Clinical pathological characteristics correlation of H3F3A gene mutation in giant cell tumor of bone: a study of 96 cases

Introduction

Giant cell tumor of bone (GCTB) is a common primary bone tumor with an estimated incidence rate of 1.2–1.7 cases per million persons per year (1,2). It accounts for nearly 3–5% and around 13.7–17.3% of primary bone tumor cases in western countries and China, respectively (3). GCTB develops mostly in the mature skeleton and arises from the epiphyseal end of long bones, for instance the distal end of the femur, proximal end of the tibia, and distal end of the radius, whereas the occurrence of GCTB in small bones such as metacarpals, metatarsals, and the jaw are relatively uncommon (1,4-6).

Regarding the histopathological morphology, GCTB is known for its mixed presence of monotonous sheets of round to oval to spindle-shaped cells, macrophage-like mononuclear cells, and osteoclast-like giant cells (1,7). Despite the seemingly benign pathological appearance and clinical behavior in most cases, GCTB is defined as an intermediate malignant neoplasm by the World Health Organization (WHO) due to its local aggressiveness and occasional metastatic biological nature. Despite the relatively low incidence, 1–9% of cases have been reported as having metastasized within 0–10 years from the diagnosis of primary GCTB (8-10). However, as for the malignancy and prognosis risk-related clinical features, there is currently no widely accepted histopathological grading scheme for GCTB. Certain morphology variations can be observed in different cases, for instance fibrosis, cystic degeneration, reactive bone formation, secondary aneurysmal bone cyst (ABC)-like changes and increased mitotic activity, with the latter being especially common in recurrent GCTB (11,12). Meanwhile, although venous involvement is also frequently observed in GCTB cases, it is not necessarily an indicator of malignancy (13). It is of clinical significance to explore the clinical features that potentially correlate with patient outcomes (14-16).

In terms of the molecular mechanisms, an inspiring breakthrough over the years has been discovery of a specific histone mutation, namely H3.3G34W (Gly34Trp), which exists in nearly 90% of GCTB cases (17). The gene discovery has led to the development of a highly sensitive and specific immunohistochemical (IHC) antibody, which has been shown in multiple studies to be highly effective in the pathological diagnosis and differentiation of GCTB from other bone diseases (18-20). Although the main mutation site of the H3F3A gene is G34W, other mutation sites have been increasingly reported, including G34V, G34L, and G34R, among others (21-24). Currently, the relationship between different H3F3A mutation sites and the histopathological morphology and clinical prognosis of GCTB is still unclear. Considering that the IHC antibody for H3.3 is specific for detecting G34W mutation (25,26), it is of clinical significance to investigate the different relationships of various H3F3A mutation sites with GCTB clinical pathological characteristics, thus assisting the clinical use of IHC as well as other sequencing experiments in the detection of gene status.

In this study, samples of 96 cases of GCTB from a local hospital were collected and used to analyze H3F3A mutation status and its association with tumor clinicopathological features. Based on the detailed evaluation by registered pathologists, the analysis results shall provide promising insights to better elucidate the mechanism and further clinical treatment of GCTB. We present this article in accordance with the STROBE reporting checklist (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2024-2564/rc).

Methods

GCTB samples collection

The retrospective cohort consisted of 96 cases with confirmed GCTB diagnosis by a registered pathologist in the Second Hospital of Shanxi Medical University Pathology Department during the period of January 2019 to December 2023. All the samples used in the study were formalin-fixed paraffin-embedded (FFPE) tissues that were stored in Second Hospital of Shanxi Medical University Biobank. The samples were originally sent from the Oncology Department after surgical removal of the tumor from the body to the Pathology Department for pathological diagnosis, and the remaining tissues were then donated to the hospital Biobank for long term storage. Informed consent regarding the potential scientific application of the samples had been obtained by the Biobank staff at the time that the donations were made. In the study, 1 representative FFPE block from each case with high tumor percentage and no necrosis, as confirmed by hospital pathologists, was selected for next-step analysis. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by Second Hospital of Shanxi Medical University Institutional Board (No. 2023YX179). Informed consent was taken from all the patients; for those under 18 years old, consent was obtained from their parents or other legal guardians.

