Fibrous dysplasia/McCune-Albright syndrome (FD/MAS) is a rare condition caused by a mutation in the GNAS locus. Apart from endocrinopathies, some cases are characterized by excessive fibroblast growth factor 23 (FGF23) production from abnormal fibro-osseous tissue in FD lesions, resulting in increased renal phosphate excretion. We present a girl with FD/MAS and severe skeletal burden, evidenced by the presence of polyostotic fibrous dysplasia, which was complicated with bone fractures. She also had hyperthyroidism and GnRH-independent precocious puberty. She received zoledronic acid infusions in preparation for hip surgery. Despite optimal conventional management with oral phosphate and alphacalcidol, which was poorly tolerated, she presented persistent hypophosphatemia. To control hypophosphatemia and its deleterious effects on bone health, treatment with burosumab off-label at a dose of 0.66mg/kg (20mg) every 2wk was initiated. Serum phosphate levels normalized within 2wk of treatment. Laboratory results showed improvement in serum alkaline phosphatase (ALP) and PTH levels. After the second injection of burosumab, phosphate and PTH rose above the normal range with normal vitamin D levels; therefore, the interval between doses was increased to 3wk, and calcium 500mg daily was added. However, phosphate levels dropped again below normal range, so she had to return to 2-weekly injections of 20mg. After 11mo on burosumab, she remains with high normal phosphate levels and normal PTH and ALP values. Burosumab is well tolerated, with no adverse events to date. Burosumab is a human monoclonal antibody against FGF23 that reduces the risk of developing FGF23-mediated hypophosphatemia and its associated complications. Burosumab should be considered as an effective and safe alternative strategy for FGF23-mediated hypophosphatemia in FD/MAS for those who either cannot tolerate or do not respond to conventional therapy. To our knowledge, this is the fourth published case worldwide describing successful treatment with burosumab in FD/MAS.

Heterotopic ossifications (HOs) are the pathologic process by which bone inappropriately forms outside of the skeletal system. Despite HOs being a persistent clinical problem in the general population, there are no definitive strategies for their prevention and treatment due to a limited understanding of the cellular and molecular mechanisms contributing to lesion development. One disease in which the development of heterotopic subcutaneous ossifications (SCOs) leads to morbidity is Albright hereditary osteodystrophy (AHO). Albright hereditary osteodystrophy is caused by heterozygous inactivation of GNAS, the gene that encodes the α-stimulatory subunit (Gαs) of G proteins. Previously, we had shown using our laboratory’s AHO mouse model that SCOs develop around hair follicles. Here we show that SCO formation occurs due to inappropriate expansion and osteogenic differentiation of cells that express alpha-smooth muscle actin and that are located within the dermal sheath. We also show in AHO patients and mice that secreted frizzled related protein 2 (SFRP2) expression is upregulated in regions of SCO formation and that elimination of Sfrp2 in male AHO mice leads to earlier development, greater severity, and acceleration of formation of SCOs. These studies provide key insights into the cellular and molecular mechanisms contributing to SCO development and have implications for potential therapeutic modalities not only for AHO patients but also for patients suffering from HOs with other etiologies.

X-linked hypophosphatemia (XLH) is a rare inherited disorder characterized by elevated levels of FGF23, chronic hypophosphatemia, impaired bone mineralization, and chronic long-term manifestations. Treatment for XLH has been mainly focused on normalizing its biochemical abnormalities. Despite treatment, patients with XLH often present impaired physical function and decreased quality of life (QoL). We hypothesize that physical functionality and QoL are more strongly associated with chronic pain and decreased muscle mass than persistent biochemical abnormalities or exposure to conventional treatment. We conducted an observational, cross-sectional study with patients with XLH. Clinical records and biochemical parameters were assessed. QoL surveys SF36v.2 and WOMAC were applied. Functional status was measured by a physiatrist and an occupational therapist. Appendicular lean mass (ALM) was measured and compared with age and sex-matched healthy controls. Enthesopathies and osteoarthritis were evaluated. Pain was assessed using the Brief Pain Inventory, the Visual Analog Scale, and the Doleur Neuropathique-4 scales. Muscle strength was evaluated by the quadriceps muscle isometric strength (QMS) and physical performance with the 6-Minute Walk Test (6MWT) and the Functional Independence Measure (FIM) scale. A total of 30 patients were included: 21 females; median age of 32yr. All participants had significant functional deficits, chronic pain, and reduced QoL. Limitations in daily activities were significantly associated with higher severity of pain, decreased ALM, lower QMS, and less distance in 6MWT (p< .05). Neither FIM scale, phosphate levels, FGF23, nor the lifetime exposure to conventional treatment was associated with these functional variables. In conclusion, impaired physical functionality in patients with XLH was associated with lower muscle mass, lower muscle strength, and severe chronic pain. These findings highlight the importance of, in addition to optimizing the biochemical control of the disease, expanding patient care including pain prevention and management as well as comprehensive physical therapy and rehabilitation.

