Prevalence of vertebral fractures and serum sclerostin levels in acromegaly

An increased prevalence of vertebral fractures (VFs) has been reported in previous studies. The aim of this study was to evaluate the association between bone mineral density (BMD), bone turnover markers, serum sclerostin levels, and vertebral fractures (VFs) in acromegaly patients. We also evaluated the effects of gonadal status, disease activity, treatment modality, age, sex, and body mass index (BMI) on skeletal endpoints. Case–control study. Seventy acromegaly patients (M/F:36/34, mean age 45.5 ± 11.9 years) and 70 controls (M/F:31/39; mean age 45.66 ± 11.9 years) were included. VFs, BMD, calcium metabolism, markers of bone turnover, and sclerostin levels were evaluated. BMD was measured by dual-energy X-ray absorptiometry (Hologic QDR 4500). Conventional lateral radiography of the spine was performed and the Genant method was used for the assessment of fractures of T4–L5 vertebrae. The prevalence of vertebral fractures was higher in acromegalic patients as compared with the control group (72.9 vs. 20%; p < 0.001). Serum phosphate (P) levels (3.46 ± 0.59 mg/dl vs. 3.11 ± 0.44 mg/dl; p < 0.001) and b-cross laps (CTx) levels (0.47 µg/l, range 0.04–2.38 vs. 0.28 µg/l, range 0.11–0.80; p < 0.001) were significantly higher in acromegaly patients than control subjects. Serum sclerostin levels were similar between either acromegaly patients and control subjects or acromegaly patients with VF and without VF. In the means of treatment modality, VFs were more frequent in patients treated with adjuvant gamma knife radiosurgery (GKS) (p = 0.07). In the binary logistic regression analysis, the age of the acromegaly patients, the presence of hypogonadism, and GKS treatment were the factors significantly correlated with the occurrence of spinal fractures. The prevalence of VFs in patients with acromegaly is higher than in control subjects. Since advanced age, the presence of hypogonadism and GKS treatment were the factors predicting VFs in acromegaly; radiological evaluations should be considered as an emerging tool especially in those patients. Although markers of bone turnover elevated in acromegaly, they were not useful for the prediction of fractures. Serum sclerostin levels showed no discrepancy between the two groups and further studies are required for assessment of sclerostin role in this form of secondary osteoporosis.


