Multiple-sectioning Study to Establish a Standardized Immunohistochemical Method for Detecting Isolated Tumor Cells in Lymph Nodes of Patients with Colorectal Cancer

To establish a standardized immunohistochemical method for detecting isolated tumor cells (ITC) in lymph nodes of patients with colorectal cancer (CRC), we examined the detection rates of ITC using sections of various thicknesses and two different kinds of cytokeratin monoclonal antibodies, CAM5.2 and AE1/AE3. According to the sixth edition of the TNM classification, ITC was defined as a single tumor cell or small cell clusters not greater than 0.2 mm. From 29 patients with stage III colorectal cancer, 149 lymph nodes diagnosed as negative for cancer metastasis by routine hematoxylin and eosin (H&E) staining were randomly selected. Twelve serial sections were cut for each lymph node. Sections one to eight were cut at a 4 μm thickness, and sections nine to 12 were cut at a 10 μm thickness. Sections one and eight, two to six and nine to 11, and seven and 12, were stained with H&E, the CAM5.2 antibody, and the AE1/AE3 antibody, respectively. Comparisons of cumulative ITC positive rates in sections of varying total thicknesses stained with the CAM5.2 antibody revealed that no significant differences were found when the total thickness of the sections examined exceeded 8 μm. There was no significant difference in ITC positive rates between CAM5.2 and AE1/AE3 staining. Our data indicate that, when monoclonal antibodies CAM5.2 or AE1/AE3 are used, ITC can be optimally detected immunohistochemically in sections with a total thickness of more than 8 μm. This practical suitable procedure can be used for standardizing the detection method for ITC.