Synovial sarcoma(SS) is a mesenchymal tumour manifested by different degrees of epithelial differentiation, including the formation of glands, presence of the specific ectopic t(X;18)(p11.2;q11.2) gene, and production of the SS18-SSX fusion gene. SS accounts for 5%-10% of soft tissue sarcomas.According to the recommendations of the NCCN and ASCO guidelines, the standardized treatment of early synovial sarcoma is mainly neoadjuvant chemotherapy combined with extensive surgical resection followed by chemotherapy or radiotherapy after surgery to strive for local radical treatment and reduce the occurrence of metastasis. However, even after standardized treatment, the 5-year survival rate of patients is only 50%-60%, and most of the patients die of lung metastasis, approximately 10% of patients have lung metastases at the first diagnosis. Even after extensive resection, approximately 50% of patients have lung metastasis during the follow-up process. Once lung metastasis occurs, the survival period is often less than 1 year, and the prognosis is extremely poor[5, 14]. The high-risk time of recurrence or metastasis of synovial sarcoma peaks in the second year after surgery. After two years, the risk of local recurrence or metastasis is significantly reduced. Several clinical trials have shown that several patients with high-grade sarcoma may benefit from immunotherapy. Thus, it is important to understand how to screen these patients and to identify relevant markers to predict the efficacy of immunotherapy. At present, the metastatic mechanism of SS is still unclear, and there are no characteristic prognostic markers. Therefore, it is important to clarify the relevant molecular metastatic mechanisms of SS and to identify new immune-related therapeutic targets and prognostic markers.
In the study, we conducted a comprehensive analysis of the GSE40021 database to identify DEIRGs associated with metastatic SS, and we performed GO function and KEGG pathway analyses on DEIRGs. We then used TCGA database to analyse the survival of DEIRGs, and we constructed a risk assessment model to predict the metastasis of synovial sarcoma patients and patients who may benefit from immunotherapy, which has important reference value for clinical practice. GO function annotation showed that the DEIRGs mainly participated in the following functions: binding between cytokines and cytokine receptors,activation of T cells,antigen processing and presentation,activation of receptors and ligands, and participation of MHC class II protein complexes. KEGG pathway analysis showed that DEIRGs were mainly involved in human leukaemia virus-1 infection, rheumatoid arthritis, antigen processing and presentation, and other related pathways. These DEIRGs were closely related to the activation of the immune system, suggesting that the metastasis of SS may occur through immune-related mechanisms. At the same time, tumour invasion and metastasis are extremely complex processes involving multiple steps and multiple genes. Therefore, the infiltration of different lymphocytes in the tumour microenvironment plays an important role in regulating tumour recurrence and metastasis. Zou et al. reported that Tregs have an immunosuppressive effect on cytotoxic CD8+ T lymphocytes through the adenosine A2A receptor pathway after activation of Tregs in the tumour microenvironment, ultimately leading to tumour recurrence or metastasis. Schiavoni et al. found that the delay in lung metastasis of malignant melanoma may be caused by the increase in the infiltration of eosinophils and CD8+ T lymphocytes in the primary tumour through the IL-33/ST2 axis .Song et al. demonstrated that ovarian cancer cells highly express the ubiquitination-related protein(UBR5), and induce the recruitment and activation of tumour-associated macrophages by regulating the secretion of key chemokines and cytokines, leading to the failure of chemotherapy and immunotherapy, which is often associated with poor prognosis. Therefore,the infiltration of immune cell that cause tumour metastasis varies in different tumour types.
