MRNA Expression Levels Of PD-L1,PD-1 And TCF-1 And Their Clinical Relevancies In Peripheral Blood Cells Of Esophageal Squamous Cell Carcinoma

Objective:Programmed death ligand-1（PD-L1）and programmed death receptor-1（PD-1）can promote or inhibit tumorigenesis under distinct contexts.The purpose of this study was to investigate the mRNA changes of PD-L1,TCF-1 and PD-1 in peripheral blood,and to explore their values in the diagnosis and prognosis of ESCC.Materials and methods:1.Peripheral venous blood samples were collected from 91 ESCC patients in Anyang Cancer Hospital between January 2019 to December 2019,including 60 males and 31 females with an age-range from 44 to 80 years old（an median age of 67±7.41years old）.According to the 8th edition of TNM staging criteria for esophageal cancer developed by the AJCC-UICC,lymph node metastasis and TNM were staged.The blood samples of 63 non-cancer controls were from Luoyang,including 27 males and 36 females with an age-range from 47 to 86 years old（an median age of 67±8.42years old）.All non-cancer controls were followed up for 12 months after peripheral blood collection to exclude the insidious tumor occurrence during this period.2.RNA extraction and c DNA reverse transcription of peripheral blood cell:Eight-hundred microliters of Trizol were added to 200μL of blood cells to extract RNA.OD260/280 ratio was determined by Nano Drop Lite Ultraviolet Spectrophotometer,and c DNA was reverse-transcribed by reverse transcription kit of Hi Script II 1 st Strand c DNA Synthesis Kit.3.Quantitative real-time-polymerase chain reaction（q RT-PCR）was performed using Ace Q q PCR SYBR Green Master Mix（Q111-02 hand 03）kit,with the reaction volume of 20μL.Triplicates for each sample were performed.The expression levels of PD-L1,PD-1 and TCF-1 mRNA in blood cells were quantified by 2^-ΔΔCt with normalization by GAPDH,4.Statistical analysis:R4.1.0 was used for statistical analyses.The different expression levels of PD-L1,PD-1 and TCF-1 mRNA in peripheral blood cell were analyzed by two independent samples t-tests.Pearson correlation analyses were carried out for the correlation analyses.The correlations of clinicopathological features were evaluated byχ² or Chi-square tests.The optimal cutoff value was determined by receiver operating characteristic curve analysis（ROC）,and the sensitivities and specificities of each molecular marker were evaluated by area under curve（AUC）.The clinicopathological data of the patients were collected through the medical record management system of Anyang Cancer Hospital,and the patients were followed up by telephone.The last follow-up time was February 2021,and the overall survival（OS）was from the operation day to the death or the last follow-up time.Univariate and multivariate Cox proportional hazard regression models were used to analyze the effects of PD-L1,PD-1,TCF-1 and clinicopathological features on OS.The survival curve was plotted by Kaplan-Meier,and the difference of survival curves among different groups were determined by log-rank test.The test level wasα=0.05.Results:1.The average expression levels of PD-L1,PD-1 and TCF-1 mRNA in peripheral blood of ESCC were 5.75±2.50,6.66±2.70,6.50±2.29,respectively.These were significantly higher than those levels in non-cancer control group（4.38±2.25,3.54±1.61,4.45±1.15,respectively,all P values<0.01）.2.The expression levels of PD-L1,PD-1 and TCF-1 in peripheral blood of ESCC were positively correlated with the correlation coefficients of PD-L1 and PD-1 of 0.61,TCF-1 and PD-L1 of 0.62,and TCF-1 and PD-1 of 0.64,respectively.3.ROC analysis showed that the optimal cutoff values of PD-L1,PD-1 and TCF-1 mRNA in ESCC peripheral blood to distinguish ESCC patients from non-cancer controls were 4.52,5.42,and 5.96,respectively.The AUCs of PD-L1,PD-1 and TCF-1 mRNA to distinguish ESCC patients from non-cancer control were 0.66,0.84 and0.78,respectively.The sensitivities and specificities of PD-L1,PD-1 and TCF-1mRNA in the diagnosis of ESCC were 0.58/0.68,0.92/0.64,0.95/0.58,respectively.4.The numbers of ESCC patients with high/low expression levels of PD-L1,PD-1 and TCF-1 mRNA in peripheral blood was 60/28,54/31 and 49/36,respectively.The expression levels of PD-1 in peripheral blood cells of female patients with ESCC was higher than those of male patients with the expression levels of 7.74±3.00 and 6.10±2.37,respectively（P=0.01）.There were no significant correlations between the expression levels of PD-L1,PD-1 and TCF-1 mRNA and other clinicopathological features.5.According to ROC,the optimal cutoff values of PD-L1,PD-1 and TCF-1mRNA for classification of high-and low-risk ESCC patients were 3.67,4.74 and 5.37,respectively.The ESCC patients with high/low expression levels of PD-L1,PD-1 and TCF-1 mRNA were 64/16,57/20,53/22,respectively.OSs were significantly longer in patients with high expression levels of PD-L1,PD-1 and TCF-1 than those patients with low expression levels in ESCC patients.6.Univariate and multivariate Cos proportional hazard regression models were used to analyze the factors affecting OS.It was found that PD-L1,PD-1,TCF-1 and lymph node metastasis were significantly correlated with the prognosis of patients with ESCC.However,no independent prognostic factors were identified in the present study.Conclusions:High expression levels of PD-L1,PD-1 and TCF-1 mRNA in peripheral blood of ESCC patients may become potential diagnostic and prognostic markers for ESCC.