去除N-连接糖基化对食管鳞状细胞癌及转移淋巴结中PD-L1表达影响

Effect of N-linked glycosylation removal on PD-L1 expression in esophageal squamous cell carcinoma and metastatic lymph nodes

目的探讨N-连接糖基化修饰对食管鳞状细胞癌(ESCC)组织程序性死亡受体-配体1(PD-L1)表达影响及其与临床病理特征关系。方法使用PD-L122C3抗体标记,ESCC组织芯片、MCF-7及NCI-H226细胞切片,分别采用标准处理或经肽N-糖苷酶F(PNGase F)处理后进行免疫组化染色。采用肿瘤细胞比例评分(TPS)、联合阳性评分(CPS)或组织化学评分(H-score)评估组织和细胞切片中PD-L1表达水平。TPS≥1%、CPS≥10定义为PD-L1表达阳性。分析原发灶与转移淋巴结组织PD-L1表达一致性;同时分析原发灶组织PD-L1表达与人口学及临床病理学特征关系。结果50例患者组织样本中有24例正常食管黏膜组织、48例原发灶组织及32例转移淋巴结组织可行PD-L1表达评价。标准处理后正常食管黏膜组织PD-L1均呈阴性表达;原发灶组织PD-L1 TPS、CPS阳性率及中位H-score分别为50.00%、29.17%和1.41分;转移淋巴结组织PD-L1 TPS、CPS阳性率及中位H-score分别为50.00%、68.75%和2.02分。PNGase F处理后5例正常食管黏膜组织出现局灶性PD-L1表达;原发灶组织PD-L1 TPS、CPS阳性率及中位H-score分别为56.25%、52.08%和3.55分;转移淋巴结组织PD-L1 TPS、CPS阳性率及中位H-score分别为59.38%、81.25%和10.33分。PNGase F处理后原发灶与转移淋巴结组织PD-L1 TPS、CPS及H-score均高于标准处理,均P<0.001。PNGase F处理后NCI-H226细胞的PD-L1 TPS和H-score均高于标准处理,t值分别为-18.087和-33.588,P值分别为0.003和0.001。标准处理和PNGase F处理后32例患者原发灶及其转移淋巴结组织PD-L1 TPS及CPS阳性表达一致率均较差。PNGase F处理后原发灶和(或)转移淋巴结组织PD-L1 TPS和CPS阳性患者比例分别为78.13%和87.50%。PNGase F处理并联合检测原发灶组织TPS阳性率高于标准处理,χ^(2)=5.497,P=0.019;CPS阳性率高于标准处理,χ^(2)=23.127,P<0.001。不同性别、年龄、肿瘤最大径、病理分级、N分期和淋巴结转移原发灶组织PD-L1 TPS和CPS阳性率差异均无统计学意义,P>0.05。结论PNGase F处理可提升PD-L122C3抗体的染色强度,PD-L1表达在食管癌原发灶与转移淋巴结组织间具有异质性,对食管癌组织进行PNGase F处理同时评估原发灶及转移淋巴结组织可以提高PD-L1阳性检出率。

Objective To investigate the effect of N-linked glycation modification on the expression of programmed death ligand 1(PD-L1)in esophageal squamous cell carcinoma tissue and its relationship with clinical and pathological features.Methods PD-L122C3 antibody was used to perform immunohistochemical staining on esophageal squamous cell carcinoma tissue chips,MCF-7,and NCI-H226 cell sections after standard treatment or peptide N-glycosidase F(PNGase F)treatment,respectively.Tumor cell proportion score(TPS),combined positive score(CPS),or histological score(H-score)were used to evaluate the expression level of PD-L1 in tissues and cell sections.TPS≥1%and CPS≥10 were defined as PD-L1 positive expression.The consistency of PD-L1 expression between the primary lesion and metastatic lymph node tissue was analyzed;The relationship between PD-L1 expression in the primary lesion tissue and demographic and clinical pathological characteristics was analyzed.Results Among the 50 patient tissue samples,24 normal esophageal mucosal tissues,48 primary lesion tissues,and 32 metastatic lymph node tissues were eligible for PD-L1 expression evaluation.After standard treatment,PD-L1 expression was negative in normal esophageal mucosal tissue;The positive rates of PD-L1 TPS,CPS,and median H-score in the primary lesion tissue were 50.00%,29.17%,and 1.41 points,respectively;The positive rates of PD-L1 TPS,CPS,and median H-score in metastatic lymph node tissue were 50.00%,68.75%,and 2.02 points,respectively.Five cases of normal esophageal mucosal tissue showed focal PD-L1 expression after PNGase F treatment;The positive rates of PD-L1 TPS,CPS,and median H-score in the primary lesion tissue were 56.25%,52.08%,and 3.55 points,respectively;The positive rates of PD-L1 TPS,CPS,and median H-score in metastatic lymph node tissue were 59.38%,81.25%,and 10.33 points,respectively.After PNGase F treatment,the PD-L1 TPS,CPS,and H-score of the primary lesion and metastatic lymph node tissue were higher than those of the standard treatment,all P<0.001.The PD-L1 TPS and H-score of NCI-H226 cells treated with PNGase F were higher than those of the standard treatment,with t values of-18.087 and-33.588,and P values of 0.003 and 0.001,respectively.The consistency of PD-L1 TPS and CPS positive expression in the primary lesion and its metastatic lymph node tissues of 32 patients after standard treatment and PNGase F treatment was poor.The proportion of PD-L1 TPS and CPS positive patients in the primary lesion and/or metastatic lymph node tissue after PNGase F treatment was 78.13%and 87.50%,respectively.The TPS positive rate of PNGase F treatment combined with detection of primary lesion tissue was higher than that of standard treatment,χ^(2)=5.497,P=0.019;The CPS positive rate was higher than the standard treatment,withχ^(2)=23.127 and P<0.001.There were no statistically significant differences in PD-L1 TPS and CPS positivity rates among different genders,ages,maximum tumor diameter,pathological grading,N-stage,and primary lymph node metastasis tissues,with P>0.05.Conclusions PNGase F treatment can enhance the staining intensity of PD-L122C3 antibody.PD-L1 expression exhibits heterogeneity between the primary and metastatic lymph node tissues of esophageal cancer.PNGase F treatment of esophageal cancer tissue while evaluating both primary and metastatic lymph node tissues can improve the PD-L1 positive detection rate.