Activating internal tandem duplications (ITD) in the juxtamembrane domain of receptor tyrosine kinase FLT3 occur frequently in patients with acute myeloid leukemia (AML). Constitutive active FLT3-ITD mutations induce aberrant signaling and promote leukemic cell transformation. Inactivation of the attenuating receptor protein tyrosine phosphatase CD45 (PTPRC) in FLT3-ITD mice resulted in the development of a severe hematopoietic phenotype with characteristics of AML. In addition, abnormal bone structures and ectopic bone formation were observed in these mice, suggesting a previously unknown role of FLT3 to control bone development and remodeling. While Ptprc knockout and Flt3-ITD mutant mice showed a largely normal bone microarchitecture, micro-CT analysis of femurs from Flt3-ITD Ptprc knockout mice revealed trabecularization of the cortical bone. This resulted in increased trabecular bone volume at the metaphysis, while the cortical bone at the diaphysis was thinner and less dense. In the metaphysis, severely reduced osteoclast and osteoblast numbers were observed. Reduced capacity of ex vivo differentiation of CD11b-positive bone marrow stem cells to mature osteoclast was accompanied by their abnormal morphology and reduced size. Transcriptome analysis revealed reduced expression of osteoclastogenic genes. Unexpectedly, cumulative resorption activity of osteoclasts was increased. Size and structure of resorption pits of differentiated osteoclasts remained similar to those observed in osteoclast cultures derived from control animals. Enhanced proliferation of cells in osteoclast cultures derived from FLT3-ITD-expressing mice was mediated by increased expression of STAT5 target genes. Transcriptome analysis of differentiated osteoclasts showed dysregulated signaling pathways influencing their differentiation as well as the coupling of bone resorption and formation. Taken together, inactivation of attenuating CD45 in mice expressing oncogenic FLT3-ITD resulted in marked abnormalities of the osteo-hematopoietic niche, which can be explained by aberrant STAT5 activation.

Vitamin D deficiency is common across the world. However, the standard clinical biomarker for vitamin D, 25OHD, may be a poor marker of vitamin D status, as most of circulating vitamin D is protein bound and not bioavailable. Free (unbound) vitamin D may therefore be a better marker of vitamin D status. We evaluated the relationship of free vitamin D with incident cardiovascular disease (CVD), heart failure (HF), kidney function decline (KFD) and fracture, among 786 participants in the Health Aging and body composition study. We used sequential models to assess hazard ratios (HRs) of each outcome that adjusted for age, sex, race, season of blood sampling, and study site, kidney function, serum calcium and phosphate, FGF 23, PTH, BMI, and vitamin D supplementation. The mean age of the 786 participants was 75 ± 3 yr, 53% were women, and 40% were Black. The median free vitamin D concentration was 5.3 (interquartile range 4.1-6.7) pg/mL. There were 157 cases of incident CVD, 123 cases of incident HF, 382 cases of incident KFD, and 178 fractures over 11 yr of follow-up. In fully adjusted models, a 2-fold greater free vitamin D was associated with lower risk of incident HF [HR 0.75, 95%CI,0.58-0.96 ] and greater risk KFD [1.25(1.03-1.52)]. We found no association between free vitamin D and incident CVD or fracture. We did not find evidence that free vitamin D was a superior marker of clinical outcomes compared to total 25OHD alone. Further studies are needed to elucidate the relationship of free vitamin D with clinical outcomes.

