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. We collected tissue samples from biopsy-proven cases of GCT of bone operated for definitive treatment. The tumor tissue isolated during the operation was named case, and the bone samples from the tumor-free margin served as controls. The osteoclastoma was graded per Campanacci radiological grading. The tumor was also graded per Enneking staging for benign bone tumors. The histological grading of the tumor was done per Jaffe et al.'s classification scheme.
Laboratory assessments
The samples were collected in RNA later from the operation theatre. They were transported at 40C and stored at -20C. The samples were taken out, and homogenization was performed for RNA extraction. The total RNA was extracted by RNA extraction kit-QIAGEN RNeasy mini kit. The extracted total RNA was quantified by spectrophotometer and expressed in ng/ul. The quality and purity of RNA were tested by UV spectrophotometer. After considering the absorbance ratio at 260 and 280 nm on the spectrophotometer, we considered a ratio above two for high-quality RNA. The quality of RNA was determined by gel electrophoresis. The samples not forming any band on gel electrophoresis were excluded from further analysis. The reverse transcriptase synthesized complementary DNA (cDNA) from the extracted RNA template. The use of one non-reverse transcriptase prevented false positive outcomes. We validated primers at different annealing temperatures, concentrations, and cDNA volumes before qPCR. An annealing temperature of 52.1°C, primer concentration of 0.5 uMol, and CDNA volume of 0.75 ul were set for qPCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as a housekeeping gene. The quantitative assessment of the samples was done by qPCR. The ratio of ROR2 and GAPDH represented normalized relative levels of ROR2 expression. We included a non-template negative control in the experiment Denaturation, annealing, and extension were performed at 95℃ for 30 secs(1 cycle), 48 ℃ for 30 secs (45X), and 72℃ (45x)for 1 min. The amplified DNA was obtained after running desired cycles.
Definitions
1. The cycle number that demonstrated the distinguishable difference between fluorescence generated by the PCR product of a given sample and background noise was fronted Ct (cycle threshold).
2. DELTA Ct (2 trials for each sample) was the difference between the cycle thresholds (Ct) of ROR2 and GAPDH genes of a particular sample given by formulae 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 formulae Delta Delta Ct = average delta Ct (sample of interest)-average delta Ct (reference sample).
4. Relative fold change expression of ROR was calculated by (Statistical Package for Sciences) SPSS software.
FOLD CHANGE IN EXPRESSION = 2^-DELTA DELTA Ct
Statistical analysis plan:
Assuming the proportion of tumor tissue with increased ROR2 expression as 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 calculation was estimated using the study of Lu et al. [1]. Demographic data, clinicopathologic findings, and PCR results for cases and controls (average Ct) were recorded. 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 cases and controls was analyzed using a paired-t test. The significance between the clinicopathological parameters and ROR 2 expression delta Ct values was analyzed using an independent t-test. The correlation between the age and duration of the disease with ROR2 expression Ct values was analyzed for cases and controls. All statistical calculations will be done using IBM SPSS Statistics for Windows, Version 19.0. IBM Corp. A Statistical significance was set at P < 0.05.