Blood test for cancer Liquid Biopsy: ctDNA in breast cancer

Liquid Biopsy: ctDNA in Breast Cancer

Introduction

In an era where medicine is moving towards precision and personalized medicine, the concept of non-invasive cancer diagnosis and monitoring is a significant change in the medical world . “Liquid Biopsy” technology is an analysis of Circulating Tumor DNA (ctDNA), which is genetic material from cancer cells that has escaped into the bloodstream.

Liquid biopsy technology enhances the potential for continuous treatment monitoring, minimal residual disease detection, and drug resistance analysis, while reducing the need for traditional tissue biopsy, which carries risks and limitations. This article presents the basic knowledge, technology, application guidelines, as well as limitations and challenges in using liquid biopsy for breast cancer patients.

1. Basic understanding of Liquid Biopsy and ctDNA

1.1 Definition and Importance of ctDNA Circulating Tumor DNA (ctDNA) is a DNA fragment released into the bloodstream by cancer cells, which often has specific biological characteristics, such as mutations, abnormal methylation patterns, or molecular size that differs from cell-free DNA (cfDNA) derived from normal cells.

1.2 Biospecimen sources

• Plasma and Serum: Centrifugation of blood is the most popular method for ctDNA testing.

• Cerebrospinal Fluid (CSF): Ideal for cancers in the central nervous system.

• Other fluids: such as urine, saliva, and pleural/peritoneal fluid can be used in some cancers.

  
  

1.3 Technology used for ctDNA testing

• PCR-based Methods: such as Digital Droplet PCR (ddPCR) and BEAMing are suitable for detecting mutations at known loci.

• Next-Generation Sequencing (NGS): A sequencing technology that can analyze multiple mutations simultaneously, including methylation and fragmentation.

• Multimodal Approaches: Integrated approaches that analyze multidimensional data (e.g. Mutation, Methylation, Copy Number Variation) to increase the sensitivity and accuracy of the test.

  
  

2. Application of Liquid Biopsy in Breast Cancer Patients

2.1 Screening and Early Diagnosis Although it is not currently approved for use as a screening test in the general population, there are studies that indicate its benefits in high-risk groups, such as those with BRCA1/2 gene mutations or those with dense breast tissue.

Limitations of using Liquid Biopsy for screening

Although early screening for breast cancer is the ultimate goal, the use of liquid biopsy in the general population or high-risk groups who are asymptomatic is not suitable. In early-stage cancer, the amount of ctDNA in the bloodstream is very low, resulting in insufficient sensitivity of the test and may lead to false negative results as follows:

1. Low sensitivity in the early stages and risk of "false negative" results (Low Sensitivity & False Negative Risk)

The amount of ctDNA in stage 1 or earlier cancers is so low that current technologies may not detect it, potentially creating a “false confidence” that could lead to the omission of standard screening tests such as mammograms, which could lead to a delayed diagnosis if cancer does develop.

2. The Problem of "False Positives" and Its Consequences

Although the specificity of mass screening is as high as 99%, when applied to millions of people, the number of "false positives" is still very high. This is mainly due to CHIP (ChIP mutations in blood cells).

• Creates anxiety in those who are positively affected

• Over Diagnostic Investigation: Patients are subjected to costly and potentially risky investigations, such as whole-body PET/CT scans, laparoscopy, or even unnecessary surgery, to look for cancers that do not actually exist.

• Burden on healthcare systems: Medical and personnel resources are being spent on tracking these false positives.

  
  

3. Identification of the location of cancer (The "Tissue of Origin" Problem)

When a ctDNA test comes back positive in a person with no history of cancer, the most important question is, "Where is the cancer?" Early liquid biopsy technologies could not pinpoint the origin of the cancer, forcing doctors to begin the process of "searching" for cancer throughout the body, which directly ties into the problem of unnecessary additional diagnostic testing in point 2. (Although newer technologies are beginning to analyze methylation patterns to better pinpoint the location, they are still not perfect.)

4. Overdiagnosis

Very sensitive tests may detect indolent cancers, which may never cause symptoms or become life-threatening to the patient, leading to "overtreatment" in which the patient may suffer side effects from surgery, radiation or chemotherapy without receiving any real life-extending benefit.