Lipocalin-2 (LCN2), a hormone produced by adipocytes, osteoblasts, and renal tubular cells, is implicated in age-related diseases, including cardio-metabolic disease. To understand the role LCN2 may play in pathological states, we first need to elucidate the relationship between circulating LCN2 with indices of cardio-metabolic health during "normal" aging. This study examined the relationship between serum levels of LCN2, age, and cardio-metabolic measures across the adult lifespan in males and females. We conducted a pooled cohort analysis including 124 community-dwelling males (n = 52) and females (n = 72) (age 20-87yr, median BMI 25.92 [23.04, 29.81] kg/m2). Serum LCN2 was analyzed using a two-step chemiluminescent microparticle monoclonal immunoassay. The relationship between LCN2 and age was evaluated by linear regression and cubic spline. Simple linear regressions were performed to investigate the relationship between LCN2 and the following variables: BMI, VO2peak, serum glucose, and body composition (DXA). For every 1yr increase in age, LCN2 levels were 0.26mg/L higher (P= .007, 95% CI [0.07, 0.45]). Each 1 unit increase in BMI (kg/m2) was associated with 0.88mg/L higher LCN2 levels (P= .027, [0.10, 1.66]) and each 1 unit increase in VO2peak (mL/kg/min) was associated with 0.38mg/L lower LCN2 (p= .003, [-0.63, -0.13]).There was no significant relationship between LCN2 and sex, glucose levels or body composition (all p> .05). LCN2 increased linearly across the adult lifespan while it decreased as fitness level increased. Future research should build on these findings to determine whether LCN2 can be used as a biomarker for chronic disease and if exercise can mitigate age-related disease associated with LCN2 changes.

Quantifying precision error for DXA, peripheral QCT (pQCT), and HR-pQCT is crucial for monitoring longitudinal changes in body composition and musculoskeletal outcomes. Agreement and associations between bone variables assessed using pQCT and second-generation HR-pQCT are unclear. This study aimed to determine the precision of, and agreement and associations between, bone variables assessed via DXA, pQCT, and second-generation HR-pQCT. Thirty older adults (mean age: 64.2±8.0yr; women: 67%) were recruited. DXA scans were performed at the total hip, lumbar spine, and whole body. Distal (4%) and proximal (30%/33%/66%) skeletal sites at the radius and tibia were scanned with pQCT and/or HR-pQCT. Root-mean-squared coefficients of variation (%CVRMS) were calculated to define precision errors, and Bland-Altman plots assessed agreement between densitometric estimates. Pearson correlations and linear regression explored relationships between bone variables at different skeletal sites and proportional bias, respectively. Precision errors ranged between 0.55% and 1.6% for DXA, 0.40% and 4.8% for pQCT, and 0.13% and 30.7% for HR-pQCT. Systematic bias was identified between pQCT- and HR-pQCT-determined radius and tibia volumetric BMD (vBMD) estimates (all p<.001). Proportional bias was not observed between vBMD measures at any skeletal site (all p>.05). pQCT- and HR-pQCT-determined total, trabecular, and cortical vBMD and estimates of bone strength at the radius and tibia were strongly correlated (all p<.05). Precision error was low for most bone variables and within the expected range for all imaging modalities. We observed significant systematic bias, but no proportional bias, between pQCT- and second-generation HR-pQCT-determined vBMD estimates at the radius and tibia. Nevertheless, measures of bone density and strength were strongly correlated at all skeletal sites. These findings suggest that although bone density and strength estimates from both imaging modalities are not interchangeable, they are strongly related and likely have similar fracture prediction capabilities.

Hormonal agents administered for metastatic castration-resistant prostate cancer (mCRPC) may lead to osteoporosis, skeletal events, reduced quality of life, and even reduced overall survival (OS). Bone-modifying agents may prevent those events but their effect on cancer-control outcomes remains uncertain. Relying on our institutional tertiary-care database, we explored the effect of bone-modifying agents (bisphosphonates such as zoledronic acid and denosumab) on OS and progression-free survival in patients with mCRPC with at least 1 bone metastasis using Kaplan-Meyer estimates and Cox regression models. Of 420 patients with mCRPC, 60% received bone-modifying agents who were younger (68 vs 69years), with more systemic treatment lines for mCRPC (3 vs 2), and a higher proportion of initial de novo metastatic disease (72% vs 62%, all p ≤ .04) than patients without bone-modifying agents. In progression-free survival analyses, no significant differences were observed between both groups. In OS analyses, significant median OS differences were observed in favor of patients with bone-modifying agents (58 vs 45months; hazard ratio [HR]: 0.66), even after multivariable adjustment (HR: 0.37; both p ≤ .01). In bone-modifying agent-stratified analyses, 57% received denosumab vs 43% bisphosphonates, with a significantly higher rate of Eastern Cooperative Oncology Group status of ≥2 in the bisphosphonates group. In progression-free and OS analyses, no significant differences were observed between bisphosphonates and denosumab patients, with numerically better results in progression-free survival analysis for denosumab after adjusting for covariates. The cumulative rate of osteonecrosis of the jaw at any treatment time was 12% in both groups and significantly decreased over time. Real-world data suggest a relatively low administration rate of bone-modifying agents in patients with osseous mCRPC. However, real-world data also suggest an OS benefit when bone-modifying agents are used, even after controlling for possible confounding patient and tumor characteristics.

