Poor prognosis of patients with triple-negative breast cancer can be stratified by RANK and RANKL dual expression

Monica E. Reyes, Takeo Fujii, Daniel Branstetter, Savitri Krishnamurthy, Hiroko Masuda, Xiaoping Wang, James M. Reuben, Wendy A. Woodward, Beatrice J. Edwards, Gabriel N. Hortobagyi, Debu Tripathy, William C. Dougall, Bedrich L. Eckhardt, Naoto T. Ueno

As clinical studies have correlated RANK expression levels with survival in breast cancer, and that RANK signaling is dependent on its cognate ligand RANKL, we hypothesized that dual protein expression further stratifies the poor outcome in TNBC. RANK mRNA and protein expression was evaluated in TNBC using genomic databases, cell lines and in a tissue microarray of curated primary tumor samples derived from 87 patients with TNBC. RANK expression was evaluated either by Mann-Whitney U test on log-normalized gene expression data or by Student's t test on FACS data. Analysis of RANK and RANKL immunostaining was calculated by H-score, and correlations to clinical factors performed using χ (2) or Fisher's exact test. Associations with RFS and OS were assessed using univariate and multivariate Cox proportional hazard models. Survival estimates were generated using the Kaplan-Meier method. In three distinct datasets spanning 684 samples, RANK mRNA expression was higher in primary tumors derived from TNBC patients than from those with other molecular subtypes (P < 0.01). Cell surface-localized RANK protein was consistently higher in TNBC cell lines (P = 0.037). In clinical samples, TNBC patients that expressed both RANK and RANKL proteins had significantly worse RFS (P = 0.0032) and OS (P = 0.004) than patients with RANK-positive, RANKL-negative tumors. RANKL was an independent, poor prognostic factor for RFS (P = 0.04) and OS (P = 0.01) in multivariate analysis in samples that expressed both RANK and RANKL. RANK and RANKL co-expression is associated with poor RFS and OS in patients with TNBC.