Abstract P6-07-11: Serum levels of RANKL are increased in primary breast cancer patients in the presence of disseminated tumor cells in the bone marrow
Authors: Kasimir-Bauer S , Bittner A-K , Goebel A , Hoffmann O , Browne AJ , Rauner M , Hofbauer LC , Wimberger P , Kimmig R , Rachner TD 
Published: March 2017

Abstract
Background: Receptor activator of nuclear factor kappa-B ligand (RANKL) is an essential protein for osteoclast regulation that is associated with benign and malignant bone disease. The activity of RANKL is controlled by its soluble decoy receptor osteoprotegerin (OPG). There is increasing evidence that RANKL may also directly affect breast cancer progression and metastasis to the bone. This study was aimed to assess the levels of RANKL and OPG in 509 newly diagnosed breast cancer patients with regard to the presence of disseminated tumor cells (DTCs) in the bone marrow (BM), circulating tumor cells (CTCs) in blood and clinical parameters.
Patients and Methods: 509 patients with first diagnosis of breast cancer between Aug 2006 and Dec 2009 were included in our study. Blood and BM sampling was performed before surgery in an adjuvant setting. Blood was collected from each patient and sRANKL as well as OPG in the serum were measured by ELISA (Immunodiagnostic, Vienna, Austria). Two BM aspirates were analyzed for DTCs by immunocytochemistry using the pan-cytokeratin antibody A45-B/B3. In a subgroup of 364 patients, 2 x 5 ml blood was studied for CTCs using the AdnaTest BreastCancer (QIAGEN, Hannover GmbH, Germany) for the detection of EpCAM, MUC-1, HER-2, and beta-Actin transcripts.
Results: Mean serum values for RANKL and OPG were 0.23 ± 0.20 pmol/l and 4.24 ± 1.70 pmol/l, respectively. RANKL levels were significantly lower in women above 60 years of age (0.19 pmol/l vs 0.26 pmol/l; p &lt; 0.0001). This finding was reflected by higher RANKL serum levels and RANKL/OPG ratios in premenopausal patients compared to peri- (p&lt;0.05) and postmenopausal patients (p&lt;0.001), respectively. RANKL/OPG ratios were also higher in patients with lymph node involvement (N1-N3, p=0.03). All other clinical parameters did not influence RANKL or OPG levels. DTCs were detected in 213/509 (42%) patients and CTCs in 81/364 (22%) patients, respectively. However, while RANKL levels were unchanged in patients with detectable CTCs, they significantly increased by 33% (p&lt;0.0001) in patients with DTCs. There was no difference in OPG levels, resulting in an increased RANKL to OPG ratio in patients with DTCs in the BM (0.087 vs. 0.060; p &lt; 0.0001).
Conclusion: In conclusion, we show that RANKL serum levels and RANKL/OPG ratios are increased in patients with detectable DTCs in the BM, prior to the establishment of detectable bone metastases. This finding warrants further investigation as it may provide a rational for novel diagnostic or therapeutic approaches.
Citation Format: Kasimir-Bauer S, Bittner A-K, Goebel A, Hoffmann O, Browne AJ, Rauner M, Hofbauer LC, Wimberger P, Kimmig R, Rachner TD. Serum levels of RANKL are increased in primary breast cancer patients in the presence of disseminated tumor cells in the bone marrow [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-07-11.