Shengwei Li＊,1, Mao Mao*,1,2

1Research & Development, TwixBio Inc, Shenzhen 518000, China

2Yonsei Song-Dang Institute for Cancer Research, Yonsei University, Seoul 03722, South Korea

Correspondence

＊Shengwei Li, Research & Development, TwixBio Inc, Shenzhen 518000, China. Email: 1298261062@qq.com

＊Mao Mao, Research & Development, TwixBio Inc, Shenzhen 518000, China. Email: maomao@yuhs.ac

Abstract

Canine cancer research is a complex subject involving many subsidiary subject areas. In this field, multiple disciplines such as biomedicine, genetics, immunology, and oncology are interwoven to deeply explore and understand the pathogenesis, prevention strategies, and treatment methods of canine cancer. Canine genomics perceives canine cancer as "a disease of the genome" and categorizes genomic alterations in canine cancer. Liquid biopsy has been described as "the next frontier in veterinary cancer care", establishing an emerging technology focused on cell-free DNA methylation profiling, cell-free DNA fragmentomics analysis, shallow whole-genome sequencing, and multi-omics analysis. In humans, given its non-invasive advantages, the utilization of liquid biopsy testing has already become integral to every step of the clinical journey of cancer screening, auxiliary diagnosis, minimal residual disease detection, therapeutic response monitoring, and recurrence monitoring. In principle, veterinary use of this technology has a similar scope of applications. The transfer of this technology from human medicine into veterinary medicine will facilitate its swift adoption, for the benefit of these veterinary patients.

KEYWORDS

canine, cancer, genomics, liquid biopsy, cell-free DNA (cfDNA), circulating tumor DNA (ctDNA)

1 INTRODUCTION

Cancer is highly prevalent in canines and represents the leading cause of mortality among them.1-3While canines and humans have a similar lifetime risk of cancer (between 1:2 to 1:4), canines have an annual incidence of cancer that is up to 10-fold higher than in humans, as their lifetime risk is compressed into a much-abbreviated lifespan.1-2There are less than 90 million canines in the United States, but about 4 to 6 million canines are newly diagnosed with cancer each year；In comparison, 1.8 million people are newly diagnosed with cancer each year in the United States, which has a population of about 330 million.4 As in humans, the cancer burden in canines increases with age: up to 50% of canines over the age of 10 will develop cancer in the rest of their lives.3 As pet ownership increases and emotional attachment to pets deepens, the enormous burden of canine cancer extends well beyond the direct health impact on canines, with significant emotional and financial implications for canine owners.5

Comparative oncology studies naturally occurring cancers in veterinary patients through the study of cancer biology, pathogenesis, and treatment for the benefit of both humans and animals. Canine and human cancers share many histological, molecular, physiological, and even epidemiological features, and this commonality provides a rationale for the field of comparative oncology, wherein a deeper understanding of cancer in one species can drive corresponding insights into the other.6 Common types of cancer in humans include: breast cancer, lung cancer, colorectal cancer, prostate cancer, stomach cancer, liver cancer, cervical cancer, esophageal cancer, thyroid cancer, bladder cancer;7 Meanwhile, common types of cancer in canines include: anal sac carcinoma, lymphoma, mammary gland cancer, mast cell tumor, oral malignant melanoma, osteosarcoma, soft tissue sarcoma, splenic hemangiosarcoma.8

The early detection methods for pan-cancer types employ next-generation sequencing (NGS) technology to identify and characterize cancer-related genomic alterations in DNA extracted from whole blood samples of canines; The related study recommends annual screening tests for all canine breeds, commencing at the age of 7.9

There are three common treatments for canine tumors: surgery, chemotherapy, and radiation therapy. Each therapy can be used alone or in combination with other therapies. When choosing a treatment, veterinarians consider the type of cancer, the rate at which it grows and spreads (the stage or grade of the cancer), and the location of the cancer.10

The field of veterinary oncology has not yet reached the point of harnessing the full power of genomics to realize the benefits of its precision medicine, and due to the current lack of regulatory standards and mechanisms for high-complexity molecular assays in the field of veterinary oncology, it is easy for low-quality assays to enter clinical applications, leading to poor prognosis. Liquid biopsy testing, which detects cancer-derived genomic alterations in cell-free DNA (cfDNA) fragments in the blood, has been used in human medicine for early cancer detection and is now also available in veterinary clinics.11

