Horizons in Veterinary Precision Oncology: Fundamentals of Cancer Genomics and Applications of Liquid Biopsy for the Detection, Characterization, and Management of Cancer in Dogs

Abstract

Cancer is the leading cause of death in dogs, in part because many cases are identified at an advanced stage when clinical signs have developed, and prognosis is poor. Increased understanding of cancer as a disease of the genome has led to the introduction of liquid biopsy testing, allowing for detection of genomic alterations in cell-free DNA fragments in blood to facilitate earlier detection, characterization, and management of cancer through non-invasive means. Recent discoveries in the areas of genomics and oncology have provided a deeper understanding of the molecular origins and evolution of cancer, and of the "one health" similarities between humans and dogs that underlie the field of comparative oncology. These discoveries, combined with technological advances in DNA profiling, are shifting the paradigm for cancer diagnosis toward earlier detection with the goal of improving outcomes. Liquid biopsy testing has already revolutionized the way cancer is managed in human medicine - and it is poised to make a similar impact in veterinary medicine. Multiple clinical use cases for liquid biopsy are emerging, including screening, aid in diagnosis, targeted treatment selection, treatment response monitoring, minimal residual disease detection, and recurrence monitoring. This review article highlights key scientific advances in genomics and their relevance for veterinary oncology, with the goal of providing a foundational introduction to this important topic for veterinarians. As these technologies migrate from human medicine into veterinary medicine, improved awareness and understanding will facilitate their rapid adoption, for the benefit of veterinary patients.

Keywords: cancer; cell-free DNA; cfDNA; circulating tumor DNA; dog; genomic; liquid biopsy; one health.

Figure 1

A brief guide to genomics. Cancer is a disease of the genome because DNA alterations provide the biological basis of cancer. Each body cell (except for mature red blood cells) contains a full copy of the organism's genome within a set of chromosomes packed in its nucleus. The DNA double-helix is formed by four nucleotides, or bases, assembled in complementary pairs via hydrogen bonds: adenine (A) is always paired with thymine (T), and cytosine (C) is always paired with guanine (G). The gene is the basic unit of heredity and consists of a long sequence of nucleotides that encodes for the synthesis of a protein by transcription to RNA (ribonucleic acid) in the cell's nucleus, followed by translation to a sequence of amino acids in the cytoplasm. The average gene comprises several thousand bases, with wide size variation. The DNA double-helix strand wraps around a set of histone proteins, forming structures known as “nucleosomes” at regular intervals along the length of the strand (Adapted from National Human Genome Research Institute, genome.gov).

Figure 2

Cellular pathways and functional processes involved in cancer. Driver mutations in cancer-related genes are responsible for cancer development. These cancer-related genes are implicated in 12 cellular signaling pathways, which can be grouped into 3 core cellular functions: cell survival, cell fate, and genome maintenance [Inspired by Hanahan & Weinberg (2011) and Vogelstein et al. (2013)] (58, 68).

Figure 3

Accumulation of small genomic alterations in cancer-related genes. Small genomic alterations in oncogenes tend to be activating mutations, which cluster at very specific locations (“hotspots”), whereas small genomic alterations in tumor suppressor genes (TSGs) tend to be inactivating mutations and may occur across the full length of the gene. The design of a high-quality genomic assay needs to account for these characteristics in order to identify relevant alterations across cancer-related genes in an efficient manner.

Figure 4

Classes of genomic alterations. Small genomic alterations include single nucleotide variants (SNVs) as well as small insertions and deletions (collectively known as “indels”). SNVs arise when one nucleotide is substituted for another, which can result in altered amino acid translation and an altered protein product. Indels involve the insertion or deletion of one or more nucleotides from the normal DNA sequence, resulting in an altered protein product. On a much larger scale, structural alterations typically involve thousands to millions of nucleotides. Copy number variants (CNVs) are a common type of structural alteration, involving gains or losses of large stretches of DNA. Translocations represent another type of structural alteration, whereby two distant, otherwise unrelated genomic regions are joined together, creating “gene fusions” that can drive tumor growth.

Figure 5

Accumulation of genomic alterations and emergence of resistance. Cancer begins with a single genomic alteration in a cancer-related gene, which provides a selective growth advantage that allows the cell with the original “clonal mutation” (also known as the “truncal mutation”) to grow and divide more quickly than neighboring healthy cells. Over time, additional genomic alterations accumulate in the DNA of these cancerous cells, leading to both linear and branched evolution from the original clonal population. This leads to a tumor comprised of various subclones, all of which share the original truncal mutation but also feature additional, unique mutations (known as “private mutations”). Administration of an efficacious treatment will typically eliminate many cells in the tumor, resulting in a reduction in tumor burden and clinical remission; however, certain subclones already harboring resistance mutations will often survive treatment at clinically undetectable levels and subsequently expand in the absence of competition. In time, this leads to the clinical observation of recurrence.

Figure 6

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 known as “DNAses.” As a result, most cfDNA fragments found in circulation are typically short, averaging 167 nucleotides in length in both humans and dogs (166, 167). 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 single stranded DNA is depicted here, for illustrative purposes).

Figure 7

Clinical use cases for liquid biopsy in cancer. Liquid biopsy can be used to inform multiple decision points along the entire continuum of cancer care: (1) Cancer screening at regular intervals in patients deemed to be at higher risk for cancer based on age and/or breed; (2) Aid in diagnosis in patients who present with clinical signs (including incidental findings on imaging or laboratory tests) that are suspicious for cancer; (3) Targeted treatment selection based on the unique mutational profile of the tumor in patients diagnosed with cancer; (4) Minimal residual disease detection following a curative-intent intervention (such as surgery); (5) Treatment response monitoring at regular intervals during extended-duration therapeutic regimens; (6) Recurrence monitoring at regular intervals after complete remission or presumed cure.