On The Prognostic Values Of Combinatorial Immune-related Markers In Primary Esophageal Small Cell Carcinoma

Background:Primary small cell carcinoma of the esophagus(PESCC)is a rare and lethal malignant tumor,and its manifestations at different sites are similar.Its high degree of malignancy,rapid progression,early metastasis,and poor prognosis has become an increasing concern.However,there is no unified standard for clinical staging,treatment and outcome evaluation.Traditional treatment which applies combined therapy of surgery and chemoradiotherapy cannot effectively improve the survival rate of patients based on the TNM stage.Therefore,seeking new biomarkers to develop a new prognostic model for outcome prediction and exploring novel therapeutic approaches to improve the overall survival time and the life quality of PESCC patients are urgently needed.The successful application of immunosuppressive agents such as PD-1/PD-L1 immune checkpoint has opened a new prospect for the treatment of small cell carcinoma.Studies in recent years showed that immune-related markers in the tumor microenvironment(TME)are recognized as biomarkers of response to monoclonal antibody drugs of the immune checkpoint of PD-1/PD-L1.Moreover,these markers are important prognostic factors for small cell lung cancer and other solid tumors in addition to clinical TNM staging.The expression of tumor?stemness?,interacts with the immune-related markers and may also affect the value of PD-L1as a biomarker for predicting immune checkpoint inhibitor response.Therefore,it is of great clinical significance to identify the mechanism of interaction between immune-related markers and tumor?stemness?expression in TME.Aims:We aim to identify biomarkers for a new nomogram that provides a better overall survival prediction than TNM staging,and analyze the potential regulatory mechanism of expression of immune-related markers in TME,and provide specific strategies for clinical trials of PESCC immunotherapy.Methods:H&E,IHC,IF,and Multi-IF were used to detect and analyze the TME status of PESCC and ESCC and the expression of"stemness"in PESCC tumors.The relationship between the expression of"stemness"in PESCC tumors and immune-related markers in TME was statistically analyzed,and independent prognostic factors were screened.Finally,the nomogram prognostic model was built with R language.Results:1)The majority of patients with PESCC were middle-aged and elderly men(61/81),and most of them were found in the middle and late stages(48/81).The average survival time was 18.3months.Kaplan-Meier analysis showed that TNM stages cover T,N,M categories,and tumor necrosis was associated with overall survival rate.2)The positive expression rate of PD-L1 in PESCC was 33.33%(27/81),PD-L1 in most positive cases were expressed exclusively in tumor stroma(25/27).Chi-square and Wilcoxon's test showed that PD-L1 expression was positively correlated with the infiltration of TIIs subtypes(CD4~+,CD8~+,CD163~+).Kaplan-Meier analysis showed that the positive expression of PD-L1 in PESCC was correlated with a long overall survival period.The positive expression rate of PD-L1in ESCC tissue samples of the control group was 30.95%(13/42),mainly expressed in tumor cells(9/13).PD-L1 expressed in stroma rather than in tumor cells was associated with the invasion of TIIs in ESCC.3)PD-L~+CD163~+TAMs were accompanied by more CD4~+T lymphocyte infiltration(20/27).The co-localization of PD-L1~+CD163~+TAMs with T lymphocytes was observed under a laser confocal microscope,and the T lymphocyte subsets co-located with them were mainly CD4~+T lymphocytes.4)In the TME of PESCC,the infiltration of T cells was mainly CD4~+subsets(68/81,CD4/CD8?1).Compared with CD8,PD-L1 expressed on T lymphocytes was more common in co-expression with CD4.FoxP3~+Tregs accounted for 13-27%of the total number of CD4~+T cells in PESC,which was much higher than that of the control group(9-13%).Kaplan Meier analysis showed that CD4,FoxP3,CD8/CD4,and FoxP3/CD8 were correlated with overall survival.5)In PD-L1 positive patients,low expression of Foxp3~+Tregs,low expression of FoxP3/CD8,and high expression of CD8/CD4 were associated with longer overall survival;in PD-L1negative patients,no necrosis,low expression of Foxp3~+Treg and CD4~+TILs were associated with longer overall survival.6)SOX2 expression was frequently observed in PESCC(53.1%,43/81).It was positively correlated late clinical stage.Kaplan-Meier analysis showed that SOX2 overexpression was associated with a worse prognosis.7)The expression of SOX2 was negatively correlated with that of PD-L1.Kaplan-Meier survival analysis showed that compared with other groups,SOX2~+PD-L1~-,SOX2~+CD4~+and SOX2~+FoxP3~+groups had a worse prognosis.8)Cox proportional hazards regression model showed that TNM stage,PD-L1,CD4,and FoxP3/CD8were independent prognostic factors for overall survival.Based on this,the nomogram model for predicting the overall survival rate of PESCC performed better than the TNM stage in prediction accuracy and goodness of fit.Conclusion:1)The low survival rate of patients with PESCC is associated with the inhibition of TME. CD4~+TILs,especially Tregs,may play an important role in tumor immune escape.2)PD-L1,CD4,FoxP3 and CD8 are predictive biomarkers for evaluating the prognosis of PESCC.The nomogram model based on these biomarkers can provide a new alternative for determining the prognosis of patients in clinical practice.3)The expression of SOX2 is association with the progression of PESCC.It regulates the expression of PD-L1 in TME and affects the prognostic role of PD-L1,CD4 and FoxP3,among other tumor immune related markers.4)The expression pattern of PD-L1 in PESCC,different from that in ESCC and other non-small cell carcinomas,is mainly expressed in immune cells.The analysis of PD-L1 expression, SOX2 expression and the TME status of PESCC can offer reference for the development of therapies with immune checkpoint inhibitors.In conclusion,for the first time ever,we build a nomogram model that is based on independent prognostic variables and superior to traditional TNM staging to predict the prognosis of PESCC.This new approach promises to be an alternative for survival prediction in clinical practice.The characterization of SOX2 expression in tumor cells and the expression of immune related markers in TME can provide theoretical fundamental for clinical trials of immunotherapy.