2. FUNDAMENTALS OF CANINE CANCER GENOMICS

2.1 Canine Cancer is a "Disease of the Genome "

The first canine reference genome was published in 2005,12 shortly after the publication of the human reference genome. 13However, progress in canine genomics have not been nearly as rapid as in humans, and most advances in genomic medicine have not yet been adopted in veterinary medicine, and there is an urgent need to bring veterinary cancer care standards up to par with human medical standards. Molecular testing based on tumor tissue has become an integral part of the "precision medicine" trend in human cancer care,14and recent innovations in this field have made possible non-invasive tests based on a simple blood drawing, called a "liquid biopsy", which is an analysis of cell-free DNA fragments released into the bloodstream by tumor cells.15-19Normal cells accumulate random genomic alterations over time due to DNA replication errors, as well as exposure to endogenous factors (such as free radicals), and to environmental carcinogens such as various forms of radiation and mutagenic chemicals in food and air,20-23and when one or more of these alterations confer an uncontrolled growth advantage to a population of cells, can lead to cancer.24Fundamentally, cancer is a disease of the genome, as it is directly caused by genomic alterations and cannot develop in the absence of such alterations.25

2.2 Classes of Genomic Alterations in Canine Cancer

1. single nucleotide variant (SNV), which involves the variation of only one letter out of several billion letters in the genome.24,26-28

2. Insertions and deletions (indels), in which one to several nucleotides are inserted into, or removed from, the normal DNA sequence.26-28

3. Structural alterations,29larger genomic events affecting thousands to millions of nucleotides can also lead to significant genomic disruption, leading to cancer. Cancer-related structural alterations include:

- copy number aberration (CNA), also known as copy number variant (CNV), in which large segments of DNA (thousands to millions of bases long, up to entire chromosomes) are either completely absent or are abnormally repeated; and

- translocations, in which DNA strands from unrelated parts of the genome are joined together and result in "fusion genes" in the RNA transcript.

The disease etiology of a given cancer is typically driven either by focal somatic alterations (SNVs, indels, and/or translocations) or by CNA, but rarely by both categories.24Cancers such as sarcomas - which are more common in canines than in humans - are mostly CNA-driven, while other cancers such as carcinomas of the lung or gastrointestinal tract, are mostly SNV and indels driven.11

3 CONTENTS, PRINCIPLES AND CLINICAL USE CASES OF LIQUID BIOPSY IN CANINE CANCER

Liquid biopsy, described as "the next frontier in veterinary cancer care", broadly refers to the sampling and analysis of analytes from various biological fluids (primarily blood, but in some cases also urine, cerebrospinal fluid, or other secretions), where sampling is performed by minimally invasive or non-invasive methods.30Blood-based liquid biopsies include: circulating nucleic acid analysis (mainly cfDNA, which includes ctDNA in patients with cancer), circulating tumor cells (CTCs), and proteins from cancer patients.30Because obtaining tissue samples for traditional tissue biopsy can be particularly dangerous or difficult, the ability to detect cancer-related analytes from blood has unique advantages of simple sampling, low risk and good repeatability, especially in cases of cancer (or suspected cancer).

3.1 Contents of Liquid Biopsy in Canine Cancer

3.1.1 cfDNA 31  

cfDNA-based approaches have emerged as a promising avenue for the diagnosis and monitoring of solid tumors, which are prevalent in both humans and canines. These approaches utilize circulating biomarkers present in cell-free DNA (cfDNA) to provide valuable insights into tumor characteristics and progression. One key advantage of cfDNA-based approaches is their non-invasive nature. Traditional methods often require invasive procedures such as tissue biopsies, which can be uncomfortable for patients and pets alike. In contrast, cfDNA can be easily obtained from blood samples, making it a convenient and less stressful option for both humans and canines undergoing tumor assessment. Furthermore, cfDNA-based approaches offer the potential for early detection of solid tumors. By analyzing specific genetic alterations or mutations found in cfDNA, researchers can identify the presence of cancerous cells even before clinical symptoms manifest. This early detection could significantly improve treatment outcomes by enabling timely intervention when tumors are still at an early stage. In addition to diagnosis, cfDNA-based approaches also hold promise for monitoring treatment response and disease progression. By regularly analyzing changes in cfDNA profiles during therapy, clinicians can assess whether a particular treatment is effective or if adjustments need to be made. This real-time monitoring allows for personalized medicine strategies that optimize patient care based on individual responses. Moreover, the use of cfDNA-based approaches extends beyond human medicine，it has shown great potential in veterinary oncology as well. Canines naturally develop spontaneous tumors that share many similarities with human cancers, making them valuable models for studying tumor biology and evaluating therapeutic interventions. The application of cfDNA analysis in canine patients not only benefits our understanding of cancer but also offers opportunities to translate findings between species. Overall, while traditional circulating biomarker techniques have had limited success thus far in solid tumor management, the emergence of cfDNA-based approaches brings renewed hope for improved diagnostics and personalized treatments in both humans and canines facing these malignancies. Continued research efforts will undoubtedly further enhance our ability to harness the full potential of this innovative approach towards better cancer care across species boundaries.

