Therapeutic efficacy monitoring in canine lymphoma patients via quantifying CDC6 plasma levels

Abstract

BACKGROUND: Liquid biopsies have gained increasing recognition as minimally invasive ways to monitor treatment response in cancer patients. They carry circulating DNA, RNA, and proteins in and outside of extracellular vesicles (EVs). We have demonstrated that Cell Division Cycle 6 (CDC6), which is involved in controlling cell proliferation, was detected in circulating EVs and increased in the plasma of canine cancer patients compared to healthy control dogs. Here, we investigated whether plasma CDC6 mRNA levels can be used to monitor treatment response in dogs diagnosed with non-Hodgkin lymphoma, and we aimed to determine whether plasma expression is related to the systemic release of EVs. METHODS: We performed longitudinal proof-of-concept studies and collected platelet-free plasma (PFP) samples of 16 canine lymphoma patients before and during their chemo treatments at various time points, as well as PFP samples of 15 healthy control dogs. The EVs were isolated from PFP by size exclusion chromatography, further purified by density gradient ultracentrifugation, and quantified by high-resolution flow cytometry. The CDC6 mRNA abundance in both the unfractionated plasma and the EVs samples was measured by digital droplet PCR (ddPCR). RESULTS: This study demonstrates that, in dogs with lymphoma, circulating CDC6 levels are increased compared to healthy controls. We show that CDC6 levels decline significantly in lymphoma patients that undergo remission in response to chemotherapy. Moreover we show that CDC6 mRNA levels correlate with the number of circulating EVs. CONCLUSIONS: Overall, our findings provides compelling evidence that plasma CDC6 mRNA expression can be used as a liquid biomarker to monitor treatment efficacy in dogs with lymphoma.