498 Co-Activation of RET and TrkA Pathways in Breast Cancer Brain Metastasis: A Pre-Clinical Study for Future Clinical Application of FDA-Approved RET and TrkA Inhibitors

INTRODUCTION: Breast cancer brain metastasis (BCBM) remains a devastating disease due to limited actionable targets and lack of brain-permeable therapies. While the Rearranged during transfection (RET) receptor tyrosine kinase and the Tropomyosin receptor tyrosine kinase A (TrkA) pathways are individually implicated in BCBM, their role as dual targets in BCBM has not been established. METHODS: We analyzed RET and TrkA pathway activation signatures and correlated the co-activation with relapse information using publicly available breast cancer patient databases (TCGA and GEO). Primary breast tumors and matched BCBM were stained using immunohistochemistry (IHC) to evaluate RET and TrkA co-expression. Western blot analysis of human breast cancer cell lines was used to determine protein expression of active RET and TrkA. Statistical analyses (Chi-square, t-test, ANOVA regression models, and Kaplan-Meier methods) were performed as appropriate. RESULTS: Analysis of 710 breast cancer patients demonstrates a significant positive correlation (R 2 = 0.2926, p < 0.001) of RET and TrkA activation signatures, suggesting pathway co-activation. The combined RET-TrkA signature was significantly higher in patients with BCBM compared to those without brain relapse (p = 0.0183), and tumors with high RET-TrkA co-activation correlated with shorter brain metastasis-free survival (74.5 vs. 96 months, p = 0.009). IHC staining of 15 BCBM patient tissue samples showed strong positive staining for both activated RET and TrkA in 13/15 samples (p = 0.0083) as well as stronger staining compared to matched primary tumor samples (p = 0.0001, p = 0.0023). Western blot analysis of breast cancer cell lines further demonstrated high expression of activated RET and TrkA in brain-tropic cell lines compared to parental lines. CONCLUSIONS: The RET and TrkA pathways are co-activated in BCBM and confer worse brain metastasis-free survival, supporting further studies using FDA-approved RET and TrkA inhibitors that show excellent cranial response rates in lung cancer patients with brain metastases.