Introduction
Acromegaly is a rare, chronic endocrine disease usually caused by growth hormone (GH) secreting pituitary adenoma; associated with a wide range of systemic complications which lead to increased mortality [1]. Vertebral fractures (VFs) are one of these complications associated with high morbidity and low quality of life [1,2]. GH and insulin-like growth factor 1 (IGF-1) play an important role in bone metabolism through their effect on osteoblasts and osteoclasts [3]. Acromegaly has been associated with increased bone remodeling which was confirmed with changes in bone turnover markers, calcium kinetics, and bone histomorphometry [4]. İt is most likely due to the effect of IGF-I on the stimulation of receptor activator of nuclear factor_B ligand (RANK-L) resulting to enhanced osteoclastogenesis [3]. Mild hyperphosphatemia and hypercalcemia might be seen in acromegaly because of increased levels of calcitriol and direct antiphosphaturic effects of IGF-1 which is independent of parathormone (PTH) action [3]. One study reported an association between high prevalence of VFs and increased bone turnover in postmenopausal women with acromegaly, likely in relationship with persistently active disease [5]. Recent studies also provided evidence of increased skeletal fragility with high prevalence and incidence of VFs in acromegaly patients despite normal bone mineral density (BMD) [5][6][7][8].
The overall median prevalence of VFs in acromegaly is about 40%, a fracture risk which is three-to eight-fold greater compared to control subjects [3,6]. The prevalence of VFs was reported in up to 63% even in remission, and the incidence was found to be 20% despite normal BMD [9]. The incidence of VFs was estimated as 42% in a prospective follow-up study, which was associated with hypogonadism, femoral neck BMD changes, previous VFs, and duration of active acromegaly [10]. As the strongest predictor of VF is shown to be the subsequent fractures, regardless of disease status; recent acromegaly guidelines suggest performing lateral spine scan imaging to assess VFs in all patients at diagnosis [11].
Sclerostin is a Wnt antagonist derived from osteocytes which reduces bone formation by suppressing Wnt signaling in osteoblast precursors and blocks osteoblast differentiation. Also, sclerostin increases osteoclastogenesis by stimulating the expression of RANK-L [12][13][14]. Based on the adverse effects of sclerostin on bone, monoclonal antisclerostin antibodies have been developed for osteoporosis treatment. Studies support that inhibition of sclerostin action results in increased bone strength and resistance to fractures. Besides, Wnt activation by the blocking effect of sclerostin leads to decreased bone resorption [15,16].
The adverse effects of sclerostin may contribute to the deteriorated bone microarchitecture in acromegaly. Nevertheless, there have been no data on sclerostin and fracture risk association in this form of secondary osteoporosis. We aimed to evaluate serum sclerostin levels associated with BMD, bone turnover markers, and VFs in acromegaly patients. We also assessed the effects of gonadal status, disease activity, treatment modality, age, sex, and body mass index (BMI) on skeletal endpoints.
Inclusion criterium was a diagnosis of acromegaly disease with the clinic and biochemical tests in line with the latest guideline. Exclusion criteria were trauma history, osteoporosis treatment other than calcium and vitamin D. The Local Ethics Committee approved the study protocol, and the patients signed informed consent.
Transsphenoidal surgery was performed as the first choice of treatment and followed by adjuvant gamma knife radiosurgery (GKS) if required. GH values at or below 0.4 μg/l after an oral glucose tolerance test and normal agerelated IGF-1 levels were defined as remission after surgery and/or irradiation. Biochemical control was defined as normal IGF-1 levels under medication. Both remission and biochemical control groups were classified as "controlled disease". Elevated levels of serum IGF-1 levels after surgery and/or radiosurgery despite medical treatment were defined as active disease. Fifty-three percent of patients had adjuvant radiosurgery (GKS) following transsphenoidal surgery, 45.7% of patients had surgery. Despite surgery and medical treatment, 22.8% of patients had elevated IGF-1 levels. Remission was achieved in 30% of patients. In respect of medical treatment; 37.1% of patients were on somatostatin receptors ligands (SRLs), 28.6% were on SRLs plus cabergoline and 4.3% were on SRLs plus pegvisomant treatment after surgery and/or radiosurgery in whom remission could not have been achieved.
Sixteen male patients and seven female patients had hypogonadotropic hypogonadism and a gonadal replacement was supplied adequately in four of them. Twelve female patients were postmenopausal. The patients on gonadal replacement at least 12 months were considered as eugonadal.

Physical evaluation
Patients with acromegaly and control subjects were evaluated for their BMI (weight/height squared) and waist circumstances (WC).
Glucocorticoid deficiency was defined as basal serum cortisol values below 3 μg/dl or inadequate response to corticotrophin stimulation test or insulin tolerance test. TSH deficiency was defined as a free T4 level below the normal laboratory reference range. Hypogonadism was evaluated by menstrual cycle history in premenopausal women, decreased gonadotrophin levels in postmenopausal women, and low plasma testosterone levels in men. GH deficiency was not assessed. Patients diagnosed with hypopituitarism were properly treated with levothyroxine, hydrocortisone, testosterone in men, or estrogen replacement in premenopausal women.

Bone mineral density (BMD)
BMD was measured at the lumbar spine (LS) (L1 to L4) and total hip using dual-energy X-ray absorptiometry (Hologic QDR 4500, Hologic Inc., Waltham, MA, USA) equipped with reference values based on the National Health and Nutrition Examination Survey (NHANES III). WHO criteria to define osteopenia (T-score between −1.0 and −2.5) and osteoporosis (T-score less than −2.5) were used. The BMD is expressed in grams per square centimeter.