Besides the tissue samples, clinical information of the cases was also retrieved from the hospital system for next-step analysis, including the following: (I) general information: age, sex, tumor site, tumor size, surgical and denosumab treatment history; (II) imaging data: Campanacci grading system evaluation results of the tumors; (III) patients postoperative follow-up information: including malignancy transformation, tumor recurrence, and metastasis status of the patients.

Tumor pathological morphology features analysis

In the study, 96 cases of GCTB samples were selected from the hospital Biobank for analysis after reconfirmation of disease diagnosis and tumor percentage by registered pathologists in the hospital’s Pathology Department. The FFPE tumor samples were firstly subjected to hematoxylin and eosin (HE) staining, and based on the HE slices, histopathological features of each case were obtained, including the proportion of multinuclear osteoclast-like giant cells, mononuclear stromal cells, intravascular tumor thrombus, reactive bone formation, spindle cell differentiation, tumor necrosis, ABCs, and surrounding soft tissues invasion (Figure 1); this information would be further used for evaluating the association GCTB clinical recurrence.

Figure 1 Representative morphology features of GCTB cases. (A-C) HE staining for showing the classic morphology features in GCTB cases, especially the mixed presence of monotonous sheets of round to oval to spindle-shaped cells, macrophage-like mononuclear cells, and osteoclast-like giant cells (A, magnification: 100×, bar represents 100 µm; B, magnification: 200×, bar represents 60 µm; C, magnification: 400×, bar represents 30 µm). HE staining for showing (D) the typical reactive bone formation in GCTB cases (magnification: 100×, bar represents 100 µm), and (E) the bones were surrounded by osteoblasts (magnification: 200×, bar represents 60 µm). (F,G) HE staining for showing the typical morphology features of aneurysmal bone cysts in the tumor (F, magnification: 100×; G, magnification: 200×; bars represent 100 and 60 µm respectively). (H-J) HE staining for showing classic morphology features for revealing spindle cells differentiation in GCTB cases, and pathological mitotic could be observed in some cases (H, magnification: 100×; I, magnification: 200×; J, magnification: 400×; bars represent 100, 60, and 30 µm respectively). (K,L) Representative HE figures of tumor necrosis in GCTB (K, magnification: 100×; L, magnification: 200×; bars represent 100 and 60 µm respectively). (M-O) HE staining for showing the surrounding soft tissues invasion by GCTB tumors (all magnification: 100×, bars represent 100 µm). GCTB, giant cell tumor of bone; HE, hematoxylin and eosin.

IHC experiment

The entire IHC experiment procedure was conducted using local hospital Pathology Department instrument and equipment, and the experiment was performed on VENTANA platform (Roche, Basel, Switzerland). All primary antibodies including H3F3AG34W (No. RM263), P53 (No. MX008), P63 (No. MX013), and Ki-67 (No. MIB-1) were purchased from MXB (Fuzhou, China), and the secondary antibody [Envision/horseradish peroxidase (HRP) kit] and diaminobenzidine (DAB) detection kit were from ZSBG-Bio (Beijing, China). Other reagents, including but not limited to, H₂O₂, antigen retrieval citrate solution, phosphate-buffered saline (PBS), and hematoxylin stain were supplied by our hospital’s Supply Department.

The procedures of the IHC experiment were same as previously reported. Firstly, the FFPE slides were deparaffinized and rehydrated using gradient ethanol, followed by incubating with 0.3% H₂O₂ with the purpose of inhibiting endogenous peroxidase activity. Then, the slides were processed according to operating manuals for antigen retrieval before the next step of primary antibodies incubation overnight at 4 ℃. Further, the slides were processed with a specific secondary antibody at 37 ℃ for 1 hour and then incubated with HRP. Finally, the slides were visualized using DAB and evaluated by local hospital registered pathologists.

