Osteoprotegerin and sRANKL serum levels in multiple myeloma patients

Introduction



Multiple myeloma (MM) is a B-cell neoplasm characterized by the clonal expansion of plasma cells in the bone marrow and the development of osteolytic bone disease.
The bone lesions result from increased osteoclastic bone resorption that occurs adjacent to the myeloma cells. The mechanism of bone destruction in myeloma appears to involve multiple osteoclast activating cytokines either produced by myeloma cells or induced by myeloma cells when they bind through adhesive interactions to marrow stromal cells [1, 2]. Osteoclastogenesis is regulated by three proteins: receptor activator of nuclear factor kappa B (RANK), its ligand (RANKL) and osteoprotegerin (OPG). Osteoclast differentiation factor, RANKL, also known as TNFa-related activation-induced cytokine (TRANCE) [3, 4] and OPG ligand [5, 6] is a member of the tumor necrosis factor family and is expressed as a membrane-bound protein on the surface of osteoblasts and marrow stromal cells. In addition, activated T cells secrete RANKL as a soluble molecule [7, 8]. RANKL appears to be cleared from the surface of cells by a TNFa- converting enzyme – like activity and is active as a soluble molecule [8]. The interaction of RANKL with RANK, which is expressed on the surface of osteoclast precursor cells and osteoclasts, stimulates the differentiation and activation of osteoclasts [9], whereas OPG blocks this process by functioning as a decoy receptor for RANKL [10]. RANKL in combination with macrophage colony-stimulating factor induces osteoclast formation in vitro [5]. Most factors that induce osteoclast formation, such as IL-1, IL-11, prostaglandin E2, and 1,25-(OH)2D3, induce osteoclast formation by acting indirectly on marrow stromal cells and upregulating RANKL production. The importance of the RANKL-OPG system in normal bone remodeling and osteoclast formation can be seen in transgenic and knockout mice. Mice with overexpression of OPG or deficiency in RANKL production or its receptor RANK, have decreased osteoclast formation and develop osteopetrosis (10, 11) whereas mice deficient in OPG have reduced bone mass and develop osteoporosis [12]. The relative levels of RANKL and OPG, a naturally occurring soluble glycoprotein, produced by many different cell types, including marrow stromal cells and osteoblasts, determine the level of osteoclast formation. Recent studies suggest that in MM patients at a tissue level there is a marked imbalance between RANKL expression and OPG levels that favor osteoclastogenesis and osteoclast activation.
Giuliani et al. [13] demonstrated that in coculture systems of human myeloma cells with marrow stromal cells, RANKL expression was upregulated and OPG production strongly downregulated at both the protein and mRNA levels. Pearse et al. [14] have examined marrow biopsy specimens from patients with myeloma and found that RANKL expression was markedly upregulated in bone marrow biopsies from patients with myeloma while OPG was expressed at very low levels compared to normal bone marrow biopsy specimens.
OPG has been shown to reduce hypercalcaemia in a murine model of colon cancer and to inhibit skeletal destruction in murine models of induced osteosarcoma [15] and bone metastasis [16]. It was also shown that the blockage of RANKL by its natural antagonist OPG could prevent the occurrence of bone disease in a murine model of MM [17].
The purpose of the present study was evaluation of serum OPG and soluble RANKL concentrations in patients with MM at the time of diagnosis and assessment of their clinical correlations. The results has already been presented at the 29th World Congress of the International Society of Hematology and XI Polish Society for Immunology Congress [18].





Patients and Methods





The study included 44 patients with MM at the time of diagnosis hospitalized in the Department of Haematology of the Institute of Haematology and Blood Transfusion in Warsaw.
In each patient the following parameters were estimated at the time of diagnosis: age, sex, complete blood examination with differential, percentage of plasma cells in the bone marrow, monoclonal protein isotype (using Beckman Paragon Immunofixation Electrophoresis Kit), urine immunoglobulin/24 hours, serum concentrations of monoclonal protein, calcium, creatinine and b2-microglobulin (using Beckman ARRAY 360), complete X-ray skeletal survey, stage of disease according to Durie and Salmon classification. The patient’s informed consent was obtained prior to performing bone marrow aspiration or biopsy.
The control group was 25 healthy subjects: 13 women and 12 men, at age ranging from 22 to 70 years.
Serum OPG and sRANKL were measured by enzyme – linked immunosorbent assay (ELISA) using kits manufactured by Biomedica GmbH (Vienna, Austria). Tests were performed according to the manufacturer’s instructions. Tests were performed in undiluted fresh or stored at -20°C serum samples. All samples were run in duplicate. Absorption was determined with an ELISA reader at 450 nm (or 405 nm) against 690 or 620 nm as reference. The standard curve was linear between 2,2 and 600 pg/ml. Assay sensitivity was 2,8 pg/ml for OPG and 8 pg/ml for sRANKL, with intra-assay and inter-assay coefficients of variation < 15%.
Statistical analyses of results were made using Statistica software – Mann- Whitney’s test and nonparametric Spearman’s correlation coefficient.





