ctDNA in Breast Cancer: Early Detection and Genetic Insights

Circulating tumor DNA (ctDNA) is a promising tool in breast cancer research, providing insights into tumor genetics through a simple blood test. It has the potential to improve early detection, monitor treatment response, and identify mutations linked to therapy resistance. Unlike traditional biopsies, ctDNA analysis offers a noninvasive way to track cancer dynamics in real time.

Understanding how ctDNA enters circulation, its composition, and the techniques used for its detection is crucial for optimizing its clinical applications.

Cellular Release Processes

ctDNA originates from tumor cells undergoing apoptosis and necrosis, the primary mechanisms responsible for its release into the bloodstream. Apoptosis, a programmed cell death process, fragments nuclear material into small, uniform DNA fragments (160–180 base pairs), which are either packaged into apoptotic bodies or released as nucleosome-associated DNA, protecting them from degradation. Necrosis, triggered by hypoxia or therapy, causes uncontrolled tumor cell breakdown, generating larger, more heterogeneous DNA fragments. The balance between these processes affects ctDNA quantity and integrity, with necrotic release contributing to a broader size distribution.

Beyond apoptosis and necrosis, tumor cells actively release DNA via extracellular vesicles such as exosomes and microvesicles, which protect ctDNA from enzymatic degradation and facilitate intercellular communication. Exosome-derived ctDNA can harbor clinically relevant mutations, making it useful for molecular profiling. The stability provided by these vesicles enhances detectability, particularly in early-stage breast cancer, where ctDNA levels are low.

The tumor microenvironment influences ctDNA release, with factors like inflammation, hypoxia, and mechanical stress modulating cellular turnover. Hypoxia can induce necrosis, increasing fragmented DNA release, while chemotherapy and radiation accelerate tumor cell death, temporarily elevating ctDNA levels before they decline as the tumor burden decreases. These fluctuations make ctDNA a valuable biomarker for monitoring treatment response.

Composition in Circulation

ctDNA exists as fragmented double-stranded DNA in the bloodstream, often bound to histones or enclosed within extracellular vesicles. Its size distribution reflects its release mechanisms, with apoptotic fragments measuring 160–180 base pairs and necrotic fragments being more heterogeneous. In breast cancer, ctDNA typically represents less than 1% of total circulating cell-free DNA (cfDNA), making detection challenging. Concentrations vary based on tumor burden, disease stage, and metastatic spread, with advanced cases exhibiting higher levels.

The mutational landscape of ctDNA mirrors that of the tumor, offering a snapshot of genetic alterations. Mutations in genes such as PIK3CA, TP53, and ESR1 are frequently detected, with some linked to therapy resistance or disease progression. For example, ESR1 mutations, which emerge under selective pressure from endocrine therapy, can be identified in ctDNA months before clinical progression. Structural variations, including copy number alterations and chromosomal rearrangements, further characterize tumor heterogeneity, enabling real-time monitoring of molecular evolution.

Epigenetic modifications also define ctDNA composition, with tumor-specific methylation patterns serving as informative biomarkers. Hypermethylation of promoter regions in genes like BRCA1, RASSF1A, and APC distinguishes cancer-derived ctDNA from normal cfDNA. These methylation signatures remain stable in circulation and can aid in early detection, particularly in cases with low mutational burden. Emerging technologies have demonstrated the feasibility of using methylation-based ctDNA assays to differentiate breast cancer subtypes.

Genetic and Epigenetic Characteristics

ctDNA carries a molecular blueprint of breast cancer, reflecting both genetic mutations and epigenetic modifications. PIK3CA mutations, found in 30–40% of hormone receptor-positive breast cancers, activate the PI3K/AKT/mTOR pathway, contributing to therapy resistance. TP53 mutations, prevalent in triple-negative and HER2-positive subtypes, drive genomic instability and indicate aggressive disease. Copy number alterations, such as HER2 and MYC amplifications, provide insights into tumor subtype classification and potential therapeutic targets.

Structural rearrangements add further complexity. Chromosomal translocations, such as ESR1 gene fusions, have been identified in endocrine-resistant breast cancer and may predict treatment response. These alterations, often undetectable in standard tissue biopsies, can be captured in ctDNA, allowing broader tumor genome assessment. Liquid biopsies enable longitudinal tracking of these changes, identifying resistance mechanisms before clinical manifestation.

Epigenetic modifications in ctDNA offer another dimension of tumor characterization. Hypermethylation of tumor suppressor genes like RASSF1A, BRCA1, and APC contributes to gene silencing and uncontrolled proliferation. These methylation patterns remain stable in circulation and have been explored as diagnostic and prognostic biomarkers. A study in Clinical Cancer Research linked RASSF1A hypermethylation in ctDNA to higher tumor stage and poorer prognosis. Unlike genetic mutations, which evolve under selective pressure, epigenetic changes often appear early in tumorigenesis, making them valuable for early detection.

Techniques for Detection

Detecting ctDNA in breast cancer requires highly sensitive methods due to its low abundance in the bloodstream. Digital PCR (dPCR) and next-generation sequencing (NGS) are widely used, each with distinct advantages. Digital PCR, including droplet digital PCR (ddPCR), provides absolute quantification of specific mutations with high sensitivity, detecting variant allele frequencies as low as 0.01%. This makes it useful for monitoring minimal residual disease (MRD) and identifying resistance mutations like ESR1 alterations. However, its targeted nature limits detection of broader genomic changes.

NGS enables comprehensive genomic profiling of ctDNA, identifying single-nucleotide variants, copy number alterations, and structural rearrangements. Hybrid-capture NGS, which enriches specific genomic regions, has been instrumental in detecting mutations in genes like PIK3CA and TP53. While NGS methods are more expensive and have longer turnaround times than digital PCR, ongoing advancements are improving efficiency and affordability.

Ultra-sensitive approaches, such as methylation-based ctDNA assays and fragmentomics, expand detection capabilities beyond mutation analysis. Methylation-specific PCR and bisulfite sequencing identify tumor-specific epigenetic changes that may precede genetic mutations, enhancing early detection. Fragmentomics, which examines ctDNA size and distribution patterns, helps distinguish tumor-derived DNA from normal cfDNA. Machine learning algorithms integrated with fragmentomic data further improve detection accuracy, particularly in early-stage breast cancer.

Sample Handling in the Laboratory

Proper handling of blood samples is essential to preserve ctDNA integrity and ensure accuracy. The choice of blood collection tubes significantly impacts ctDNA yield and stability. Standard EDTA tubes require immediate processing to prevent leukocyte lysis, which can contaminate samples with non-tumor cfDNA. Specialized tubes containing preservatives, such as Streck or Cell-Free DNA BCT tubes, stabilize nucleic acids and allow delayed processing without compromising quality. These tubes maintain ctDNA stability for up to seven days at room temperature, making them useful in clinical settings where immediate centrifugation is not feasible.

Plasma separation is critical, as improper centrifugation can introduce genomic DNA contamination from lysed blood cells. A two-step centrifugation protocol—first at low speed to remove cellular components, then at high speed to clarify plasma—minimizes unwanted DNA fragments. Isolated plasma should be stored at -80°C to prevent nuclease-mediated degradation, as prolonged storage at higher temperatures reduces ctDNA recovery. Extraction methods, such as silica-based columns or magnetic bead-based approaches, must be highly efficient, especially for early-stage breast cancer cases where ctDNA concentrations are minimal. Ensuring high-quality extraction maximizes detection success.