Tumor-derived DEFB1 induces immune tolerance by inhibiting maturation of dendritic cell and impairing CD8+T cell function in esophageal squamous cell carcinoma

Objective:CD8+T cells are the key effector cells in the anti-tumor immune response.The mechanism underlying the infiltration of CD8+T cells in esophageal squamous cell carcinoma(ESCC)has not been clearly elucidated.Methods:Fresh ESCC tissues were collected and grouped according to the infiltration density of CD8+T cells.After the transcriptome sequencing on these samples and the combined analyses with The Cancer Genome Atlas(TCGA)ESCC data,a secreted protein DEFB1 was selected to explore its potential role in the infiltration of CD8+T cells.Bioinformatics analyses,histological verification and in vitro experiments were then performed.Results:DEFB1 was highly expressed in ESCC,and the high expression of DEFB1 was an independent risk factor for overall survival.Since the up-regulation or down-regulation of DEFB1 did not affect the proliferation,migration and apoptosis of ESCC cells,we speculated that the oncogenic effect of DEFB1 was achieved by regulating microenvironmental characteristics.Bioinformatics analyses suggested that DEFB1 might play a major role in the inflammatory response and anti-tumor immune response,and correlate to the infiltration of immature dendritic cell(imDC)in ESCC.Histological analyses further confirmed that there were less CD8+T cells infiltrated,less CD83+mature DC(mDC)infiltrated and more CD1a+imDC infiltrated in those ESCC samples with high expression of DEFB1.After the treatment with recombinant DEFB1 protein,the maturation of DC was hindered significantly,followed by the impairment of the killing effects of T cells in both 2D and 3D culture in vitro.Conclusions:Tumor-derived DEFB1 can inhibit the maturation of DC and weaken the function of CD8+T cells,accounting for the immune tolerance in ESCC.The role of DEFB1 in ESCC deserves further exploration.

Correlation of matrix metalloproteinase suppressor genes RECK,VEGF,and CD105 with angiogenesis and biological behavior in esophageal squamous cell carcinoma

AIM: To explore the expression of reversion inducing cysteine-rich protein with Kazal motifs (RECK), vascular endothelial growth factor (VEGF) and endoglin (CD105) protein and its correlation with occurrence, development, invasion and metastasis in esophageal squamous cell carcinoma (ESCC). METHODS: Streptavidin-peroxidase (SP) immunohisto- chemistry was used to detect expression of RECK and VEGF in 62 cases of ESCC, 31 cases of adjacent atypical hyperplastic epithelium and 62 cases of normal esophageal epithelium. CD105 Mb was used to assess microvessel density (MVD). RESULTS: The expression of RECK was closely correlated with histological grade, infiltrative depth and lymphatic metastasis in ESCC (P < 0.05). The expression of RECK decreased during cancer development: normal esophageal epithelium (85.5%, 53/62), adjacent atypical hyperplastic epithelium (71.0%, 22/31), and carcinoma (59.7%, 37/62). There was a significant difference among the groups (P < 0.05). The expression of VEGF protein was closely correlated with infiltrative depth and lymphatic metastasis in ESCC (P < 0.05). The expression of VEGF protein increased during cancer development: normal esophageal epithelium (29.0%, 18/62), adjacent atypical hyperplastic epithelium (54.8%, 17/31), and carcinoma (67.7%, 42/62). There was a significant difference among the groups (P < 0.05). MVDCD105 increased in accordance with histological grade, butthere was no significant difference (grade Ⅰ, 36.92 ± 10.85; grade Ⅱ, 37.65 ± 9.50; and grade Ⅲ, 38.06 ± 12.19). The MVDCD105 was closely correlated with infiltration and lymphatic metastasis in ESCC (P < 0.05). The expression of RECK was inversely correlated with the expression of VEGF and CD105. CONCLUSION: RECK, VEGF and CD105 play important roles in the infiltration, metastasis and carcinogenesis in esophageal carcinoma. Angiogenesis in ESCC may be promoted by over-expression of CD105.

Effect of a cancer vaccine prepared by fusions of hepatocarcinoma cells with dendritic cells

AIM: To prepare a cancer vaccine (H(22)-DC) expressing high levels of costimulatory molecules based on fusions of hepatocarcinoma cells (H(22)) with dendritic cells (DC) of mice and to analyze the biological characteristics and induction of specific CTL activity of H(22)-DC. METHODS: DCs were isolated from murine spleen by metrizamide density gradient centrifugation, purified based on its characteristics of semi-adhesion to culture plates and FcR-,and were cultured in the medium containing GM-CSF and IL-4. A large number of DC were harvested. DCs were then fused with H(22) cells by PEG and the fusion cells were marked with CD11c MicroBeads. The H(22)-DC was sorted with Mimi MACS sorter. The techniques of cell culture, immunocytochemistry and light microscopy were also used to test the characteristics of growth and morphology of H(22)-DC in vitro. As the immunogen, H(22)-DC was inoculated subcutaneously into the right armpit of BALB/C mice, and their tumorigenicity in vivo was observed. MTT was used to test the CTL activity of murine spleen in vivo. RESULTS: DC cells isolated and generated were CD11c+ cells with irregular shape, and highly expressed CD80, CD86 and CD54 molecules. H22 cells were CD11c- cells with spherical shape and bigger volume, and did not express CD80, CD86 and CD54 molecules.H(22)-DC was CD11c+ cells with bigger volume, being spherical, flat or irregular in shape, and highly expressed CD80, CD86 and CD54 molecules, too. H(22)-DC was able to divide and proliferate in vitro, but its activity of proliferation was significantly decreased as compared with H(22) cells and its growth curve was flatter than H(22) cells. After subcutaneous inoculation over 60 days, H(22)-DC showed no tumorigenecity in mice, which was significantly different from control groups (P【0.01). The spleen CTL activity against H(22) cells in mice implanted with fresh H(22)-DC was significantly higher than control groups (P 【 0.01). CONCLUSION: H(22)-DC could significantly stimulate the specific CTL activity of murine spleen, which suggests that the fusion cells have already obtained the function of antigen presenting of parental DC and could present H(22)specific antigen which has not been identified yet, and H(22)-DC could induce antitumor immune response; although simply mixed H(22) cells with DC could stimulate the specific CTL activity which could inhibit the growth of tumor in some degree, it could not prevent the generation of tumor. It shows that the DC vaccine is likely to become a helpful approach in immunotherapy of hepatocarcinoma.