At present, screening prognostic-related genes and constructing predictive models through multiomics sequencing have shown great predictive potential in a variety of tumour types. Li et al. constructed 14 immune-related gene prognostic risk assessment models, which showed good predictive performance for the prognosis of patients with osteosarcoma. Huang et al. screened 15 alternative splicing genes related to prognosis and two splicing molecules related to bone metastasis to construct a risk assessment model, which showed that the prognosis of breast cancer patients and the occurrence of bone metastasis also have good predictive performance. To the best of our knowledge, there is currently no risk assessment model for immune-related genes in patients with metastatic SS. The present study aimed to provide clinicians with accurate and important references for the prognosis of SS patients.The model contained six genes, namely, GREM2, CTSS, TINAGL1, ACKR1, HLA-DRB1 and STC2. GREM genes belong to the members of the DAN family, including Grem1 and Grem2, which are a large family that encode bone morphogenetic protein (BMP) antagonists.The secreted glycosylated protein encoded by this gene exerts an antagonistic effect by directly binding to the BMP protein, which plays an important regulatory role in organ formation and tissue differentiation[22, 23]. GREM2 is significantly highly expressed in gastric cancer tissues. Wang et al. demonstrated that it GREM2 maintains the stemness of gastric cancer cells through the JNK signalling pathway, ultimately promoting tumour invasion and metastasis. Several studies have shown that the GREM2 gene also regulates the proliferation and differentiation of human pluripotent stem cell-derived cardiac progenitor cells by regulating the BMP signalling pathway[25, 26], suggesting that this gene is closely related to the stemness of cells. The cathepsin S protein encoded by the CTSS gene is an important part of the family, and it is significantly different from other members of the cysteine protease family, mainly due to its limited tissue distribution and its ability to maintain good conformational stability at neutral and weakly alkaline pH. Several studies have shown that CTSS is involved in the occurrence and development of tumours, such as tumour angiogenesis and metastasis[27, 28]. CTSS has also been found to be significantly highly expressed in tumour tissues or cell lines. At the same time, clinical evidence indicates that the upregulation of CTSS protein levels is related to the poor prognosis of tumour patients. Lee et al. found that the expression level of CTSS protein in the tumour tissues of breast cancer patients is significantly negatively correlated with the BRCA1 gene. These results suggest that the CTSS protein is activated after radiotherapy, which hydrolyses the BRCA1 protein, thereby inhibiting the repair of intracellular DNA double-strand break damage and eventually promoting tumour cell proliferation, invasion, and migration. The STC2 gene encodes a secreted glycoprotein that regulates a variety of biological processes, such as the transport of calcium and phosphate in the kidney and intestine, cell metabolism, or cellular calcium/phosphate homeostasis. Several studies have shown that the STC2 gene regulates a variety of signalling pathways, and it is also closely related to cell proliferation, apoptosis, tumour metastasis, and treatment resistance. Wang et al. found that the STC2 gene is highly expressed in the tissues and metastatic lymph nodes of patients with nasopharyngeal carcinoma and that it is associated with poor prognosis. At the same time, other studies have confirmed that the STC2 gene is closely related to the occurrence and development of hepatocellular carcinoma. In vivo experiments have confirmed that tumour cells activate the APAF1, APC, and PTEN pathways as well as reduce STC2 expression by knocking down the Mus81 gene, thereby inhibiting tumour cell proliferation and metastasis. However, the above research results contradict our analysis. At present, the STC2 gene is mainly studied in epithelial-derived tumours. Considering that the role of STC2 in tumours of mesenchymal origin may be different from tumours of epithelial origin, subsequent biological experiments are still needed to confirm the inference.
To characterize the infiltration of immune cells in the tumour microenvironment, we further explored the relationship between immune-related prognostic models and immune cell infiltration. We found that activated B lymphocytes, effector memory CD4+ lymphocytes, effector memory CD8+ T lymphocytes, eosinophils, mast cells, monocytes, NK cells, plasma-like DC cells, and TH1 helper T lymphocytes had significantly low infiltration in patients with high-risk scores. Among them, eosinophils, monocytes, mast cells, and NK cells showed a significant difference in the metastasis-free survival time of low-risk patients, further suggesting that the immune-related prognostic model has a good ability to assess the infiltration of certain immune cells. At the same time, we found that there was a significantly low infiltration of macrophages and CD4+ T lymphocytes in patients with metastatic synovial sarcoma in the GSE40021 dataset. At present, there are few studies on mast cells and eosinophils in tumour metastasis. It remains unclear whether mast cells and eosinophils are involved in the regulation of the immune microenvironment of synovial sarcoma. Therefore, we focused on NK cells, macrophages, and CD4+ T lymphocytes.
