The analysis of circulating tumor DNA (ctDNA) is rapidly changing the landscape of cancer diagnosis and management. This approach uses a non-invasive “liquid biopsy” to analyze genetic material shed by tumors directly into the bloodstream. By providing a real-time molecular snapshot of a patient’s cancer, ctDNA sequencing helps physicians personalize treatment strategies. The technique analyzes the tumor’s unique genetic fingerprint from a simple blood sample, bypassing the need for invasive tissue procedures and offering distinct advantages for monitoring disease progression.
Understanding Circulating Tumor DNA
Circulating tumor DNA (ctDNA) is a specific subset of cell-free DNA (cfDNA) found in the blood. Cell-free DNA refers to small fragments of genetic material released into the bloodstream from dying cells across the body. The unique feature of ctDNA is that it originates specifically from cancerous cells, distinguishing it from the vast majority of cfDNA released by healthy cells. Tumor cells release these DNA fragments through processes like apoptosis and necrosis. Because ctDNA is shed directly from the tumor, it carries the cancer’s unique genetic alterations, allowing researchers to identify the fragments as tumor-derived, even though ctDNA typically constitutes a very small fraction of the total cfDNA pool. In early-stage cancers, this fraction can be less than 0.1%, making its detection a significant technical challenge.
The Liquid Biopsy and Sequencing Process
The process begins with the liquid biopsy, a standard blood draw that is minimally invasive and easy to repeat. The collected blood is processed to separate the plasma, which contains the cell-free DNA, from the intact blood cells. This step typically involves two rounds of high-speed centrifugation to prevent contaminating DNA from healthy white blood cells from interfering with the analysis.
Once the cfDNA is isolated, the sequencing technology must be sensitive enough to detect the tiny percentage of ctDNA within the abundant normal DNA background. Two main technologies address this challenge: digital PCR (dPCR) and Next-Generation Sequencing (NGS). Digital PCR is highly sensitive, capable of detecting variants at a frequency as low as 0.01%, but it can only screen for a limited number of known mutations. Conversely, NGS is less sensitive, with a variant allele frequency threshold often around 0.1% to 1%, but it provides a comprehensive profile by simultaneously screening a broad panel of genes for both known and unknown alterations.
How ctDNA Guides Cancer Treatment
The analysis of ctDNA provides oncologists with dynamic, actionable information across the entire arc of cancer care. A primary application is monitoring the effectiveness of therapy in real-time. A rapid decrease in ctDNA suggests the tumor is shrinking and responding well to the drug. Conversely, if ctDNA levels remain high or begin to rise, it signals treatment failure or disease progression months before conventional imaging, such as CT scans, can confirm the change.
ctDNA is also a tool for detecting minimal residual disease (MRD) following curative treatments like surgery. Even if a patient appears cancer-free on imaging, the persistence of ctDNA fragments indicates that microscopic clusters of tumor cells remain. The presence of MRD is a strong predictor of future relapse and can be detected up to 16 months earlier than clinical recurrence, allowing doctors to stratify high-risk patients for intensive adjuvant therapy.
Repeated ctDNA sequencing is also used to identify the emergence of resistance mutations that cause targeted therapies to fail. As a tumor evolves under the selective pressure of a drug, it can develop new genetic changes, such as the EGFR T790M mutation in lung cancer, that render the current treatment ineffective. A liquid biopsy can identify these resistance-driving mutations, guiding the physician to switch the patient to a more appropriate second-line therapy.
Current Hurdles and Expanding Uses
Despite its promise, the widespread use of ctDNA sequencing faces several significant challenges that require standardization. The primary hurdle is sensitivity, particularly in early-stage cancers where the tumor may not shed enough DNA for current tests to reliably detect it. This low shedding rate can result in false negative results, which is a major concern when using the test for screening or minimal residual disease detection.
Another challenge is the need for standardization across laboratories, as differences in blood collection tubes, processing protocols, and sequencing technologies can all impact the accuracy and comparability of results. Furthermore, the high cost of the most sensitive sequencing platforms limits its accessibility for routine, large-scale screening.
The future application of ctDNA lies in Multi-Cancer Early Detection (MCED) tests, which aim to screen for dozens of cancer types simultaneously from a single blood draw in high-risk or general populations. These tests often analyze epigenetic changes, such as DNA methylation patterns, in addition to mutations, to improve sensitivity and predict the tissue of origin for a detected tumor.