Abstract 695: Significant loss of nuclear expression of Actinin-4 in metastatic breast carcinoma and lymph nodes: A novel biomarker for metastatic breast carcinoma

Background: Actinin-4 is an actin cross-linked protein. The expression and subcellular distribution of actinin-4 (ACTN4) has been associated with multiple roles in tumorigenicity and cancer metastasis. In the present study, we developed a novel antibody against human actinin-4 and investigated the clinical utility of actinin-4 as a biomarker in metastatic breast cancer. Methods and findings: We generated a novel monoclonal antibody (AB-Actinin-4) using hybridoma technology and native ACTN4 protein as an immunogen. The antibody specifically recognized both native and denatured human Actinin-4 protein and displayed high affinity as assessed by ELISA, immunohistochemistry assay, western blot and flow cytometry. Using this antibody and ELISA as well as immunohistochemical techniques, we examined plasma levels and tissue expression of actinin-4 in healthy controls’ and breast cancer patients’ samples. 50 plasma samples from healthy controls and breast cancer patients were analyzed for the presence of soluble actinin-4. ELISA results showed significantly decreased actinin-4 levels in plasma samples from patients with breast carcinomas compared to healthy control plasma. A total of 156 cases, including 70 normal, 36 non-metastatic breast carcinoma, 50 breast carcinoma tissues with regional metastasis, and 50 matched metastatic lymph nodes were examined. The immunoreactivity of actinin-4 was located mainly in nucleus and cytoplasm of cells. To assess the difference between groups, the staining intensity and intracellular distribution were compared and the intensity in nucleus and cytoplasm was separately scored as 0, 1+, 2+ and 3+. Grades 0 and 1+ were defined as negative and 2+ and 3+ as positive. Significant differences between the groups were found in nuclear staining. 92% of normal and 89% of non-metastatic breast carcinoma samples displayed positive nuclear staining. In contrast, only 29% of metastatic lymph nodes and 51% of metastatic breast cancer tissues showed strong nuclear staining. Conclusion: Actinin-4 demonstrated significantly decreased expression in plasma from breast cancer patients. Metastasis leads to significant loss of the nuclear expression of Actinin-4 in breast cancer tissues, in comparison to normal and non-metastatic breast carcinoma tissues. Our data suggests that Actinin-4 might have significant impact in breast cancer metastasis. Actinin-4 may serve as a reliable biomarker for the diagnosis and prognosis of metastatic breast cancer as