Cancer risk in patients treated with denosumab compared with alendronate: A population-based cohort study.

BACKGROUND: Antiresorptive treatment is currently used in millions of patients with osteoporosis and cancer worldwide. Early studies of denosumab suggested a small signal in ovarian cancer incidence and emerging data suggest that denosumab stimulates germ cell proliferation in the gonads. This study aims to determine the association between the use of denosumab and the risk of reproductive cancers compared with the use of alendronate. RESEARCH DESIGN AND METHODS: Using a cohort study design, we used the Danish nationwide registries to identify a population of subjects ≥50 years of age during 2010-2017 who started denosumab after being on alendronate treatment for at least six months. The cohort was matched 1:2 with patients who had been treated with alendronate alone for at least six months. The risk of reproductive cancers and the risk difference between groups were estimated using the Longitudinal Targeted Maximum Likelihood Estimation (L-TMLE) method. RESULTS: We identified 6054 Danish individuals who underwent treatment with denosumab. These individuals were matched with 12,108 receiving alendronate. The absolute risk of reproductive cancer was 1.05 % (95 % CI 0.75-1.34) after three years for denosumab users and was not different 0.03 % (-0.34-0.39) than for alendronate users. In supplemental analyses, there was no increased risk of non-reproductive cancers associated with the use of denosumab (risk difference of 0.54 % (-0.41-1.19). Analysis comparing denosumab users with the general population gave similar results. CONCLUSION: There was no difference in the risk of cancer following treatment with denosumab compared to treatment with alendronate assessed after a short follow-up of 3 years.

Low serum anti-Müllerian hormone is associated with semen quality in infertile men and not influenced by vitamin D supplementation.

BACKGROUND: Anti-Müllerian hormone (AMH) is released by testicular Sertoli cells and of great importance during fetal male sexual development, but less is known about the role of circulating AMH during adulthood. In vitro studies have shown that vitamin D may induce AMH transcription, but a controlled trial investigating the possible effect of vitamin D on serum AMH has not been conducted in men. METHODS: A single-center, double-blinded, randomized placebo-controlled clinical trial (NCT01304927) conducted in Copenhagen, Denmark. A total of 307 infertile men were included and randomly assigned (1:1) to a single dose of 300,000 IU cholecalciferol followed by 1400 IU cholecalciferol + 500 mg of calcium daily (n = 151) or placebo (n = 156) for 150 days. Difference in serum AMH was a predefined secondary endpoint. Explorative outcomes were associations between serum AMH and gonadal function in infertile men. The primary endpoint was difference in semen quality and has previously been published. RESULTS: Infertile men in the lowest AMH tertile had significantly lower sperm concentration (∆T3-1 16 mill/mL (228%); P < 0.001), sperm count (∆T3-1 55 million (262%); P < 0.001), motile sperm count (∆T3-1 28 million (255%); P < 0.001), progressive motile sperm count (∆T3-1 18 million (300%); P < 0.001), testis size (∆T3-1 2.7 mL (16%); P < 0.001), serum inhibin B (∆T3-1 72 pg/mL (59%); P < 0.001), inhibin B/FSH ratio (∆T3-1 48 (145%); P < 0.001), and higher FSH (∆T3-1 2.6 (38%); P < 0.001) than the tertile of infertile men with highest serum AMH. Vitamin D supplementation had no effect on serum AMH compared with placebo treatment. CONCLUSIONS: In infertile men, low serum AMH is associated with severely impaired gonadal function illustrated by poor semen quality and lower testosterone/LH ratio. Serum AMH in infertile men was not influenced by vitamin D supplementation.

RANKL regulates testicular cancer growth and Denosumab treatment has suppressive effects on GCNIS and advanced seminoma.

BACKGROUND: Testicular germ cell tumours (TGCTs) have a high sensitivity to chemotherapy and a high cure rate, although with serious adverse effects. In the search for tumour suppressive drugs, the RANKL inhibitor Denosumab, used to treat osteoporosis, came up as a candidate since RANKL signalling was recently identified in the testis. METHODS: Expression of RANKL, RANK and OPG, and the effects of RANKL inhibition were investigated in human TGCTs, TGCT-derived cell-lines, and TGCT-xenograft models. Serum RANKL was measured in TGCT-patients. RESULTS: RANKL, RANK, and OPG were expressed in germ cell neoplasia in situ (GCNIS), TGCTs, and TGCT-derived cell lines. RANKL-inhibition reduced proliferation of seminoma-derived TCam-2 cells, but had no effect on embryonal carcinoma-derived NTera2 cells. Pretreatment with Denosumab did not augment the effect of cisplatin in vitro. However, inhibition of RANKL in vivo reduced tumour growth exclusively in the TCam-2-xenograft model and Denosumab-treatment decreased proliferation in human GCNIS cultures. In TGCT-patients serum RANKL had no prognostic value. CONCLUSIONS: This study shows that the RANKL signalling system is expressed in GCNIS and seminoma where RANKL inhibition suppresses tumour growth in vitro and in vivo. Future studies are needed to determine whether RANKL is important for the malignant transformation or transition from GCNIS to invasive tumours.

Treatment options for hypercalcemia after cosmetic oil injections: Lessons from human tissue cultures and a pilot intervention study.

