Abstract P1-08-25: RANK expression is prognostic and predictive in primary breast carcinoma: Analysis of samples from the GeparTrio study
Authors: Pfitzner BM , Branstetter D , Mergler B , Loibl S , Denkert C , Fasching PA , Dougall WC , von Minckwitz G 
Published: August 2014

Abstract
Purpose: RANK ligand (RANKL), a key factor for bone remodeling and metastasis, is crucial for the development of mouse mammary glands during pregnancy. RANKL functions as a major paracrine effector of the mitogenic action of progesterone in mouse and human mammary epithelium via its receptor RANK and has a role in ovarian hormone-dependent expansion and regenerative potential of mammary stem cells. Pharmacologic inhibition of RANKL attenuates the development of mammary carcinoma and inhibits metastatic progression in multiple mouse models. Many published expression analyses of RANK and RANKL have been performed using immunohistochemistry (IHC) without documented validation of antibody specificity. This study analyzed primary breast carcinoma samples from the GeparTrio study to correlate the expression of human RANK and RANKL (using specific, monoclonal antibodies validated and optimized for IHC) with pathological complete response (pCR: ypT0 ypN0), disease-free (DFS), and overall (OS) survival after anthracycline-taxane-based neoadjuvant chemotherapy.
Methods: Pre-treatment FFPE core biopsies (n = 601) from participants of the neoadjuvant GeparTrio study were analyzed for percentage and intensity (H-score) of immunohistochemical RANK and RANKL expression. Antibodies against human RANK (N-1H8; Amgen) and human RANKL (M366; Amgen) were used for IHC staining. The overall expression measured by IHC was calculated using the H scoring method. The specificity of the antibodies was substantiated by concordant signals observed using multiple independent analyses, including IHC, flow cytometry and Western blots of positive and negative control cells and xenograft samples.
Results: RANK protein was expressed within the tumor epithelium in 160 (27%) patients. A cutoff of an H-score of &gt;8.5 was identified as optimal for prediction of pCR, DFS, and OS using an automated cut-off finder (http://molpath.charite.de/cutoff). High RANK expression (&gt;8.5 H-Score) was observed in 14.5% of patients and was strongly correlated with high tumor grade (p&lt;0.001) and negative hormone-receptor status (p&lt;0.001). Patients with high RANK expression showed a higher pCR rate (23.0% vs 12.6%, p = 0.010), shorter DFS (p = 0.038) and OS (p = 0.011) compared to patients with low RANK expression. However, prognostic and predictive information was not independent from other baseline parameters (age, histologic type, stage, grade, and hormone/HER2-receptor) in multivariate analyses. Only 6% of samples expressed RANKL within the tumor epithelium, which was not correlated with any clinical features.
Conclusion: Immunohistochemical RANK and RANKL protein expression was observed in the epithelial carcinoma in human primary breast cancers. Higher RANK expression in the primary tumor is associated with a higher sensitivity to chemotherapy, but also a higher risk of relapse and death. However, due to its strong correlation with grade and hormone-receptor status, RANK does not provide independent predictive and prognostic information. Denosumab, a clinically available antibody against RANKL, might have direct anti-tumor activity and should be further explored in this group of patients with high RANK expression.
Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-08-25.