What is ctDNA (Circulating Tumor DNA)?
Circulating tumor DNA (ctDNA) is fragmented DNA released into the bloodstream from tumor cells through apoptosis and necrosis, which can be detected and analyzed through liquid biopsy to identify cancer-specific genetic and epigenetic alterations. 1, 2
Core Biological Concept
ctDNA represents tumor-derived cell-free DNA fragments circulating in peripheral blood and other bodily fluids (CSF, ascites, urine). 1, 3 Unlike normal cell-free DNA present in all individuals, ctDNA carries the specific genetic mutations, amplifications, and alterations characteristic of the patient's cancer. 2, 4
Detection Technologies
Two main technological approaches exist for ctDNA detection:
Tumor-informed (customized panel): Involves whole-exome sequencing of tumor tissue first, followed by personalized detection with ultra-high-depth sequencing; provides highest sensitivity and accuracy but at greater cost and cannot detect new mutations. 5
Tumor-agnostic (fixed panel): Uses standardized plasma testing panels with medium-to-high depth sequencing; offers broader applicability at lower cost but with reduced sensitivity. 5
Detection methods range from single-gene PCR assays to comprehensive next-generation sequencing (NGS) panels. 5 Digital PCR, real-time PCR, and mass spectrometry-based platforms are also utilized. 4
Clinical Applications Across Cancer Types
Established Uses
ctDNA testing is FDA-approved and guideline-recommended for specific clinical scenarios:
EGFR mutation detection in NSCLC: The first FDA-approved ctDNA application for predicting response to EGFR tyrosine kinase inhibitors, particularly useful when tissue is unavailable. 5, 6
PIK3CA mutation testing in breast cancer: Validated for selecting patients eligible for alpelisib therapy, with equivalent performance whether using tissue or liquid biopsy. 5
HER2 amplification and resistance mechanism detection: Useful when tissue sampling is not feasible. 5, 7
Emerging Applications
ctDNA shows promise for minimal residual disease (MRD) monitoring in early-stage disease:
Post-surgical surveillance: Detection of ctDNA after stage II colon cancer resection provides direct evidence of residual disease and identifies patients at very high risk of recurrence. 5
Risk stratification: Enables more precise identification of patients who may benefit from adjuvant chemotherapy versus those who can safely avoid it. 5
Treatment response monitoring: Serial ctDNA measurements can detect disease progression earlier than imaging in metastatic settings. 5, 2
Critical Limitations and Pitfalls
Several important caveats must be understood when interpreting ctDNA results:
Sensitivity varies dramatically: Depends on cancer stage, tumor burden, proximity of sampling site to disease, and biomarker type. 5, 7 Approximately 25% of NSCLC patients are "non-shedders" with DNA levels below detection limits. 5
Negative results require caution: A negative ctDNA test does NOT exclude the presence of tumor mutations or amplifications and should never be considered definitive. 5, 7
Cannot replace tissue biopsy for: Histologic diagnosis, transformation assessment (e.g., small cell transformation in EGFR-mutant NSCLC), or initial cancer staging. 5
Detection challenges: MET amplification and oncogenic fusions remain difficult to detect reliably in ctDNA compared to tissue. 5
CSF ctDNA considerations: More abundant in CSF than plasma for CNS malignancies, but provides "net output" from both parenchymal and leptomeningeal disease rather than distinguishing between them. 5
Quality and Validation Requirements
Only clinically validated ctDNA testing methods performed in CLIA-certified laboratories should be used for clinical decision-making. 5, 7 Standardization remains a critical challenge, particularly for NGS-based analyses. 5
The European Liquid Biopsy Society emphasizes that interpretation and reporting are more complex for ctDNA than conventional tissue-based NGS, requiring specific guidelines for optimal communication between laboratories and oncology teams. 5
Optimal Clinical Integration
ctDNA is most appropriate when:
- Adequate tissue cannot be safely obtained or is insufficient for analysis 5, 7
- Monitoring disease progression in patients unable to undergo repeat biopsies 7
- Serial monitoring for treatment response or early recurrence detection 5, 2
- Capturing tumor heterogeneity and genomic evolution in metastatic disease 1, 2
ctDNA should NOT replace tissue biopsy when tissue can be safely obtained for initial diagnosis, staging, or when histologic subtype classification is needed for treatment planning. 7