The Prognostic Role Of Alternative Splicing Events In Soft Tissue Sarcomas And The Correlation With Tumor Microenvironment

Purpose: Alternative splicing(AS)event is one of the most vital procedures of post-transcription modification for pre-RNA,and it has been reported that over 90% of genes in humans were modified by AS event.In recent years,a large number of studies have reported that AS events play an important role in diseases,especially malignant tumors.Besides,AS event was considered to be related with tumorigenesis,progression,metastasis,and drug resistance of tumor.With the development of high-throughput sequencing,many studies focused on the comprehensive genome-wide profiling of AS events in cancer and confirmed that AS events can be used as robust prognostic biomarkers.However,the role of AS events in the prognosis of patients with soft tissue sarcoma(STS)remains unclear.Material and Methods: RNA-seq,clinicopathologic data and follow-up information of the STS cohort were extracted from TCGA database.The data of AS events were downloaded from the TCGASplice Seq database and the PSI was selected to quantify the AS events.Univariate Cox analysis,LASSO regression analysis,and multivariate Cox analysis were performed to determine the independent prognostic AS events related to overall survival(OS)-and disease-free survival(DFS),and two signatures were established.Subsequently,Cox regression analysis was used to determine the clinical variables that independently associated with STS patients’ prognosis,and nomograms were established by integrating the AS-based signature and independent prognostic variables.The receiver operating characteristic(ROC)curve with area under the curve(AUC),calibration curve,and decision curve analysis(DCA)were used to evaluate the nomogram.Afterward,we used the CIBERSORT and ESTIMATE algorithm to quantify the immune cell proportion and tumor microenvironment score,respectively.The association between AS events-based clusters and TME as well as immune cells were studied.Results: A total of 195 patients were included.Totally,1945 and 1831 AS events were identified as OS-and DFS-related AS events,respectively.Nine AS events independently related to OS and eight AS events independently related to DFS were identified by LASSO regression analysis and multivariate Cox regression analysis.In the clinical data,tumor multifocal indicator and surgical margin resection status were identified as independent OS-and DFS-related variables,respectively.Two nomograms based on the AS events and clinical data were established.The AUC values of OS nomogram at 3-,5-,and 7-year were 0.894,0.834 and 0.810,respectively,while the AUC values of DFS nomogram at 3-,5-,and 7-year were 0.808,0.833 and 0.807,respectively.The AUC values of both nomograms were higher than that of different clinical variables or the prognostic signatures based on AS events.The calibration curve and DCA showed excellent performance of nomograms.Furthermore,three subtypes were identified by unsupervised cluster analysis,and there was significantly different prognosis among the three subtypes.The differences on macrophages,mast cells,natural killer cells,memory T cells,B cells and neutrophils among the three subtypes were statistically significant.In the TME score,the differences on immune score and stromal score among the three subtypes were also statistically significant.Conclusions: AS events are reliable prognostic biomarkers in patients with STS.The clinical prediction nomograms based on AS events can effectively predict the OS and DFS of STS patients.AS events may be involved in the remodeling of immune cell composition and immune microenvironment in STS patients.