3.1.2 Protein markers32

In humans, a number of blood-based protein biomarkers have been used for human cancer screening and monitoring using immunoassays, including: carcinoembryonic antigen (CEA) for colorectal cancer, prostate-specific antigen (PSA) for prostate cancer, cancer antigen 125 (CA-125) for ovarian cancer, and alpha-fetoprotein (AFP) for hepatocellular carcinoma. In veterinary medicine, protein biomarkers such as thymidine kinase type 1 (TK1), canine C-reactive protein (cCRP) and alpha-fetoprotein receptor (RECAF) have been used for canine cancer detection. Despite these advancements, it is important to acknowledge that current protein biomarker tests still face limitations when it comes to sensitivity and specificity in detecting cancer. False positives or negatives can occur due to factors such as individual variations or overlapping expression patterns with non-cancerous conditions. Therefore, ongoing research aims at identifying additional biomarkers or combining multiple markers to enhance accuracy and reliability. Overall, the field of protein biomarker research continues to evolve rapidly both in humans and animals alike. With further exploration into new markers and improved detection techniques, we hope to achieve more accurate early diagnosis and effective monitoring of cancers across species boundaries.

3.1.3 Circulating tumor cells 33

Circulating tumor cells (CTCs) have emerged as a promising area of research in the field of oncology. These intact tumor cells, originating from solid tumors, can sometimes be found circulating in the bloodstream. The discovery and study of CTCs have opened up new avenues for understanding cancer metastasis and monitoring disease progression. Researchers have been actively investigating the clinical implications of detecting CTCs in circulation. By analyzing these cells, scientists hope to gain insights into tumor biology, identify potential therapeutic targets, and develop personalized treatment strategies. However, despite significant progress in this field, the translation of CTCs research into clinical practice remains limited at present. One challenge lies in reliably isolating and characterizing CTCs from blood samples due to their rarity compared to other blood components. Various techniques such as immunomagnetic separation or microfluidic devices are being explored to improve the sensitivity and specificity of CTCs detection methods. Moreover, researchers are also working towards developing standardized protocols for analyzing CTCs that can be easily implemented across different laboratories and healthcare settings. This would ensure consistency in results and facilitate comparison between studies. Another aspect that needs further investigation is determining the clinical significance of detecting CTCs at different stages of cancer progression. It is crucial to understand whether the presence or absence of these cells correlates with prognosis or response to treatment. Such knowledge could potentially guide clinicians in making informed decisions regarding patient management. Furthermore, efforts are underway to explore additional biomarkers associated with CTCs that could provide valuable information about tumor heterogeneity or drug resistance mechanisms. Integrating multiple molecular markers alongside traditional imaging techniques may enhance our ability to accurately diagnose cancers and monitor treatment responses over time.

In conclusion, while there has been remarkable progress made in studying circulating tumor cells (CTCs), their full clinical potential is yet to be realized. Continued research endeavors aimed at improving detection methods, standardizing protocols, elucidating their biological significance, and exploring novel biomarkers will pave the way for future advancements in utilizing CTCs analysis for precision medicine approaches against cancer. 

3.2 Principles of Liquid Biopsy in Canine Cancer

3.2.1 Origin and Characteristics of cfDNA (Figure 1)31

FIGURE 1 Origins of cell-free DNA. When a cell dies through either programmed cell death (apoptosis) or necrosis, its cellular contents (including DNA from the nucleus) are released into the bloodstream. At this point, the DNA becomes “cell-free DNA” and is rapidly degraded into small fragments through the action of circulating enzymes. As a result, most cfDNA fragments found in circulation are typically short, averaging 167 nucleotides in length in both humans and canines.31 While both healthy cells and tumor cells contain DNA that becomes cfDNA in circulation, only tumor cells will harbor somatic genomic alterations in cancer-related genes. Detection of such genomic alterations in the cfDNA of a patient is thus indicative of the presence of tumor cells in the body, providing the rationale for “liquid biopsy” testing approaches.(Note: cfDNA exists as both single stranded DNA and double stranded DNA; only double stranded DNA is depicted here, for illustrative purposes.)

