumor DNA is the fraction of cell-free DNA released by tumor cells.

It can contain tumor-specific mutations, copy-number alterations, and methylation patterns.

In advanced disease, circulating tumor DNA can sometimes be detected at relatively higher levels.

However, early-stage tumors may release very small quantities.

This creates a major technical challenge for early detection.

Highly sensitive sequencing and molecular detection methods are therefore required.

DNA Methylation Biomarkers

DNA methylation is an important epigenetic mechanism regulating gene expression.

Cancer-associated methylation changes can occur early during malignant transformation.

Tumor-derived DNA with abnormal methylation patterns can potentially enter the circulation.

Methylation-based assays are therefore being investigated for early breast cancer detection.

One advantage of methylation markers is that they may detect molecular alterations without requiring a known tumor mutation.

Circulating Tumor Cells

Circulating tumor cells are malignant cells that enter the bloodstream.

Their detection can provide direct evidence of tumor-associated cellular activity.

However, circulating tumor cells are often extremely rare, particularly in early-stage disease.

Highly sensitive enrichment and detection technologies are therefore required.

Their potential role in early detection remains an active area of research.

Protein Biomarkers

Proteins are among the most extensively investigated biomarker classes.

Several serum proteins have been evaluated in breast cancer.

CA 15-3 and carcinoembryonic antigen are used clinically in certain contexts, particularly for disease monitoring, but they do not have sufficient sensitivity and specificity to function as stand-alone screening biomarkers for early breast cancer.

Research continues to identify more specific protein signatures.

Combining multiple proteins may improve diagnostic performance.

Extracellular Vesicles

Extracellular vesicles are membrane-bound particles released by cells.

They can contain proteins, lipids, messenger RNAs, and microRNAs.

Tumor-derived extracellular vesicles may carry molecular information reflecting the biology of malignant cells.

Because the vesicle membrane can protect RNA and other molecular components from degradation, extracellular vesicles have attracted considerable attention as liquid biopsy candidates.

Metabolic Biomarkers

Cancer cells undergo metabolic reprogramming to support proliferation and survival.

Changes in glucose metabolism, lipid synthesis, amino-acid metabolism, and mitochondrial activity can alter circulating metabolites.

Metabolomic profiling may therefore identify molecular patterns associated with breast cancer.

However, metabolic biomarkers can be influenced by age, diet, medication, body composition, and other systemic factors.

Genetic Biomarkers

Inherited genetic variants can contribute to breast cancer susceptibility.

Variants in genes involved in DNA repair and genomic stability can influence individual risk.

However, susceptibility markers primarily indicate predisposition rather than the presence of an early tumor.

Combining genetic risk assessment with molecular biomarkers may provide a more comprehensive screening strategy.

Epigenetic Biomarkers

Epigenetic alterations can occur before extensive morphological changes become detectable.

Abnormal methylation of tumor-associated genes can therefore provide an early molecular signal.

Epigenetic biomarkers may be detected in tissue or circulating DNA.

Their stability and measurable nature make them attractive candidates for liquid biopsy applications.

Molecular Biomarkers and Breast Cancer Subtypes

Breast cancer is biologically heterogeneous.

Major molecular categories include hormone receptor-positive, HER2-positive, and triple-negative breast cancers.

Molecular biomarker profiles may differ among these subtypes.

Consequently, a biomarker panel that performs well in one subtype may not have equivalent performance in another.

Future biomarker studies should therefore consider molecular subtype and tumor heterogeneity.

Biomarker Panels

The use of a single molecular biomarker is often limited by insufficient sensitivity or specificity.

Combining several biomarkers may overcome some of these limitations.

For example, a diagnostic panel could combine microRNAs, DNA methylation markers, proteins, and clinical variables.

A multimarker strategy may provide stronger discrimination between healthy individuals and patients with early disease.

Integration with Imaging

Molecular biomarkers should not necessarily replace imaging.

Instead, they may complement established screening approaches.

A possible future workflow could involve molecular risk assessment followed by targeted imaging.

This could potentially improve screening efficiency and reduce unnecessary investigations.

Review Design

The present article was developed as a narrative review of molecular biomarkers investigated for the early detection of breast cancer.

Literature Search

Relevant scientific publications were considered from major biomedical literature databases and peer-reviewed journals.

Search terms included combinations of:

“breast cancer,” “early detection,” “molecular biomarkers,” “microRNA,” “circulating tumor DNA,” “cell-free DNA,” “DNA methylation,” “circulating tumor cells,” “extracellular vesicles,” “proteomic biomarkers,” and “metabolomics.”

