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Factors Affecting the Recurrence of Giant Cell Tumor of Bone After Surgery: A Clinicopathological Study of 80 Cases from a Single Center

Background/Aims: This aim of the present study was to identify specific markers determining the recurrence of the giant cell tumor of bone (GCTB). Methods: This study involved the clinicopathological analysis of 80 cases. All of the clinical features, pathological fracture, Campanacci grade, histological features and surgical methods were reviewed. Immunohistochemistry was used to detect the expression of Ki-67, CD147, mutant p53 and p63 in GCTB. Comparisons between different groups were performed using the Chi-square test. The risk factors affecting recurrence were analyzed using a binary logistic model. Kaplan-Meier analysis was employed for the survival analysis between the groups. Cell proliferation assays, migration and invasion assays were used to detect the function of CD147 on GCTB in vitro. Results: The univariate analysis showed that Ki-67 and CD147 expression, pathological fracture, Campanacci grade and surgical method were associated with recurrence. The multivariate analysis revealed that CD147 expression, Campanacci grade and surgical method were the factors affecting GCTB recurrence. In addition, the Kaplan-Meier analysis revealed that these factors affected tumor-free survival time. In vitro study revealed that the CD147 knockdown by small interfering RNA (siRNA) technique dramatically reduced the proliferation, migration and invasion of GCTB. Conclusion: Our results suggest that CD147 may serve as an adequate marker for GCTB recurrence. Campanacci grade is a risk factor for GCTB recurrence, which is also affected by the surgical method used.


Introduction
Giant cell tumor of bone (GCTB) is a challenging clinicopathological entity, accounting for 5% of all primary bone tumors [1]. The occurrence of GCTB is primarily observed in individuals between 20 and 40 years old. The site of predilection for GCTB is the extremity of long bone in skeleton of mature adults. Histologically, it is composed of oval and spindle mononuclear cells, uniformly distributed and large multinucleated osteoclast-like giant cells. Although the term "giant cell tumor" implies that the multinucleated giant cells are responsible for the proliferative capacity of this tumor, there is evidence that the stromal cells, the major components of the mononuclear cell population, represent the true neoplastic components of GCTB [2]. Due to its local recurrence and rare pulmonary metastases [3], GCTB is considered to have a low malignant potential [4].
At present, surgery is the primary treatment method for GCTB. When GCTB is unresectable, or when surgery is likely to result in severe morbidity, denosumab, a receptor activator of nuclear factor kappa-B ligand (RANKL) inhibitor can be used [5]. Denosumab functions by inhibiting osteoclastogenesis. GCTB patients can survive for a long time after proper treatment, therefore the impact of surgical treatment on limb function should be fully considered. The choice of surgical method depends on many factors, such as pathological fracture, tumor size, bone cortex damage, soft tissue involvement, and articular cartilage damage [6,7]. Nowadays, curettage and prosthetic replacement are the main methods to treat GCTB.
To date, defining cellular and molecular characteristics of more aggressive GCTB has been difficult to elucidate. Studies have shown that mutations in p53 mutation are associated with local GCTB recurrence, and the overexpression of C-myc oncogene has been associated with GCTB metastasis [8,9]. Telomeric reduction and telomerase activity may act as oncogenic events, promoting and sustaining the transformed GCTB phenotype [10]. Some studies have shown that a high Ki-67 proliferative index is associated with the malignancy of bone tumors [11]. In some tumors, CD147 acts as a prognostic marker and therapeutic target [12]. It has been suggested that mutations in p53 are correlated with the lung metastasis in GCTB [13]. In addition, the overexpression of p63 has been implicated in GCTB progression [14]. Thus, these proteins might play a regulatory role in the progression of GCTB. However, the prognostic value of these proteins in GCTB has not been evaluated.
In this study, we detected the expression and localization of Ki-67, CD147, mutant p53, and P63 in 80 cases of GCTB and examined the relationship of these results to the recurrence of GCTB. In addition, we observed and followed-up these patients, evaluating their corresponding clinical features, pathological fracture, Campanacci grade and the used surgical methods to explore the factors associated with the recurrence of GCTB. In vitro study was performed to investigate the function of CD147 on GCTB. We hope to elucidate the factors influencing the recurrence of GCTB.

