Oncology

Research Abstract

O03 - Comprehensive Analysis of Gene Mutations in Canine Histiocytic Sarcoma by Whole Exome Sequencing

Thursday, June 14
2:00 PM - 2:15 PM
Location: WSCC 615

Gene mutations in various canine tumors have been explored. In canine histiocytic sarcoma (HS), the mutations of TP53 and PTPN11 genes have been reported to be frequently observed. However, there has been no report on the comprehensive analysis of gene mutations in canine HS. The objective of this study was to conduct the comprehensive analysis of mutations of protein coding genes in dogs with HS by whole exome sequencing.


Genomic DNA was extracted from the tumor tissues of three dogs that were histologically diagnosed with HS. For the analysis of germline DNA sequences, genomic DNA was also extracted from normal tissues or peripheral blood of these dogs. Whole exome sequencing was performed using Illumina NextSeq 500. The alignment of processed reads to the canine reference genome (CanFam 3.1) was carried out using Genome Analysis Toolkit. VarScan was used for calling of the somatic variants in each dog and SnpEff was used for the annotations of the variants. The extracted somatic variants were filtered by the read depth and the putative impact for the gene functions. The variants in the genes known to be associated with the pathogenesis of human tumors were validated using Sanger sequencing.


Whole exome sequencing generated the mean read depth of 630×, and 99.2 % of the unique reads could be mapped to the canine reference genome on average. After the analysis of the data, 11 variants in 11 genes, 17 variants in 16 genes, and 14 variants in 13 genes were extracted as the somatic mutations in Dogs 1, 2, and 3, respectively. Of these variants, the mutations of TP53 (Dog 1), PDGFRB and N4BP2 (Dog 2), and SH3KBP1 (Dog 3) were confirmed by Sanger sequencing.


This study revealed somatic mutations of TP53, PDGFRB, N4BP2, and SH3KBP1 genes in canine HS. We previously reported the high incidence of TP53 gene mutations in canine HS. N4BP2 was reported to interact with BCL3, a target of PI3K/Akt and MAPK/ERK pathways, and SH3KBP1 was shown to induce down-regulation of protein tyrosine kinase that activates PI3K/Akt and MAPK/ERK pathways (Fig. 1). Although further studies are needed to know the frequencies of the gene mutations, findings obtained in the present study will lead to the introduction of molecular targeted therapy directed to PI3K/Akt, MAPK/ERK, and p53 pathways in canine HS.

Hajime Asada, DVM

Graduate student
The University of Tokyo

Education:
2019 Expected to graduate with Ph.D. (Doctor of Veterinary Medicine)
2015 B.S. in Veterinary Medicine, The University of Tokyo

Publications:
1. Takahashi, M., Tomiyasu, H., Hotta, E., Asada, H., Fukushima, K., Kanemoto, H., Fujino, Y., Ohno, K., Uchida, K., Nakayama, H. and Tsujimoto, H.
“Clinical characteristics and prognostic factors in dogs with histiocytic sarcomas in Japan.”
J. Vet. Med. Sci. 76. p661-666. 2014
2. Asada, H., Tomiyasu, H., Goto-Koshino, Y., Fujino, Y., Ohno, K. and Tsujimoto, H.
“Evaluation of the drug sensitivity and expression of 16 drug resistance-related genes in canine histiocytic sarcoma cell lines.”
J. Vet. Med. Sci. 77. p677-684. 2015
3. Asada, H., Tsuboi, M., Chambers, J.K., Uchida, K., Tomiyasu, H., Goto-Koshino, Y., Ohno, K. and Tsujimoto, H.
“A 2-base insertion in exon 5 is a common mutation of the TP53 gene in dogs with histiocytic sarcoma.”
J. Vet. Med. Sci. 79. P1721-1726. 2017

Presentation(s):

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