What’s the Big Deal with Liquid Biopsy and cfDNA?

If you keep hearing about cfDNA (cell-free DNA), liquid biopsy, MRD (minimal/molecular/measurable residual disease), or precision medicine, and you want a quick overview of how these things all fit together in this rapidly developing field, this article is for you.

A (Very) Brief Overview of Disease Detection and Diagnostics

Ideally, a physician should be able to diagnose a patient’s disease and — based on that diagnosis — know the best course of treatment. For millennia, diagnoses relied almost exclusively on reported symptoms and what a physician could hear, see, feel, or taste. A heart murmur may be heard, a tumor might be felt, cataracts might be seen, or babies might be licked (cystic fibrosis causes salt to build up on the skin, though until Dr. Dorothy Andersen characterized it in the late 1940s, it was not recognized as a disease).

The development of modern diagnostic tools, such as biopsies or various imaging techniques like CT or PET scans, has allowed physicians to “see” more deeply into the human body. Yet these methods have severe limitations — tumors may be too small to see, cancers may be located in areas impossible to biopsy, or diseases may not manifest symptoms in the patient until advanced stages, which wouldn’t be a problem if constant monitoring was possible, but constant protein analysis or scans are not feasible for continuous monitoring. Liquid biopsy, which is the collection of cfDNA and/or cfRNA via minimally invasive techniques (blood draw, urine collection, etc.), has emerged as a promising alternative to traditional diagnostics and disease detection because it addresses these concerns and boasts several other crucial advantages.

The Rise of Cell-Free DNA Technologies

Cell-free nucleic acids (cfNAs, which include not only cfDNA/cfRNA produced by healthy tissues in the body, but also those produced by tumor cells and pathogens) have the power to overcome the limitations of traditional diagnostics by providing a much less invasive diagnostic with more sensitivity. cfDNA was first discovered in 1948, when researchers observed the presence of cfDNA in the bloodstream. Subsequently, in 1966, the link between high cfDNA levels and risk of autoimmune disease was made, though the mechanism wasn’t described until decades later.

An illustration of increased levels of cfDNA increasing inflammation, a hallmark of many diseases, including autoimmune diseases. Image taken from Andargie et al. 2023.

In 1977, cfDNA was first used for tumor detection, but molecular technology was not yet advanced enough to take advantage of cfDNA’s full potential. Despite advances in technology, including the advent of PCR in 1985, the first commercially available cfDNA test did not arrive until 2000, and the arrival of the first FDA-approved cfDNA test was 16 years later, in 2016.

Around this time, the use of cfNAs exploded.

Some of the clinical applications of cfDNA. Figure taken from Silva de Miranda et al., 2021

What Are Cell-Free Nucleic Acids and How Are They Used

Cell-free nucleic acids refer to the fragmented DNA and RNA molecules that are constantly released into the bloodstream by various cells in the body, including healthy and diseased cells (circulating tumor DNA, known as ctDNA, is the subset of cfDNA that comes from tumor cells). It is thought that cfDNA is released from cells going through apoptosis and necrosis, but perhaps also through active secretion. Usually, the fragments are ~167 bases long, exactly the length of a strand wrapped around a histone plus the length of the linker piece of DNA that connects histones. It’s important to note that the size of the cfDNA depends on the disease state and specific disease.

An image taken from Shi et al. 2020 showing the different size distributions of cfDNA depending on which cells they come from.

One of the key advantages of cfNA diagnostics is that the collection of cfDNA/cfRNA is minimally invasive (this is generally referred to as the emerging field of liquid biopsy, particularly in cancer applications). Since these DNA/RNA fragments are floating around in the bloodstream, a simple blood test can capture them. Urine, drainage from wounds, and other non-invasive analytes also contain cfNAs with useful assay applications.

It is important to note that the terms used in this space can be murky, and “categories” of applications can be overlapping and ill-defined. Common terms you may encounter are:

1. Liquid biopsy: a minimally invasive alternative to traditional biopsy technologies spanning many applications, but largely used in cancer detection and monitoring
2. Non-invasive prenatal testing/screening (NIPT/NIPS): a minimally invasive application for screening fetal health covering a large number of tests, all with different targets
3. Precision medicine: the broad field of medical care designed to optimize efficiency or therapeutic benefit to a patient by using genetic or molecular profiling
4. Minimal/measurable/molecular residual disease (MRD): the medical condition in which a small number of cancer cells persist in a patient either during or after treatment at levels undetectable by traditional detection methods

Looking at the cfDNA/cfRNA also allows diseases to be detected with high sensitivity. Since they are nucleic acids, amplification techniques like PCR can find even a single copy of the DNA and amplify it so that it can be read. Then, by analyzing the unique genetic signatures present in an individual’s cfDNA, healthcare providers can tailor treatments and preventive strategies to the specific needs of each patient, ultimately improving outcomes and reducing the risk of adverse reactions. While this is already being done at the genome level (for example, the TPMT gene test can inform the right dosage of medicine for children with leukemia), many diseases cannot be diagnosed at the genome-wide level. Therefore, looking at cfDNA can provide a much more targeted diagnosis.