Patients
From 2005 to 2009, 96 cases of GCTB were treated at Shanghai Jiao Tong University Affiliated Sixth People's Hospital. Complete clinical and pathological data was generated for each patient, and 80 patients had complete follow-up data. Two Chief Pathologists confirmed all pathological diagnoses after surgery. GCTB was most commonly located in the femur (38 cases), followed by the tibia (25 cases), the radius (7 cases), the humerus (5 cases), the fibula (3 cases) and some rare sites, such as the talus (1 case) and the ischium (1 case). 30 cases of pathological fracture occurred in this series. A total of 15 cases of recurrence were observed at the time of final consultation. The specimens from all cases were fixed with 10% formalin, decalcified when necessary, and embedded in paraffin. The imaging classification was determined according to Campanacci's grading system [15]. The patients were followed up at 3-month intervals for the first 3 years, every 6 months until the 5th year, and every year until the time of final consultation.

Immunohistochemistry
A standard immunohistochemistry staining procedure was used in the present study. The paraffinembedded sections were cut at 4 μm, dewaxed in xylene and treated with microwave heating at 60°C for 20 min in a EDTA buffer (pH 9.0) for antigen retrieval. Each slide was blocked for endogenous peroxidase activity through incubation in 0.3% H 2 O 2 for 10 min, followed by overnight incubation at 37°C with a mAb Ki-67 (1:200, DAKO), CD147 (1:100, Santa Cruz, USA), mutant p53 (1:200, DAKO) or p63 (1:80, DAKO). The slides were subsequently rinsed three times in PBS, incubated for 30 min with Dako REAL TM EnVision TM (DAKO, USA), followed by three washes in PBS and color development for 3-10 minutes in a moist chamber at room temperature using DAB. The slides were counterstained with hematoxylin and dehydrated in graded ethyl alcohol (70%, 90%, and 100%). In the sections used as negative controls, the primary antibody was substituted with PBS.

Evaluation of Immunohistochemistry
A semi-quantitative counting standard was used to measure Ki-67 proliferation. Two blinded observers determined the labeled cell count (Ki-67 proliferation index) in ten high-power fields. The Ki-67 proliferation index was defined as the ratio of labeled cells to total cells, and the median of Ki-67-positive cells was calculated. When the number of CD147-positive cells was ≤5%, the tissue was considered negative; 6-25%, weak; 26-50%, moderate; and ≥51%, strong [16]. When the number of nuclei positively stained for p63 or mutant p53 was >10%, the tissue was considered positive, otherwise the tissue was considered negative [17].

Establishment of GCTB Cell Line and Cell Culture
Mak et al [18,19] established primary cell cultures of GCTB stromal tumor cells from fresh GCTB tissue. According to their method, we got GCTB stromal tumor cells from the fresh specimens. They were cultured in Dulbecco's modified Eagle's medium and supplemented with 10% fetal bovine serum (Biowest, South America Origin), 100 U/ml penicillin (Sigma-Aldrich, St Louis, MO, USA) and 100 mg/ml streptomycin (Sigma-Aldrich).
siRNA Transfection and Quantitative Real-time PCR Analysis (qRT-PCR) CD147-specific siRNA (sense, 5-UUC UCC GAA CGU GUC ACG UTT-3, antisense, 5-ACG UGA CAC GUU CGG AGA ATT-3) and negative control siRNA (sense, 5-UUC UCC GAA CGU GUC ACG UTT-3, antisense, 5-ACGU GAC ACG UUA GAA TT-3) were selected and synthesized by GenePharma. A total of 5 × 10 4 cells were seeded into each well of a 6-well plate. The next day, the cells were transfected with the siRNAs using Lipofectamine 2000 Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. For Western blot assays, migration and invasion, the cells were collected 48h after transfection. The mRNA expression levels were quantified using a 7500 Real-Time PCR System with 7500 software, version 2.0.5 (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. β-Actin was used as the endogenous control for quantifying mRNA levels. Three independent assays were performed.
Cell Proliferation Assays 5000 cells were seeded into each well of a 96-well plate and incubated after 48 h transfection. A 10-μL aliquot of CCK-8 (Dojindo, Japan) was added to quintuplicate wells and incubated for 2 h. To calculate the number of viable cells in each well, the absorbance at 450 nm was measured. Each measurement was performed in quintuplicate, and the experiments were repeated twice.