The presence of cfDNA in humans was first reported in 1948, and while cfDNA was hypothesized to be linked to metastatic cancer in the mid-1960s, it took until 1977 for the first results evaluating cfDNA concentrations in patients with cancer compared to normal controls to be published, and neoplastic characteristics were reported in circulation in 1989.11In 1996, two landmark papers reported the detection of cancer-derived alterations in the plasma or serum of cancer patients in the form of ctDNA.34-35Since then, efforts have been made to develop molecular tests to detect the presence of cancer-derived alterations in the blood and to use this information for cancer detection, characterization, treatment, and monitoring.11In 1997, fetal origin cfDNA was found in maternal plasma,36 leading to the first widely adopted clinical application for cfDNA testing: The use of maternal blood samples to screen for common fetal chromosomal abnormalities such as trisomy 21 (Down syndrome)37 is a revolutionary development -- invasive diagnostic tests previously used such as chorionic villus sampling or amniocentesis carry a risk of miscarriage.38 The introduction of cfDNA-based non-invasive prenatal testing (NIPT) in 201137 has fundamentally changed the delivery of prenatal care and has screened tens of millions of pregnant women, leading to a significant reduction in the number of invasive diagnostic procedures for detection of fetal chromosomal abnormalities.39

Xing Ji et al. 40 utilized advanced genetic testing techniques to detect any abnormal cell-free DNA patterns that could indicate the presence of cancer. The findings were significant as they highlighted the potential benefits of using noninvasive prenatal screening tests for early detection and monitoring of maternal cancers during pregnancy. This approach could lead to earlier diagnosis and treatment, improving outcomes for both mother and baby. However, further research is needed to determine the optimal management strategies for pregnant women with suspected or confirmed malignancies detected through noninvasive prenatal screening tests.

Zuowei Meng et al. 41 focuses on the utilization of ctDNA features and α-Fetoprotein (AFP) levels in blood samples to detect hepatocellular carcinoma (HCC) noninvasively. CtDNA refers to small fragments of tumor DNA that are released into the bloodstream by cancer cells. By analyzing these ctDNA features, researchers aim to identify specific genetic alterations or mutations associated with HCC. In addition to ctDNA analysis, this study also investigates the use of AFP levels in blood samples for HCC detection. AFP is a protein produced by fetal liver cells but its elevated levels have been observed in patients with HCC. Combining both ctDNA analysis and AFP measurements could potentially enhance the accuracy and sensitivity of HCC detection.

3.2.2 Methylation Profile Analysis of cfDNA

The attachment of methyl (CH3) groups to the DNA strand at specific locations throughout the genome is associated with cancer, and methylation of the promoter region of tumor suppressor genes can inactivate the expression of these genes, thereby allowing oncogene-driven cancers to proliferate unhindered.42Moreover, DNA in cells from specific organs has methylation profiles specific to those organs,43and when the DNA from cancer cells in a particular organ is released into the circulation as circulating tumor DNA (ctDNA), its methylation "signature" carries information about the presence of cancer and the organ of origin of that cancer.16 Therefore, analysis of cfDNA methylation profiles based on NGS has emerged as one of the most promising methods for detecting cancer and assigning it to specific organs of origin, which has obvious clinical benefits.16

