Abstract P6-01-01: RANK/RANKL expression by immunohistochemistry (IHC) in young breast cancer (BC) patients and during pregnancy: Association with clinicopathologic features, gene expression profiles and patient outcome
Authors: Azim Hatem A , Peccatori Fedro A , Brohee Sylvain , Branstetter Daniel , Pruneri Giancarlo , Loi Sherene , Viale Giuseppe , Dougall Bill , Sotiriou Christos 
Published: May 2015

Abstract
Background & Objectives: RANKL is a major paracrine effector of the mitogenic action of progesterone in mammary epithelium via its receptor RANK. Increased mammary tumor formation following pregnancy was observed in transgenic mice with gain of function in RANK, a process that was arrested using a RANKL inhibitor. Based on epidemiological studies, pregnancy increases BC risk on the short term with pregnancy-associated BC associated with poor prognosis. Here, we report for the first time the expression of RANK/RANKL in young BC patients using IHC, and its association with diagnosis during pregnancy and prognosis. We also evaluate genes and pathways that are activated in RANK/RANKL expressing tumors.
Patients & Methods: 195 young BC patients were included; of whom 65 were diagnosed during pregnancy. All patients had central pathologic review and 85% had available gene expression data using Affymetrix. RANK/RANKL expression by IHC on the primary tumor and adjacent normal tissue was performed at Amgen laboratories, blinded for clinical data. IHC was performed with antibodies against human RANK (N-1H8) and human RANKL (M366) using the H-score. The difference in expression of RANK/RANKL between pregnant and non-pregnant patients and the association with clinicopathologic features were examined. We evaluated genes and pathways that are associated with RANK/RANKL expression as a continuous variable in a linear regression model. Finally, we tested the association between RANK/RANKL expression and disease-free survival (DFS).
Results: Median age was 36 years (range: 28-47). RANKL expression was more prevalent in the pregnant group independent of other pathologic features; both on the tumor (mean H score: 32 vs. 8) and adjacent normal tissue (mean H score: 87.3 vs. 32.9, both p&lt;0.001). 18.7% of pregnant and 5.3% of non-pregnant patients had ≥10% of cells with RANKL expression 3+. RANKL expression was significantly higher in PgR+, well differentiated, and luminal-A tumors, with negative correlation with Ki-67 (all p&lt;0.001). RANK expression was higher on normal compared to tumor (23.6 vs. 14.2, p=0.003), with no differences according to pregnancy status. RANK expression was higher in triple negative and poorly differentiated tumors (all p&lt;0.001). Using FDR&lt;0.05, 151 and 1207 genes were significantly correlated with RANKL and RANK expression by IHC, respectively. A positive correlation was observed between mRNA and IHC expression of RANKL (r=0.89, p&lt;0.001) and RANK (r=0.19, p=0.01). High RANKL expression was associated with pathways related to mammary gland development, bone resorption, T-cell proliferation and regulation of chemotaxis, while RANK expression was associated with immune response and proliferation pathways. At a median follow-up of 65 months, neither RANK nor RANKL expression was associated with DFS.
Conclusions: RANKL expression is higher during pregnancy both in normal breast tissue and primary tumor and is associated with important biological processes. These results support the preclinical data suggesting RANKL as a key player in the crosstalk between pregnancy and BC guiding further development of RANKL-targeted therapy.
Citation Format: Hatem A Azim Jr, Fedro A Peccatori, Sylvain Brohee, Daniel Branstetter, Giancarlo Pruneri, Sherene Loi, Giuseppe Viale, Bill Dougall, Christos Sotiriou. RANK/RANKL expression by immunohistochemistry (IHC) in young breast cancer (BC) patients and during pregnancy: Association with clinicopathologic features, gene expression profiles and patient outcome [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-01-01.