Soluble RANKL and OPG levels were measured in the serum of 15 hypercalcemic patients at baseline and on 5 consecutive days following treatment with the ...
ANTICANCER RESEARCH 25: 3607-3612 (2005)
Bisphosphonate Treatment does not Affect Serum Levels of Osteoprotegerin and RANKL in Hypercalcemic Cancer Patients NIKLAS ZOJER1, KARIN BRENNER1, DORA BEKE1, STEFAN KUDLACEK2,4, GERHARD HAWA3, WOLFGANG WOLOSZCZUK3,4, LORENZ C. HOFBAUER5 and MARTIN PECHERSTORFER1 1First
Department of Medicine and Medical Oncology, Wilhelminenspital der Stadt Wien, Vienna; 2Department of Medicine, Krankenhaus der Barmherzigen Brüder, Vienna; 3Biomedica Group, Vienna; 4Boltzmann Institut für Altersforschung, Vienna, Austria; 5Department of Gastroenterology, University of Marburg, Germany
Abstract. Bisphosphonates are the standard treatment for hypercalcemia of malignancy. We hypothesized that bisphosphonate treatment and the subsequent fall in serum calcium might induce changes in the RANK/RANKL/OPG system, which plays a pivotal role in the regulation of bone resorption. Soluble RANKL and OPG levels were measured in the serum of 15 hypercalcemic patients at baseline and on 5 consecutive days following treatment with the aminobisphosphonate ibandronate. At day 0, the median soluble OPG level was elevated (p=0.0021) in the hypercalcemic group as compared to normal controls, while the median serum RANKL level was not significantly different. Ibandronate treatment and the resulting decrease (p2.7 mmol/L) cancer patients were treated with 6 mg ibandronate on day 0 of days 0-5. The duration of the study was determined by the fact that the majority of hypercalcemic patients are normocalcemic by the 5th day after treatment with bisphosphonates. The median patient age was 68 years (range 41-78 years); 7 patients were male, 8 female. Five patients had breast cancer and 5 lung cancer. The other 5 patients suffered from esophageal cancer, prostate cancer, renal cancer, endometrial cancer and B-cell-non-Hodgkin’s lymphoma, respectively. Bone metastases were present in 9 patients and absent in 4; bone involvement had not been determined in the remaining 2 patients. Nine patients had been previously treated with bisphosphonates, the last bisphosphonate treatment having been administered at least one month before day 0 of the study period. Six mg of ibandronate (1-hydroxy-3-[1-methylpentylamino]propylidene bisphosphonic acid; Roche, Basel, Switzerland) was dissolved in 500 mL of normotonic saline solution and infused over the course of 1 hour. Blood samples were drawn at baseline (day 0) and on each of the next 5 days. Serum creatinine and serum calcium were determined by routine automatic methods. To measure OPG, sRANKL and PTH, aliquots were centrifuged and frozen immediately after the blood samples were collected. The study was performed in accordance with the Declaration of Helsinki, amended by the 29th (Tokyo) and the 35th (Venice) World Medical Assembly, and in accordance with pertinent Austrian laws. The protocol was approved by the local ethics committee. Patients and healthy controls gave their informed consent prior to enrolment in the study. Healthy controls. Sex- and age-matched healthy adults, acting as controls in the present investigation, participated in a large investigation of normative values of bone metabolism in the Austrian population. The criteria for recruitment of individuals for this investigation have been published elsewhere (10-11). Normative values stated below in the sections on OPG and sRANKL assays refer to these publications. The 15 control persons in our study were selected from the entire cohort based on agreement in sex and age with the cancer patients.
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OPG assay. The osteoprotegerin test kit (Biomedica Group, Vienna, Austria) is an enzyme immunoassay designed to determine the total amount (bound and unbound) of OPG in biological fluids (serum, plasma, cell culture supernatants). In the first step, the assay buffer, the sample and the biotinylated OPG detection antibody were added simultaneously to the wells. Osteoprotegerin, if present in the sample, binds to the precoated capture antibody and is sandwiched by the detection antibody. After a washing step, which removes all non-specific bound material, streptavidin-HRP conjugate was added to the wells. After removal of unbound conjugate by washing, tetramethylbenzidine (TMB) was added to the wells as substrate. Osteoprotegerin was quantitated by an enzyme-catalyzed color change detectable by a standard ELISA reader. The intensity of the color developed is directly proportional to the amount of osteoprotegerin present in the sample. In 447 healthy males, the median serum OPG concentration was 1.74 pmol/L. In 687 healthy females, the median serum OPG was 1.95 pmol/L. Based on the entire collective of healthy adults, the upper limit of normal range (mean + 2SD) of serum OPG was 7.67 pmol/L for males and 7.73 pmol/L for females (10). sRANKL assay. The sRANKL test kit (Biomedica Group) is an enzyme immunoassay designed to determine soluble, uncomplexed human RANKL in biological fluids (serum and cell culture supernatants). In the first step, samples and the biotinylated anti-sRANKL detection antibody were pipetted into the wells. Human sRANKL, if present in the sample, binds to the precoated recombinant OPG and forms a sandwich with the detection antibody. Following a wash to remove all non-specific bound material, streptavidin-HRP conjugate was added to the wells. After removal of unbound conjugate by washing, TMB was added to the wells as substrate. sRANKL was quantitated by an enzyme-catalyzed color change. The intensity of the color developed is directly proportional to the amount of sRANKL present in the sample. The color development was stopped after 30 minutes and the intensity of the color was measured with a standard ELISA reader. In 394 healthy males, the median sRANKL concentration was 0.94 pmol/L. In 635 healthy females, the median sRANKL was 0.37 pmol/L. Based on the entire collective of healthy adults, the upper limit of normal range (mean + 2SD) of sRANKL was 1.38 pmol/L for males and 1.09 pmol/L for females (11 and unpublished results). Statistical analysis. At baseline, the comparison of laboratory values in cancer patients and in healthy adults was performed using the nonparametric Mann-Whitney rank sum test. The intraindividual changes in laboratory values after ibandronate treatment were analyzed with the nonparametric Friedman test followed by Dunn’s test. All tests were two-sided. A p-value
