Evaluation of the Prognostic Value of RANK, OPG, and RANKL mRNA Expression in Early Breast Cancer Patients Treated with Anthracycline-Based Adjuvant Chemotherapy

Abstract

BACKGROUND: Prevention of bone metastases is a major issue for breast cancer patients, as it would improve quality of life in a population where long survival is anticipated. PATIENTS AND METHODS: Early breast cancer patients, who had been treated with anthracycline-based chemotherapy within two randomized trials, were included in the study. We evaluated, by quantitative reverse transcription–polymerase chain reaction, 819 formalin-fixed paraffin-embedded tumor tissue samples for mRNA expression of RANK, OPG, and RANKL, as well as their ratios, for potential prognostic significance for the development of bone metastases and also for disease-free survival (DFS) and overall survival. RESULTS: Median age was 52.7 years, whereas 54.2% of the patients were postmenopausal and 78.3% estrogen receptor/progesterone receptor positive. After a median follow-up of 119.9 months, 226 patients (27.6%) had died and 291 patients (35.5%) had disease progression. Low mRNA expression of RANKL was associated with postmenopausal status and greater number of positive lymph nodes (P = .002 and P < .001, respectively). In the univariate analysis, low RANKL mRNA expression was found to be an unfavorable factor for DFS [hazard ratio (HR) = 1.33, 95% confidence interval (CI) 1.05-1.68, Wald's P = .018] and bone metastasis–free survival (HR = 1.67, 95% CI 1.09-2.56, P = .018), although it did not retain its significance in the multivariate analysis. CONCLUSIONS: Low RANKL mRNA expression in early breast cancer patients is of prognostic significance for increased risk for relapse and bone metastases and might potentially guide clinical decision-making for the use of anti-RANKL agents in the treatment of early breast cancer patients at high risk for metastatic spread, provided that our findings are validated in independent cohorts.

Evaluation of the prognostic value of CD3, CD8, and FOXP3 mRNA expression in early‐stage breast cancer patients treated with anthracycline‐based adjuvant chemotherapy

“…More than one FFPE section (2‐8 sections, 10 μm thick) was used for RNA extraction when the tumor surface of a given sample was less than 0.25 cm 2 . From each FFPE section or macrodissected tissue fragments, RNA was extracted using a standardized fully automated isolation method for total RNA from FFPE tissue, based on germanium‐coated magnetic beads (XTRAKT kit, STRATIFYER Molecular Pathology GmbH, Cologne, Germany) in combination with a liquid handling robot (XTRAKT XL, STRATIFYER Molecular Pathology GmbH), as previously described in detail . The method involves extraction‐integrated deparaffinization and DNase I digestion steps.…”

“…For each primer/probe set, the amplification efficiency was tested, aiming to reach comparable efficiency of >90% (efficiency range from 91% to 108%). Primers and hydrolysis probes were diluted to 100 μmol/L, using a stock solution with nuclease‐free water (Life Technologies GmbH, Darmstadt, Germany) . qRT‐PCR was applied for the relative quantification (RQ) of RANK, OPG, and RANKL.…”

“…From each FFPE section or macrodissected tissue fragments, RNA was extracted using a standardized fully automated isolation method for total RNA from FFPE tissue, based on germanium-coated magnetic beads (XTRAKT kit, STRATIFYER Molecular Pathology GmbH, Cologne, Germany) in combination with a liquid handling robot (XTRAKT XL, STRATIFYER Molecular Pathology GmbH), as previously described in detail. 19,32,34,35,43,44 The method involves extraction-integrated deparaffinization and DNase I digestion steps. The quality and quantity of RNA were checked by measuring CALM2 expression as a surrogate for amplifiable mRNA by qRT-PCR.…”

“…Moreover, the majority of breast cancer patients develop bone metastases, with the microenvironment of the host and its interactions with tumor cells playing a pivotal role in their progression . Receptor activator of nuclear factor‐kB (RANK) with its ligand (RANKL) and the RANKL decoy receptor osteoprotegerin (OPG) is highly involved in many cell functions associated with formation and progression of bone metastases; we have recently demonstrated that low RANKL mRNA expression in tumor tissue of early breast cancer patients is of prognostic significance for increased risk of relapse and bone metastases . RANK and RANKL are expressed not only in tumor tissue but also in immune cells.…”

“…18 Receptor activator of nuclear factor-kB (RANK) with its ligand (RANKL) and the RANKL decoy receptor osteoprotegerin (OPG) is highly involved in many cell functions associated with formation and progression of bone metastases; we have recently demonstrated that low RANKL mRNA expression in tumor tissue of early breast cancer patients is of prognostic significance for increased risk of relapse and bone metastases. 19 RANK and RANKL are expressed not only in tumor tissue but also in immune cells. For example, RANK is expressed in tumor-associated macrophages (TAMs) and NK cells, while RANKL has been reported to be expressed in activated T cells.…”