Immunohistochemical Mapping of Bcl9 Using Two Antibodies that Recognize Different Epitopes Is Useful to Characterize Juvenile Development of Hepatocellular Carcinoma in Myanmar

B-cell lymphoma 9 (Bcl9) is the core component of Wnt/β-catenin signaling and overexpressed in nuclei of various tumors, including hepatocellular carcinoma (HCC). However, the extent of Bcl9 expression relative to HCC differentiation stage and its functional aspects are poorly understood. In this study, we examined the expression pattern of Bcl9 immunohistochemically, using two anti-Bcl9 antibodies; one was a conventional polyclonal-antibody (anti-Bcl9(ABC)) against amino acid no.800–900 of human-Bcl9, while the other (anti-Bcl9(BIO)) was against amino acid no.50–200, covering Pygopus-binding sites of Bcl9. Immunohistochemistry using anti-Bcl9(BIO) demonstrated distinctive staining in the cytoplasm, while the anti-Bcl9(ABC) signal was detected in both cytoplasm and nuclei of HCC cells, reflecting different states of Bcl9 function because Pygopus-binding to Bcl9 is essential to exert its function together with β-catenin in nucleus. Quantitative analysis revealed a significantly higher immunohistochemical-score by anti-Bcl9(BIO) in normal liver comparing various differentiation grades of HCC (P < 0.004), whereas no significant difference was noted with anti-Bcl9(ABC). Interestingly, immunohistochemical-score of anti-Bcl9(BIO) in patients aged < 40 years was significantly lower than that of ≥ 40 years group (P < 0.01). The results indicated that anti-Bcl9(BIO) detected cytoplasmic Bcl9, which does not bind to Pygopus suggesting it could be a useful indicator for development of HCC in young Myanmar patients.