3.2.3 cfDNA Fragmentomics44-45

cfDNA fragmentomics for canine cancer is the application of cfDNA fragmentomics in the study and diagnosis of cancer in canines. Cell-free DNA (cfDNA) in bodily fluids has rapidly transformed the development of noninvasive prenatal testing (NIPT), cancer liquid biopsy, and transplantation monitoring. The study of the fragmentation patterns of cfDNA, also referred to as ‘fragmentomics’, is now an actively pursued area of biomarker research. Clues that cfDNA fragmentation patterns might carry information concerning the tissue of origin of cfDNA molecules have come from works demonstrating that circulating fetal, tumor-derived, and transplanted liver-derived cfDNA molecules have a shorter size distribution than the background mainly of hematopoietic origin. Liquid biopsy has created paradigm shifts in diagnostics and management, including noninvasive prenatal testing (NIPT) and oncology. It is notable that a rapid global success in NIPT using cfDNA in maternal plasma has promoted the parallel achievement for cancer detection. To push the frontiers of this field, many efforts have been made to investigate the biological properties of cfDNA molecules in the prenatal and oncology contexts. An improved understanding of the biological characteristics of cfDNA often catalyzes new diagnostic tools. For instance, the realization of the size difference between maternal and fetal DNA leads to a novel approach for detecting fetal chromosomal aneuploidies using fragment sizes. The synergistic use of the size-based and count-based approaches could improve the accuracy of data interpretation, such as the fetal/maternal origin of copy number alterations seen in maternal plasma. Similarly, the characteristic size profiles of tumor-derived DNA molecules could aid in cancer detection. With the advent of new sequencing technologies and bioinformatics tools, significant progress has been made in understanding the biological characteristics of cfDNA molecules. Fragmentation patterns of cfDNA molecules have attracted a lot of recent research interest, including fragment sizes, nucleosome relationships, endpoints, end motifs, and topological forms, forming a field of cfDNA fragmentomics. Fragmentomic features of cfDNA molecules bear a wealth of molecular information pertaining to the tissues of origin, paving the crucial foundation for exploring and developing potential diagnostic tools based on cfDNA fragmentomics. Such development could accelerate the development of high-performance diagnostic tools for NIPT, cancer detection, monitoring of organ transplantation, as well as detection of other diseases (e.g., autoimmune diseases).

3.2.4 Shallow Whole-Genome Sequencing (sWGS)46

Shallow whole-genome sequencing for canine cancer refers to the use of shallow whole-genome sequencing technology to study and analyze genes related to canine cancer. This technology can quickly and accurately identify specific gene variations in the canine genome that may be associated with the occurrence and development of canine cancer. The process typically involves collecting a blood sample from a canine and then sequencing the cell-free DNA (cfDNA) in the blood using shallow whole-genome sequencing techniques. In general, sWGS can only detect copy number alterations, fragment size and end motifs, but not mutations such as SNVs, indels, and structural variants(SVs). Additionally, shallow whole-genome sequencing can be used to monitor the effectiveness of treatment for canine cancer. For example, if the treatment is effective, the levels of specific cfDNA fragments associated with cancer should decrease over time; If the treatment is not effective, these fragment levels may remain high or even increase. In summary, shallow whole-genome sequencing for canine cancer offers a non-invasive and informative approach to studying and treating canine cancer, this technique can help veterinarians make more informed decisions about treatment options and monitor the effectiveness of treatment.

3.2.5 Multi-omics Analysis47

Currently, the only technology that can simultaneously detect all major classes of genomic alterations in cfDNA, as well as features such as methylation and fragmentation patterns, is next-generation sequencing (NGS), which takes a "pan-cancer" approach to accurately analyze and detect changes in the somatic genome. In the past few years, multi-omics liquid biopsy methods have been introduced, combining genomic and proteomic methods, injecting new vitality into protein analysis and becoming a valuable adjunct to cfDNA analysis.15,17 Successful development of pan-cancer liquid biopsy tests in canines may require a similar combination strategy.

Multi-omics analysis is a technique that comprehensively analyzes multiple molecular levels for the study of molecular mechanisms, biomarker discovery and drug target identification of canine cancer. This method of analysis can provide detailed information on disease development, progression, and response to treatment. Multi-omics analysis usually includes multiple aspects such as genomics, transcriptomics, proteomics and metabolomics. Genomics is concerned with the structure and variation of the entire genome, transcriptomics is concerned with the expression level of genes, proteomics is concerned with protein expression and post-translational modification, and metabolomics is concerned with the composition and changes of metabolites. These data can be combined to provide a more comprehensive molecular profile and help scientists better understand the mechanisms and treatment strategies of canine cancer. Multi-omics analysis of canine cancer requires specialized techniques and equipment, such as high-throughput sequencing, mass spectrometry, and nuclear magnetic resonance, as well as relevant bioinformatics analysis methods. Through these technologies, scientists can study the molecular characteristics of canine cancer and provide new ideas and methods for the diagnosis, treatment and prevention of the disease.

