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

ABSTRACT

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.