Background: Fibrous dysplasia/McCune-Albright syndrome (FD/MAS) is a rare genetic bone disease caused by a somatic mutation in the GNAS gene. Currently used bone turnover markers (BTMs) do not... Show moreBackground: Fibrous dysplasia/McCune-Albright syndrome (FD/MAS) is a rare genetic bone disease caused by a somatic mutation in the GNAS gene. Currently used bone turnover markers (BTMs) do not correlate with the clinical picture and are not useful to predict or monitor therapy success. This study assessed the correlation of RANKL, OPG, RANKL/OPG ratio, IL-6 and sclerostin with the classic BTMs alkaline phosphatase (ALP), procollagen type 1 propeptide (P1NP) and beta crosslaps (CTX), with pain, skeletal burden score (SBS) and response to bisphosphonate or denosumab treatment.Methods: Ninety-six serum samples of adult patients >18 years of age with any subtype of FD/MAS were included from the biobank facility of the Leiden University Medical Center, Center for Bone Quality between 2015 and 2021. Standard laboratory assessments were assessed as part of usual care. The concentrations of potential biomarkers RANKL, OPG, sclerostin, IL-6 were analyzed. Data on FD/MAS subtype, age, pain, treatment history and treatment response were retrieved from the electronic patient files. Baseline characteristics were summarized by descriptive statistics. Correlations of the concentrations of the potential biomarkers with classic bone turnover markers, SBS and pain scores were cross-sectionally assessed by Spearman rank order correlation. Correction for multiple testing was performed by Benjamini and Hochberg False Discovery Rate. A sensitivity analyses was performed by excluding patients with SBS below 15 and patients using antiresorptive medication at the time of blood withdrawal or within the wash-out period. In patients treated with bisphosphonates or denosumab after blood withdrawal, pre-treatment concentrations were compared in patients with and without therapy response by Mann Whitney U test.Results: The median age of the patients was 41.2 (Q1-Q3 25.9-52.2) years, 62.5 % was female. Median SBS was 2.5 (Q1-Q3 0.5-7.8). RANKL level correlated weakly with ALP (Spearman rho 0.309, p = 0.004, n = 84), but not with P1NP or CTX. The RANKL/OPG ratio, OPG, IL-6 and sclerostin did not correlate with ALP, P1NP or CTX. None of the potential biomarkers correlated with SBS or pain. Results of the sensitivity analyses were comparable. Pre-treatment biomarker levels were similar in patients with and without improvement in pain scores following bisphosphonate therapy. Pre-treatment RANKL and sclerostin were comparable between patients with and without improvement in pain scores after denosumab therapy. Pre-treatment IL-6 level and the RANKL/OPG ratio seemed to be higher in patients with response to denosumab (IL-6: median 0.64 (Q1-Q3 0.53-0.74) pg/mL, n = 6, RANKL/OPG: median 0.062 (Q1-Q3 0.016-0.331), n = 5) compared to patients without response (IL-6: median 0.35 (0.20-0.54) pg/mL, n = 5, RANKL/OPG: 0.027 (0.024-0.046), n = 4). Pre-treatment IL-6 correlated with the improvement in maximum pain scores (rho 0.962, p < 0.001, n = 9) and average pain scores (rho 0.895, p = 0.001, n = 9) reported during denosumab therapy.Conclusion: Increased concentrations of RANKL, IL-6, sclerostin and of the RANKL/OPG ratio do not indicate severity of FD/MAS, as no correlation was observed of these potential biomarkers with the classic BTMs and SBS. Biomarker levels did not correlate with pain and had no value in predicting bisphosphonate treatment response. These biomarkers are not superior over the currently used methods of assessing ALP, P1NP and CTX or evaluating SBS to establish disease extent or activity and provide no reliable results.Yet, possibly pre-treatment IL-6 and the RANKL/OPG ratio may have some predictive value for clinical response to denosumab. Therefore, studies investigating disease activity and treatment response should include lesional imaging and patient-reported outcome measures. Show less
Meier, M.E.; Hagelstein-Rotman, M.; Majoor, B.C.J.; Geels, R.E.S.; Appelman-Dijkstra, N.M.; Bravenboer, N. 2023
Background: In fibrous dysplasia/McCune-Albright syndrome (FD/MAS), mosaic mutations in the GNAS gene lead to locally abnormal bone turnover. Additionally, patients with FD/MAS, particularly with... Show moreBackground: In fibrous dysplasia/McCune-Albright syndrome (FD/MAS), mosaic mutations in the GNAS gene lead to locally abnormal bone turnover. Additionally, patients with FD/MAS, particularly with thoracic lesions, have an increased risk for breast cancer. Development and progression of breast cancer has been associated with expression of Receptor Activator of NF-kappa B ligand (RANKL) in mammary tissue, and due to the GNAS mutation, RANKL is systemically increased in patients with FD/MAS. Yet it is unknown whether breast cancer in FD/MAS is also dependent on RANKL. We hypothesized that the GNAS mutation might induce RANKL overproduction and an oncogenic niche in mammary tissue, and examined RANKL expression in breast cancer tissue of patients with FD/MAS compared to controls. Methods: Nine patients with FD/MAS and breast cancer were included and clinical data were retrieved. Patients were matched to controls with breast cancer without FD/MAS based on age and tumor type. Three pregnant breast cancer patients were included as positive controls. Immunohistochemical detection of RANKL was per-formed on formalin-fixed paraffin-embedded breast cancer specimens. Staining intensity was classified as weak, moderate or intense. The area of positive RANKL staining divided by the total ductal-lobular area was assessed (positive area