Significance of β-catenin expression for the incidence of pathological fractures in giant cell tumors of bone

Introduction

Giant cell tumor of bone (GCTB) is a primary bone tumor composed of two types of cells: mononuclear and osteoclast-like giant multinuclear cells [1, 2]. It usually involves the end of a long bone in middle-aged patients. GCTB most commonly occurs in patients 20-40 years of age, and can be found in many sites of the body; however, a half of GCTBs occur around the knee [3, 4]. Taking into consideration its unclear biological behavior, high recurrence rate and possible pulmonary metastasizing, GCTB is one of the most controversial and widely discussed bone tumors. Pathological fractures occur at first presentation in 9.0% to 30.0% of all patients with giant cell tumor of bone. The importance of understanding the incidence of pathological fractures in GCTB is evidenced by the fact that its presence determines the therapeutic approach, primarily the type and extent of surgical intervention [5, 6, 7].
Our research involved determining the possible role of p53, cyclin D1, B-catenin and Ki 67 in the increase in risk of fractures in patients with giant cell tumor of bone.

Material and methods

The analysis included a total of 164 GCTB samples diagnosed at the Reference Center for Bone and Joint Tumors within the Institute for Pathology of the Belgrade Faculty of Medicine. All samples belong to patients registered in the Bone Tumor Registry and treated at the Institute for Orthopedic Surgery “Banjica”.
Pathological fractures were confirmed on the basis of relevant radiological and surgical data.

Immunohistochemistry

Serial sections, 5 µm thick, were cut and immunohistochemical techniques were carried out using the avidin-biotin-peroxidase complex method with an LSAB2 kit (Dako, Glostrup, Denmark). The primary antibodies used in this study were: p53 (M3629Clone 318-6-11, dilution 1 : 25; Dako), β-catenin (M3539 Clone β-catenin1, dilution 1 : 200; Dako), Ki 67 (M7240 Clone MIB-1, dilution 1 : 100; Dako), and cyclin D1 (EPR2241, ab134175, dilution 1 : 200; Abcam).
Tumor cells with nuclear staining were considered positive in immunohistochemistry for p53, cyclin D1 and Ki-67. Giant cells with nuclear and stromal cells with cytoplasmic staining were considered positive in immunohistochemistry for β-catenin. The labeled-cell count of all antibodies was determined in ten high-power fields at 200× magnification, by two blinded independent observer-pathologists. The p53 expression was evaluated qualitatively in mononuclear tumor cells. For cyclin D1 and β-catenin we observed qualitative positivity separated in giant cells to less than 15 nuclei (GC < 15) and cells to 15 or more than 15 nuclei (GC ≥ 15). The percentage of positive cells for Ki-67 was recorded semi-quantitatively and used to obtain a score on a scale of 0-3 as follows: score 0 if no positive cells; score 1 if ≤ 5% cells were positive; score 2 if 6-19% of cells were positive; score 3 if ≥ 20% positive cells. In statistics, we used uni- and multivariate analyses, and a p-value of less than 0.05 was accepted as statistically significant.

Results

Demographic characteristics

Out of 164 patients with primary GCTB, 76 were men and 88 were women, making the gender ratio of 19 : 22. The mean age of first diagnosis was 30.8 ±13.2 years. Duration of symptoms varied from 1 day (in patients with sudden pathological fractures) to 12.6 months (Table I).

Symptoms and localization

The main symptoms were pain and swelling. The most frequent localization was the knee region – 63.4% (distal femur 42.7% and proximal tibia 20.7%) (Table I).

Fractures

Our study included 21 patients with pathological fractures and typical microscopic tumor presentation (Fig. 1A, 2). They represent 12.8% of all patients (Table I).

Immunohistochemistry

According to the immunohistochemical expression of p53 and Ki-67 in mononuclear stromal cells, as well as of cyclin D1 in multinuclear giant cells, there was no significant association with immunopositivity or risk of fractures (Tables II-IV).
Our sample confirmed a twice higher frequency of pathological fractures in patients with p53 expressed in stromal cells, but statistical analysis showed no significance of this difference (χ2 = 2.705; p = 0.100) (Table II).
Distributions of patients with pathological fractures were extremely similar, almost identical in each of the categories of Ki 67 positivity (χ2 = 0.026; p = 0.872) (Table III).
Cyclin D1 expression in giant cells with less than 15 nuclei was most commonly associated with pathological fractures (in 16.5% of patients). Patients with cyclin D1 positivity in giant cells with more than 15 nuclei had no pathological fractures. Nevertheless, the difference was not statistically significant (χ2 = 0.007; p = 0.933) (Table IV).
By means of correlating β-catenin expression with the incidence of pathological fractures, we found a highly statistically significant correlation between these two features in our sample (Table V).
Our research revealed that patients with cytoplasmic expression of beta-catenin in stromal cells (Fig. 1B) had three times more frequent incidence of pathological fractures, which was highly statistically significant (χ2 = 7.065; p = 0.008).
Moreover, a highly statistically significant correlation between the nuclear expression of β-catenin in giant cells (Fig. 1C) and the incidence of pathological fractures was also found (χ2 = 8.824; p = 0.003).

