Twofold Expression of Receptor Tyrosine Kinase 2 (ROR2) in Giant Cell Tumors of Bone: Outcome of a Case‒Control Study

Abstract

Suppression or overexpression of transmembrane proteins of the Wnt family and receptor tyrosine kinases (ROR1 and ROR2) is implicated in the causation of cancer. The objective of this study was to determine the expression of ROR2 in patients with giant cell tumor of bone (GCT) by quantitative PCR (qPCR). In this case‒control study, samples of tumor tissue (patients) and bone from the tumor-free margin (controls) were subjected to qPCR in patients who underwent definitive treatment. The GCTs were classified per radiologic classification and histologic grading. Eleven cases and controls, consisting of six men and five women with a mean age of 33.18 ± 12.35 (20–50) years, were included over the study period of 2 years. The median duration since diagnosis was 12 (IQR 9) months. There was a 2.51-fold change (upregulation of ROR2 expression) in cases compared with controls, which was significant (0.00). There was an increase in the expression of ROR2 with tumor grade. However, these differences were not significant (Campanacci (P 0.05 cases and 0.84 controls), Jaffe (P 0.07 cases and 0.44 controls), or Enneking (0.07 cases and 0.44 controls)). Treatment with bisphosphonates (P = 0.17) or denosumab (P = 0.75) had no significant effect on ROR2 expression. Patients with GCT exhibit more than twofold upregulation of ROR2 expression, confirming its role in causing osteoclast-mediated bone destruction. Therefore, ROR2 may be a target for drug development in the treatment of GCT.

Introduction

Giant cell tumors (GCTs) of bone account for 4–5% of all bone tumors. It accounts for approximately 18% of all benign bone tumors. Identifying membrane proteins expressed exclusively on tumor cells is the goal of cancer drug development. Denosumab, a RANKL inhibitor monoclonal antibody, is an agent that acts through membrane proteins. The receptor tyrosine kinase-like orphan receptors type 1 and 2 (ROR1/2) are type-I transmembrane proteins. These proteins are expressed in cancerous and adult normal tissues. In mammals, ROR2 has been shown to act as a receptor or coreceptor for Wnt5a to mediate non-canonical Wnt signaling. ROR2 mediates noncanonical Wnt5a signaling by inhibiting the β-catenin-TCF pathway and activating the Wnt/JNK pathway. This results in polarized cell migration. The Wnt5a/ROR2 axis has been shown to increase the invasion of the osteosarcoma cell lines SaOS and U2OS in vitro [1]. ROR2 was also demonstrated to be a therapeutic target for osteosarcoma [2]. Non-canonical Wnt signaling impairment causes bone loss in arthritis and osteoporosis patients. Wnt5a-ROR2-mediated signaling between osteoblast lineage cells and osteoclast precursors enhances osteoclastogenesis [3]. In vivo, both Wnt5a and ROR2 deficiency and osteoclast precursor-specific ROR2 deficiency or osteoblast lineage cell-specific Wnt5a deficiency led to impaired osteoclastogenesis in mouse models. Considering the above findings, we studied the expression of the ROR2 gene through quantitative PCR (qPCR) in patients with GCTs who underwent definitive treatment.

Methods

This case‒control study was conducted at a regional cancer center to treat musculoskeletal tumors from 2021 to 2023. The study was funded by the institute and approved by its review board vide DHR Reg no.EC/new/nst/2020/331″ dated 13/07/2021. We collected tissue samples from biopsy-proven cases of bone GCT for definitive treatment. The tumor tissue isolated during the operation was referred to as the case, and the bone samples from the tumor-free margin served as controls. Osteoclastoma was graded according to the Campanacci radiological grading system. The tumor was also graded per the Enneking staging system for benign bone tumors. The histological grading of the tumor was performed according to the Jaffe et al.’s classification scheme.

Laboratory Assessments

The samples were collected from the operation theater and stored in RNA later. They were transported at room temperature and stored at – 80 °C. We had facility of storage near the operation theater.

The samples were removed, and homogenization was performed for RNA extraction. Total RNA was extracted using an RNA extraction kit (QIAGEN RNeasy Mini Kit). The extracted total RNA was quantified by a spectrophotometer and is expressed in ng/µL. The quality and purity of the RNA were tested via a UV spectrophotometer. After determining the absorbance ratio at 260 and 280 nm on the spectrophotometer, we considered a ratio above two to indicate high-quality RNA. The quality of the RNA was determined by gel electrophoresis. The samples that did not form any band on gel electrophoresis were excluded from further analysis. Reverse transcriptase was used to synthesize complementary DNA (cDNA) from the extracted RNA template. The use of one nonreverse transcriptase prevented false-positive outcomes. We validated the primers at different annealing temperatures, concentrations, and cDNA volumes before qPCR. An annealing temperature of 52.1 °C, primer concentration of 0.5 µL, and cDNA volume of 0.75 µL were used for qPCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as a housekeeping gene. The samples were quantified by qPCR. The ratio of ROR2 to GAPDH represents the normalized relative level of ROR2 expression. We included a nontemplate negative control in the experiment. Denaturation, annealing, and extension were performed at 95 °C for 30 s (1 cycle), 48 °C for 30 s (45 ×), and 72 °C (45 ×) for 1 min. The amplified DNA was obtained after running the desired cycles. The Fig. 1 illustrates a flow diagram for PCR estimation method in this study.