Inclusion Criteria

Studies were considered relevant when they investigated molecular markers associated with:

• Early breast cancer detection;
• Liquid biopsy;
• Circulating nucleic acids;
• Circulating proteins;
• Extracellular vesicles;
• Metabolic changes;
• Epigenetic alterations; or
• Multimarker diagnostic strategies.

Exclusion Criteria

Studies that focused exclusively on advanced-stage disease monitoring without relevance to early detection were not emphasized.

Data Synthesis

The available evidence was organized according to biomarker class, biological basis, diagnostic potential, limitations, and future clinical applications.

Results

The reviewed evidence indicates that multiple molecular biomarker classes have potential for early breast cancer detection.

Circulating microRNAs are among the most extensively investigated candidates because of their stability and biological relevance.

Cell-free DNA and circulating tumor DNA provide opportunities to detect tumor-associated genetic and epigenetic alterations.

DNA methylation markers may be particularly valuable because epigenetic abnormalities can occur relatively early during carcinogenesis.

Protein biomarkers can provide complementary information but generally lack sufficient performance as independent early screening markers.

Extracellular vesicles offer an additional source of tumor-associated molecular information.

Metabolomic profiling may also distinguish cancer-associated metabolic states.

Overall, multimarker approaches appear more promising than individual biomarkers because breast cancer is molecularly heterogeneous.

The search for reliable molecular biomarkers for early breast cancer detection is motivated by the limitations of conventional screening and the biological complexity of breast cancer.

A successful biomarker should ideally be detectable before clinical progression, demonstrate high sensitivity and specificity, be reproducible, and be measurable through a practical and minimally invasive method.

No single biomarker currently satisfies all of these requirements.

MicroRNAs have several characteristics that make them attractive.

They can be detected in circulation and may remain relatively stable because of their association with proteins or extracellular vesicles.

However, many microRNAs are altered in several cancers and non-malignant conditions.

Consequently, their diagnostic specificity can be limited.

Cell-free DNA provides another promising source of information.

Tumor-derived DNA can contain mutations, copy-number changes, and methylation abnormalities.

The major challenge is that the tumor-derived fraction can be extremely low during early disease.

This makes analytical sensitivity essential.

DNA methylation may offer an advantage because cancer-associated methylation changes can be abundant even when tumor-derived DNA concentration is low.

Nevertheless, methylation patterns may vary according to tumor subtype and individual patient characteristics.

Protein biomarkers have the advantage of established laboratory technologies.

However, traditional serum markers such as CA 15-3 have limited utility for population-based early screening when used alone.

Future research is therefore increasingly focused on protein combinations and integration with other molecular markers.

Extracellular vesicles represent an emerging source of biomarkers.

Because they transport nucleic acids and proteins between cells, their contents may reflect tumor biology.

However, standardized isolation and characterization methods are still needed.

The heterogeneity of breast cancer represents another important challenge.

Different molecular subtypes may release different biomarker profiles.

A universal biomarker may therefore be difficult to identify.

Instead, subtype-specific or multimarker approaches may provide greater diagnostic performance.

Clinical Potential of Molecular Biomarkers

Molecular biomarkers may eventually contribute to several aspects of breast cancer management.

Challenges in Biomarker Development

Several barriers must be addressed before molecular biomarkers can be incorporated into routine screening.

Biological Heterogeneity

Breast tumors differ considerably in their genetic, epigenetic, and metabolic characteristics.

This heterogeneity can reduce the performance of universal biomarkers.

Low Biomarker Concentration

Early tumors may release only small quantities of tumor-associated molecules into the circulation.

Sensitive analytical methods are therefore essential.

Lack of Standardization

Differences in sample collection, processing, storage, extraction, and analytical platforms can affect biomarker measurements.

Standardized protocols are required.

False-Positive Findings

Some molecular markers can be altered by inflammation, benign disease, aging, or other cancers.

Biomarker specificity must therefore be carefully evaluated.

Validation

Many promising biomarkers have been identified in small observational studies.

Large prospective studies involving diverse populations are needed to determine their actual clinical utility.

Future Perspectives

Future molecular screening strategies are likely to involve combinations of biomarkers rather than a single molecular marker.

Integration of circulating DNA, microRNAs, proteins, extracellular vesicles, and metabolites may provide a more comprehensive representation of early tumor biology.

Artificial intelligence and machine-learning methods may help identify complex patterns that are difficult to recognize using conventional statistical approaches.

Another important development is the integration of molecular biomarkers with imaging.

Combining molecular risk scores with mammographic or other imaging findings may improve diagnostic decision-making.

Longitudinal sampling may also provide additional value.