Discussion

GCT is a locally aggressive and purely osteolytic skeletal lesion [1, 3, 4, 7]. Pathological fractures can be caused by tumor invasion and associated with stress. They represent a relatively frequent complication found in 9-30% of patients with GCTB [4, 8]. The prevalence of pathological fractures in this study was 12.8%, which was in compliance with the findings of other authors.
Causes of pathological fractures are multifactorial and still insufficiently known. An additional possible parameter leading to the incidence of pathological fractures is the activation of the Wnt pathway of carcinogenesis described in GCTB [9, 10, 11, 12]. Individual genes included in the Wnt pathway influence the destructivity of GCTB and promote cell growth. It is widely known that β-catenin has an important role in the Wnt signaling pathway, as well as in the incidence of numerous primary mesenchymal tumors of bone and soft tissue [11, 12, 13, 14].
Our research reaffirmed that β-catenin expression in the cytoplasm of mononuclear cells, as well as in giant cell nuclei, was associated with a high risk of fracture incidence with GCTB. Tumors with stromal, mononuclear cells displaying cytoplasmic positivity to β-catenin were three times more frequently manifested, both clinically and radiologically, by the incidence of pathological fractures. Nuclear expression of β-catenin in giant multinuclear, osteoclast-like cells was highly statistically correlated with the incidence of fractures. Positivity to β-catenin in GCTB cells indicated activation of the Wnt pathway in GCTB, as well as the possible importance of this signaling pathway, both for the pathogenesis and for locally aggressive behavior of this primary bone tumor.
Bearing in mind that Shuxin et al. [15] confirmed that the JUN gene, which is a part of the Wnt pathway activated in GCTB, was in charge of promoting stromal cell growth and increasing the destructivity of this tumor, this could be used for explaining the association with pathological fractures. It is also known that the JUN gene plays a very important role in the canonical Wnt signaling pathway, primarily by interacting with β-catenin – TCFs transcriptional complex. Therefore, β-catenin expression indicates increased transcription in GCTB, as well as possible hyperactivation of the JUN gene [14, 15, 16].
In addition, this gene, which is closely linked to β-catenin, correlates with positive regulation of matrix metalloproteinases, which have a great significance for dyscoherence of tumor cells, and therefore locally aggressive biological behavior. The following matrix metalloproteinases are highly positive in GCTB: MMP-2, MMP-13 and MMP-9. The role of MMP-13 is to optimize bone resorption by giant osteoclast-like cells, whereas its high expressivity in GCTB could be responsible for the occurrence of osteolysis and pathological fractures [15, 16, 17, 18].
Both the JUN gene and β-catenin could also impact certain physiological processes in extracellular matrix, by promoting growth and emphasizing destructivity in GCTB.

Conclusions

The importance of understanding the occurrence of pathological fractures in GCTB is evident in the fact that its presence determines the therapeutic approach, primarily the type and extent of surgical intervention. The study showed that β-catenin expression highly correlates with the incidence of pathological fractures in GCTB patients. Taking into account that β-catenin is closely linked to activation of the Wnt signaling pathway in GCTB pathogenesis, one could postulate that activation of the Wnt pathway is one of the contributing factors for locally destructive behavior of this tumor, as well as for the incidence of pathological fractures.
All procedures followed were in accordance with the ethical standards and approved by the Ethics Committee (Belgrade University; School of Medicine), number of solution: 29/V-1 (11.10.2012.)

This work was supported by the following grants: III 45005, 173049 from the Ministry of Education, Science and Technological Development of the Republic of Serbia.
The authors declare no conflict of interest.

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Address for correspondence

Jelena Sopta
Institute for Pathology
Medical Faculty
University Belgrade
Dr Subotica 1
11000 Belgrade, Serbia
e-mail: jslabic@yahoo.com