Iron deficiency anemia is treated by iron supplementation. Increasing evidence has shown that the carbohydrate components in iron infusions can cause hypophosphatemia and subsequent osteomalacia due to excess intact fibroblast growth factor 23 (iFGF23). We here undertook an in-depth characterization of 13 patients with iron infusion-induced osteomalacia (IIIO). Patients were characterized (monocentric institutional practice) by means of laboratory, bone density, HR-pQCT, and virtual osteoid volume estimation. We additionally report a patient who was treated with burosumab because iron infusions had to be continued despite osteomalacia. All 13 patients received ferric carboxymaltose (FCM) infusions and presented with low phosphate levels. Stopping the FCM infusions and supportive treatment by substitution of phosphate, calcium, native, and/or active Vitamin D was the chosen therapeutic approach. Pain, mobility, and biochemical data, such as serum phosphate levels, BMD, bone microstructure, and virtual osteoid volume, were the main outcome measures. Our results indicate biochemical normalization (eg, phosphate levels pre: 0.50mmol/L ± 0.23mmol/L, post: 0.93mmol/L ± 0.32mmol/L, p<.001) after stopping the FCM infusion and establishing supportive treatment. Additionally, pain levels on the visual analog scale (VAS) decreased (VASpre 7.31 ± 1.22, VASpost 2.73 ± 1.27, p<.0001) and areal BMD (expressed by T-score) improved significantly (T-scorepre: -1.85 ± 1.84, T-scorepost: -0.91 ± 2.13, p<.05). One patient requiring ongoing FCM infusions experienced significant additional benefits from burosumab treatment. In conclusion, our results highlight the importance of monitoring phosphate in patients treated with FCM infusions. Stopping FCM infusions is effective in addressing the excess of iFGF23 and thereby phosphate wasting. Supportive therapy enables quick recovery of the musculoskeletal system and improves pain levels in these patients.

Nitrogen bisphosphonates, such as zoledronic acid, target the enzyme farnesyl diphosphate synthase (FDPS) in the isoprenoid biosynthetic pathway (IBP), and are the frontline treatment for osteolytic bone diseases. A strong affinity of these agents for bone limits their distribution out of the skeleton. Geranylgeranyl diphosphate synthase (GGDPS) is directly downstream to FDPS in the IBP and novel GGDPS inhibitors such as RAM2061 have been shown to have key drug-like features including prolonged half-life, metabolic stability, and systemic distribution. Furthermore, RAM2061 exerts anti-neoplastic benefits in mouse models of multiple myeloma and Ewing sarcoma. Therefore, we are interested in determining the potential impact of RAM2061 on osteoclast biology and bone remodeling. Studies utilizing undifferentiated RAW264.7 cells demonstrated that treatment with RAM2061 depletes cells of geranylgeranyl diphosphate, impairs protein geranylgeranylation, and induces markers of the unfolded protein response pathway and apoptosis. Differentiation of RAW264.7 cells to mature osteoclasts is disrupted by RAM2061, resulting in decreased numbers of mature osteoclasts, altered morphology, and decreased tartrate-resistant acid phosphatase activity. Treatment of fully differentiated RAW264.7 cells with RAM2061 led to decreased resorptive activity. Confocal microscopy studies revealed that RAM2061 disrupts Cdc42 localization, inhibiting proper actin ring formation in osteoclasts. No significant impact on bone turnover markers or bone histomorphology was observed following a 3-week treatment of CD-1 mice with RAM2061, although decreased numbers of osteoclasts were observed. Liquid chromatography-tandem mass spectrometry studies confirmed accumulation of RAM2061 in bone from the in vivo studies as well as hydroxyapatite binding in vitro. In conclusion, these studies are the first to demonstrate the anti-osteoclastic activity of GGDPS inhibitor treatment and support future studies exploring the therapeutic benefit of this novel therapy in the setting of pathological bone remodeling.