Abstract: There have been many reports on the overexpression of human epidermal growth factor receptor 2 (HER2) in patients with colon cancer. However, the role and frequency of HER2 overexpression have not been clearly defined. Anti-HER2 therapy has been shown to improve the prognosis of HER2-positive patients with breast and stomach cancers. In this study, we explored HER2 expression in patients with colon cancer at stages II and III by immunohistochemistry (IHC) and dual-color in situ hybridization (DISH), and examined the correlation between HER2 expression and clinicopathological factors. Moreover, we examined the correlation between HER2 expression and mucin 1 (MUC1) expression. The subjects were 121 patients with colon cancer at stages II and III who underwent surgery in our hospital during the period from 2007 to 2009. Sections containing the deepest part of a lesion were subjected to immunostaining for HER2 and MUC1. HER2 expression was assessed in accordance with Ventana's Guidelines for HER2 Testing in Stomach Cancer, with sections comprising less than 10% of weakly to moderately stained tumor cells scored as 1 > 2. HER2 expression scored as 2 was defined with sections comprising more than 10% of the weakly to moderately stained tumor cells. Patients with a score of 1 > 2 and 2 were also subjected to DISH using a Dual ISH HER2 kit. MUC1 expression was scored according to the percentage of stained area as follows: 0, 0 to 5%; 1, 5 to 50%; and 2, 50% and higher. Patients with a score of 1 and 2 were defined as MUC1-positive. The analysis of HER2 by IHC yielded the following scores: 45 patients (37.2%), 0; 38 patients (31.4%), 1; 14 patients (11.6%); 1 > 2; 24 patients (19.8%), 2; and 0 patients (0%), 3. For the 38 patients with a score of 1 > 2 and 2, DISH returned ratios of HER2 to Chr17 expression (HER2: Chr17 ratio) from 1.13 to 1.93 (mean = 1.46). There was no significant correlation between HER2 expression and clinicopathological factors. The numbers of MUC1-positive patients according to HER2 score were as follows: 22 patients (48.9%) in the score 0 group (45 patients); 25 patients (65.8%) in the score 1 group (38 patients); 10 patients (71.4%) in the score 1 > 2 group (14 patients), and 22 patients (91.7%) in the score 2 group (24 patients). There was a positive correlation between HER2 expression and MUC1 expression. Specifically, MUC1 expression levels increased with HER2 expression level, and the percentage of MUC1-positive patients was significantly higher in the HER2 score 2 group than in the HER2 score 0 group (P < 0.01). Rates of HER2 positivity by DISH or fluorescence in situ hybridization (FISH) in patients who had an HER2 score of 2+ by IHC were 45% and 24% in the patients with stomach and breast cancers, respectively. However, the positivity rate was 0% in the patients with colon cancer in this study. This result indicates that patients with colon cancer who have an IHC HER2 score of 2+ are more likely to be HER2 negative by DISH than patients with breast and stomach cancers, although larger cohort studies are required before a definitive conclusion can be made. There was a positive correlation between HER2 expression and MUC1 expression in this study, although further examination is required because there were no patients who had an HER2 score of 3+ or 2+ by IHC and were HER2 positive by DISH in this study. HER2 expression in colon cancer should be cautiously assessed by both IHC and DISH.