Chromosomal Abnormalities Associated With Local Recurrence or Pulmonary Metastasis of Giant Cell Tumor of Bone in Thai Adults: A Prospective Cohort Study With 6 Years of Follow-Up


 Background: Giant cell tumor (GCT) of bone demonstrates chromosomal abnormalities. This study aimed to investigate the prognostic role of chromosomal abnormalities of primary GCT of bone relative to local recurrence or pulmonary metastasis.Methods: This prospective longitudinal cohort study with 6 years of follow-up included consecutive patients with primary GCT of bone that were surgically treated during 2011 to 2013 at the Department of Orthopaedic Surgery, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand. All patients underwent surgical resection with extended intralesional curettage and phenol local adjuvant therapy. Systematic cytogenetic analysis compared cytogenetic abnormalities between patients with and without local recurrence or pulmonary metastasis. Fifteen patients were eligible, enrolled, and had successful cytogenetic analysis.Results: The median follow-up time was 46 months (interquartile range [IQR]: 32-58). Five patients experienced local recurrence or pulmonary metastasis with a median time to recurrence of 6 months (IQR: 3.25-10.5). The mean number of abnormal cells in the primary culture compared between those with local recurrence or pulmonary metastasis and those without was 24.4 vs. 9.6 cells, respectively (p=0.04). A similar pattern was observed in the cultures of the subsequent four passages (all p<0.05). Forty-five patterns of clonal telomeric association (tas) were observed in passaged cultures. Six tas patterns were associated with local recurrence or pulmonary metastasis, including tas(11;19)(p15;q13.4), tas(15;19)(q26.3;q13.4), tas(15;22)(p13;p13), tas(16;19)(p13.3;q13.4), tas(17;19)(p13;q13.4), and tas(19;22)(q13.4;q13).Conclusions: The mean number of abnormal cells and the six identified TAS patterns may be valuable prognostic factors for local recurrence or pulmonary metastasis of GCT tumor of bone.


Background
Giant cell tumor (GCT) of bone is the most common benign aggressive tumor of bone. The incidence of this tumor in Asian population is approximately 15% [1][2][3][4], and postoperative recurrence rates ranging from 18-65% have been reported [5][6][7]. The recurrence rate is approximately 17.4% within the rst ve years of follow-up [8]. Intralesional curettage with the subsequent application of different adjuvant substances to control local recurrence of GCT of bone have been studied, including warm Ringer's lactate solution, phenol, bone cement, zoledronic acid-loaded bone cement, and liquid nitrogen [9][10][11][12].
Campanacci, et al. reported the presence of tumor at the surgical margin to be the prognostic factor that most strongly predicts recurrent GCT [13]. Other potential predictors have been extensively studied; however, radiographic ndings, histologic appearance, and immune-histochemical stain, such as Ki-67, were not able to predict local aggressiveness and/or the metastatic potential of the tumor after surgery [14][15][16][17][18][19]. Conversely, abnormalities found in genetic studies of DNA or chromosomal study of the tumor were strongly associated with local recurrence and lung metastasis [20][21][22][23]. Bridge, et al. found chromosomal abnormalities in GCT of bone, particularly telomeric fusion. As a consequence of that nding, cytogenetic analysis has been suggested as a potentially useful tool for predicting the aggressiveness of a tumor [24]. However, few cytogenetic studies have been reported to date. Accordingly, the aim of this study was to identify chromosome abnormalities in GCT of bone that are signi cantly associated with the primary outcomes of our study by subjecting tissue taken from primary lesions to cytogenetic analysis and then comparing cytogenetic abnormalities between patients with and without local recurrence or pulmonary metastasis.

