Clinical validation of a blood-based liquid biopsy test integrating cell-free DNA quantification and next-generation sequencing for cancer screening in dogs.

OBJECTIVE: To validate the performance of a novel, integrated test for canine cancer screening that combines cell-free DNA quantification with next-generation sequencing (NGS) analysis. SAMPLE: Retrospective data from a total of 1,947 cancer-diagnosed and presumably cancer-free dogs were used to validate test performance for the detection of 7 predefined cancer types (lymphoma, hemangiosarcoma, osteosarcoma, leukemia, histiocytic sarcoma, primary lung tumors, and urothelial carcinoma), using independent training and testing sets. METHODS: Cell-free DNA quantification data from all samples were analyzed using a proprietary machine learning algorithm to determine a Cancer Probability Index (High, Moderate, or Low). High and Low Probability of Cancer were final result classifications. Moderate cases were additionally analyzed by NGS to arrive at a final classification of High Probability of Cancer (Cancer Signal Detected) or Low Probability of Cancer (Cancer Signal Not Detected). RESULTS: Of the 595 dogs in the testing set, 89% (n = 530) received a High or Low Probability result based on the machine learning algorithm; 11% (65) were Moderate Probability, and NGS results were used to assign a final classification. Overall, 87 of 122 dogs with the 7 predefined cancer types were classified as High Probability and 467 of 473 presumably cancer-free dogs were classified as Low Probability, corresponding to a sensitivity of 71.3% for the predefined cancer types at a specificity of 98.7%. CLINICAL RELEVANCE: This integrated test offers a novel option to screen for cancer types that may be difficult to detect by physical examination at a dog's wellness visit.

Next-generation sequencing-based liquid biopsy may be used for detection of residual disease and cancer recurrence monitoring in dogs.

OBJECTIVE: The purpose of this study was to evaluate the performance of a next-generation sequencing-based liquid biopsy test for cancer monitoring in dogs. SAMPLES: Pre- and postoperative blood samples were collected from dogs with confirmed cancer diagnoses originally enrolled in the CANcer Detection in Dogs (CANDiD) study. A subset of dogs also had longitudinal blood samples collected for recurrence monitoring. METHODS: All cancer-diagnosed patients had a preoperative blood sample in which a cancer signal was detected and had at least 1 postoperative sample collected. Clinical data were used to assign a clinical disease status for each follow-up visit. RESULTS: Following excisional surgery, in the absence of clinical residual disease at the postoperative visit, patients with Cancer Signal Detected results at that visit were 1.94 times as likely (95% CI, 1.21 to 3.12; P = .013) to have clinical recurrence within 6 months compared to patients with Cancer Signal Not Detected results. In the subset of patients with longitudinal liquid biopsy samples that had clinical recurrence documented during the study period, 82% (9/11; 95% CI, 48% to 97%) had Cancer Signal Detected in blood prior to or concomitant with clinical recurrence; in the 6 patients where molecular recurrence was detected prior to clinical recurrence, the median lead time was 168 days (range, 47 to 238). CLINICAL RELEVANCE: Next-generation sequencing-based liquid biopsy is a noninvasive tool that may offer utility as an adjunct to current standard-of-care clinical assessment for cancer monitoring; further studies are needed to confirm diagnostic accuracy in a larger population.

Detection of Age-Related Somatic Alterations in Canine Blood Using Next-Generation Sequencing-Based Liquid Biopsy: An Analysis of over 4800 Dogs.

Age-related somatic genomic alterations in hematopoietic cell lines have been well characterized in humans; however, this phenomenon has not been well studied in other species. Next-generation sequencing-based liquid biopsy testing for cancer detection was recently developed for dogs and has been used to study the genomic profiles of blood samples from thousands of canine patients since 2021. In this study, 4870 client-owned dogs with and without a diagnosis or suspicion of cancer underwent liquid biopsy testing by this method. Copy number variants detected exclusively in genomic DNA derived from white blood cells (WBC gDNA-specific CNVs) were observed in 126 dogs (2.6%; 95% CI: 2.2-3.1); these copy number variants were absent from matched plasma cell-free DNA, and from tumor tissue in dogs with concurrent cancer. These findings were more common in older dogs and were persistent in WBC gDNA in over 70% of patients, with little to no change in the amplitude of the signal across longitudinal samples. Many of these alterations were observed at recurrent locations in the genome across subjects; the most common finding was a partial loss on CFA25, typically accompanied by a partial gain on the same chromosome. These early findings suggest that age-related somatic alterations may be present at an appreciable frequency in the general canine population. Further research is needed to determine the clinical significance of these findings.