Fig. 1.

Illustration of quantitative PCR estimation method for cases and controls

Definitions

1. The cycle number that demonstrated the distinguishable difference between the fluorescence generated by the PCR product of a given sample and the background noise was fronted Ct (cycle threshold).

2. DELTA Ct (2 trials for each sample) was calculated as the difference between the cycle threshold (Ct) of ROR2 and that of the GAPDH gene in a particular sample according to the formula DELTA Ct = Ct (ROR2) – Ct (GAPDH gene).

3. Averages of all DELTA Ct values were tabulated, called DELTA DELTA Ct, for cases and controls given by the formula Delta delta Ct = average delta Ct (sample of interest) − average delta Ct (reference sample).

4. The relative fold change in the expression of ROR was calculated by Statistical Package for Sciences (SPSS) software.

$$\mathrm{Fold}\mathrm{change}\mathrm{in}\mathrm{expression}=2^{\wedge }-\mathrm{delta}\mathrm{delta}\mathrm{Ct}$$

Statistical analysis plan

Assuming that the proportion of tumor tissue with increased ROR2 expression was 72 for cases and 23 for controls with a possible alpha error of 5% and 80% power, the sample size was estimated to be 38 (19 each for cases and controls). The approximate sample size was estimated using the method of Lu et al. [1]. Demographic data, clinicopathologic findings, and PCR results for cases and controls (average Ct) were recorded. The delta Ct was calculated for cases and controls. The distribution of delta Ct values among cases and controls was determined using the Shapiro‒Wilk test. The difference in ROR2 expression between patients and controls was analyzed using a paired t test. The significance between the clinicopathological parameters and the delta Ct values of ROR 2 expression was analyzed using an independent t test. The correlations between age and duration of disease and between the ROR2 expression Ct values were analyzed for the patients and controls. All the statistical calculations will be performed using IBM SPSS Statistics for Windows, version 19.0, IBM Corp. The statistical significance was defined by a P value < 0.05.

Results

We operated on 11 patients with GCT during the study period. There were six males and five females. The mean age of the patients was 33.18 ± 12.35 (20–50) years. The median disease duration from diagnosis was 12 (IQR 9) months. One patient each received bisphosphonate and denosumab therapy before definitive surgery. There were nine primary and two recurrent patients in this study. The demographic outcome variables are presented in Table 1. We confirmed the normality of the distribution of ROR2 expression in patients and controls by the Shapiro‒Wilk test (Fig. 2). There was a 2.51-fold change (upregulation) in the expression of ROR2 in patients compared with that in controls (0.00). Table 2 shows the average values of ROR2 and the housekeeping gene GAPDH between the patients and controls. The expression of ROR2 in cases and controls did not significantly differ between the Campanacci grade (P = 0.05 cases and 0.84 controls), Jaffe grade (P = 0.07 cases and 0.44 controls), and Enneking grade (0.07 cases and 0.44 controls). Treatment with bisphosphonates (P = 0.17) or Denosumab (P = 0.75) did not significantly influence ROR2 expression.

Table 1.

Demographic outcomes in patients with GCT

Parameter	Result
Site of GCT	Distal femur = 2 Proximal tibia = 6 Proximal femur = 1 1 each distal humerus, calcaneum, and proximal humerus
Campanacci radiological grade	Grade 2 = 8 Grade 3 = 3
Enneking grade	Grade 2 = 7 Grade 3 = 4
Jaffe histological grade	Grade 1 = 7 Grade 2 = 4
Operative procedure	Curettage with high-speed burr and cement = 8 Amputation = 2 Resection and reconstruction = 1

Fig. 2.

Shapiro Wilk histogram showing distribution of delta Ct values in the cases and controls

Table 2.

Average values of ROR2, housekeeping gene GAPDH delta Ct, and delta delta Ct between cases and controls

Sample no	Average ROR2 Ct cases	Average GAPDH Ct cases	Average ROR2 Ct controls	Average GAPDH Ct controls	Delta Ct cases1	Delta Ct controls2	Delta delta Ct3
1	22.92	27.81	29.59	33.89	− 4.89	− 4.31	− 0.59
2	23.35	28.69	29.97	34.16	− 5.34	− 4.19	− 1.15
3	22.89	29.18	28.03	32.60	− 6.29	− 4.57	− 1.73
4	23.33	30.15	29.13	33.76	− 6.82	− 4.64	− 2.18
5	22.20	28.11	29.88	34.50	− 5.91	− 4.62	− 1.29
6	22.19	28.04	30.50	34.96	− 5.85	− 4.46	− 1.39
7	24.42	28.95	30.97	35.10	− 4.53	− 4.13	− 0.41
8	25.08	30.72	28.79	33.38	− 5.64	− 4.59	− 1.05
9	23.64	29.17	28.34	32.15	− 5.53	− 3.81	− 1.72
10	22.44	28.23	30.07	34.19	− 5.79	− 4.13	− 1.67
11	22.74	28.17	28.87	32.82	− 5.43	− 3.95	− 1.48
MEAN	23.20	28.83	29.46	33.77	− 5.64	− 4.31	− 1.33
± SD	1.11	1.15	0.99	1.13	0.62	0.29	0.49

