Evaluation of the kinase domain of c-KIT in canine cutaneous mast cell tumors

Abstract

Background: Mutations in the c-KIT proto-oncogene have been implicated in the progression of several neoplastic diseases, including gastrointestinal stromal tumors and mastocytosis in humans, and cutaneous mast cell tumors (MCTs) in canines. Mutations in human mastocytosis patients primarily occur in c-KIT exon 17, which encodes a portion of its kinase domain. In contrast, deletions and internal tandem duplication (ITD) mutations are found in the juxtamembrane domain of c-KIT in approximately 15% of canine MCTs. In addition, ITD c-KIT mutations are significantly associated with aberrant KIT protein localization in canine MCTs. However, some canine MCTs have aberrant KIT localization but lack ITD c-KIT mutations, suggesting that other mutations or other factors may be responsible for aberrant KIT localization in these tumors.

Methods: In order to characterize the prevalence of mutations in the phospho-transferase portion of c-KIT's kinase domain in canine MCTs exons 16-20 of 33 canine MCTs from 33 dogs were amplified and sequenced. Additionally, in order to determine if mutations in c-KIT exon 17 are responsible for aberrant KIT localization in MCTs that lack juxtamembrane domain c-KIT mutations, c-KIT exon 17 was amplified and sequenced from 18 canine MCTs that showed an aberrant KIT localization pattern but did not have ITD c-KIT mutations.

Results: No mutations or polymorphisms were identified in exons 16-20 of any of the MCTs examined.

Conclusion: In conclusion, mutations in the phospho-transferase portion of c-KIT's kinase domain do not play an important role in the progression of canine cutaneous MCTs, or in the aberrant localization of KIT in canine MCTs.

Figure 1

Schematic diagram of the receptor tyrosine kinase KIT.

Figure 2

Immunohistochemical staining patterns of canine cutaneous MCTs staining with anti-KIT antibodies. A. KIT staining pattern 1: peri-membrane KIT protein localization; B. KIT staining pattern 2: focal or stippled cytoplasmic KIT protein localization; C. KIT staining pattern 3: Diffuse cytoplasmic KIT localization. Canine MCTs with KIT staining patterns 2 and 3 that lacked ITD c-KIT mutations were screened for mutations in c-KIT exon 17. B and C show MCTs with aberrant KIT localization that lack ITD c-KIT mutations.

Figure 3

Schematic Diagram of Primer Design for Polymerase Chain Reaction. Forward and reverse primers (arrows) were designed in introns (black boxes) flanking exons 16, 17, 18, 19, and 20 (white boxes).

Figure 4

Amino acid alignment of the kinase domain (exons 16–20) of canine and human KIT. Canine and human amino acid alignments demonstrate a 99.5% identity between the phospho-transferase portion of the kinase domain of these species. Codon 816, which is commonly mutated in human mastocytosis patients (D816V; shaded residue) is also conserved in canine KIT.