In vitro migration and invasion assays
Cell migration and invasion assays were performed in a 24-well plate with 8-mm pore size chamber inserts (Corning, New York, NY, USA). For the migration assays, after transfection with siRNA, 5 × 10 4 cells per well were placed into the upper chamber on an uncoated membrane. For the invasion assays, 1 × 10 5 cells per well were placed into the upper chamber on a Matrigel-coated membrane. The cells were diluted with serum-free culture medium. In both assays, when the cells were seeded into the upper chamber, they were suspended in 200 μl of Dulbecco's modified Eagle's medium without fetal bovine serum. The lower chambers contained 800 μL of medium with 10% FBS. The cells were incubated at 37℃ in 5% CO 2 for 18 h and 20 h for the migration and invasion assays, respectively. Then, the membrane inserts were removed

Basic Patient Information
A total of 49 males and 31 females were included in this study. The median age was 32 years old (range: 16-74 years). The follow-up period ranged from 32 to 86 months, and the median time was 58 months. According to Campanacci's grading system, 19 cases were stage 1, 37 cases were stage 2, and 24 cases were stage 3. The time to recurrence ranged from 1 to 48 months. The recurrence rate in the series was 18.75%. The recurrence occurs in the femur (7/38 cases), the tibia (5/25 cases), the radius (2/7 cases) and the humerus (1/5 cases). No significant difference was found between the different locations. A total of 57 patients received curettage and blurring and the remaining 23 patients received tumor resection and prosthesis replacement. Curettage was performed through a large cortical window to remove the entire visible tumor. The cavity was subsequently blurred using  a high-speed bone drill and washed with distilled water until all pathological tissue was removed. Lastly, the cavity was filled with autologous bone, allogeneic bone or bone cement. Indications for prosthesis replacement rather than curettage included pathological fractures with joint invasion, a large tumor with soft tissue extension or unstable fracture pattern.

Immunohistochemistry and Univariate Analysis Results
p63, Ki-67 and mutant p53 were observed only in the nuclei of mononuclear stromal cells, and no staining was present in the multinucleated giant cells. CD147 was detected in the multinuclear osteoclast-like giant cells and stromal cells, with strong immunostaining on cell membrane. The immunohistochemical staining of two typical cases of GCTB with or without recurrence is shown in Fig. 1. The experimental data also revealed that the recurrence rate was higher in the Ki-67 >15% group than that in the Ki-67 ≤15% group, consistent with the result obtained in previous reported literature [11]. The mean Ki-67 index in non-recurrent group is 8.73%, while the mean Ki-67 index in recurrent group is 24.17%, which is much higher than that of non-recurrent group. Similarly, there was a significant difference in the recurrence rate between the CD147-positive and CD147negative groups (χ2=6.65, P=0.010). The local recurrence rate was 4.35% (1/23 cases) in patients treated through tumor resection and prosthesis replacement. Conversely, the local recurrence rate was higher in patients treated through tumor curettage and blurring, with a 24.56% incidence. The recurrence rate was improved with improvements in the Campanacci grade, and a significant difference was observed (χ2=13.32, P=0.000). Similarly, the local recurrence rate was higher in patients with pathological fracture (χ2=10.11, P=0.002). No significant statistical effect on the local recurrence rate was observed for gender, age, mutant p53, p63, and tumor diameter. The results of the univariate analysis are shown in Table 1.

Multivariate Analysis
Independent factors associated with the recurrence of GCTB were assessed using a logistic regression model. The following factors were assessed in the model: Ki-67, CD147, surgical method and Campanacci grade. The factors independently associated with GCTB recurrence through binary logistic regression were CD147 (OR=5.446, 95% CI 1.163-25.498,   Ki-67 was not significantly associated with recurrence in the multivariate analysis. Therefore, CD147 and Campanacci grade were the risk factors for GCTB recurrence, while the surgical method was a protective factor. The results of the multivariate analysis are shown in Table 2.

Tumor-free Survival Time
As shown in Fig. 2, the Kaplan-Meier analysis revealed a significant difference in tumorfree survival time between the curettage and blurring group and prosthesis replacement    (Fig. 3). A similar result was observed for the tumor-free survival time between the CD147-negative and CD147-positive groups (χ2=7.362, P=0.007) (Fig. 4).

In Vitro Study of CD147 Function
We established primary GCTB stromal tumor cells from fresh GCTB tissue (Fig. 5A and  5B). siRNA technique was used to knockdown CD147. The experimental data revealed that CD147 was down-regulated after transfected for 48 h (Fig. 5C). Cell proliferation assays revealed that knockdown of CD147 dramatically reduced the proliferation of GCTB (Fig. 5D). Moreover, the migration and invasion assays showed that inhibition of CD147 could inhibit the migration and invasion of GCTB in vitro, as shown in Fig. 5E. In conclusion, CD147 plays an important role in the progression of GCTB in vitro.

