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February 2024
Liquid Biopsy
Sometimes cancer is easy to diagnose and sometimes it takes months to arrive at an answer. To tackle an opponent as widespread and insidious as cancer, we need all the tools that modern science has to offer – and now, we have one more – liquid biopsy.
A New Tool in the Fight Against Canine Cancer
Cancer is a big problem in veterinary medicine. In fact, it’s probably on your list of differential diagnoses multiple times each week—think of that senior ADR dog, the patient with hypercalcemia, or the dog that arrives at your office limping. Sometimes cancer is easy to diagnose and sometimes it takes months to arrive at an answer. To tackle an opponent as widespread and insidious as cancer, we need all the tools that modern science has to offer—and now, we have one more—liquid biopsy. In short, liquid biopsy is the long-awaited “blood test for cancer,” finally allowing us to say “yes” when asked “will the blood work test for cancer?”
How It Works
So, what is liquid biopsy and how does it work? Liquid biopsy involves the analysis of biomarkers in various biological fluids. For this article, we will be focusing on blood-based liquid biopsy analyzing a biomarker called “cell-free DNA” using an advanced technology called “next-generation sequencing (NGS).” When thinking about how this technology works, there are a few basic principles to keep in mind. First, abnormalities in DNA are the root cause of cancer. Second, cells of the body (including cancer cells, if present) are constantly turning over (dying and being replaced) and releasing pieces of DNA into the bloodstream. Third, these pieces of DNA can be isolated from a blood sample and analyzed using NGS to look for abnormalities in the DNA.
This technology, often referred to as “NGS-based liquid biopsy,” evaluates the quality of the DNA circulating in blood—looking for abnormalities in the DNA that are specific to the presence of cancer. It’s important to distinguish NGS-based liquid biopsy from protein-based blood tests that examine the quantity of select proteins in the body (such as nucleosomes), as protein-based tests are not specific for cancer and may frequently return moderate (indeterminate) or false positive results due to conditions that cause protein elevation, like inflammation or infection.
A liquid biopsy starts with a blood draw in the exam room.
For the veterinary team, the NGS-based liquid biopsy process is simple: blood is drawn into specialized tubes that help stabilize the DNA at room temperature for up to seven days, the sample is shipped to the lab, and results are returned in about a week. On the laboratory side, when the sample arrives, DNA is extracted and sequenced using NGS. If abnormalities are identified in the DNA, it is likely that cancer is present in the patient and a positive result is issued. If no abnormalities are identified, it is unlikely that cancer is present, and a negative result is issued. Similar to a traditional biopsy, liquid biopsy tells whether cancer may be present at the time the sample is taken, not whether cancer may develop in the future.
How Good Is It?
At this point you may be wondering: What type of cancer can be found using blood, and how good is this blood test at finding cancer? The answers to these questions come from the test’s clinical validation study, which was published in 2022.
In this large study (involving over 1,000 dogs with and without cancer), 30 different types of cancer were detected using NGS-based liquid biopsy. Some cancer types had very high detection rates; for example, for lymphoma, hemangiosarcoma, and osteosarcoma, the test had a high detection rate of 85%, but for cancer types more readily detectable on a physical exam, such as anal gland adenocarcinoma and mast cell tumor, detection rates were closer to 25%. So, as a general rule of thumb, if you see something that you can easily sample for cytology or histopathology (like a cutaneous or subcutaneous mass)—go ahead and sample it! Liquid biopsy doesn’t replace your need for a tissue biopsy, so if you can access a mass you suspect to be cancer, you will generally get more information from a tissue biopsy (or fine-needle aspiration) than a liquid biopsy.
Liquid biopsy is highly specific for cancer, because it is looking for DNA abnormalities that should not typically be found in healthy dogs or in dogs with conditions other than cancer.