Results





Fig. 1. illustrates serum OPG concentration in particular MM patients and healthy individuals.
The OPG serum levels in the whole group of MM patients were 111±69 pg/ml (mean±SD) and in healthy age- and sex- matched controls 77±22 pg/ml. The median OPG concentrations in myeloma and control sera were 87 pg/ml (range 42–346) and 74 pg/ml (range 49-130), respectively. This difference was statistically significant (p=0.0208).
Results of serum OPG concentration in MM patients in relation to gender, stage of disease, presence of osteolysis, hypercalcaemia, renal failure and monoclonal protein isotype are presented in table 1.
OPG serum levels were higher in myeloma patients with renal failure as compared with patients with normal renal function (p=0.0094) (Fig. 1C).
Increased (> 130 pg/ml) serum OPG level occurred with the same frequency in myeloma patients with osteolysis (18% of cases) as in those with it (18% of cases) (Fig.1D).
In healthy subjects serum level of OPG increased significantly with age (r=0.78; p=0.000004) (Fig. 2). Similarly in MM patients serum concentrations of OPG increased with age (r=0.303; p=0.046) (Fig. 3). There was a positive correlation with OPG and b2-microglobulin serum concentrations (r=0.43; p=0.0039) (Fig. 4).
Among 4 myeloma patients with increased OPG serum level above 250 pg/ml, 2 had renal failure, 1 immunoglobulin light chain amyloidosis and 1 generalized skin lesions with lymphocytes infiltration. Only in 2 of all myeloma patients OPG level was slightly below 49 pg/ml, the lowest level measured in the control group (Fig. 1A).
Figure 5 illustrates serum sRANKL concentration in particular MM patients and healthy individuals. The sRANKL serum levels in the group of MM patients were 3.4±4.9 pg/ml and in healthy subjects 5.0±10 pg/ml. This difference was statistically significant (p=0.01) (Table 2).





