Academic Fellow Veterinary Teaching Hospital, Hokkaido University Sapporo, Hokkaido, Japan
Abstract:
Background: Canine multicentric high-grade lymphoma (MHGL) patients usually benefit from chemotherapy, but some have poorer prognosis. DNA methylation analysis may identify prognostic CpG sites for canine MHGL.
Objective: Identify prognostic CpG sites of MHGL through genome-wide DNA methylation analysis with pyrosequencing validation.
Animals: Test group: 24 dogs. Validation group: 86 dogs. All client-owned dogs were diagnosed with MHGL and treated with CHOP chemotherapy.
Methods: Genome-wide DNA methylation analysis using Digital Restriction Enzyme Analysis of Methylation (DREAM) was performed on the test group to identify differentially methylated CpG sites (DMCs). Bisulfite pyrosequencing was used to measure the methylation status of candidate CpG sites in the validation group. Survival curves were generated and analyzed using the Kaplan-Meier (log-rank) product limit method.
Results: DNA methylome analysis of 101,576 CpG sites hierarchically clustered the test group into 4 groups with different mean survival times (MST). The longest MST group (463 days, P = 0.0015) had 278 hyper- and 441 hypo- DMCs. Among significant DMCs, DMCs near FAM213 and PHLPP1 genes were identified as candidates . Pyrosequencing results of the validation group showed that the group with DMC methylation near the FAM213 gene at < 40% had a longer MST (499 days, P = 0.0102). Dogs grouped with combined DMC methylation near genes of FAM213 at < 40% and PHLPP1 at < 10% had an even longer MST (694 days, P = 0.00024).
Conclusion and clinical importance: Methylation status of prognostic CpG sites can delineate canine MGHL cases with longer MST, warranting active pursuit of CHOP chemotherapy.