While we think of the human body as having one definitive genome, this is grossly simplified. As our cells replicate and divide, they slowly accumulate errors over our lifetime. One cell’s genome may differ subtly from another cell, even within the same tissue type in a person’s body. With diseases like cancer, cfDNA shed from the cancer may hold different signatures than cfDNA shed by the rest of the healthy cells, thereby allowing for detection. The first FDA-approved cfDNA test did exactly this with non-small cell lung cancer patients, allowing physicians to detect which mutations were present and inform a personalized, effective treatment plan in less than four hours, a massive leap forward in the field of precision medicine.

A brief overview of the differences between traditional medicine and the growing field of precision medicine.

However, even cfDNA isn’t always enough to paint the whole picture, and the field turns to cfRNA. Placental-associated hypertensive disorders of pregnancy, like preeclampsia, affect 1 in 6 pregnancies. Traditionally, clinicians look at a person’s previous medical history, their family’s medical history, and the person’s race to determine a risk factor. This leads to unequal care and is not sufficiently effective at predicting when problems will arise and how serious the symptoms will be. A recent study looking at the cfRNA of 9,102 pregnant people shows that we can monitor and treat these disorders with high accuracy and earlier than ever before, and now a test is being commercialized by Mirvie, a biotechnology company focused on pregnancy health.

Challenges to Progress and Adoption

While the use of cfNAs is accelerating, two broad challenges have significantly slowed progress and adoption in clinical settings.

First, cfNAs are not abundant, which poses severe technical challenges. There are not many cell-free nucleic acids floating around at any one time, and the body is very good at cleaning up fragments of DNA/RNA outside of cells, so detecting cfNAs is a little like trying to grab a handful of snowflakes in mid-air on a warm day. This creates a myriad of pre-analytical variations that can be difficult to manage, and we’ll dive deeper into this in our next blog post.

An overview of some of the technical difficulties of working with cfDNA from Tabrizi et al. 2024

Second, put simply, there is so much more to learn, which compounds both the difficulty in making these assays and in getting them approved. The promise of precision medicine, one of the largest applications for cfNAs, is that by detecting the mutation(s) present, you can personalize the treatment for more effective disease management. However, this requires large studies to understand how to effectively treat each mutation profile, and then, after the research is done, yet another study to achieve regulatory approval. Coupled with the fact that many mutations are exceptionally rare, and there may be many treatment options available, these studies can take a long time and many resources. Furthermore, with each disease and treatment, standardized protocols and robust quality control frameworks must be agreed upon by a large consensus of practitioners, which is an incredibly important and challenging task on its own.

An overview of circulating tumor DNA (ctDNA) assay development workflows, which is only a subset of the cfDNA applications, demonstrates the complexity of the field and how much needs to be developed and standardized at every step. Image taken from Bronkhorst et al. 2023.

Who is Working on These Challenges?

These challenges are well known, and a community focused on addressing them is growing. Aside from a large number of academic and industry research groups tackling the technical challenges, several groups have risen to address the regulatory and clinical implementation challenges, speeding up adoption in the clinic.

Large consensus initiatives are the key, and the International Society of Liquid Biopsy (ISLB) is one such group emphasizing the need for standardized methodologies and diagnostic workflows. By forming the Quality Control and Accreditation Committee, they have focused on mitigating pre-analytical variability and are ensuring adherence to international standards like ISO 15189 and CLIA/CAP. However, they are just one such player striving towards this goal, and finding consensus with other groups like the European Society of Medical Oncology (ESMO), American Society of Clinical Oncology (ASCO), and International Liquid Biopsy Standardization Alliance (ILSA) is crucial for integrating these new technologies into the clinical setting.

Consortia like BLOODPAC are another example of how the community is coming together to determine best practices and standardize workflows. Formed in 2016, BLOODPAC has brought together 68+ members across academia, non-profits, industry, government agencies, and pharma, and aims to keep collaborating with diverse consortia “to accelerate the development, validation, and clinical use of liquid biopsy assays to better inform medical decisions and ultimately improve patient care and outcomes.”

Where is the Field Now?

The cfDNA diagnostic market is rapidly expanding (CAGR 25.9%), with numerous companies at the forefront of this revolution. Some of the leading players in this space include Guardant Health, Foundation Medicine, and Natera, all of which have developed FDA-approved cfDNA tests for various applications, such as cancer detection, prenatal testing, and organ transplant monitoring. Companies like Karius are taking it one step further and looking at non-human cfDNA, specifically looking at pathogen cfDNA found in a patient’s blood, which can be invaluable for quickly diagnosing and treating infections.

Figure from Decibio, published July 2024 with a selection of liquid biopsy players.

We’re still in the early days of this new wave of cell-free technology. Though only a handful of cfDNA tests are FDA-approved so far, more are on the horizon as they move through the regulatory process. Groundbreaking research, like the cfRNA study mentioned above, is also fueling more and more diagnostics for diseases in dire need of better testing. However, the highly complex nature of working with cfDNA, including challenges in sample collection, processing, and storage, has presented significant technical obstacles for researchers and developers. Stay tuned for our upcoming deep dive into some of these technical complexities!

In sum, as the field continues to evolve and overcome the existing barriers, the advent of cell-free testing has ushered in a new era of healthcare, offering unprecedented opportunities for earlier detection, personalized treatment, and improved patient outcomes.