Discussion





OPG was identified in 1997, and the term was coined for its protective effect on bone (Latin: os, bone; protegere, to protect) [10]. In healthy people, osteoclastic activity is regulated by a balance between OPG and its ligand (RANKL). Abnormalities of the OPG/RANKL system have been implicated in the pathogenesis of postmenopausal osteoporosis, benign and malignant bone tumours, bone metastases, and hypercalcemia of malignancy, while administration of OPG has been demonstrated to prevent or mitigate these disorders in animal models [15–17, 19–22].
In our control group of healthy subjects, the serum OPG concentrations ranged from 49 to 130 pg/ml and the arithmetic mean ±SD was 77±22 pg/ml. Our results are consistent with a study of Szulc et al. [23] conducted in 252 healthy men with an age range of 19–85 years in which the mean OPG concentration was 62±43 pg/ml. Similarly, in a study of Jung et al. [21] which included 36 male controls, the serum OPG concentrations were 7.1–74.4 ng/l, mean 39±15 ng/l. It should be noted that serum OPG concentrations of 5–130 ng/ml [13, 24] and 1–3 mg/l [25] in healthy adults and mean values of 230 ng/l in women in the age more than 65 years have also been reported [1]. The reasons for these discrepancies are unknown. It has been assumed differential detection of OPG forms leading to the use of OPG ligand as a capture protein in another ELISA test [26].
Studies designed to assess OPG serum levels in MM patients have yielded contradictory results [13, 24, 27].
Seidel et al. [24] reported the results of analysis of OPG concentrations in available 225 sera of MM patients at diagnosis who entered the Nordic Myeloma Study Group randomized a-interferon trial in the period from June 1990 to November 1992. OPG concentrations in particular patients ranged from 2.6 to 80 ng/ml with a mean concentration of 9.9±9 ng/ml, median 7.4 ng/ml while in healthy age and sex–matched controls (n=40) OPG levels ranged from 5.1 to 130 ng/ml. In approximately 20% of the MM patients, OPG levels were below 5.1 ng/ml. In this study long term storage of myeloma serum samples could influence the results. In a study of Giuliani et al. [13], an ELISA performed for MM patients (n=30) as compared with a panel of 24 sex-matched healthy subjects showed that OPG serum levels were reduced (mean ±SD: 18.5±6.0 ng/ml versus 26.6±4.8; p=0.009). The levels of OPG detected in Seidel et al. [24] and Giuliani et al. [13] panels of sera were higher than those obtained by us and others [21, 23, 27], possibly because of the use of different antibodies to perform the ELISA.
The present study showed that serum OPG levels are not reduced in MM patients (mean 111±69, median 88 pg/ml versus 77±22, median 74 pg/ml in healthy subjects; p=0.0208) and the values are higher in patients with renal failure (mean 164±78, median 130 pg/ml versus 101±64, median 85 pg/ml in MM patients with normal renal function; p= 0.0094). It was found positive correlation of serum concentrations of OPG and age (r=0.303; p=0.046) and serum b2M concentrations (r=0.43; p=0.0039). Our results confirm the observation of Kyrstonis et al. [27] who found in MM patients elevated OPG serum concentrations (median 50 pg/ml versus 35 pg/ml in healthy subjects; p=0.03) which fluctuated depending on disease activity.
The increase in the antiresorptive factor OPG in MM and its correlation with aging, both of which are associated with an increase in bone resorption may reflect a protective mechanism of the skeleton to compensate for increased bone resorption and bone loss.
OPG has recently been found to be overexpressed in bone metastases of prostate cancer patients. OPG inhibits prostate cancer – induced osteoclastogenesis and prevents prostate tumor growth in the bone [21, 22]. The results of study performed by Jung et al. [21] suggest that increased serum OPG is a marker of bone metastatic spread in prostate cancer patients.
An age – dependent increase in circulating serum concentrations of the OPG was reported by Szulc et al. [23]. Additionally, OPG concentrations in postmenopausal women with a high rate of bone turnover were higher than in those with a low rate of bone turnover; they were substantially higher in postmenopausal women with the most severe degrees of osteoporosis [25].
The present study showed that serum sRANKL levels are not increased in MM patients (mean 3.4±4.9, median 0 pg/ml versus 5.0±10, median 0 pg/ml in healthy individuals; p=0.65). This is in contrast to cellular study which clearly demonstrated that there is an imbalance between OPG and RANKL levels (in favor of RANKL) in the bone marrow environment of patients with MM [13, 14].
Several studies have unambiguously demonstrated that myeloma cells enhance RANKL expression by bone marrow-residing stromal cells [13] and even endothelial cells [28] through direct cell-to-cell contact. Croucher et al. [17] and Sezer et al. [29, 30] have recently found that murine and human myeloma cells express RANKL. Furthermore, Heider et al. [31] demonstrated that the level of RANKL expression by myeloma plasma cells significantly correlates with osteolytic bone disease in multiple myeloma.
Using a coculture transwell system Giuliani et al. [7] found that human myeloma cell lines increased the expression and secretion of RANKL in activated T lymphocytes and similary purified MM cells stimulated RANKL production in autologous T lymphocytes. They also found that RANKL mRNA was up-regulated in bone marrow T lymphocytes of MM patients with severe osteolytic lesions, suggesting that T cells could be involved in MM-induced osteolysis through the RANKL overexpression.





Conclusions





In some (20%) patients with MM at diagnosis serum OPG level is elevated suggesting that it may be secreted in mechanism of bone self-protection. Significantly increased OPG concentrations in MM patients with renal failure may be related to its decreased elimination. In half of MM patients serum sRANKL is undetectable.





References



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Correspondence: prof. Maria Kraj, Klinika Hematologiczna, Instytut Hematologii i Transfuzjologii, Chocimska 5, 00-957 Warszawa, tel. +48 22 849 84 32, faks +48 22 849 84 32, e-mail: mkraj@ihit.waw.pl