OBJECTIVE: Granuloma formation following self-administered cosmetic oil injections can lead to severe hypercalcemia and renal calcifications due to extra-renal vitamin D activation. This translational study aims to identify Prednisolone sparing therapeutics for hypercalcemia after development of granulomatous disease secondary to paraffin oil injections. MATERIALS AND METHODS: Granuloma tissue isolated from five men were cultured ex vivo and treated with selected drugs to block generation of activated vitamin D (1,25(OH)2D3). In a retrospective study, we included data before and during different treatments of 21 men with paraffin oil induced granulomatous hypercalcemia (46 treatment courses) where serum calcium, parathyroid hormone, vitamin D metabolites, creatinine and inflammatory markers were measured. RESULTS: Addition of Ketoconazole or Ciclosporin to granuloma tissue ex vivo culture, significantly suppressed production of 1,25(OH)2D3 after 48 h (both p < 0.05). Prednisolone was the first treatment option in most men and lowered serum levels of ionized calcium after 1, 2, 3 and 6 months compared with baseline (p < 0.05). Ketoconazole or Hydroxychloroquine had no significant effect on serum calcium levels and were unable to reduce the concomitant daily Prednisolone doses (p > 0.05). Azathioprine did not reduce calcium levels. However, addition of Tacrolimus to Prednisolone treatment enabled a reduction in Prednisolone dose after 3 months (p = 0.014), but with no additional effect on calcium homeostasis. CONCLUSION: This study verifies that Prednisolone is an effective treatment and suggests that calcineurin inhibitors may be used as Prednisolone sparing treatment for paraffin oil-induced granulomatous hypercalcemia. Randomized clinical trials are needed to determine clinical efficacy.

Calcium transport in male reproduction is possibly influenced by vitamin D and CaSR.

Vitamin D is important for gonadal function in rodents, and improvement of vitamin D status in men with low sperm counts increases live birth rate. Vitamin D is a regulator of transcellular calcium transport in the intestine and kidney and may influence the dramatic changes in the luminal calcium concentration in epididymis. Here, we show spatial expression in the male reproductive tract of vitamin D receptor (VDR)-regulated factors involved in calcium transport: transient receptor potential vanilloid 5/6 , sodium/calcium exchanger 1, plasma membrane calcium ATPase 1, calbindin D9k, calcium-sensing receptor (CaSR), and parathyroid hormone-related peptide (PTHrP) in mouse and human testis and epididymis. Testicular Casr expression was lower in Vdr ablated mice compared with controls. Moreover, expression levels of Casr and Pthrp were strongly correlated in both testis and epididymis and Pthrp was suppressed by 1,25(OH)2D3 in a spermatogonial cell line. The expression of CaSR in epididymis may be of greater importance than in the gonad in mice as germ cell-specific Casr deficient mice had no major reproductive phenotype, and coincubation with a CaSR-agonist had no effect on human sperm-oocyte binding. In humans, seminal calcium concentration between 5 and 10 mM was associated with a higher fraction of motile and morphologically normal sperm cells, and the seminal calcium concentration was not associated with serum calcium levels. In conclusion, VDR regulates CaSR and PTHrP, and both factors may be involved in the regulation of calcium transport in the male reproductive tract with possible implications for sperm function and storage.

RANKL regulates male reproductive function.

Infertile men have few treatment options. Here, we demonstrate that the transmembrane receptor activator of NF-kB ligand (RANKL) signaling system is active in mouse and human testis. RANKL is highly expressed in Sertoli cells and signals through RANK, expressed in most germ cells, whereas the RANKL-inhibitor osteoprotegerin (OPG) is expressed in germ and peritubular cells. OPG treatment increases wild-type mouse sperm counts, and mice with global or Sertoli-specific genetic suppression of Rankl have increased male fertility and sperm counts. Moreover, RANKL levels in seminal fluid are high and distinguishes normal from infertile men with higher specificity than total sperm count. In infertile men, one dose of Denosumab decreases RANKL seminal fluid concentration and increases serum Inhibin-B and anti-Müllerian-hormone levels, but semen quality only in a subgroup. This translational study suggests that RANKL is a regulator of male reproductive function, however, predictive biomarkers for treatment-outcome requires further investigation in placebo-controlled studies.

Luteinizing Hormone Receptor Is Expressed in Testicular Germ Cell Tumors: Possible Implications for Tumor Growth and Prognosis.

Luteinizing hormone/choriogonadotropin receptor (LHCGR) regulates gonadal testosterone production and recent studies have suggested a growth-regulatory role in somatic cancers. Here, we established that LHCGR is expressed in a fraction of seminoma cells and germ cell neoplasia in situ (GCNIS), and the seminoma-derived cell line TCam2 released LHCGR into the medium. LH treatment induced proliferation of TCam2 cells in vitro, while hCG treatment induced a non-significant 51% increase in volume of tumors formed in a TCam2 xenograft model. A specific ELISA was used to detect a soluble LHCGR in serum. Serum concentrations of soluble LHCGR could not distinguish 4 patients with GCNIS and 216 patients with testicular germ cell tumors (TGCTs) from 297 infertile or 148 healthy young men. Instead, serum LHCGR levels were significantly higher in 112 patients with a seminoma > 5 cm or elevated serum lactate dehydrogenase (LDH) compared with men harboring smaller seminomas < 2 cm or normal LDH levels. Serum LHCGR levels in TGCT patients could not predict relapse irrespective whether determined pre- or post-orchiectomy. Combined, these novel findings suggest that LHCGR may be directly involved in the progression and growth of seminomas, and our retrospective pilot study suggests that serum LHCGR may have some prognostic value in men with seminoma.