5. Cost-Effectiveness

Currently, Liquid Biopsy is still very expensive. Using it to screen the entire population or even risk groups requires a huge budget. In order to push for it to become a public health policy, there must be clear evidence that the benefits of early cancer detection outweigh the cost of testing and the cost of following up on false positives. Such data still has to wait for large-scale, long-term studies. Due to these important limitations, the use of Liquid Biopsy for screening is still mainly in the "research area" and is not yet recommended as a replacement for existing standard screening.

2.2 Monitoring of residual disease after treatment (Minimal Residual Disease - MRD)

• Early detection of recurrence: ctDNA can detect residual disease an average of 3.4–18.5 months earlier than radiological examination.

• Prognosis: Patients with no ctDNA detected after treatment completion tend to have a longer disease-free survival, while those with ctDNA still detected have a high risk of relapse of up to 85%.

Although the use of ctDNA for MRD monitoring has great potential to detect recurrence early and help with prognosis, there are still important limitations and challenges to consider, including:

1. Sensitivity and risk of false negatives

Very low residual disease (MRD) releases extremely low levels of ctDNA, possibly below the limit of detection by current technologies. This can lead to “false negative” results , where the patient still has cancer cells but the test result is negative, leading to misconceptions among patients and physicians, and potentially missed opportunities to start treatment early.

2. Specificity and false positives from CHIP

Some of the mutations detected are not necessarily from cancer cells, but rather from Clonal Hematopoiesis of Indeterminate Potential (CHIP) , a blood cell mutation that is becoming more common with age. This can lead to a "false positive" result , in which the patient may no longer have cancer cells but the test result is positive, leading to anxiety, unnecessary further testing, or even overtreatment.

3. Tumor Biology Heterogeneity

Each type of cancer or even each patient has a different ability to release DNA into the bloodstream (Shedding). Some patients may be "Low-shedder" or "Non-shedder", meaning that even though there is remaining disease, ctDNA cannot be detected. Therefore, Liquid Biopsy cannot be used effectively in all patients. And a negative result may not always mean that there is no remaining disease.

4. Lack of Clear Clinical Actionability

There is currently no consensus or standardized guideline on what to do next when ctDNA is detected in MRD. Evidence from large trials supporting that prompt treatment initiation can prolong overall survival remains limited. Clinicians and patients face difficult decisions about whether to continue close monitoring or initiate treatment with potentially serious side effects without clear evidence of benefit.

Summary table of limitations of using ctDNA for MRD monitoring

Limitations	Description	Impact on patients/treatment
False Negative	The amount of ctDNA is too low to detect.	Misconceptions may occur, which may lead to delayed detection of recurrence.
False Positive	Mutation detected from blood cells (CHIP)	Anxiety, unnecessary testing/treatment
Cancer biology	The ability to release DNA varies from person to person.	This method may not work in some patients (low-shedders).
Treatment guidelines	There is no clear evidence on what to do if the result is positive.	Uncertainty in deciding on the next treatment option

2.3 Evaluation of preoperative chemotherapy (Neoadjuvant Chemotherapy) is very important in triple-negative breast cancer (TNBC):

• Decline in ctDNA levels during treatment is associated with pathological complete response (pCR).

• Patients with still detectable ctDNA after surgery had a significantly higher rate of disease recurrence.

The use of ctDNA to monitor response to preoperative chemotherapy is a potential approach, particularly in TNBC. However, it has limitations and issues that need to be carefully considered before it can be used for practical clinical decision-making, including:

1. Imperfect Prediction of pCR

Although clearance of ctDNA is a very good sign, it does not guarantee a 100% pathological complete response (pCR). Conversely, some patients may achieve pCR despite low levels of ctDNA. Therefore, using ctDNA results alone to decide to stop or reduce chemotherapy prematurely is highly risky and is not yet accepted in standard practice.

2. Limited Actionability for Treatment Modulation

This is the most important limitation at present. Although ctDNA is not found to decrease (indicating that it may not respond to drugs), there is still no clear evidence from large trials that adjusting the chemotherapy regimen based on ctDNA results can actually improve overall survival. The decision to change treatment based on ctDNA results is still in the realm of clinical trials only. It is not yet possible to use it as a standard practice.

3. Lack of Standardized Timing

There is currently no standard consensus on the best time to perform a ctDNA blood test (e.g., after the first, second chemotherapy doses, or before surgery). Chemotherapy itself may affect total cfDNA levels in the blood, complicating interpretation. Results can vary depending on the timing of blood draws, making it difficult to compare data between institutions and set a clear cut-off point for decision-making.