As for the IHC evaluation criteria of the five genes including H3F3A, H3F3B, P53, P63, and Ki-67, any percent of nuclear staining in mononuclear stromal cells was considered positive for H3F3A, H3F3B, and P63 genes (the staining in other cells, for instance, multinuclear giant cells was not treated as positive). Both diffuse strong nuclear staining and no staining were considered positive for P53 gene (scattered moderate or weak staining was treated as negative). Meanwhile, the evaluation of Ki-67 was based on the percent of nuclear staining mononuclear stromal cells in the whole tissue slide. All the evaluation processes were conducted by registered pathologists in the local hospital (Figure 2).

Figure 2 Representative IHC staining results in GCTB cases. (A-C) IHC staining of H3.3 G34W in different GCTB cases with various morphology features (all positive staining, A,B, magnification: 200×, bars represent 60 µm; C, magnification: 100×, bar represents 100 µm). (D-F) IHC staining of P53 protein in different GCTB cases, including (D) for showing a case with sporadic P53 staining which was classified as wild type staining, (E) revealing a case with non P53 staining which was classified as nonsense mutation, and (F) displaying a case with strong P53 staining which was classified as missense mutation (D-F, magnification: 200×, bars represent 60 µm). (G-I) Representative IHC staining of P63 in different GCTB cases with various morphology features (all positive staining, G,H, magnification: 200×, bars represent 60 µm; I, magnification: 100×, bar represents 100 µm). GCTB, giant cell tumor of bone; IHC, immunohistochemistry.

Sanger sequencing analysis

Before next-step Sanger sequencing analysis, nucleic acid was isolated from the FFPE GCTB tumor samples using a TIANGEN FFPE DNA Kit (Beijing, China); all the procedures were operated according to the manufacturer’s instructions of the kit. Before the extracted DNA was applied for the sequencing experiment, the extracted DNA was quantified using Qubit 3.0 Fluorometer (Thermo Fisher Scientific, Carlsbad, CA, USA).

Moreover, Sanger sequencing was used for detection of H3F3A gene mutations in the samples. Polymerase chain reaction (PCR) analysis was performed on Roche Cobasz480, and the bidirectional Sanger sequencing of the PCR products was performed using ABI3500DX (Thermo Fisher Scientific). All the DNA sample processing and experimental procedures were carried out in accordance with the H3F3A commercial kit instructions (Jingzhun Medical Technology, Beijing, China). The sequencing results were analyzed by Sequencing Analysis 5.4 software (Thermo Fisher Scientific).

Statistical analysis

Statistical analysis was conducted using the software SPSS 23.0 (IBM Corp., Armonk, NY, USA); the enumeration data were analyzed using t-test or Mann-Whitney U test, and the measurement data were analyzed by Chi-squared test. A P value <0.05 was considered statistically significant (for all analysis results, * represents P<0.05, ** represents P<0.01, *** represents P<0.001).

Results

General characterization of GCTB samples

The 96 GCTB cases included 44 males and 52 females (the male/female ratio: 0.85). As for the patient age distribution, 6 cases were <20 years old, 42 cases were between 20 and 40 years old, and 48 cases were >40 years old. Meanwhile, most of the samples were from the limbs of patients (71 cases, 73.96%), 9 cases were originated from vertebrae, and the other 16 cases were from irregular bones. Moreover, the maximum tumor diameter of 10 cases was <3 cm, 44 cases were 3–6 cm, and 42 cases were >6 cm. In terms of the imaging Campanacci classification of the tumor, 32 cases belonged to Grade I, 40 cases were Grade II, and the other 24 cases were classified as Grade III. As for the tumor recurrence frequency, 15 cases of the 96 GCTB samples were further discovered with postoperative recurrence, no distal metastasis was observed by the end of the study, and all patients were alive (Table 1).

