A 64-year-old woman complaining of left shoulder pain with resulting impairment in daily activities underwent radiography and computed tomography. The imaging examinations revealed an expanding lytic lesion of the left clavicle along with multiple osteolytic lesions in other regions, including the skull, right scapula, humeri, ribs, cervical and thoracic vertebra, right ilium and femora. On tumor staging studies, there was no evidence of pulmonary metastasis or other primary tumors. The patient became increasingly bed-confined due to multifocal bone pain. An iliac bone tumor biopsy revealed atypical spindle cell proliferation with active mitosis (>20/10 high-power fields) and partial necrosis. Osteoclast-like giant cells were occasionally observed (Fig. 1). Immunohistochemical analyses revealed expression of alpha-smooth muscle actin (smooth muscle-specific actin), h-caldesmon and vimentin (Fig. 1). These observations confirmed the diagnosis of bone leiomyosarcoma.
Denosumab (120 mg/day) was administered subcutaneously for 4 weeks, in addition to daily supplementation with calcium and vitamin D. Furthermore, the right scapula, cervical and thoracic vertebrae, left clavicle, femora and right humerus were subjected to standard radiation therapy as palliative care. The patient did not undergo adjuvant chemotherapy or surgery.
After 10 weeks of denosumab administration, the pain symptoms resolved and the patient recovered from the bedridden status. There were no reported denosumab-related side effects. The first follow-up computed tomography examination, performed 2 months post-denosumab treatment, revealed that all tumor growth had stabilized. Osteosclerotic changes were observed in multiple osteolytic lesions following denosumab and radiation combination therapy and denosumab therapy alone (Figs. 2 and 3). However, after 14 months of denosumab administration, a new lumbar vertebral metastasis was detected. At 56 months since denosumab administration, the patient had developed multiple metastases that had led to multiple organ failure, and finally succumbed to gastrointentinal bleeding.
RANKL gene expression was evaluated in a biopsy of the iliac bone metastasis in the present case, and in surgically resected samples from giant cell tumors of bone (GCTB, n=6), aneurysmal bone cysts (ABC, n=2), fibrous dysplasia (FD, n=6) and bone metastases from breast cancer (BC; n=3).
Total RNA was prepared from iliac bone biopsies using ISOGEN reagent (Nippon Gene; Tokyo, Japan) according to the manufacturer's recommendations. cDNA synthesis was performed using the PrimeScript  RT reagent kit (TaKaRa Bio; Tokyo, Japan). Quantitative polymerase chain reaction analysis was performed using SYBR Premix Ex Taq II in a Thermal Cycler Dice Real-Time System TP800 (TaKaRa Bio; Otsu, Japan). RANKL was selected as the target gene (forward primer: 5'-GCCTTTCAAGGAGCTGTGCAA-3', reverse primer: 5'-ATCTAACCATGAGCCATCCACCAT-3') and GAPDH was selected as the reference gene (forward primer: 5'-GCACCGTCAAGGCTGAGAAC-3', reverse primer: 5'-TGGTGAAGACGCCAGTGGA-3'). A standard curve was generated using the SaOS2 osteosarcoma cell line, and was used to calculate gene copy numbers. RPMI8226, a myeloma cell line, was used as the calibrator. The target gene expression level was calculated as the ratio of the copy number of the target gene to that of the reference gene. Finally, the relative level of expression was calculated as [copy number of the target gene (RANKL)/copy number of the reference gene (GAPDH)]/copy number of the target gene (RANKL) in the RPMI8226 cell line.
In the present case, RANKL gene expression was 747-fold higher compared with that of the calibrator. The results for the other tumor types are shown in Table I. The median fold changes for RANKL relative expression levels were 994-, 1,030-, 616- and 69-fold for GCTB, ABC, FD and bone metastasis from BC, respectively (Fig. 4).
All the procedures were conducted following international and national regulations, in accordance with the Declaration of Helsinki.
Informed consent was obtained from all subjects for participation in the study, the use of their tissue and publication of the data.