4. Interference from CHIP

As in other contexts, the detection of low levels of mutations after initiation of treatment may be a sign of CHIP, not necessarily of drug-resistant cancer cells, which may lead to misinterpretation of the patient as not responding to treatment and may lead to unnecessary treatment escalation.

Summary table of limitations of using ctDNA in NACT follow-up.

Limitations	Description	Impact on clinical decision making
Prediction of pCR is incomplete.	A negative ctDNA result does not guarantee 100% that a pCR will occur.	It cannot be used to make decisions to prematurely stop standard treatment.
There is no standard for changing treatment.	There is insufficient evidence that changing medications based on ctDNA results will prolong survival in patients.	The decision to change the drug formula still needs to be within the research scope.
Lack of time standards	There is no consensus on when is the best time to get a blood test.	It is difficult to compare results and set clear guidelines.
CHIP interference	The signs found may not be drug-resistant cancer cells.	This may lead to inaccurate interpretations of drug unresponsiveness.

2.4 Monitoring of metastatic disease and drug resistance mechanisms

• Tumor Burden Monitoring: Blood ctDNA levels are often associated with changes in tumor size.

• Detection of resistance mutations: New mutations that arise during treatment, such as ESR1 gene mutations, can be detected, which are associated with resistance to aromatase inhibitors.

  
  

  The use of liquid biopsy to monitor disease volume and detect drug resistance mechanisms is very useful and is one of the accepted applications in practice. However, there are still limitations to consider, including:

1. Discordance with Radiographic Imaging

A situation may arise where blood ctDNA levels rise (indicating that the disease may be worsening) but CT scans or PET scans still show stable disease. This phenomenon, known as "biochemical progression without radiographic progression," creates significant decision-making difficulties. Physicians must consider whether to change treatment based on worsening blood results or wait until clear changes are seen on imaging, for which there is no standard consensus.

2. Limitations from Tumor Biology and Site of Metastasis

• Low/Non-Shedders: Some patients have tumors that do not release high enough amounts of ctDNA, resulting in blood ctDNA levels that do not reflect the true amount of the tumor.

• Lesion location: Metastases to certain organs, such as the brain (due to the blood-brain barrier) or bones, may release less ctDNA into the bloodstream than metastases to the liver or lungs.

  
  

  Patients may have metastatic disease in the brain or bones, but blood ctDNA levels are not elevated, leading to false confidence and reaffirming that liquid biopsy cannot replace routine radiological examinations.

  
  

3. Complexity of Mutation Interpretation

• Subclonal Mutations: Mutations detected may originate from a small subclone of cancer cells, which may not be the primary cause of drug resistance.

• CHIP interference: It remains a problem to distinguish cancer cell mutations from blood cell mutations

  
  

  The discovery of a new mutation does not necessarily mean a change in treatment. Interpretation requires expertise to assess the clinical significance of the mutation.

  
  

4. Non-Actionable Findings

Technology may be able to accurately detect drug-resistant mutations, but there are currently no targeted therapies approved for that mutation in breast cancer, leaving doctors and patients with worrying information that may not immediately lead to treatment changes, leading to stress without further treatment options.

Summary table of limitations of using ctDNA for monitoring metastatic disease.

Limitations	Description	Impact on treatment planning
Inconsistency with Imaging	Blood test results are getting worse, but CT scan results are stable.	Uncertainty in deciding when to change treatment
Biology and location of disease	Some cancers/some sites release less ctDNA.	It cannot be used as a substitute for imaging and may underestimate the actual disease.
Complexity of interpretation	The mutation found may not be the main cause of drug resistance	It requires a high level of expertise to interpret the results and may lead to inappropriate treatment.
Unable to lead to treatment	Mutations found, but no specific drugs available	Creating concern without further treatment options

3. Using Liquid Biopsy to Guide Treatment

• Identification of Actionable Mutations: The detection of mutations in PIK3CA , HER2 , and ESR1 genes allows physicians to select appropriate targeted therapy.

• Comparison of the roles between Tissue Biopsy and Liquid Biopsy:

  • Tissue Biopsy: Still necessary for initial diagnosis, accurate testing of Fusion genes and Copy Number Alterations.