Recurrence-related clinical and pathological GCTB characteristics

Among the included 96 GCTB cases, 15 cases were further developed with postoperative recurrence (recurrence rate: 15.62%), and the analysis of the clinical and pathological characteristics that were related to the recurrence revealed a statistically significant correlation between the imaging Campanacci grading system and recurrence. Nearly all of the recurrences (14/15 cases, 93.33%) had occurred in patients with Campanacci II and III grade tumors, and the recurrence in Campanacci III cases was higher than that in the Campanacci II cases (6/24, 25% vs. 8/40, 20%).

Meanwhile, the correlation between surgical methods and recurrence was also statistically significant; a higher recurrence rate was discovered in the patients who underwent segmental resection surgeries than in those with tumor curettage operations, although the result was possibly relating with the preoperative tumor size and grading system results which have represented major determinants in surgical decision-making. As for the other clinical characteristics, although more recurrence was discovered in male than female patients, the difference was not statistically significant. No specific correlation was revealed between patients age, tumor site, tumor size, or denosumab treatment history and tumor recurrence, although some of the results might be due to the very limited case number in the group, for instance only 1 of the patients underwent denosumab treatment and he had not experienced recurrence.

Additionally, in terms of pathological features, tumor invasion of surrounding soft tissues, higher expression of P53, and lower expression of P63 were indicated to be statistically significantly correlated with GCTB recurrence, and tumors with surrounding soft tissue invasion had a much higher risk of recurrence than the others. Meanwhile, although tumor necrosis, spindle cells differentiation, intravascular tumor thrombus, and pathological nuclear mitosis in the recurrence group seemed higher than they were in the non-recurrence group, the difference was not statistically significant (Figure 3, Table 2).

Figure 3 The results of 2 representative H3F3A IHC positive and negative staining cases respectively. (A) HE staining of a representative GCTB case with H3F3A IHC-positive result, classic morphology features could be observed in the case with mixed presence of monotonous sheets of round to oval to spindle-shaped cells, macrophage-like mononuclear cells, and osteoclast-like giant cells (magnification: 100×, bar represents 100 µm). (B) HE staining of the same case as in A with larger magnification for showing the evenly arranged osteoclast-like giant cells and the mixed monotonous cells (magnification: 200×, bar represents 60 µm). (C) IHC analysis revealed H3.3 G34W negative in the first case (magnification: 200×, bar represents 60 µm). (D) HE staining of a representative GCTB case with H3F3A IHC negative result, less osteoclast-like giant cells were observed in the case, and the tumor cells were more spindle cell differentiated (magnification: 100×, bar represents 100 µm). (E) HE staining of the same case as in D with larger magnification for showing the spindle cell differentiation and less giant cells presented in the case (magnification: 200×, bar represents 60 µm). (F) IHC analysis revealed H3.3 G34W positive staining in the second case (magnification: 200×, bar represents 60 µm). GCTB, giant cell tumor of bone; HE, hematoxylin and eosin; IHC, immunohistochemistry.

H3F3A gene mutation status in GCTB samples

After analyzing the potential tumor recurrence-related clinical and pathological characteristics, the H3F3A gene status was detected in the GCTB samples by both IHC and Sanger sequencing experiments. The results revealed that among the 96 GCTB cases, the H3F3A gene mutation rate was 88.54% (85 cases), with the most common mutation being H3F3A G34W (76 cases, 89.41%), and other mutation sites including G34V (4 cases), G34L (2 cases), and Y41H (3 cases) (Figure 4).

Figure 4 Sanger sequencing results of 4 representative cases with H3F3A gene mutations in different sites. (A) HE staining and (B) Sanger sequencing result of a representative GCTB case with H3F3A G34W mutation. (C) HE staining and (D) Sanger sequencing result of a representative GCTB case with H3F3A G34V mutation. (E) HE staining and (F) Sanger sequencing result of a representative GCTB case with H3F3A G34L mutation. (G) HE staining and (H) Sanger sequencing result of a representative GCTB case with H3F3A Y41H mutation (for all the HE staining in A, C, E, and G figures, magnification: 100×, bars represent 100 µm). GCTB, giant cell tumor of bone; HE, hematoxylin and eosin.