percentage, PAP). Number of patients with RANKL expression was compared between FD/MAS and control group by chi-square (chi 2) test, the PAP by Mann-Whitney U test (MWU). Results: RANKL expression was observed in 3 patients with FD/MAS (38 %), mainly in healthy tissue, and none of the control patients (chi 2 p = 0.055). The FD/MAS group demonstrated considerably more intense staining than the control group, comparable to positive controls. The median PAP was 0.64 % (range 0.14-2.04 %) in the 3 FD/MAS patients with RANKL expression, 0.01 % (Q1-Q3: 0.0003-0.514 %) in the entire FD/MAS group, 0.006 % (Q1-Q3: 0.001-0.012 %) in the control group (MWU = 0.574), and 0.19 % (0.08-0.32 %) in the pregnant patients. All patients with FD/MAS and RANKL expression had thoracic bone lesions, but no correlation was observed between RANKL expression and presence of the GNAS mutation or FD disease burden. Conclusions: The triad of a higher number of patients, higher positive area percentage and stronger intensity in the FD/MAS compared to the control group indicates that RANKL may be upregulated in mammary tissue in a subset of patients with FD/MAS, which may explain the increased risk for breast cancer, although the clinical significance remains unclear. Further research is needed to establish risk profiles for the development of RANKL-positive breast cancer and to improve early screening and treatment. Show less
Schreuder, W.H.; Lipplaa, A.; Cleven, A.H.G.; Berg, H. van den; Bisschop, P.H.; Jongh, R.T. de; ... ; Lange, J. de 2022
Background: In all giant-cell-rich lesions (GCRL) occurring in bone, a common underlying excessive RANKL expression is held responsible for the osteolytic activity. Apart from giant cell tumour of... Show moreBackground: In all giant-cell-rich lesions (GCRL) occurring in bone, a common underlying excessive RANKL expression is held responsible for the osteolytic activity. Apart from giant cell tumour of bone (GCTB), systematic outcome analysis of RANKL inhibition in other GCRL is unavailable. The aim of this study is to assess the efficacy and safety of a 1-year denosumab protocol in giant cell lesions of the jaw (GCLJ).Methods: A retrospective cohort study was conducted compromising patients treated with a 1year protocol of monthly subcutaneously administered 120 mg denosumab. Objective tumour response based on histology and imaging was used to calculate objective tumour response rate, progression-free survival (PFS) and time to progression. Type, severity and frequency of adverse events were recorded in a standardised way to assess safety.Results: Twenty patients, predominantly female (90%), were included. Fifty-five per cent of lesions were located in the mandible; most classified as aggressive lesions (90%). Thirty-five per cent (7/20) of cases were either recurrent after prior treatment or progressive, while on other drug treatment. Objective tumour response rate was 100% after 12 months of treatment. Median PFS was 50.4 months (95% CI 38.0-62.8) with a cumulative PFS rate of 22.6% (95% CI 1.8-43.4) at 5 years follow-up. Median time to progression was 38.4 months (95% CI 26.0 -50.8). Treatment was well tolerated, and none of the patients had to interrupt therapy for toxicity.Conclusion: High-dose denosumab is effective and safe in achieving a complete response in GCLJ within 12 months. The high long-term relapse rate after treatment cessation is the main obstacle for denosumab to become standard treatment for GCLJ. 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Show less
Beekman, K.M.; Zwaagstra, M.; Veldhuis-Vlug, A.G.; Essen, H.W. van; Heijer, M. den; Maas, M.; ... ; Bravenboer, N. 2019
Estrogen deficiency induces bone loss by increasing bone resorption, in part through upregulation of receptor activator of nuclear factor-kappa B ligand (RANKL). RANKL is secreted by osteoblasts... Show moreEstrogen deficiency induces bone loss by increasing bone resorption, in part through upregulation of receptor activator of nuclear factor-kappa B ligand (RANKL). RANKL is secreted by osteoblasts and osteocytes, but more recently bone marrow (pre)adipocytes have also been shown to express RANKL. Estrogen deficiency increases bone marrow adipose tissue (BMAT). The aim of this study was to determine the effect of ovariectomy (OVX) on RANKL protein expression by bone marrow adipocytes in C3H/HeJ mice. Fourteen-week-old female C3H/HeJ mice (n = 20) were randomized to sham surgery (Sham) or OVX. After 4 wk animals were euthanized. BMAT volume fraction (BMAT volume/marrow volume) was quantified by polyoxometalate-based contrast-enhanced nano-computed tomography. The percentage of RANKL-positive bone marrow adipocytes (RANKL-positive bone marrow adipocytes/total adipocytes) and the percentage of RANKL-positive osteoblasts covering the bone surface (bone surface covered in RANKL-positive osteoblasts/total bone surface) were quantified in the distal metaphysis of immunohistochemically stained sections of the left femur. The effects of OVX were analyzed by Student's t test or Mann-Whitney U test. RANKL was detected in osteoblasts, osteocytes, and bone marrow adipocytes. OVX significantly increased mean percentage of RANKL-positive bone marrow adipocytes [mean (SD): Sham 42 (18)%; OVX 64 (12)%; P = 0.029] as well as BMAT volume/marrow volume [median (interquartile range): Sham 1.4 (4.9)%; OVX 7.2 (7.3)%; P = 0.008] compared with Sham. We show that OVX increased both the percentage of RANKL-positive bone marrow adipocytes and the total BMAT volume fraction in C3H/HeJ mice. Therefore, RANKL produced by bone marrow adipocytes could be an important contributor to OVX-induced bone loss in C3H/HeJ mice. Show less