Liquid biopsy is highly specific for cancer, because it is looking for DNA abnormalities that should not typically be found in healthy dogs or in dogs with conditions other than cancer. Because of this, the false positive rate of the test is very low, only 1.5%, which is critically important when testing for a condition as important and impactful as cancer. If these DNA abnormalities are found, indicating that cancer cells are present in the body, this is called a “cancer signal.”
Results from liquid biopsy are a straightforward positive or negative, which are reported out as “Cancer Signal Detected” or “Cancer Signal Not Detected,” respectively. In some cases, the pattern of DNA abnormalities identified in the sample indicates that a specific type of cancer is likely to be present—particularly, a hematological malignancy (lymphoma or lymphoid leukemia). This is known as a “Cancer Signal Origin” prediction and, when encountered, is included in the report.
Following a positive result, the next step is a workup to find where the cancer signal may be coming from, which may include a thorough history and physical exam, routine labs (if not recently performed), imaging (chest X-rays and/or abdominal ultrasound), and sampling of any detected masses or enlarged lymph nodes. In rare cases, advanced imaging (such as echocardiogram and/or CT) may be indicated.
Nearly 90% of patients with positive results from liquid biopsy had their cancer readily found, with the vast majority of these diagnoses achieved using in-house capabilities within two weeks.
At first, the idea of going on a “cancer hunt” may seem a bit overwhelming, but data tell us otherwise. In a 2023 study published in the Journal of the American Veterinary Medical Association involving real world clinical samples submitted for liquid biopsy testing, nearly 90% of patients with positive results from liquid biopsy had their cancer readily found, with the vast majority of these diagnoses achieved using in-house capabilities (without referral to a specialist) within two weeks.
Practical Applications
NGS-based liquid biopsy can be used in a variety of clinical scenarios to detect and monitor cancer in dogs: as a screening tool for healthy dogs, as an aid in diagnosis for dogs in which cancer is suspected, and to monitor dogs who have received a cancer diagnosis.
Screening for Healthy Dogs
Arguably, one of the most exciting uses of liquid biopsy is for screening apparently healthy dogs at high risk of cancer, such as older dogs and those belonging to certain breeds. The goal of cancer screening is to detect cancer earlier, at a time when outcomes are better and treatment is more likely to be successful. Cancer screening is a routine practice in human medicine, with tests like mammograms and colonoscopies regularly employed for the early detection of cancer. Such screening tools do not exist for dogs, and so we rely heavily on the annual or biannual wellness visit to help detect cancer in our canine patients.
DNA is extracted and analyzed in the lab.
Unfortunately, no matter how thorough an exam is, some cancers hide in areas of the body that are difficult (or sometimes impossible) to assess by physical exam. In fact, in a recent study published in the Journal of Veterinary Internal Medicine involving over 350 dogs with cancer, only 4% had their disease detected at a wellness visit. The ability to identify cancer in a larger proportion of asymptomatic patients has the potential to be a game changer in terms of patient outcomes, as it is well established that preclinical detection of cancer is associated with improved outcomes for a variety of cancer types. As a general recommendation, all dogs should begin cancer screening at age 7, but dogs of certain breeds and sizes should start screening earlier, in some cases as early as age 4.
Suspicion of Cancer
When cancer is already suspected in a patient, liquid biopsy can be used as an aid in diagnosis to help move cancer up or down on the list of differential diagnoses. There are a wide range of scenarios in which a noninvasive tool for cancer detection can offer utility in the workup of patients in which there is a clinical suspicion of cancer. For example, a patient may present with clinical or nonspecific findings (e.g., unexplained weight loss, reduced appetite, or lethargy) with no obvious mass or lesion to sample for traditional diagnostics; changes on a patient’s lab results may warrant further studies, such as unexplained findings on a complete blood count (e.g., cytopenias), hypercalcemia, or inconclusive lymph node cytology; or, a patient’s imaging results may be suspicious for malignancy, but in a location that poses a challenge to using traditional diagnostics (e.g., an abnormality of the spleen, an aggressive bone lesion, or nodules, masses, or enlarged lymph nodes on a chest X-ray).