Meanwhile, high consistency was discovered between the IHC experiment and Sanger sequencing result, especially for the H3F3A G34W mutation. Based on the IHC experiment, 76 cases were positive staining, and 74/76 cases were G34W mutations (97.3%), and the other 2 cases were G34V mutations. Meanwhile, as for the other rare mutation sites including 2 cases of G34V, 2 cases of G34L, and 3 cases of Y41H, the IHC results were all negatively staining, indicating the specificity of the H3F3A antibody for detecting G34W mutation (Table 3).

Association between H3F3A gene mutation and GCTB clinical and pathological features

To preliminarily elucidate the association between H3F3A and GCTB clinical and pathological features, firstly, based on whether the gene was mutated, the 96 GCTB cases were divided into two groups for comparative analysis. The results revealed a statistically significant correlation between spindle cell differentiation as well as P63 expression and H3F3A mutation; both the spindle cell differentiation ratio and P63 expression were much higher in the cases with H3F3A mutation than they were in other cases. Meanwhile, there was neither a significant correlation between H3F3A mutation and clinical characteristics including patient gender, age, tumor location, tumor size, and Campanacci grading results, nor between H3F3A mutation and other pathological indicators such as osteogenesis, tumor necrosis, intravascular tumor thrombus, and nuclear mitosis, indicating the necessity of H3F3A gene detection by IHC or sequencing experiments in clinical application (Table 4).

Although no specific correlation has been found between H3F3A mutation and GCTB characteristics, the different sites of the H3F3A gene seemed to be of clinical significance. Based on different H3F3A mutation sites, the 85 GCTB cases with H3F3A mutation were divided into two groups, namely H3F3A G34W mutation group and rare mutation sites group (the 9 GCTB cases with G34V, G34L, and Y41H mutations were classified into one group considering the limited number of cases harboring these mutations), and a comparative analysis was conducted between the groups. The results revealed that compared with the G34W mutation group, the cases with rare mutation sites had a higher risk of postoperative recurrence and surrounding soft tissue invasion, pathological nuclear mitosis, and P53 expression were also higher in this group, indicating the different biological nature, and more importantly, different prognosis risk, between the two groups of GCTB cases (Table 5).

Case presentations and clinical association of H3F3A Y41H mutation samples

Among the 85 GCTB cases with H3F3A mutation, besides the common G34W mutation, three types of other mutation sites including G34V, G34L, and Y41H were detected, and the Y41H mutation was firstly reported in this study. Considering the potential clinical significance of the samples and to assist further studies, the detailed information of the 3 cases was independently displayed.

Of the 3 cases, 2 were females and the other was male; all 3 patients were older than 20 years. The tumors had all developed in the patients’ limbs; the tumor size of 1 case was between 3 and 6 cm, whereas those of the other 2 were over 6 cm. Campanacci grading results of all 3 cases were Campanacci II or III, and all 3 cases recurred after surgery, which is in consistency with the above deduction that the cases with rare H3F3A sites mutation harbored increased prognosis risk.

As for the pathological morphology characteristics, the spindle cells differentiation were observed in all 3 cases, and big proportion of mononuclear stromal cells, tumor necrosis, and reactive bone formation were discovered in 2/3 cases. Meanwhile, none of the 3 cases developed ABCs (Figure 5).