Methods
This prospective longitudinal cohort study with 6 years of follow-up included consecutive patients with primary GCT of bone that were surgically treated during 2011 to 2013 at the Division of Orthopaedic Oncology of the Department of Orthopaedic Surgery, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand. The Siriraj Institutional Review Board (SIRB) approved our study protocol (COA no. 418/2556 (EC4)), and all patients provided written informed consent to participate. All methods were carried out in accordance with relevant guidelines and regulations.
Complete history, physical examination, and imaging investigations, including plain radiograph at the lesion, plain radiograph of the chest (posteroanterior and lateral views), computed tomography (CT) chest, bone scan, and magnetic resonance imaging/CT scan at the lesion, were performed in all patients.
Open biopsy was performed for de nite diagnosis in all patients. If the pathological diagnosis was conventional GCT of bone, the patient was enrolled in the study. Demographic and clinical data, including age, gender, location of the tumor (axial/nonaxial skeleton), and preoperative Campanacci GCT tumor classi cation, were recorded in all patients. All patients then underwent de nitive treatment using extended intralesional curettage, and 1 cubic mm of the solid parts of the tumor was sent for cytogenetic study. The primary tumor tissues derived intraoperatively were added into sterile tubes containing fetal bovine serum cell culture media, and then they were immediately transferred to our cytogenetic laboratory. Local treatment with high-speed burr, electrocautery, and phenol was performed, and bone cement was used to ll the bony lesions. After surgery, patients were followed-up every month during the rst year, and then every three months after that for six years consistent with routine practice at our The specimens were subsequently mechanically disaggregated by mincing with a scalpel blade, and enzymatically by incubation in collagenase (Biochrom AG). Short-term culture was performed following the Mandahl protocol. After removal of the collagenase solution, the cells were resuspended in DMEM with 20% fetal calf serum. The cultures were placed in a humidi ed 37°C, 5% CO 2 incubator. After 10-14 days of cultivation, the cells were in log phase. Three hours before harvesting, the cells were exposed to colchicine (0.25 µg/mL). The cell suspension was then resuspended in KCI Solution (hypotonic) 0.075 M (Sigma-Aldrich) twice, and then xed three times with a 3:1 mixture of methanol and acetic acid. The cell suspension was dropped on slides, and Q-banding was performed. Serial passaging was then performed on the cultures of all patients to study the dynamics of chromosome changes in GCT of bone. The culture conditions were as previously described, and the cell cultures were split 1:2 at subcon uency. The samples were studied from primary culture to ve passages [25]. The 50 metaphases in every passage were analyzed according to the International System for Human Cytogenetic Nomenclature 2013. The number of abnormal cells and the total number of abnormalities and types of abnormalities, including numerical, structural, and telomeric association, were recorded. Association of the short arm of the acrocentric chromosome was also recorded as telomeric association. Nonclonal and clonal chromosome changes, and the frequencies of the telomerase associations were calculated.

Study size and statistical analysis
An a priori sample size was calculated for this study. All eligible patients were included. Categorical variables are presented as frequency and percentage, and normally distributed continuous variables are given as mean plus/minus standard deviation. Unpaired t-test was used to compare the means of the local recurrence or pulmonary metastasis group with the group that didn't have either of those outcomes. A p-value of less than 0.05 was considered statistically signi cant, and all data analyses were performed using SPSS Statistics version 18 (SPSS, Inc., Chicago, IL, USA).

Results
A total of 15 patients were screened for eligibility and enrolled. No patients were lost to follow-up or died during the study period. All participants completed the study protocol, including successful cytogenetic analysis. Patient demographic and clinical characteristics are shown in Table 1. The mean age of included patients was 35.4 years. Of the 15 included patients, 10 (66.7%) were female. Most of our GCT cohort had non-axial skeletal involvement (86.6%) and Campanacci stage 2 (66.7%). There was only one case (6.7%) of con rmed pulmonary metastasis prior to operation. Five patients (33.3%) had either local recurrence or pulmonary metastasis during the 6-year postoperative follow-up period. The mean number of abnormal cells in the primary culture of patients with either study outcome of interest was signi cantly higher than in those without (24.4 vs. 9.6 cells, respectively; p=0.04). Similar patterns were found in subsequent cultures for four passages (p<0.05) (  .4;q13), were consistently found in the cultured samples from primary GCT of bone from patients with recurrence (Figures 1-6). Similar abnormal observations were found in all passaged cultures.