Discussion
GCTB is a primary bone tumor that predominantly occurs in young adults. According to 2013 WHO classification of the bone tumors, GCTB is considered to be intermediate (locally aggressive, rarely metastasizing) tumor. To date, no prognostic marker for the risk of GCT recurrence has been identified. To identify a prognostic marker for the risk of GCTB recurrence, we detected the expression of Ki-67, mutant p53, p63 and CD147 using immunohistochemistry in 80 cases. Univariate analysis revealed that Ki-67 and CD147 affected recurrence, while CD147 expression was independently associated with recurrence of GCTB in the multivariate analysis. Notably, p63 and mutant p53 were not associated with the recurrence of GCTB in this study.
CD147, also called extracellular matrix metalloproteinase inducer (EMMPRIN) or Basigin, is a transmembrane glycoprotein of the immunoglobulin superfamily. The relative molecular mass of CD147 is 50-60 kDa, and this protein is widely present in human tissues and organs [20]. The gene encoding CD147 is located on chromosome 19p13.3 [21]. CD147 is a multifunctional membrane protein involved in many malignant tumors, such as lung and breast cancers [22,23]. CD147 can induce MMP synthesis in stromal cells, resulting in tumor progression and extracellular matrix protein degradation to regulate tumor cell biological behaviors, including invasion and metastasis [24]. These findings suggest that CD147 plays an important role in tumorigenesis and might be a potential target for anti-tumor therapy. In the present study, the results of the Chi-square and Kaplan-Meier analyses showed that the level of CD147 expression was positively correlated with the recurrence of GCTB and negatively correlated with the tumor-free survival period of GCTB patients. In addition, in vitro study showed that knockdown of CD147 dramatically reduced the proliferation, migration and invasion of GCTB. Therefore, CD147 might be a risk factor for the recurrence of GCTB and might be a novel therapeutic target for the treatment of this tumor.
It has been suggested that the Campanacci classification is associated with GCTB recurrence, and the higher the grade, the higher the rate of recurrence [25]. However, other studies have shown that the recurrence of GCTB is not associated with the Campanacci grade, and the surgical method is the primary factor influencing postoperative recurrence. Prosser et al retrospectively analyzed the factors influencing the recurrence of GCTB in 193 patients. These authors showed that the recurrence of GCTB was closely associated with the involvement of the cortical bone. When the tumor did not break through the cortical bone, the recurrence rate was only 7%; however, when the tumor broke through the cortical bone, the rate was as high as 29% [25]. Balke et al reported that the involvement of soft tissue was an independent predictor for GCTB recurrence. When the soft tissue surrounding the tumor was not affected, the recurrence rate was 16.2%, but when the soft tissue was affected through tumor invasion, the recurrence rate was nearly twice as large as the former (29.7%).
These authors suggested that before determining the surgical method for the treatment of GCTB patients, the destruction of cortical bone and the involvement of soft tissue should be evaluated as predictive values for tumor recurrence [26]. In the present study, we observed that the Campanacci grade was an independent factor influencing recurrence. The recurrence rate in the Campanacci grade 1 group was 0, whereas that in grades 2 and 3 was 13.51% and 41.67%, respectively.
The influence of surgical method on GCTB recurrence remains unclear. The recurrence rate of simple tumor curettage with or without bone graft is approximately 17% to 31% [27,28]. The recurrence rate observed in the present study was 18.75%. For Campanacci grade 3 GCTB, where both the bone and soft tissue are affected, conventional tumor curettage and bone grafting are difficult to guarantee the complete removal of lesions, resulting in a high rate of recurrence after operation. For these patients, tumor resection and prosthesis replacement can resolve the problem. Some studies have shown that after tumor-type prosthesis replacement, GCTB could be thoroughly treated, and the survival index was significantly improved, showing an adequate curative effect [29]. Therefore, the use of tumor type prosthesis replacement to treat GCTB has received much attention. However, the unavoidable clinical complications of prosthesis replacement (prosthetic loosening, allergy and secondary infection) and the limited service life of the prosthesis (10-15 years), makes prosthesis replacement inappropriate for young patients with a Campanacci grade of 1-2. For these patients, tumor curettage with inner wall blurring and repeated hypotonic distilled water washing is used to reduce postoperative GCTB recurrence [30].
In conclusion, CD147 might be an adequate marker for GCTB recurrence. Campanacci grade is a risk factor for the recurrence of GCTB. An appropriate surgical method should be decided for patients with a Campanacci grade of 2 or 3 to both remove the tumor and preserve joint function.