Abstract
Giant cell tumor of the bone (GCTB) is a common primary benign tumor, but in some cases, it behaves aggressively, resulting in tumor recurrence. The standard treatment for GCT is thorough curettage with adjuvant treatment such as phenol, liquid nitrogen, high-speed burr, or methylmethacrylate cement. This article presents the case of a 30-year-old male with GCT of the right distal femur, which demonstrated a complete necrosis of GCTB. Interestingly, the specimen also showed adipocytic lineage, and strong expression of apoptotic markers by [terminal deoxynucleotidyl-transferase dUTP nick-end labelling (TUNEL) and caspase-3] and peroxisome proliferator-activated receptor gamma (PPARγ). To the Authors' knowledge, this is the first reported case of complete necrosis of GCTB concurrent with adipocytic lineage and high expression of PPARγ. PPARγ is a master regulator of fat differentiation. PPARγ possesses antitumor activity through suppression of tumor proliferation and invasion and induction of differentiation and apoptosis. Although we could not conclude on the exact cause of complete necrosis and high expression of PPARγ in this case, we focused on the medical history, where this patient took zaltoprofen (240 mg/day) for four weeks before the biopsy to alleviate his pain. Zaltoprofen is a propionic-acid derivative non-steroidal anti-inflammatory drug, and it is reported to act as a direct ligand for PPARγ. We speculated that one of the possible mechanisms of PPARγ activation in this case was induction by zaltoprofen, at least in part. Although further analysis using cultured tumor cells with ligands specific to the receptor is necessary, PPARγ may be a novel therapeutic target in GCTB.
Giant cell tumor (GCT) of the bone is a common primary benign tumor; however, it exhibits aggressive behavior and occasionally gives rise to pulmonary metastases (1). The standard treatment for GCT is thorough curettage with adjuvant treatment such as phenol, liquid nitrogen, high-speed burr, or methylmethacrylate cement (2-6) to reduce the local recurrence. The treatment for recurrent lesions is complicated and sometimes requires the sacrifice of adjacent joints (6). Although GCT is also known to exhibit necrosis with hemorrhage (7), complete necrosis is quite rare. There have been only a few reports on spontaneous necrosis of GCTs, and the underlying mechanism has not been fully- clarified (8-10).
Peroxisome proliferator-activated receptor-gamma (PPARγ) is a master regulator of fat differentiation (11). It is also expressed in various types of cancer and possesses antitumor activity through suppression of tumor proliferation and invasion and induction of differentiation and apoptosis. PPARγ ligands have been investigated and include synthetic ligands such as thiazolidinediones (TZDs), widely used to treat type-2 diabetes mellitus (12), and endogenous ligands, such as fatty acids and prostaglandin-D2 metabolite 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) (13). It has also been reported that some non-steroidal anti-inflammatory drugs (NSAIDs), including indomethacin, act as direct ligands for PPARγ (14). In some types of cancer-including liposarcoma (15), and cancer of the colon (16), breast (17), and prostate (18), targeted-therapy for PPARγ has been performed.
In this study, we present the case of a 30-year-old man with GCT of the right distal femur, which showed complete necrosis and high expression of PPARγ. The patient was informed that data of his case would be submitted for publication, and gave his consent.
Case Report
A 30-year-old man was referred to our University Hospital with pain in the right knee of two months duration without any history of associated trauma. Physical examination revealed no soft tissue swelling or mass around the knee joint, despite his limp. A plain radiograph showed an osteolytic lesion around the right distal femur (Figure 1a and b). Magnetic resonance images (MRI) of the lesion showed mixed low- and iso-intensities on T1-weighted images (Figure 2a) and mixed iso- and high-intensities on T2-weighted images (Figure 2b). There was no abnormal soft tissue mass (Figure 2c). Technetium-99m scintigraphy revealed an area of strong accumulation in the right distal femur (Figure 2d). The patient had taken zaltoprofen (240 mg/day) for four weeks before the biopsy to alleviate his pain.
Anteroposterior (AP) (a) and lateral (b) radiographs of the patient at presentation, revealing an osteolytic lesion of the right distal femur. AP (c) and lateral (d) radiographs taken 24 months after surgery, showing stable artificial bone graft without evidence of tumor recurrence.
a: Coronal T1-weighted magnetic resonance imaging (MRI) (TR 675 ms, TE 15.7 ms) scan revealing mixed low- and iso-intensity. b: T2-Weighted MRI (TR 4500 ms, TE 106 ms) images showing areas of mixed low- and high-intensities. c: Axial T2-weighted MRI (TR 4500 ms, TE 106 ms) showing areas of mixed low and high-intensity. No abnormal soft tissue mass can be seen. d: Bone scintigram (99m-technetium) showing areas of high accumulation in the right distal femur.
An open biopsy was performed, and the lesion was diagnosed as necrotic GCT with fatty change (Figure 3a and b), which was strongly-positive for CD68 (Figure 3c), a marker of monocyte–macrophage lineage cells. Two-thirds of the tumor cells were necrotic. Three weeks after the biopsy, thorough curettage was performed using a high-speed burr, adjuvant phenol, and artificial bone paste (α-tricalcium phosphate; BIOPEX, Mitsubishi Materials Corporation, Tokyo, Japan). In the permanent section, no viable tumor cells were seen after surgery. The patient was permitted to place partial weight on the right leg one week after surgery. Full weight-bearing was permitted one month after surgery. The patient was free of disease for 24 months after surgery. Although the artificial bone had not been replaced by newly formed bone, incorporation with the host bone was achieved (Figure 1c and d). The patient also showed normal functionality of his right knee joint.
