Primer-walking sequencing identifies predominantly noncoding TP53 variants in canine cancers

Tumor protein p53 (TP53) alterations are frequently reported in canine cancers but remain incompletely characterized due to differences in reference genomes and sequencing cost.

To evaluate a cost-efficient primer-walking approach for TP53 variant screening in dogs with spontaneous tumors.

Tumor tissue (n = 10) or blood (n = 16) was collected from 26 dogs with suspected neoplasia. Interpretable TP53 sequences spanning introns and exons 2-11, excluding long intron 1 and non-protein-coding exon 1, were obtained by primer-walking polymerase chain reaction and Sanger sequencing. Bidirectional reads were compared with the Ensembl ROS_Cfam_1.0 v114 reference sequence.

TP53 gene sequence variations were observed in 18/26 sequenced tumor cases, including one exonic frameshift insertion and 17 noncoding variants: 13 intronic variants and 4 variants in the 5' untranslated region. Recurrent substitutions and deletions occurred in the intron 8-10 region. Variants were detected in lymphomas and carcinomas, including cases from several small breeds, but no significant association with tumor type or breed was demonstrated.

Primer-walking Sanger sequencing can screen TP53 regions beyond coding exons in canine tumor samples. Matched tumor-normal sequencing is needed to determine somatic versus germline origin and to evaluate noncoding TP53 variants as potential biomarkers or drivers.