3.3 Clinical Use Cases of Liquid Biopsy in Canine Cancer

Liquid biopsy promises the convenience of a blood draw combined with the power of genomic technology. It is unlikely to fully replace the key role that traditional tissue biopsy plays in veterinary cancer diagnosis and management, but the non-invasive nature of liquid biopsy, coupled with its ability to detect tumor signal from any malignant mass in the body, should allow it to provide immediate value in several clinical scenarios once it becomes commercially available. In humans, liquid biopsy has demonstrated feasibility and great clinical potential across multiple use cases, spanning the entire continuum of cancer care; a similar spectrum of applications is in principle available for veterinary uses of the technology.11

3.3.1 Screening

Certain canine breeds are known to be more predisposed to cancer than others, presumably due to cancer-predisposing mutations that have become concentrated in certain populations over time as a result of the breeding process. Liquid biopsy-based screening paradigm focused on high-risk groups, such as canines from predisposed breeds or from geriatric populations, and their solutions are nearing commercialization,48some of which have also shown potential for predicting the organ of origin of the tumor, facilitating the path to a definitive diagnosis.16-17

3.3.2 Auxiliary Diagnosis

In the veterinary clinics, when cancer is suspected due to clinical signs or a clinical history, a liquid biopsy can be used as an aid in diagnosis. Compared to the current standard of care, liquid biopsy testing can promote early detection of cancer and reduce the economic burden of treatment, with treatment costs for human cancer patients diagnosed early in the disease course being 2 to 4 times less than for those diagnosed at later stages.48-49

3.3.3 Minimal Residual Disease (MRD) Detection

After curative-intent treatment (such as surgery) has been performed to remove the tumor, adjuvant therapy is often considered because of the risk of malignant deposits remaining in the body and resulting in relapse (or recurrence) in the future.50MRD is defined as occult malignant disease that exists immediately after surgery and undetectable by conventional methods, usually detected by the presence of ctDNA in the circulation.51The short half-life of cfDNA (minutes to hours in both humans and canines) makes it an ideal analyte for MRD testing, as detection of any amount of ctDNA starting within a few days after surgery would point to the persistent presence of malignant disease in the body.11 Many cancer types in humans have been studied in the context of MRD detection, including breast, pancreatic, lung, nasopharyngeal, and colorectal, as well as hematological malignancies.52 Liquid biopsy-based MRD testing in canine patients can inform the relative risk of such recurrence following curative-intent interventions and thereby guide decisions regarding initiation of adjuvant treatment as soon as the patient has recovered from surgery.11

3.3.4 Treatment Response Monitoring

Traditionally, treatment response monitoring has been performed by clinical observation and by imaging (mainly ultrasound and radiography, in the veterinary setting), which has significant shortcomings.53 Real-time monitoring of tumor dynamics via serial liquid biopsy testing may help the clinicians differentiate among different scenarios and obtain more frequent updates on the patient’s response to treatment than might be feasible with imaging alone. The non-invasive nature of liquid biopsy compared to current methods could pave the way for it to become a routine surveillance test during cancer treatment in canines.11

3.3.5 Recurrence monitoring

Sequential cfDNA testing during the post-treatment period is designed to identify "molecular relapse" many months before clinical or radiological relapse becomes apparent.52Early identification of cancer relapse may help guide treatment and management decisions in canine patients to improve clinical outcomes through earlier adjuvant therapeutic intervention.

4.CONCLUSION11

The genomic revolution has already had a significant impact on cancer care for human patients and is poised to revolutionize veterinary medicine in a similar manner in the coming years. As genomics becomes a routine part of veterinary care, expansion into multi-omics liquid biopsy approaches is likely to follow, including epigenomics (methylation and histone modification analysis), transcriptomics (gene expression, micro RNAs), proteomics (tumor markers, other peptides), metabolomics, fragmentomics, etc. The introduction of high-quality, clinically validated pan-cancer liquid biopsy tests into the realm of veterinary medicine has the potential to have a significant impact on every step along the clinical journey of a canine cancer patient, from early detection to recurrence monitoring.

AUTHOR CONTRIBUTIONS

Shengwei Li: Drafting and revising the manuscript；Mao Mao: Conception and design, revising the manuscript.

ACKNOWLEDGEMENTS

The authors thank Yi Zhang for his contributions to the illustration.

The cost of publication was provided by TwixBio Inc, Shenzhen 518000, China.

FUNDING INFORMATION

The authors received no funding for this work.

CONFLICT OF INTEREST

The authors have no conflict of interest to declare.

ORCID

Shengwei Li https://orcid.org/0009-0003-5533-9140

Mao Mao https://orcid.org/0000-0002-8570-8571

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