Appropriate epitope retrieval for c-kit protein immunostaining in routinely prepared specimens of gastrointestinal stromal tumor

Gastrointestinal stromal tumor (GIST), immunohistochemically characterized by c-kit protein (CD117) expression, is known to be resistant to chemotherapy and radiotherapy when it is malignant. Recently, Gleevec (STI571) has been introduced for the therapy of metastatic GISTs, and appropriate evaluation of c-kit protein has thus been clinically relevant to confirm the indication of Gleevec therapy. However, we often experienced the instability of immunohistochemical staining for c-kit protein. The aim of the present study is to establish a reproducible immunohistochemical procedure for c-kit protein detection in paraffin sections. Regarding the fixation period, formalin fixation for 24 to 72 hours gave the best result among several fixation conditions. For the purpose of epitope retrieval, hydrated heating in EDTA solution, pH 8.0, employing a pressure cooker gave the most reproducible immunostaining result. For visualization of the reaction products, diaminobenzidine solution available from DakoCytomation Co. was the most sensitive. Under these conditions, c-kit protein immunoreactivity was clearly observed in 20 (95%) of 21 GISTs, routinely processed for histologic preparation. Two tumors were proven to be false negative by the routine diagnostic procedure.