Liquid Biopsies: Understanding the Impact of ctDNA in Modern Medicine

By Anvi Singh¹ and Lipika Chablani², Ph.D. 

¹Wilsonville High School, 6800 SW Wilsonville Rd, Wilsonville, OR 97070, singha@wvhs.wlwv.k12.or.us 
²Department of Pharmaceutical Sciences, Wegmans School of Pharmacy, St. John Fisher University, Rochester, NY 14618. lchablani@sjf.edu

Tissue biopsies have proven their clinical utility over decades and remain the most common method for cancer detection. Annually, they aid in the diagnosis of nearly two million cancer cases in the United States [1]. However, despite their contributions to healthcare, tissue biopsies might not be the optimal cancer detection method for long. They have several drawbacks, including the fact that they are time-consuming, invasive, and require skilled practitioners to execute. In recent years, the focus has shifted towards molecule-based cancer diagnosis, which utilizes components that originate from the tumor [2]. These components include circulating tumor cells (CTCs), cell-free DNA (cfDNA), and circulating tumor DNA (ctDNA), which are all released by the tumor and can be isolated and analyzed through a liquid biopsy process. Cancer detection using this new technology is a potential solution to many of the limitations of core biopsies. 

Graphical Abstract

While deriving specific information from biomarkers like CTCs and cfDNA can be challenging, ctDNA provides a more detailed insight into the developing tumor [3]. It can reveal the approximate size, location, and tumor type. A few different mechanisms are responsible for the release of circulating tumor DNA into the bloodstream, which allows the healthcare team to understand the developing tumors. The proliferation of tumor cells can cause inflammation, nutrient depletion, and oxidative stress/hypoxia, which occurs when the amounts of oxygen reaching the cells are inadequate [4]. This results in a violent tumor cell death termed as necrosis, leading to an explosion of cell content/fragments into the bloodstream. Similarly, apoptosis, a programmed cell death, also occurs in cancer cells but is less frequently the cause of spewing cell content in the bloodstream. Both necrosis and apoptosis eventually contribute to floating ctDNA in the bloodstream, where it has a half-life of just under two hours [2]. Due to the limited half-life, ctDNA sampling always provides the real-time data regarding the progression and state of the tumor as opposed to the delayed data that tissue biopsies provide.

Blood draws serve as the primary source of ctDNA samples. Such samples are analyzed in the lab using a variety of techniques. They offer easy cancer screenings for individuals who may be at risk or have a genetic predisposition to the disease, allowing for early tumor detection, early therapeutic intervention, and the possibility of an increased survival rate [5]. This is especially beneficial for cancers that are found in the internal organs, which are difficult to detect during routine examinations and have a late onset of symptoms [6]. Along with early detection, ctDNA analysis can also predict the failure of a treatment regimen before it is evident from the recurring symptoms or metastasis. Based on these factors, identifying common genetic mutations in the DNA associated with previous therapy failures or resistances can allow the healthcare team to remain vigilant and manage treatments as needed [2]. With each round of successful therapy, the amount of ctDNA decreases [7]. However, in case of an unsuccessful therapy, monitoring ctDNA levels provides information regarding the residual disease and a potential relapse [8]. As an example, in a study conducted on patients with stage II colon cancer, ctDNA was detected in 7.9% of the patients following tumor resection. 80% of those ctDNA-positive patients relapsed, whereas only 9.8% of those with negative ctDNA tests relapsed, suggesting that a patient who tests ctDNA-negative after treatment has a higher probability of being cancer-free [9]. This demonstrates the role of ctDNA in predicting future cancer therapies to minimize relapses, especially in cases where the patient has developed resistance to existing therapies and the mutated tumor is populating more aggressively. For all stages of cancer, whether it be helping with early detection of the disease, mid-therapy for monitoring treatment and identifying therapy resistance, or post therapy for predicting relapse, ctDNA can prove to be an extremely valuable tool.

Even though liquid biopsies using ctDNA are promising, there are still some challenges to overcome before they can be used as a common clinical practice. Perhaps the most evident one is the analyses of these molecules, which are sparsely available in the blood, have limited half-life, and are extremely small in size. Additionally, during therapy, the amounts of tumor DNA that are released depend on how fast the treatment kills the cancer, resulting in fluctuating DNA levels [10]. There are several methods that have already been established for the purpose of extracting ctDNA from samples, but they struggle with cost and specificity. Table 1 provides a summary of ctDNA detection/analytical methods and their pros and cons respectively.

Finally, false positive results can also create an extra burden on the patient’s body as treatments are continued in the absence of the tumor. Often, these treatments damage healthy cells while treating the cancer, and can have detrimental effects if used unnecessarily.

The use of ctDNA sampling for cancer detection offers several advantages over the current standard of care, tumor biopsies, and several ctDNA testing kits that utilize liquid biopsies have even gained FDA approval (as shown in Table 2). These are available for clinical use, while many others are being evaluated. Despite this, ctDNA detection and analysis is still an evolving technology with various drawbacks that are yet to be addressed. Table 3 further provides a comparison of the advantages and drawbacks of ctDNA as a cancer detection tool.

While circulating tumor DNA is most commonly derived from the blood, there are several other bodily fluids that serve as sampling sources of ctDNA and can be useful for cancer detection. In some cases, these alternate non-blood sources are much more precise compared to the blood, due to their proximity to the tumor. Non-blood sources can also serve as an important resource for sampling patients who have anemia, which often develops in advanced-stage cancers [15]. Some of the most common alternatives that are being researched include urine, stool, cerebrospinal fluid (CSF), saliva, sputum, and pleural fluid. The sampling process for these sources is similar to that of ctDNA from the bloodstream; a sample of the source is needed, and then the circulating tumor DNA is extracted and analyzed. An additional advantage of some of these procedures is that they can be sampled at home (example: urine, stool, and saliva), without assistance from a medical professional [15]. Table 4 provides a summary of such alternate non-blood sources for ctDNA sampling. The table further includes the ongoing research using these sample sources in regards to cancer detection, and the pros and cons of using these sources.

In conclusion, ctDNA is a fairly new technology that has the potential to make remarkable advances in the future of cancer detection. Addressing the current issues with ctDNA through improved techniques and rigorous testing will allow liquid biopsies to become cheaper and more reliable, increasing the likelihood of them replacing core biopsies in cancer detection and monitoring. Once implemented and integrated into widespread clinical use, it is probable that they will lower mortality rates from the disease through more sensitive detection, efficient monitoring, and simple post-treatment checks to rule out cancer recurrence. Harnessing the capabilities of ctDNA can improve countless lives, paving the way for new innovations to support several other facets of the medical field. 

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