Clinical experience with next-generation sequencing-based liquid biopsy testing for cancer detection in dogs: a review of 1,500 consecutive clinical cases.

OBJECTIVE: To review ordering patterns, positivity rates, and outcome data for a subset of consecutive samples submitted for a commercially available, blood-based multicancer early-detection liquid biopsy test for dogs using next-generation sequencing at 1 laboratory. SAMPLE: 1,500 consecutively submitted blood samples from client-owned dogs with and without clinical suspicion and/or history of cancer for prospective liquid biopsy testing between December 28, 2021, and June 28, 2022. PROCEDURES: We performed a retrospective observational study, reviewing data from 1,500 consecutive clinical samples submitted for liquid biopsy testing. Outcome data were obtained via medical record review, direct communication with the referring clinic, and/or a patient outcome survey through October 16, 2022. RESULTS: Sixty-four percent (910/1,419) of reportable samples were submitted for cancer screening, 26% (366/1,419) for aid in diagnosis, and 10% (143/1,419) for other indications. The positivity rate was 25.4% (93/366) in aid-in-diagnosis patients and 4.5% (41/910) in screening patients. Outcome data were available for 33% (465/1,401) of patients, and outcomes were classifiable for 428 patients. The relative observed sensitivity was 61.5% (67/109) and specificity was 97.5% (311/319). The positive predictive value was 75.0% (21/28) for screening patients and 97.7% (43/44) for aid-in-diagnosis patients, and the time to diagnostic resolution following a positive result was < 2 weeks in most cases. CLINICAL RELEVANCE: Liquid biopsy using next-generation sequencing represents a novel tool for noninvasive detection of cancer in dogs. Real-world clinical performance meets or exceeds expectations established in the test's clinical validation study.

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.

The goal of cancer screening is to detect disease at an early stage when treatment may be more effective. Cancer screening in dogs has relied upon annual physical examinations and routine laboratory tests, which are largely inadequate for detecting preclinical disease. With the introduction of non-invasive liquid biopsy cancer detection methods, the discussion is shifting from how to screen dogs for cancer to when to screen dogs for cancer. To address this question, we analyzed data from 3,452 cancer-diagnosed dogs to determine the age at which dogs of certain breeds and weights are typically diagnosed with cancer. In our study population, the median age at cancer diagnosis was 8.8 years, with males diagnosed at younger ages than females, and neutered dogs diagnosed at significantly later ages than intact dogs. Overall, weight was inversely correlated with age at cancer diagnosis, and purebred dogs were diagnosed at significantly younger ages than mixed-breed dogs. For breeds represented by ≥10 dogs, a breed-based median age at diagnosis was calculated. A weight-based linear regression model was developed to predict the median age at diagnosis for breeds represented by ≤10 dogs and for mixed-breed dogs. Our findings, combined with findings from previous studies which established a long duration of the preclinical phase of cancer development in dogs, suggest that it might be reasonable to consider annual cancer screening starting 2 years prior to the median age at cancer diagnosis for dogs of similar breed or weight. This logic would support a general recommendation to start cancer screening for all dogs at the age of 7, and as early as age 4 for breeds with a lower median age at cancer diagnosis, in order to increase the likelihood of early detection and treatment.

Cancer detection in clinical practice and using blood-based liquid biopsy: A retrospective audit of over 350 dogs.