Figure 5 Cases presentation of the 3 H3F3A Y41H mutation samples. (A) HE staining for observing the morphology feature in case 1 (magnification: 100×, bar represents 100 µm). (B) HE staining with larger magnification for showing the reactive bone formation in the tumor area of case 1 (magnification: 200×, bar represents 60 µm). IHC staining results for showing the (C) H3.3 G34W(−), (D) H3F3B(−), (E) P53 (+, mutated staining), and (F) Ki67 (+, 60%) in case 1 (all magnification: 200×, bars represent 60 µm). (G) HE staining for observing the morphology feature in case 2 (magnification: 100×, bar represents 100 µm). (H) HE staining with larger magnification for showing the tumor necrosis area in the tumor (magnification: 200×, bar represents 60 µm). IHC staining results for showing the (I) H3.3 G34W(−), (J) H3F3B(−), (K) P53 (sporadic+, wild type staining), and (L) Ki67 (+, 40%) in case 2 (all magnification: 200×, bars represent 60 µm). (M) HE staining for observing the morphology feature in case 3 (magnification: 100×, bar represents 100 µm). (N) HE staining for showing the tumor necrosis area in the tumor (magnification: 200×, bar represents 60 µm). (O) HE staining for showing the surrounding soft tissues invasion of the tumor in this case (magnification: 200×, bar represents 60 µm). IHC staining results for showing the (P) H3.3 G34W(−), (Q) H3F3B(−), and (R) Ki67 (+, 50%) in case 3 (all magnification: 200×, bars represent 60 µm). HE, hematoxylin and eosin; IHC, immunohistochemistry.

Discussion

GCTB is a common primary bone tumor with local aggressiveness and occasional metastasis; it comprises nearly 4–5% of all primary bone tumors and 20% of benign bone tumors (27,28). Despite its benign histological appearance and clinical behavior in most cases, nearly 10–40% of cases may experience postoperative recurrence, and around 1% of cases might transform into malignant tumors and even distal metastasis (10,29). In 1926, Finch and Gleave reported the first case of lung metastasis in GCTB, and the average incidence of lung metastasis has been reported to be 3% (1–9%) in different studies (1). However, there is currently no widely accepted histopathological grading scheme for predicting tumor recurrence and metastasis, partly due to the fact that there is an average 3.5 years of interval (0–10 years) between the diagnosis of primary GCTB and occurrence of distal metastasis (30,31), making it difficult to identify effective metastasis prediction factors.

At present, the commonly acknowledged factors affecting patient prognosis and tumor development in GCTB include the location of tumor, tumor size, tumor recurrence history, clinical treatment, and patient’s physical condition (32-35). The location of tumor has been an important factor affecting treatment operations; tumors located near joints or critical anatomical structures such as the spine may face more complex surgical challenges and harbor higher risks of recurrence, and the location also has critical impact on functional recovery after surgery. Tumor size is another critical clinical factor associated with patient prognosis; in the case of tumors that are so large that complete surgical removal becomes very difficult, even if the tumor were to be removed, the surgery might cause more bodily injury or even function loss. Meanwhile, the type of surgery, for instance curettage and extensive resection, also has significant impacts on patients prognosis (36). Although extensive resection may reduce the risk of recurrence, it may also result in greater functional loss. The use of adjuvant therapy, such as denosumab or radiation therapy, may affect the likelihood of tumor recurrence and the patient’s quality of life (16,37-42).

In this study, besides the above factors, the clinical and pathological characteristics of 96 cases of GCTB were collected to analyze the potential factors associated with tumor recurrence, and we discovered that higher imaging Campanacci grading result, pathological invasion to surrounding soft tissues, P53 mutation, and lower P63 expression were statistical significantly correlated with GCTB recurrence. Especially for Campanacci grading result, nearly all of the recurrence cases (14/15 cases) occurred in Campanacci II and III grade tumors, and the recurrence in Campanacci III cases was higher than that in the Campanacci II cases. Meanwhile, although the difference was not statistically significant, bigger tumor size, more necrosis, higher intravascular tumor thrombus, and pathological nuclear mitosis were discovered in the recurrence group of cases than the other case groups. These results shall be of clinical value for predicting GCTB recurrence risk.