Vertebral fractures
Conventional lateral radiography of the thoracic and LS was performed by an experienced radiology technician according to a standardized protocol in all patients. To avoid the overestimation of fractures due to spinal deformities, the radiographs were evaluated by two experienced observers (D.G.Y. and O.B.), one of whom is a radiologist, qualified in musculoskeletal radiology. The semi-quantitative method proposed by Genant et al. for the assessment of fractures of T4-L5 vertebrae was used [17]. Both physicians were blinded to the patient's characteristics. The fractures were defined as grade 1 (mild fracture), grade 2 (moderate fracture), and grade 3 (severe fracture) based on a height ratio reduction of 20%-25%; 25%-40%; and >40%; respectively.

Statistics
All analyses were performed using commercial statistical software (version 22.0; IBM SPSS). Descriptive statistics were given as the median and range for continuous data, and as percentages and frequency for categorical data. Continuous variables were analyzed for homogeneity of variance using the Kolmogorov-Smirnov test, and those with normal distribution were analyzed with the t-test, while those with uneven distribution were analyzed with the Mann-Whitney U test. The Chi-square test or Fisher's exact test were used to analyze categorical data. Pearson correlation analysis was performed between serum sclerostin and IGF-1, GH, and bone turnover marker. The influence of risk factors for the prevalence of VFs in patients with acromegaly was assessed using binary logistic regression. p values < 0.05 were considered statistically significant.
No difference was seen in fracture prevalence between active and controlled disease (18.75 vs. 22.2% p = 0.5). Both groups showed no difference in serum Ca, P, PTH, 25 (OH) vitamin D levels, bone turnover markers, sclerostin levels, and BMD measurements ( Table 2).
The figure shows the prevalence of VFs in acromegaly patients that were stratified according to disease activity and gonadal status (Fig. 1). The highest prevalence of VF was seen in the group with active disease and hypogonadism (100%). It was followed by the group with hypogonadal controlled acromegaly patients (84.6%). The prevalence was decreased in eugonadal active and eugonadal controlled acromegaly patients (63.6% and 57.1%, respectively) (p:0.03).
Patients with VFs were significantly older than those without fractures (p = 0.01). The disease duration was slightly longer in patients with fracture but did not attain a statistical significance (6.4 ± 5.4 vs. 4.1 ± 4.0 years, p = 0.12). The prevalence of hypogonadism was higher in acromegalic patients with fractures (p = 0.01). In the means of treatment, VFs were more frequent in patients treated with adjuvant GKS compared with patients treated only   (Table 3).
In the binary logistic regression analysis, the age of acromegaly patients, the presence of hypogonadism, and GKS treatment were the factors significantly correlated with the spinal fracture occurrence (R 2 : 17.5; p = 0.002) ( Table 4).

