Analysis of Liquid Biopsy Performance for the Detection of Lymphoma in Dogs with a Focus on Recurrent Copy Number Variants in Matched Plasma and Lymph Node Aspirates: Implications for a Comparative Oncology Model

Abstract

Introduction Liquid biopsy has shown great promise for the detection and monitoring of lymphoma in humans. Recently, similar technology has been developed and made commercially available for dogs. Because the canine and human cancer genomes share such high homology (>90%), and certain lymphoma subtypes are highly similar in terms of their biologic and molecular features between the two species, the study of lymphoma in dogs affords unique opportunities for the genomic characterization of the disease, comparative oncology studies, and the development and assessment of novel therapies. The current study describes the performance of a next-generation sequencing-based liquid biopsy assay for the detection of canine lymphoma, and in a subset of patients, analyzes concordance for prognostic copy number variants between matched plasma samples and lymph node aspirate specimens. Methods As part of a larger clinical research collection program for the CANcer Detection in Dogs (CANDiD) study (Flory, 2022), blood samples were collected following diagnosis but prior to undergoing therapy from a cohort of 116 client-owned dogs with lymphoma (63 intermediate to large B-cell, 21 intermediate to large T-cell, 6 T-zone, and 26 unphenotyped). Lymphomas with inconclusive immunophenotype or other types of indolent lymphomas were excluded. A subset of dogs analyzed for the current study also had matched cytology specimens available from lymph node fine needle aspirates (FNA). Blood samples and FNA specimens 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. Specific analysis focused on FNA-plasma concordance for recurrent copy number variants that have been previously identified in canine lymphoma patients and have prognostic potential (i.e., gains in chromosomes 13 and 31 and losses in chromosome 14) (Richards, 2015; Thomas, 2003). Results The overall detection rate for lymphoma by liquid biopsy was 92.2% (107/116); with detection rates of 57% for localized/regional disease (Stages I and II) and 95% for disseminated disease (Stages III, IV, and V). By immunophenotype, the detection rate for B-cell lymphoma was 97%, T-cell 91%, T-zone 50%, and unphenotyped 92%. In the subset of lymphoma-diagnosed dogs with matched plasma and FNA samples, the majority of cases had at least one of the prognostic copy number alterations previously reported in canine lymphoma patients (13G, 14L, 31G), demonstrating the potential of liquid biopsy to noninvasively detect clinically important biomarkers. A variety of genomic alterations were identified in patients with positive liquid biopsy results, including single nucleotide variants in common oncogenes and tumor suppressor genes (e.g., TP53 mutations), and copy number variants across the genome. Some of these variants are also important biomarkers in human lymphoma, in particular TP53 mutations (Chakravarty, 2017), highlighting the potential for canine liquid biopsy testing to support comparative oncology studies for the benefit of both species. Conclusion A novel next-generation sequencing-based liquid biopsy tool has demonstrated the ability to identify genomic alterations in blood samples of dogs with lymphoma, including recurrent CNVs previously described in canine lymphoma patients, and variants in genes previously described in human and canine lymphoma tissue. These findings, along with the high degree of homology between human and canine lymphomas, support the utility of liquid biopsy for the study of lymphoma in dogs as a comparative oncology model for accelerated biomarker discovery and therapeutic development in both species.