Meanwhile, as for the diagnosis and management of GCTB, over the past few decades, there has been a significant evolution regarding the RANK/RANKL/OPG signaling pathways which have been gradually been understood to play vital roles in GCTB development (43,44). In addition, a specific histone mutation, namely H3.3G34W, has been discovered to be present in almost all GCTBs. In 2013, Behjati et al. first reported the presence of H3F3A mutation in nearly 92% of GCTB, and it has been increasingly accepted to be the driving mutation of the tumor (17). The discovery of the H3.3G34W mutation leads to the identification of a highly sensitive and specific IHC antibody, which has been shown to be very useful in the diagnosis of GCTB and differentiating it from other bone tumors. The antibody has been widely applied in clinical medical diagnosis.

Besides H3F3A G34W, increasing studies have found other rare (<1–2%) mutations in the H3F3A gene, such as H3F3A G34M, G34L, G34V, and so on, and the mutations are mostly limited to monocytes and have not been detected in osteoclasts or their precursor cells (45,46). There is increasing experimental evidence about the specificity of the H3.3G34W antibody being used for only detecting H3F3A G34W mutation; the antibody is not suitable for the other mutation sites. In this study, IHC experiments based on the H3F3A G34W antibody and Sanger sequencing were combine used to detect the gene status in GCTB. A consistent result was obtained in relation to the specificity of the IHC experiment for G34W mutation; of the 76 cases that were revealed to be positively staining by IHC, 74 cases were G34W mutations, meanwhile, only 2 of the 9 rare site mutations (2 cases of G34V, 2 cases of G34L, and 3 cases of Y41H) were detected by IHC experiment.

Different studies have been showing that the rare mutation sites of H3F3A, for instance G34V, G34M, and G34L, are mainly found in the small bones of hands and feet, patella, and axial bone, which seems different from the G34W mutation locations. In this study, among the 4 cases of G34V, 1 case had originated in the first metatarsal bone of the left foot, 1 case was from the left calcaneus bone, and the other 2 cases of G34V mutation as well as all the 2 cases of G34L mutation and 3 cases of Y41H mutation were all occurred at the end of long tubular bone. Bigger clinical studies containing larger cohort of patients shall be needed to better clarify the problem. A notable discovery of the study is that when compared with the common G34W mutation group, the cases with mutations in rare sites had a higher risk of postoperative recurrence and surrounding soft tissue invasion; for instance, all 3 cases with the newly discovered Y41H mutation recurred after surgery. Meanwhile, the pathological nuclear mitotic rate was also higher in this group, indicating the potential different prognosis risk between the GCTB cases with G34W mutation and other rare sites mutations.

There are certain limitations in this study. Only 96 cases from our hospital, instead of a multi-center cohort, were included in the study. Although we only detected mutations in the H3F3A gene and did not include the H3F3B gene or other related gene mutations, the current results of the study still contribute to deepening the understanding of the mechanism behind GCTB development.

Conclusions

Several clinicopathological features including Campanacci grading system and soft tissues invasion were shown to be associated with tumor recurrence. Patients with GCTB H3F3A rare mutation sites may experience recurrence more frequently than those with the common G34W mutation. Our findings warrant further bigger cohort evaluation of the association between H3F3A and GCTB characteristics.

Acknowledgments

We sincerely appreciate the sample donors who were all local hospital patients for donating their postoperative tissues to our BioBank, their samples were very important resources for the study, it is our honor to acknowledge their contributions.

Footnote

Reporting Checklist: The authors have completed the STROBE reporting checklist. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2024-2564/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2024-2564/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2024-2564/prf

Funding: The work was supported by China Central Government Fund for Guiding Local Scientific and Technological Development (No. YDZJSX2021A042), the Science Project from Health Commission of Shanxi Province (No. 2023103) and grants of Natural Science Foundation of Shanxi Province in China (Nos. 202203021222393, 202303021222333, and 202403021211135).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2024-2564/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by Second Hospital of Shanxi Medical University Institutional Board (No. 2023YX179). Informed consent was taken from all the patients; for those under 18 years old, consent was obtained from their parents or other legal guardians.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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