  • Liquid Biopsy (CGP): This is particularly useful when insufficient biopsy material is available, the patient is in a pre-clinical condition, or in situations where rapid results are required.

    
    

    “Using Liquid Biopsy to Guide Treatment” is very important because it directly affects the decision of choosing drugs for patients. Using Liquid Biopsy to detect actionable mutations is one of the most tangible benefits of this technology. However, its practical application faces several limitations and challenges.

Limitations:

1. Risk of False Negatives and Missed Treatment Opportunities

This is the most important limitation in this context. A patient may have a drug-responsive mutation (e.g. PIK3CA ) in their tumor, but because the tumor is a low-shedder, it is not detected in the blood. A “negative” result from a liquid biopsy does not 100% confirm the absence of the mutation, and may result in a “missed opportunity” for the patient to receive an effective targeted therapy. For this reason, most guidelines still recommend a tissue biopsy if the blood test is negative but clinically suspicious.

2. Discordance between Tissue and Liquid Results

There may be cases where the results of tissue and blood tests give inconsistent results, which is due to the biological diversity of cancer (Tumor Heterogeneity) or changes of cancer over time (Temporal Evolution), creating a difficult clinical dilemma as to which test result to believe more. Generally, if the Liquid Biopsy gives a "positive" result, it is usually reliable, but if it gives a "negative" result, it is less reliable and more weight is given to the result of the tissue test.

3. Limitations in Detecting Certain Mutation Types

Liquid Biopsy is highly effective in detecting point mutations but has limited ability to detect other types of genetic changes such as:

• Gene Fusions : Difficult to detect from broken DNA fragments in the blood.

• Copy Number Alterations (Group gains or losses of copies of a gene): Particularly in the assessment of HER2 amplification , where biopsy-based testing (IHC/FISH) remains the gold standard.

  
  

  Liquid biopsy cannot replace all genetic testing from tissue samples. The choice of testing method therefore depends on the type of mutation to be found.

4. Drug Access and Regulatory Hurdles

Even if an “Actionable Mutation” is detected, targeted drugs that specifically target that mutation may not yet be approved for breast cancer in Thailand, or may be very expensive and not covered by the treatment plan. This gap between the “found data” and the “actual treatment available” can create expectations and disappointment for patients.

Summary table of limitations of using liquid biopsy to guide treatment

Limitations	Description	Impact on treatment choice
False Negative	No actual mutations were detected in the tumor.	Patients may miss out on opportunities to receive effective targeted medications.
Inconsistent test results	Blood test results and biopsy results conflict.	There is confusion in decision making and the discretion of an expert is required.
Technical limitations	Poor detection of certain types of changes (e.g. Gene fusions, HER2 amp)	Tissue biopsy is required for selecting certain therapeutic targets.
Access to medicines	Targets are detected but there is no medicine available or the medicine is not covered by the treatment rights.	The information obtained cannot lead to any truly useful treatment.

5. Approval status and international guidelines

• FDA Approval (US):

  • (✓) Approved: For use in metastatic setting patients to detect drug-specific mutations.

  • (✗) Not yet approved: for screening or use as a standard MRD monitoring tool.

• Guidelines from international guidelines such as ESMO, NCCN:

  • Liquid Biopsy is recommended when tissue examination is difficult.

  • It is recommended to repeat the test if the result is negative but the disease is still suspected.

6. Summary

Dimension of challenge	details
Sensitivity	It is still limited in early-stage cancer due to low ctDNA abundance.
Specificity	False positive results may occur from clonal hematopoiesis (CHIP), which is a mutation in non-cancerous blood cells.
Standardization	The analysis process, reporting and interpretation still lack universal standards.
Cost-Effectiveness	The cost of testing remains high, and further studies are needed to demonstrate its cost-effectiveness in different contexts.

Future Directions

• AI-based Algorithms: Using AI to analyze ctDNA data to predict diseases and track changes in real time

• Multi-omics Platforms: Integrating multiple data types (genetic, proteomic, methylation) to create highly accurate prognostic models.

• Large-scale clinical trials: To generate robust evidence and push Liquid Biopsy into standard practice.

ctDNA analysis or Liquid Biopsy is an important tool in the personalized care of breast cancer patients. Although it cannot replace the primary diagnosis, it is very useful for monitoring treatment, indicating the use of targeted drugs, and monitoring drug resistance. In the future, it is likely to become the standard of care for patients.