Sutherland et al. found that the decrease in infiltration of NK cells rather than cytotoxic CD8+T lymphocytes in the tumour microenvironment is the main reason for the metastasis and spread of primary tumours by conducting a transcriptome analysis of the primary tumours of small cell lung cancer patients and transgenic mice. Ren et al. found that the metastasis regulation of tumour-infiltrating neutrophils is mediated by the activation state of NK cells in a mouse model of breast cancer. The infiltration of NK cells in tumours plays a crucial role in regulating tumour metastasis. Kuo et al. cocultured tumour cells with macrophages and collected the supernatant for proteomic analysis, and they showed that the tumour cells secrete succinate into the microenvironment and are activated by binding to succinate receptors on macrophages. Polarization to M2-type macrophages ultimately promotes tumour invasion and metastasis. Ruffell et al. reported that macrophages are important participants in the regulation of tumour microenvironment homeostasis. Tumour cells promote the polarization of macrophages to the M2-type by secreting different mediators, which may be driven by local hypoxia and fibrosis of the tumour microenvironment. The increased infiltration of these M2-type macrophages leads to tumour recurrence and metastasis caused by inhibiting the recruitment of cytotoxic T lymphocytes and regulating other aspects of tumour immunity. Sun et al. found that the high expression of the A2AR gene is closely related to the poor prognosis of patients, and high expression of the A2AR gene is also related to the high infiltration of CD4+ regulatory T lymphocytes (Tregs) by analysing the immunohistochemical results of large samples of head and neck squamous cell carcinoma tissues. A recent review published by Lam et al. proposed that CD4+ T lymphocytes have a variety of different cell subpopulations. The balance between immunosuppressive Tregs and the proinflammatory TH17 cell subset plays an important regulatory role in the occurrence and metastasis of lung cancer. Several studies have shown that these two CD4+ T cell subgroups play an active role in promoting lung cancer progression and metastasis. The infiltration of NK cells, eosinophils, macrophages, and CD4+ T lymphocytes in tumours plays a crucial role in regulating tumour metastasis. To further verify the role of these three immune cell subpopulations in patients with metastatic synovial sarcoma, we enrolled 16 synovial sarcoma patients with follow-up data in our centre from January 2018 to January 2019 in the study. Among them, 5 patients had metastases during the two-year follow-up period, and 11 patients had no metastases. In patients with metastatic SS, the infiltration of macrophages was significantly increased, and the infiltration of NK cells was significantly reduced. Furthermore, we also found that the infiltration of M2-type macrophages was the main infiltration, while the infiltration of CD4+ T lymphocytes was not significantly different. Combined with the analysis of the GEO database, we found that most of the immune checkpoints were not significantly different between metastatic and nonmetastatic SS patients. Moreover, there was low expression of LAG3 and PD-L1 in metastatic patients. These findings suggested that the metastasis of synovial sarcoma is not triggered by evading immune checkpoint surveillance. Combined with our analysis data, it is reasonable to speculate that the immune mechanism of metastasis may be caused by tumour cells inhibiting the secretion of certain cytokines, resulting in a decrease in the infiltration of NK cells and macrophages in the tumour microenvironment. We also found that the infiltrating macrophages were mainly polarized to the M2 type, which formed an immunosuppressive tumour microenvironment, thereby promoting lung metastasis.
Our study also had several limitations. First, the survival analyses of the identified DEIRGs were validated for all sarcomas and not specifically synovial sarcoma in TCGA because there is currently no database of large samples of synovial sarcoma patients with follow-up information. Peng et al. found that several biomarkers related to metastatic synovial sarcoma are also associated with prognosis through all sarcomas in TCGA. Because Peng and colleagues also found that the expression of the screening markers is significantly higher than that of normal tissues in the GEPIA dataset, they believe that this method of screening still has a certain reference value. Existing evidence shows that immune-related prognostic models can predict SS metastasis and the infiltration of immune cell in the tumour microenvironment. However, the number of clinical samples was small, and future research will require a large number of clinical samples and multiomics analysis.