Elsevier

The Veterinary Journal

Volume 207, January 2016, Pages 118-123
The Veterinary Journal

Analysis of c-KIT exon 11 mutations in canine gastrointestinal stromal tumours

https://doi.org/10.1016/j.tvjl.2015.10.051Get rights and content

Highlights

  • We assessed c-KIT exon 11 mutations in canine gastrointestinal stromal tumors.

  • RT-PCR had a higher detection rate (34/46; 73.9%) than conventional PCR (15/46; 32.6%).

  • Diffuse or partial KIT immunostaining was detected in the tumor.

  • Neither pattern was significantly associated with c-KIT exon 11 mutation status.

  • RT-PCR was more sensible for detecting c-KIT mutations than conventional PCR.

Abstract

The aim of this study was to determine the type and frequency of c-KIT exon 11 mutations in canine gastrointestinal stromal tumours (GISTs) and investigate the association between the c-KIT mutation status and KIT immunohistochemical staining pattern. Mutations in exon 11 of c-KIT were examined in 46 formalin-fixed paraffin-embedded canine GISTs using PCR of genomic DNA and reverse transcription-PCR (RT-PCR) of cDNA.

Exon 11 c-KIT mutations were detected in 15/46 (32.6%) cases by conventional PCR and 34/46 (73.9%) cases by RT-PCR; the mutation detection rate was significantly higher for RT-PCR (P = 0.004, Fisher's exact test). Ten different mutations, including deletion, internal tandem duplication and point mutations, were identified by RT-PCR. Immunohistochemistry was performed using an anti-KIT antibody; diffuse KIT staining was detected in the tumour cell cytoplasm in 32/46 (69.6%) cases and partial or stippled cytoplasmic staining of KIT was observed in 14/46 (30.4%) cases. Neither pattern was significantly associated with c-KIT exon 11 mutation status (P = 1.000, chi-square test). These data indicate that c-KIT exon 11 mutations occur frequently in canine GISTs, similar to human GISTs; however, there is no association between c-KIT mutations and the KIT expression pattern in canine GISTs. This study suggests that RT-PCR is more sensitive than conventional PCR for the detection of c-KIT mutations in canine GISTs.

Introduction

The molecular basis of human gastrointestinal stromal tumours (GIST) is relatively well characterised (Hirota et al, 1998, Heinrich et al, 2008). In human GISTs, mutations in c-KIT occur frequently on exon 11 and also infrequently on exons 9, 13 and 17 (Corless and Heinrich, 2008). It has been reported that c-KIT mutations also occur in canine GISTs (Frost et al, 2003, Gregory-Bryson et al, 2010). Canine GISTs have received little analysis, though a small number of c-KIT mutations were previously identified on exons 9 and 11 (Frost et al, 2003, Gregory-Bryson et al, 2010, Irie et al, 2015). In canine GISTs, Frost et al. (2003) detected a single c-KIT exon 11 point mutation and single c-KIT exon 11 deletion mutation in four cases (50%), and Gregory-Bryson et al. (2010) detected c-KIT exon 11 deletion mutations in six of 17 cases (35.3%). There is a lower reported frequency of c-KIT exon 11 mutations in canine GISTs than in human GISTs. Therefore, it is necessary to analyse c-KIT mutations in canine GISTs more thoroughly to investigate this difference.

Although a general treatment option for human GIST is surgical resection, there is no effective therapy for unresectable or metastatic GISTs and the prognosis in both cases may be poor. Thus, imatinib mesylate is the first-line therapy for those GISTs carrying the c-KIT mutation (Rubin et al., 2007). As with human GIST patients, a favourable response to imatinib mesylate has been reported in dogs with unresected or metastatic GISTs carrying a c-KIT mutation respectively, suggesting the possibility of predicting a better response to imatinib mesylate (Kobayashi et al, 2012, Irie et al, 2015). Interestingly, a metastatic GIST reported by Irie et al. (2015) showed complete remission after imatinib mesylate treatment. Therefore, it is important to assess c-KIT mutations in canine GISTs so that appropriate therapeutic options can be identified.

One recent study reported that the immunohistochemical staining patterns of KIT in human GISTs were different from the patterns observed in normal tissues (Jaramillo et al., 2012). In canine mast cell tumours, three different KIT staining patterns have been reported (Webster et al., 2006). It has been suggested that these tumour-staining patterns are related to c-KIT mutations (Webster et al, 2006, Jaramillo et al, 2012). However, debate remains on this issue, as tumours with c-KIT mutations do not always display aberrant KIT staining patterns.

We hypothesised that the frequency of c-KIT exon 11 mutations in canine GISTs is comparable to that of human GISTs, and that the c-KIT mutation status would be associated with the immunohistochemical staining pattern for KIT. In this study, the type and frequency of c-KIT exon 11 mutations in canine GISTs were examined using genomic DNA and cDNA prepared from formalin-fixed paraffin-embedded (FFPE) sections, and the relationship between the c-KIT exon 11 mutation status and the immunohistochemical staining pattern for KIT was investigated.

Section snippets

Sample processing

The study was conducted at North Lab and approved by the Institutional Animal Care and Use Committee of the North Lab (Certificate Number 14-001, 5 May 2014). Spontaneously occurring intestinal masses from client-owned dogs were surgically resected by veterinarians, immersed in 10% phosphate buffered formalin and submitted to North Lab for pathological examination. Tissue samples were embedded in paraffin and 4 µm-thick sections were stained with haematoxylin and eosin. GIST was diagnosed

Type and frequency of c-KIT exon 11 mutations

Results of mutation analysis from 46 cases are shown in Table 1. All mutation types (and frequencies) detected in this study are summarised in Table 2. Mutations in exon 11 of c-KIT were detected in 15/46 (32.6%) cases of GIST by analysis of genomic DNA. The most frequent mutation was c.1666_1671del (p.W556_K557del), which was observed in 11/46 cases (23.9%). The other mutations identified by genomic DNA analysis were: c.1724T>C (p.L575P) in 1/46 cases (2.2%); c.1665_1671delinsTCAT

Discussion

Mutations in exon 11 of c-KIT were detected in 73.9% of the cases of canine GIST in the present study. This is comparable to that of human GIST cases (69%; Heinrich et al., 2008). The previously reported c.1667_1672del (p.W556_K557delinsF) and c.1724T>C (p.L575P) mutations (Frost et al., 2003) were also observed in our study. A p.W556_K557del mutation has also been reported previously (Gregory-Bryson et al., 2010), and although a slightly different deletion was observed in our study

Conclusions

Mutations in exon 11 of c-KIT were detected in 73.9% of cases of canine GISTs and RT-PCR analysis of cDNA was the preferred method over conventional PCR amplification of genomic DNA. However, RT-PCR analysis of FFPE sections is more complicated and costs more than conventional PCR. Detection of c-KIT mutations in canine GISTs is an important clinical laboratory test required to select optimal therapeutic strategies such as targeted therapy. Further study is necessary to identify the optimal

Conflict of interest statement

None of the authors of this paper have a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper.

Acknowledgements

We are grateful to member practitioners of North Lab who submitted intestinal masses for pathological examination. We are also grateful to Dr. Tsuyoshi Takahashi, Department of Surgery, Osaka University Graduate School of Medicine, for his kind advice for this study.

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