Immunohistochemical Analysis of HOXA5 Expression

The technical process of immunohistochemistry (IHC) was performed as previously described^{7 (link)}. The HOXA5 staining strength was categorized into different groups depending on the positive cell percentage and positive cell staining density. The positive cell percentage was categorized into five grades: 0–3% (0), 3–25% (1), 26–50% (2), 51–75% (3), and 76–100% (4). The positive cell staining density was also categorized into four grades: negative (0), weak brown (1), moderate brown (2), and strong brown (3). The final immunohistochemical score was calculated as follows: immunoreactivity score (IRS) = intensity score × positive score. The final IRS were categorized into three groups: negative (≤3), weak positive (>3 but ≤6), and strong positive (>6). The antibodies used were as follows: anti-HOXA5 (1:100, sc-365784, Santa Cruz); anti-Ki67 (1:100, sc-23900, Santa Cruz); anti-cyclinD1 (1:100, sc-8396, Santa Cruz); anti-p21 (1:50, sc-817, Santa Cruz); anti-β-catenin (1:50, sc-7963, Santa Cruz); anti- p53 (1:50, sc-7963, Santa Cruz). The technical process of immunocytochemistry was performed as previously described^{22 (link)}.