Immunohistochemistry assay

The antigen retrieval was conducted using a microwave method, and then, TPM1 antibody (1:500, Abcam) was added for immunostaining. After being incubated with biotinylated secondary antibody, the specimens were transferred to a DAB detection system. Hematoxylin was used to counterstain nuclei, according to the manufacturer’s protocol (DaKo, Glostrup, Denmark).

The visual evaluation of immunostaining degree was based on a four grade scoring system (ranging from 0–3). If the proportion of cells with positive staining was less than five percent, the score was zero. If the proportion was between five percent and twenty-five percent, the score was one. If the proportion was between twenty-five percent and fifty percent, the score was two. If the proportion was higher than fifty percent, the score was three. Each sample, which contained approximately 500 cells, was observed by two pathologists, and then, the average count was calculated based on the observation values. Clinical samples were divided into two groups, a high- expression group and a low-expression group. The high-expression group contained clinical samples with staining scores of 2 or 3 points. Conversely, the low-expression group contained clinical samples with staining scores of 0 or 1 points.