Feasibility of Shallow Whole‐Genome Sequencing‐Based Circulating Tumour DNA Analysis in Canine Multicentric Lymphoma: A Pilot Study

Lymphoma is a common canine malignancy with high morbidity and variable prognosis. Circulating tumour DNA (ctDNA) has emerged as a minimally invasive biomarker for tumour burden, treatment response and prognosis in human lymphoma. However, the feasibility of applying shallow whole-genome sequencing (sWGS)-based ctDNA analysis to canine lymphoma remains unclear. In this pilot study, plasma cell-free DNA (cfDNA) was quantitatively assessed and additional genomic features were evaluated using sWGS in dogs with multicentric lymphoma. cfDNA concentration was significantly higher in dogs with lymphoma (n = 13) compared with healthy controls (n = 24) (median 3.18 vs. 0.25 ng/mL; Mann-Whitney U test, p = 0.004). However, total cfDNA concentration was not significantly associated with time to progression (TTP) (p = 0.055). Tumour fraction (TF), a surrogate for ctDNA, showed preliminary association with clinical outcome, with a shorter median TTP in the high-TF group than in the low-TF group (77 vs. 302 days; p = 0.006). Genomic analysis further revealed copy number aberrations (CNAs); notably, gains on chromosome 13 containing the MYC locus were identified in three dogs, all of which belonged to the high-TF group. Dogs with lymphoma also displayed distinct cfDNA fragmentation profiles, characterized by an enrichment of shorter fragments within the analysed size range and a shift in the highest mean-profile peak compared with healthy controls. These findings demonstrate the feasibility of sWGS-based cfDNA analysis in canine lymphoma and suggest that TF may provide clinically relevant information beyond total cfDNA concentration. Validation in a substantially larger, independent cohort is warranted.