Repeated measurement of circulating biomarkers could potentially identify changes occurring before a tumor becomes clinically apparent.

Molecular biomarkers represent a promising area of research for improving the early detection of breast cancer.

Circulating microRNAs, cell-free DNA, circulating tumor DNA, DNA methylation patterns, circulating tumor cells, proteins, extracellular vesicles, and metabolites can provide molecular information associated with malignant transformation.

However, individual biomarkers generally have limitations in sensitivity, specificity, or reproducibility.

Multimarker approaches that combine different molecular classes may provide greater diagnostic accuracy.

Integration of molecular biomarkers with conventional imaging, clinical risk factors, and artificial intelligence may further improve early detection.

Before widespread clinical implementation, prospective validation, standardized laboratory protocols, cost-effectiveness assessment, and evaluation across diverse populations are required.

Continued advances in liquid biopsy technologies and molecular profiling may ultimately contribute to more sensitive, minimally invasive, and personalized strategies for early breast cancer detection.

1. Harbeck N, Penault-Llorca F, Cortes J, et al. Breast cancer. Nature Reviews Disease Primers. 2019;5:66.
2. Sung H, Ferlay J, Siegel RL, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: A Cancer Journal for Clinicians. 2021;71:209–249.
3. Heer E, Harper A, Escandor N, Sung H, McCormack V, Fidler-Benaoudia MM. Global burden and trends in premenopausal and postmenopausal breast cancer. Breast Cancer Research. 2020;22:1–11.
4. Henry NL, Hayes DF. Cancer biomarkers. Molecular Oncology. 2012;6:140–146.
5. Schwarzenbach H, Hoon DSB, Pantel K. Cell-free nucleic acids as biomarkers in cancer patients. Nature Reviews Cancer. 2011;11:426–437.
6. Dawson SJ, Tsui DWY, Murtaza M, et al. Analysis of circulating tumor DNA to monitor metastatic breast cancer. New England Journal of Medicine. 2013;368:1199–1209.
7. Dawson SJ, Rosenfeld N, Caldas C. Circulating tumor DNA to monitor metastatic breast cancer. New England Journal of Medicine. 2013;368:1199–1200.
8. Mitchell PS, Parkin RK, Kroh EM, et al. Circulating microRNAs as stable blood-based markers for cancer detection. Proceedings of the National Academy of Sciences. 2008;105:10513–10518.
9. Schwarzenbach H, Nishida N, Calin GA, Pantel K. Clinical relevance of circulating cell-free microRNAs in cancer. Nature Reviews Clinical Oncology. 2014;11:145–156.
10. Hayes J, Peruzzi PP, Lawler S. MicroRNAs in cancer: biomarkers, functions and therapy. Trends in Molecular Medicine. 2014;20:460–469.
11. Cortez MA, Bueso-Ramos C, Ferdin J, Lopez-Berestein G, Sood AK, Calin GA. MicroRNAs in body fluids—the mix of hormones and biomarkers. Nature Reviews Clinical Oncology. 2011;8:467–477.
12. Heitzer E, Haque IS, Roberts CES, Speicher MR. Current and future perspectives of liquid biopsies in genomics-driven oncology. Nature Reviews Genetics. 2019;20:71–88.
13. Ignatiadis M, Sledge GW, Jeffrey SS. Liquid biopsy enters the clinic—implementation issues and future challenges. Nature Reviews Clinical Oncology. 2021;18:297–308.
14. Turchinovich A, Weiz L, Langheinz A, Burwinkel B. Characterization of extracellular circulating microRNA. Nucleic Acids Research. 2011;39:7223–7233.
15. Théry C, Witwer KW, Aikawa E, et al. Minimal information for studies of extracellular vesicles 2018: MISEV2018. Journal of Extracellular Vesicles. 2018;7:1535750.
16. Kalluri R, LeBleu VS. The biology, function, and biomedical applications of exosomes. Science. 2020;367:eaau6977.
17. Wan JCM, Massie C, Garcia-Corbacho J, et al. Liquid biopsies come of age: towards implementation of circulating tumour DNA. Nature Reviews Cancer. 2017;17:223–238.
18. Bardelli A, Pantel K. Liquid biopsies, what we do not know yet. Cancer Cell. 2017;31:172–179.
19. Pharoah PDP, Antoniou A, Bobrow M, et al. Polygenic susceptibility to breast cancer and implications for prevention. Breast Cancer Research. 2002;4:1–7.
20. Mavaddat N, Michailidou K, Dennis J, et al. Polygenic risk scores for prediction of breast cancer and breast cancer subtypes. Nature Genetics. 2019;51:128–141.