Discussion
Prediction of local recurrence or pulmonary metastasis of GCT of bone after surgery is a major challenge for orthopedic oncology surgeons. The recurrence rate of GCT and/or pulmonary metastasis was reported to range from 0% to 4% [26][27][28][29][30]. Early detection of patients at high risk for recurrence would improve outcomes; however, a proven and accepted list of independent predictive factors has not yet been established. Some predictors of recurrent GCT of bone have been explored and reported, including serum alkaline phosphatase, serum calcium, serum phosphate, serum vitamin D level, and bone mineral [14][15][16][17][18][19][20][21][22][23], yet none of these are used in clinical practice. Some centers use Campanacci staging, which is a system that was designed to stage only GCT [13]. Gorunova, et al. suggested that local recurrence probably depends more on the adequacy of surgical treatment than on the intrinsic biology of the tumors [32]. In contrast, other authors proposed that the intrinsic biology of the tumors, such as DNA, may be associated with the recurrence of GCT of bone [33][34][35]. Jeffrey, et al. found telomeric association and del(11)(p11) to have strong association with local recurrence of GCT of bone [36]. Bridge, et al. also stated that telomeric fusion and cytogenetic analysis might be useful in predicting the biologic behavior of GCT of bone [24]. Therefore, the present study was conducted with cytogenetic study to identify chromosomal abnormalities that may predict recurrent GCT of bone.
The results of the cytogenetic analysis in the present study showed a signi cantly higher number of abnormal cells in the recurrent group compared with the nonrecurrent group. This nding could be clearly established at the primary culture or the rst passage of the tumor. Furthermore, we found six consistent patterns of cytogenetic abnormality only in our recurrent cases. The cytogenetic analysis results were also similar in the later passages (the second to fth passages of the DNA culture). The duration of each passage was approximately 1 week to cytogenetically analyze the aggressiveness of the tumor. These ndings are very important because the mean number of cells in the primary culture was adequate for identifying the aggressiveness of the intrinsic tumor biology. The results of this study provide credible evidence that cytogenetic analysis may be a dependable method for predicting the likelihood of tumor recurrence in GCT of bone. Our study had a similar result to that of Unni, et al. who reported the most common abnormal karyotypic nding in recurrence of GCT of bone to be tas11p, 15p, 19q and 21p [36].

Limitations
This study has some mentionable limitations. First, this study may have been limited by interobserver variation during the cytogenetic study due to the high technical skill required. Second, we did not conduct a genome sequencing study because it is necessary that we identify chromosomal abnormalities via cytologic study rst. Third, the sample size was small, so adjustment for confounding factors or prognostic modeling could not be performed. Fourth and last, the patients that were enrolled in this study were recruited from a single center. The strength of this study is its prospective longitudinal cohort design with a 6-year follow-up period.

Conclusion
The mean number of abnormal cells and six tas patterns, including tas ( All methods were carried out in accordance with relevant guidelines and regulations. This study was retrospectively registered at gov (TCTR20210427003) Consent to participate: Informed consent was obtained from all individual participants included in the study.
Consent for publication: All authors have read and approved the nal submitted manuscript.
Authors' contributions: Pakpoom Ruangsomboon and Rapin Phimolsarnti provided research questions, conducted data collection, analyzed data, discussion and developed the full manuscript. Worapa Heepchantree, PhD. And Pissanu Reingrittha, MD examined all data analysis, detailed the results, statistical calculation and collected and monitored data. Saranatra Waikakul, MD also provided the research question and useful advice Con ict of interest declaration All authors declare no personal or professional con icts of interest, and no nancial support from the companies that produce and/or distribute the drugs, devices, or materials described in this report.

Availability of data and material
The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Consent for publication
All authors have read and approved the nal version of the manuscript, and all authors agree with the decision to submit this manuscript for journal publication.