Detection of exon-11 mutation in c-kit using PCR technique from canine cutaneous mast cell tumors obtained by fine-needle aspiration method

Abstract

This research has been objectively set up to study the usage of MCT cells collected by fine-needle aspiration (FNA) method for investigating mutant exon-11 in proto-oncogene c-kit from 30 MCT dogs. All studied MCT dogs were performed tissue biospsy for MCT histopathologic diagnosis and grading using Patniak histopathologic grading system. All biopsied tissues were also studied the staining patterns of KIT (CD117) by CD117-immunohistochemitry. The result was used as the standard criteria for CD117-immunocytochemistry interpretation. For fine-needle aspirated MCT cells (FNA-MCT cells) in cell suspensions, they were used further for study in detection of mutant exon-11 in c-kit. From flow cytometric quantitative analysis in fifteen FNA-MCT cell suspensions, the result suggested that there were approximately 6,345 FNA-MCT cells in 1 μl of cell suspensions (6.345 x 10⁶ cells/ ml). FNA-MCT cells were used for 2 purposes; the first for CD117-immunocytochemistry study and the last for DNA extraction used in PCR study. The result from CD117-immunocytochemistry indicated that the staining patterns of KIT mimic to the staining patterns observed in CD117-immunohistochemistry consisting of perimembrane, paranuclear and cytoplasmic diffuse. Moreover, the consequence also indicated that each specimen possessed the same staining pattern both in CD117-immunocytochemistry and CD117-immunohistochemistry except in one specimen having the different result. This suggested that FNA-MCT cells should be the representative of neoplastic cells in biopsied tissues. For PCR analysis, one FNA-MCT specimen classified as MCT-grade II by histopathology gave the positive outcome by detecting the mutant exon-11 in c-kit with PCR. In the comparative study of PCR analysis for mutant exon-11 in c-kit from FNA-MCT cells collected directly from the tumor mass with FNA-MCT cells sorted through flow cytometric cell sorter in one case, the results were not different from each other indicating that FNA-MCT cells directly obtained from the tumor mass should be the representative of MCT-cell populations in the biopsied tissue. In addition, they could be preliminarily applied for clinical detection of mutant exon-11 in c-kit by PCR. However, a mass study for further investigating the consistency of this method should be executed before this method will be clinically applied for MCT diagnosis and therapeutic planning.