Circulating tumor DNA (ctDNA) sequencing is a method for detecting and analyzing small fragments of DNA released by cancerous tumors into the bloodstream. This analysis, often called a liquid biopsy, provides a non-invasive way to gather genetic information about a tumor from a simple blood sample. This approach allows for the identification of genetic alterations associated with the cancer, helping to paint a clearer picture of the disease.
The Biological Origin of Circulating Tumor DNA
Every tumor is composed of cancer cells that grow and divide. As these cells die from natural processes or by outgrowing their blood supply, they break apart. When this happens, they release fragments of their DNA into the patient’s bloodstream, which is known as circulating tumor DNA, or ctDNA.
These small DNA fragments circulate throughout the body in the blood plasma. The amount of ctDNA can vary significantly from person to person and depends on the type, size, and stage of the tumor, as well as the rate at which its cells are shedding DNA.
It is important to understand that ctDNA is a specific type of cell-free DNA (cfDNA), a broader term for any DNA found freely circulating in the bloodstream. Healthy cells also release their DNA into the blood when they die. A challenge of ctDNA analysis is to distinguish the DNA fragments from tumor cells from the much larger background of cfDNA released by normal cells.
The ctDNA Sequencing Process
The process begins with a standard blood draw. In the laboratory, the first step is to separate the plasma—the liquid portion of the blood—from the red and white blood cells and platelets. This is accomplished by spinning the blood sample in a centrifuge, which causes the heavier cellular components to separate from the plasma.
Following separation, the next task is to isolate the cell-free DNA from the plasma. Because cfDNA, including any ctDNA that may be present, exists in very small quantities, specialized kits are used to extract these DNA fragments. This process filters out other components of the plasma, resulting in a purified sample of cfDNA.
This isolated DNA is then prepared for analysis using Next-Generation Sequencing (NGS). NGS platforms are capable of reading millions of small DNA fragments simultaneously, allowing for a comprehensive analysis of the genetic code. The sequencer deciphers the precise order of the nucleotide bases (A, C, G, and T) that make up the DNA, generating a large amount of raw genetic data.
The final step involves bioinformatic analysis to interpret the sequencing data. Computer programs compare the genetic information to a reference human genome to identify specific mutations that are known hallmarks of cancer. This analysis can pinpoint the low-frequency variants characteristic of ctDNA amidst the background of normal cfDNA, revealing the tumor’s genetic profile.
Clinical Applications in Oncology
The information gathered from ctDNA sequencing has several practical applications in managing cancer care.
- Early detection: Because ctDNA can be shed by tumors at an early stage, sequencing this DNA could identify the presence of cancer before a person develops symptoms, offering a new avenue for screening individuals at high risk.
- Guiding targeted therapy: Many modern cancer treatments target specific genetic mutations. By identifying the mutations in a patient’s tumor DNA, ctDNA analysis helps oncologists select the most effective drug for that individual’s cancer, a core principle of precision medicine.
- Monitoring treatment response: The levels of ctDNA in the bloodstream often correlate with the tumor’s size and activity. By taking periodic blood samples, doctors can track these levels; a decrease may indicate the treatment is working, while an increase could signal that the cancer is growing or has become resistant.
- Detecting cancer recurrence: After a tumor has been treated, trace amounts of cancer can remain, known as minimal residual disease (MRD). ctDNA analysis can detect these microscopic remnants long before they would be visible on imaging scans, providing an early warning that the cancer may be returning.
Comparison with Traditional Tissue Biopsies
A primary difference from a traditional tissue biopsy is the level of invasiveness. A tissue biopsy is a surgical procedure that involves inserting a needle or making an incision to remove a physical piece of the tumor, which can involve risks and recovery time. In contrast, a liquid biopsy requires only a simple, low-risk blood draw.
Another distinction is the comprehensiveness of the genetic picture each method provides. A tissue biopsy analyzes a sample from a single location within a tumor, but tumors are often genetically diverse. A liquid biopsy, on the other hand, captures DNA shed from all tumor sites, potentially offering a more complete profile of the cancer’s overall genetic landscape.
The ease of repeatability also sets the two methods apart. Performing multiple tissue biopsies over time to monitor a cancer’s evolution is often impractical for the patient. Because liquid biopsies are minimally invasive, they can be performed frequently, allowing clinicians to track genetic changes in the tumor in response to treatment or as the disease progresses.