Discussion
This study confirms that acromegaly patients have a high risk of VFs despite increased BMD values. The patient's age and hypogonadism were the predictive risk factors of VFs. GKS treatment was also associated with VF occurrence in this study. In terms of calcium metabolism and bone turnover markers, CTx, and serum P levels were elevated in acromegaly patients but did not differ in acromegaly patients with and without fracture. Serum sclerostin levels showed no difference between acromegaly and control groups. There was no correlation between serum sclerostin and IGF-1, Ca, P, 25 (OH) vitamin D, CTx, osteocalcin, PTH levels.
Elevated GH/IGF-1 levels in acromegaly are associated with high bone turnover in favor of bone resorption that can result in increased skeletal fragility [4]. BMD is not reliable to predict VFs as confirmed several studies showing high prevalence and incidence of VFs despite normal BMD values [5,7,8,18]. Since it is difficult to diagnose VFs according to the clinic or BMD; morphometric and radiological evaluations are emerging tools for the assessment of VFs, causing increased mortality and morbidity [6,19]. However, the data are limited considering the relationship between VFs and bone turnover markers. Sclerostin reduces osteoblast differentiation and increases osteoclastogenesis by stimulating the expression of RANK-L [12][13][14]. Sclerostin is reported to be elevated in X-linked hypophosphatemia, characterized by elevated fibroblast growth factor 23 levels; decreased phosphate reabsorption, and skeletal mineralization [16]. In contrast to the effects of sclerostin on phosphate metabolism, mild hyperphosphatemia is reported due to direct antiphosphaturic effects of IGF-1 in acromegaly [3]. A study showed significantly higher serum sclerostin levels in active acromegaly patients compared to controls and sclerostin levels correlated with the GH and IGF-1; nevertheless, VFs and bone turnover markers were not evaluated [20]. İn another study which included active and controlled acromegaly patients, patients with acromegaly had significantly lower sclerostin levels than the control group [21]. There was no significant difference in serum sclerostin levels among active and controlled acromegaly patients. We found no significant difference in serum sclerostin levels between nor  acromegaly and control groups, neither acromegaly patients with VF and without VF.
In contrast to previous case-control studies [8,10], there was a statistically significant relationship between the patient's age and the prevalence of VFs. There was no significant difference between acromegaly patients with and without VF in terms of disease activity, disease duration in this study similar to the other studies [8,9]. When acromegaly patients were classified according to the disease status and gonadal status, the most prevalent fracture rate was seen in hypogonadal active patients. Also, there was a significant correlation between VF occurrence and the presence of hypogonadism in univariate analysis in line with previous studies [5,8,9] that persisted in multivariate analysis in this study.
Despite higher levels of serum CTx and P in acromegaly patients compared to controls, no significant difference was found in bone turnover markers and calcium metabolism between patients with VF and without VF as similar to the data on previous studies [8,9]. Serum 25 (OH) vitamin D levels were higher in acromegaly patients compared to controls, and in patients with VF than without VF. This finding confirmed that standard modifications, e.g., vitamin D replacement were not adequate in acromegaly-related osteoporosis as they were in primary osteoporosis to prevent VF [22]. The role of vitamin D supplements in the prevention of VFs in acromegaly needs to be studied with further studies [19].
Although, there are studies that evaluated the effects of medical treatment on the bone [23], the association between GKS treatment and VFs has not been evaluated so far. We found that the prevalence of VFs is higher in acromegaly patients treated with GKS and there was a significant correlation in multivariate analyses between GKS treatment and VFs. We think that this relation might be associated with secondary hypogonadism or GH deficiency related to GKS but we were not able to analyze the correlation being limited by the small GKS patient size. On the other hand, in a recent study evaluating GKS, the new-onset anterior pituitary deficiency rate was found to be low [24]. Further studies are needed for clarification whether GKS; as a modern radiosurgical approach is associated with a reduced impact on morbidity than conventional radiotherapy [19].
A possible limitation of the present study was the assessment of the VFs with semi-quantitative method instead of quantitative morphometric analysis. To avoid the overestimation of fractures due to spinal deformities, the radiographs were evaluated by two experienced observers, one of whom is a qualified musculoskeletal radiologist.
In conclusion, the prevalence of VFs in acromegaly patients is higher independently of disease activity. Advanced age, hypogonadism, and GKS treatment were the factors predicting VFs, so radiological evaluations should be considered as an emerging tool, especially in those patients. Besides, whether GKS treatment per se a risk factor for VFs or not is needed to be evaluated with further studies. There was no relation between serum sclerostin levels, disease activity, and VFs in this study. Given the novel osteoporosis agents inhibiting the adverse effects of sclerostin on bone, serum sclerostin levels should be investigated with more studies in this form of secondary osteoporosis to prevent vertebral fractures.