In each of these scenarios, liquid biopsy offers an alternative for determining whether the patient could have cancer—by simply drawing blood. When liquid biopsy is incorporated early in a patient’s workup, concurrent with other tests, it can provide valuable information with the potential to help shorten the path to a diagnosis. The quicker a diagnosis can be made, the sooner that family can consider treatment options—hopefully, at a time when interventions are more likely to be successful.
One of the most exciting uses of liquid biopsy is for screening apparently healthy dogs at high risk of cancer, such as older dogs and those belonging to certain breeds.
After a Diagnosis
After cancer has been diagnosed and a treatment plan has been put into place, liquid biopsy can be used for cancer monitoring to help answer some important questions like “did we get it all?” and “will it come back”? For patients that have a positive liquid biopsy result followed by excisional surgery to diagnose and treat cancer, liquid biopsy can be drawn seven to 30 days following surgery to determine if residual disease remains in the body (either at the surgery site or at a distant metastatic site).
Those patients can also be monitored with periodic liquid biopsy tests throughout treatment (typically every two to three months, but frequency may vary by cancer type) to determine whether cancer is recurring. In some cases, liquid biopsy can detect recurrent cancer months prior to the re-emergence of clinical signs, alerting the clinician to perform a thorough workup to confirm the recurrence, hopefully before the patient gets sick from the return of the disease.
Changing the Future of Cancer
NGS-based liquid biopsy is positioned to change how cancer is detected and monitored in dogs—by helping to identify cancer in healthy dogs, to aid in a quick and efficient diagnosis for dogs suspected of having cancer, and to help monitor dogs with cancer to identify signs of residual disease and disease recurrence. By combining liquid biopsy with the existing tools in the diagnostic toolbox, the veterinary team becomes an even more powerful force in the fight against canine cancer.
References
- Flory A, Kruglyak KM, Tynan JA, McLennan LM, Rafalko JM, Fiaux PC, et al. Clinical validation of a next-generation sequencing-based multi-cancer early detection “liquid biopsy” blood test in over 1,000 dogs using an independent testing set: The CANcer Detection in Dogs (CANDiD) study. PLOS One. 2022; 17(4):e0266623.
- O’Kell AL, Lytle KM, Cohen TA, Wong LK, Sandford E, Rafalko JM, et al. Clinical experience with next-generation sequencing–based liquid biopsy testing for cancer detection in dogs: a review of 1,500 consecutive clinical cases. J Am Vet Med Assoc. 2023; 1–10.
- Flory A, McLennan L, Peet B, Kroll M, Stuart D, Brown D, et al. Cancer detection in clinical practice and using blood-based liquid biopsy: A retrospective audit of over 350 dogs. J Vet Intern Med. 2023; 37(1):258–67.
- Rafalko JM, Kruglyak KM, McCleary-Wheeler AL, Goyal V, Phelps-Dunn A, Wong LK, et al. Age at cancer diagnosis by breed, weight, sex, and cancer type in a cohort of more than 3,000 dogs: Determining the optimal age to initiate cancer screening in canine patients. Plos One. 2023; 18(2):e0280795.
- McCleary-Wheeler A, Fiaux P, Ruiz-Perez C, McLennan L, Tynan J, Hicks S, et al. Next-generation sequencing-based liquid biopsy can be used for detection of residual disease and cancer recurrence monitoring in dogs. bioRxiv [Internet]. 2023 Sep 8; Available from: biorxiv.org/content/10.1101/2023.09.08.556935v1
Andi Flory, DVM, DACVIM (Oncology), has worked as an oncologist in the US and Australia, In 2019, Flory co-founded PetDx to bring noninvasive cancer detection to veterinary medicine. |
Photo credits: Chalabala/iStock via Getty Images Plus, Infographic and photo courtesy of PetDx