To evaluate the cause of total necrosis, we performed terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay and immunohistochemical analysis for caspase-3. A specimen obtained from another patient (59-year-old female) was used as a control. A cell death kit (Roche, Mannheim, Germany) and antibody to active caspase-3 (Promega, Madison, WI, USA) were used according to the manufacturers' instructions to detect the TUNEL reaction and active caspase-3. Nuclear staining was performed using 4’,6’-diamidino-2-phenylindole (DAPI) (Vectashield; Vector Laboratories, Inc., Burlingame, CA, USA), and a fluorescence microscope (BZ-9000; Keyence, Osaka, Japan) was used to obtain images. A very strong TUNEL response (Figure 4a and b) and caspase-3-positive cells (Figure 4c and d) were detected, whereas no positive cells were detected in the control (Figure 5a-d). We also investigated fatty de-generation, in which PPARγ is a key factor. Expression of PPARγ in the tumor specimen was examined. A mouse monoclonal antibody against PPARγ (1:250; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and rat anti-mouse immunoglobulin-G fluorescein isothiocyanate (1:400 dilution; eBioscience, San Diego, CA, USA), as the secondary antibody were used for detection. Strong expression of PPARγ was observed (Figure 4e and f) in the specimen of this patient, whereas no positive cells were observed in the control (Figure 5e and f).
Histological biopsy analysis revealing a necrotic giant cell tumor (a) with fatty change (b) (Hematoxylin and eosin stain, the scale bar indicates 100 μm.). c: Immunohistochemisty for macrophage-associated antigen (CD68) in a biopsy specimen. Strong expression of CD68 can be seen. The scale bar indicates 100 μm.
Immunofluorescence confocal microsocopy. The expression patterns of terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive cells determined in the biopsy specimen (a); merged with 4’,6-diamidino-2-phenylindole (DAPI) staining (b). The expression pattern of caspase-3-positive cells determined (c), merged with DAPI staining (d). The expression pattern of peroxisome proliferator-activated receptor gamma (PPARγ)-positive cells (e), merged with DAPI staining (f). The scale bar indicates 100 μm.
Discussion
As far as we are aware, this is the first reported case of massive apoptosis with fat differentiation of bone GCT strongly expressing PPARγ. The initial MRI and bone scintigraphy before treatment showed no apparent necrosis, although MRI demonstrated small hemorrhagic foci. More interestingly, at the time of biopsy, one-third of the tumor cells were still viable; however, the specimen obtained during curettage performed three weeks after biopsy showed complete necrosis. GCT occasionally exhibits spontaneous hemorrhage and necrosis (7), but we found only two cases reporting complete necrosis of GCTs (8, 9). In both cases, the causes were unknown. Immunohistochemical analyses showed a very strong TUNEL response (Figure 4a and b) and caspase-3-positive cells (Figure 4c and d), which meant that massive apoptosis was induced in this patient. Also of note in the present case was the presence of fatty degeneration in the tumor. To our knowledge, there have been no reports of fatty degeneration in GCT. We considered the possibility of fat differentiation not fatty degeneration. These findings (apoptosis and fat differentiation) suggested that PPARγ may play a role in the underlying pathology. Strong expression of PPARγ was observed (Figure 4e and f) in the specimen of this patient. PPARγ is a key transcriptional factor involved in fat differentiation (10). It possesses antitumor activity through suppression of tumor proliferation and invasion and in induction of differentiation and apoptosis. PPARγ ligands have been investigated and include synthetic ligands such as TZDs (12) and 15d-PGJ2 (13). It has also been reported that some NSAIDs, including indomethacin, act as direct ligands for PPARγ (14). We then focused on the medical history of this patient. He was prescribed the NSAIDs zaltoprofen (240 mg/day) for four weeks before the biopsy to alleviate his pain. Zaltoprofen inhibits bradykinin-induced nociceptive responses more potently than other NSAIDs such as indomethacin (19). Yamazaki reported that zaltoprofen induced apoptosis in rheumatoid synovial cells through the activation of PPARγ (20). Although we could not conclude the exact cause of complete necrosis and high expression of PPARγ in this case, we speculated that one of the possible mechanism of PPARγ activation was induction by zaltoprofen, at least in part. In some types of cancer including liposarcoma (15), and cancer of the colon (16), breast (17), and prostate (18), targeted-therapy for PPARγ has been performed. These findings suggest that activation of PPARγ could be a novel therapeutic tool against GCT.
Immunofluorescence confocal microsocopy of control. No terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive cells can be seen in the control case (a); image merged with 4’,6-diamidino-2-phenylindole (DAPI) staining (b). Caspase-3-positive cells cannot be seen in the control case (c); image merged with DAPI staining (d). PPARγ-positive cells cannot be seen in the control case (e); image merged with DAPI staining (f). The scale bar indicates 100 μm.
In conclusion, GCT of the distal femur exhibited massive apoptosis and fat differentiation with strong expression of PPARγ, which may have been induced completely or partially by zaltoprofen. Although further analysis using cultured tumor cells with ligands specific to the receptor is necessary, PPARγ may be a novel therapeutic target in bone GCT.
Acknowledgements
The Authors would like to acknowledge and thank Ms. Keiko Nagashima for her great contribution.
Footnotes
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Disclosure Statement
The Authors have declared that no competing interests exist.
- Received March 22, 2013.
- Revision received April 14, 2013.
- Accepted April 17, 2013.
- Copyright© 2013 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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