BACKGROUND: Guidelines-driven screening protocols for early cancer detection in dogs are lacking, and cancer often is detected at advanced stages. HYPOTHESIS/OBJECTIVES: To examine how cancer typically is detected in dogs and whether the addition of a next-generation sequencing-based "liquid biopsy" test to a wellness visit has the potential to enhance cancer detection. ANIMALS: Client-owned dogs with definitive cancer diagnoses enrolled in a clinical validation study for a novel blood-based multicancer early detection test. METHODS: Retrospective medical record review was performed to establish the history and presenting complaint that ultimately led to a definitive cancer diagnosis. Blood samples were subjected to DNA extraction, library preparation, and next-generation sequencing. Sequencing data were analyzed using an internally developed bioinformatics pipeline to detect genomic alterations associated with the presence of cancer. RESULTS: In an unselected cohort of 359 cancer-diagnosed dogs, 4% of cases were detected during a wellness visit, 8% were detected incidentally, and 88% were detected after the owner reported clinical signs suggestive of cancer. Liquid biopsy detected disease in 54.7% (95% confidence interval [CI], 49.5%-59.8%) of patients, including 32% of dogs with early-stage cancer, 48% of preclinical dogs, and 84% of dogs with advanced-stage disease. CONCLUSIONS/CLINICAL IMPORTANCE: Most cases of cancer were diagnosed after the onset of clinical signs; only 4% of dogs had cancer detected using the current standard of care (i.e., wellness visit). Liquid biopsy has the potential to increase detection of cancer when added to a dog's wellness visit.

Blood-Based Liquid Biopsy for Comprehensive Cancer Genomic Profiling Using Next-Generation Sequencing: An Emerging Paradigm for Non-invasive Cancer Detection and Management in Dogs.

This proof-of-concept study demonstrates that blood-based liquid biopsy using next generation sequencing of cell-free DNA can non-invasively detect multiple classes of genomic alterations in dogs with cancer, including alterations that originate from spatially separated tumor sites. Eleven dogs with a variety of confirmed cancer diagnoses (including localized and disseminated disease) who were scheduled for surgical resection, and five presumably cancer-free dogs, were enrolled. Blood was collected from each subject, and multiple spatially separated tumor tissue samples were collected during surgery from 9 of the cancer subjects. All samples were analyzed using an advanced prototype of a novel liquid biopsy test designed to non-invasively interrogate multiple classes of genomic alterations for the detection, characterization, and management of cancer in dogs. In five of the nine cancer patients with matched tumor and plasma samples, pre-surgical liquid biopsy testing identified genomic alterations, including single nucleotide variants and copy number variants, that matched alterations independently detected in corresponding tumor tissue samples. Importantly, the pre-surgical liquid biopsy test detected alterations observed in spatially separated tissue samples from the same subject, demonstrating the potential of blood-based testing for comprehensive genomic profiling of heterogeneous tumors. Among the three patients with post-surgical blood samples, genomic alterations remained detectable in one patient with incomplete tumor resection, suggesting utility for non-invasive detection of minimal residual disease following curative-intent treatment. Liquid biopsy allows for non-invasive profiling of cancer-associated genomic alterations with a simple blood draw and has potential to overcome the limitations of tissue-based testing posed by tissue-level genomic heterogeneity.

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

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.

De novo structure prediction and experimental characterization of folded peptoid oligomers.

Peptoid molecules are biomimetic oligomers that can fold into unique three-dimensional structures. As part of an effort to advance computational design of folded oligomers, we present blind-structure predictions for three peptoid sequences using a combination of Replica Exchange Molecular Dynamics (REMD) simulation and Quantum Mechanical refinement. We correctly predicted the structure of a N-aryl peptoid trimer to within 0.2 Å rmsd-backbone and a cyclic peptoid nonamer to an accuracy of 1.0 Å rmsd-backbone. X-ray crystallographic structures are presented for a linear N-alkyl peptoid trimer and for the cyclic peptoid nonamer. The peptoid macrocycle structure features a combination of cis and trans backbone amides, significant nonplanarity of the amide bonds, and a unique "basket" arrangement of (S)-N(1-phenylethyl) side chains encompassing a bound ethanol molecule. REMD simulations of the peptoid trimers reveal that well folded peptoids can exhibit funnel-like conformational free energy landscapes similar to those for ordered polypeptides. These results indicate that physical modeling can successfully perform de novo structure prediction for small peptoid molecules.