1. Delta Ct cases = Average ROR2 Ct cases − Average GAPDH Ct cases

2. Delta Ct controls = Average ROR2 Ct controls − Average GAPDH Ct controls

3. Delta delta Ct = Average delta Ct cases − Average delta Ct controls

Discussion

We found an upregulation of ROR2 in GCTs. There was no difference in ROR2 expression according to radiological grade, histological stage, or dose of denosumab or bisphosphonate. However, there was an increase in the histological grade of the tumor, which failed to reach statistical significance. The role of ROR2-mediated signaling through noncanonical Wnt5a has been studied in mouse models. Osteoclasts responsible for bone resorption are under the control of osteoblast lineage cells—the latter express the cytokines RANKL colony-stimulating factor 1 (CSF1) and osteoprotegerin. Osteoclast precursors express RANK and CSF1 receptors and differentiate into osteoclasts after interacting with osteoblast lineage cells. Therefore, osteoblast lineage cells provide a suitable microenvironment for osteoclastogenesis. Osteoblasts and osteoclast precursor cells express Wnt5a and ROR2, respectively. ROR2 signaling in osteoclast precursors enhances RANKL-induced osteoclastogenesis [3]. Overexpression of Wnt5a signaling and inhibition of ROR2 signaling in the synovium lead to bone loss and suppression of bone loss, respectively [4]. Based on the findings in mice, we hypothesized that ROR2 would be overexpressed in GCTs and estimated its expression in vivo. A study showed that the osteosarcoma cell lines SaOS-2 and U2OS invade in vitro by activating Wnt5a/Ror2 signaling in a cell-autonomous manner. Investigations of the osteosarcoma cell lines SaOS-2 and U2OS revealed the role of Wnt5a as a tumor suppressor protein [5]. Wnt5a-ROR2 signaling through the noncanonical Wnt/c-Jun N-terminal kinase (JNK) pathway and Wnt/Ca2 +  + pathway affects cell migration and polarity. Thus, overexpression of Wnt5a-ROR2 has been implicated in tumorigenesis and osteoclastogenesis. Matrix metalloproteinase 13 (MMP13) gene expression has shown suppression in presence of ROR2 suppression on gene expression profiling study. Consequent to it reduced invasiveness of SaOS-2 cells lines has been demonstrated. The role of Src family protein tyrosine kinases (SFKs) has shown causation in MMP13 expression through the Wnt5a-ROR2 pathway. Therefore, the roles of Wnt5a in human cancers are controversial. The differential effects of over and under expression may be attributable to differences in receptors or cell contexts in tumors.

Since both the upregulation and downregulation of ROR2 are associated with tumorigenesis, it is plausible to question the reliability of these quantitative assessments to draw definitive conclusions. However, a comparison of the outcomes of our study with those of other studies demonstrated coherence in the results.

We designed cases and controls in this study from paired samples from the same patient to limit confounding factors. Our study provides valuable insight into the expression of ROR2 in GCTs, which could lead to future drug development. Our finding of upregulation of ROR2 expression is consistent with the findings of other studies investigating ROR2 expression in mouse models and in osteosarcoma. However, the findings of our study should be viewed from the perspective of the limited number of high-quality studies and discrepancies in the historical data on ROR2 for bone tumors.

Failure to generalize the findings of this study due to undesired sample size is the main limitation of this study. However, findings of this study do lay down a foundation for further studies estimating the role of ROR2-mediated osteoclast bone resorption through giant cell tumor cell lines or dedicated multi-institutional studies. Further a study investigating the effects of bisphosphonates and denosumab on ROR2 expression can support the findings of this study.

Author Contribution

Dr. Sandeep Kumar Nema contributed to conceptualization, funding acquisition, methodology and supervision, manuscript preparation, and review. Dr. Shiva S. contributed to formal analysis, investigation, project administration, resources, and software. Dr. Shyam Kumar Tripathi contributed to formal analysis, resources, and manuscript preparation. Dr. Kirubakaran Saraswathy P. contributed to validation and manuscript review.

Funding

This study was funded by Jawaharlal Institute of Postgraduate medical education and research Pondicherry India under Intramural funding program of the institute.

Data Availability

The data that support the findings of this study are available from the corresponding author, [SKN], upon reasonable request.

Declarations

Conflict of Interest

The authors declare no competing interests.