Expression of human chorionic gonadotropin, CD44v6 and CD44v4/5 in esophageal squamous cell carcinoma

AIM： To study the relationship between the expression of human chorionic gonadotropin （HCG）, CD44v6, CD44v4/5 and the infiltration, metastasis of esophageal squamous cell carcinoma. METHODS： By labeled streptavidin-biotin technique, the expressions of HCG, CD44v6, and CD44v4/5 in 42 patients with esophageal squamous cell carcinoma were examined. RESULTS： The positive rate of HCG expression in patients with lymph node metastasis was 85.71% （18/21）, higher than that （57.14%, 12/21） in those without lymph node metastasis （P〈0.05）. The positive rate of CD44v6 expression was 71.43% （15/21） in lymph node metastasis group, and 38.09% （8/21） in nonmetastasis group; there was a significant difference between the two groups （P〈0.05）. The positive rate of CD44v4/5 expression was 76.19% （16/21） in lymph node metastasis group, and 42.86% （9/21） in non-metastasis group; there was also a significant difference between them （P〈0.05）. From grade Ⅰ to grade Ⅲ in differentiation, the positive rate of HCG expression was 84.62% （11/13）, 70.59% （12/17） and 58.33% （7/12）, respectively, there was no significant difference among them （P〉0.05）. The positive rate of CD44v6 expression in grades Ⅰ-Ⅲ of cancer tissues was 76.92% （10/13）, 52.94% （9/17）, and 33.33% （4/12） respectively; there was no significant difference among them. The positive rate of CD44v4/5 expression in grades Ⅰ-Ⅲ of cancer tissues was 69.23% （9/13）, 64.71% （11/17）, and 41.67% （5/12） respectively; there was no significant difference among the three groups. There was no correlation between the positive rates of HCG and CD44v6, CD44v4/5 expression. Cancer cells in carcinomatous emboli and those infiltrating into vascular wall strongly expressed HCG, CD44v6, and CD44v4/5. CONCLUSION： Expression of HCG, CD44v6, and CD44v4/5 in esophageal squamous cell carcinoma is related to its infiltration and metastasis. HCG, CD44v6, and CD44v4/5 have different effects on the infiltration and metastasis of esophageal squamous cell carcinoma.

Association and prognostic significance of alpha-L-fucosidase-1 and matrix metalloproteinase 9 expression in esophageal squamous cell carcinoma

BACKGROUND Alpha-L-fucosidase-1(FUCA1)has been demonstrated to play opposing regulatory roles in adenocarcinoma and non-adenocarcinoma.Moreover,recent studies reported that FUCA1 could decrease the invasion capability by downregulating matrix metalloproteinase 9(MMP-9)expression.However,the potential role and prognostic significance of FUCA1 in esophageal squamous cell carcinoma(ESCC)have not yet been explored.AIM To evaluate the status,association,and prognostic value of FUCA1 and MMP-9 expression in ESCC.METHODS Patients who underwent esophagectomy for ESCC between January 1,2014,and December 31,2014 at Sun Yat-Sen University Cancer Center were enrolled.The expression status of FUCA1 and MMP-9 in cancerous tissues was detected using immunohistochemistry.In addition,the expression profiles of the FUCA1 and MMP-9 genes in non-metastatic ESCC were extracted from The Cancer Genome Atlas(TCGA)database.RESULTS High expression of FUCA1 and MMP-9 was found in 90 patients(75.6%)and 62 patients(52.1%),respectively.In the high FUCA1 expression group,the constituent ratios of patients with stage III disease(61.1%vs 37.9%,P=0.029),lymphatic invasion(62.2%vs 31.0%,P=0.003),and high MMP-9 expression(60.0%vs 27.6%,P=0.002)were significantly higher than those in the low FUCA1 expression group.In Kaplan-Meier univariate analysis,advanced tumor-nodemetastasis stage(III,P=0.001),positive regional lymph node metastasis(N+,P=0.002),high FUCA1 expression(P=0.001),and high MMP-9 expression(P=0.002)were potential predictors of shorter overall survival(OS),which was similar to the results analyzed based on the TCGA database.Further Cox multivariate regression analyses still demonstrated that FUCA1 and MMP-9 expression levels were independent prognostic factors of OS[hazard ratio(HR):0.484,95%confidence interval(CI):0.239-0.979;P=0.044;and HR:0.591,95%CI:0.359-0.973,P=0.039,respectively].CONCLUSION FUCA1 cooperation with MMP-9 may have a major role in affecting the ESCC invasion and metastatic capability,and serve as a valuable prognostic biomarker in ESCC.

Expressions and the clinical significances of p53, p57(Kip2) and CD68 in esophageal squamous cell carcinoma

Objective： The aim of our study was to investigate the expression of p53, p57（Kip2） and CD68 in esophageal squamous coil carcinoma （ESCC） and their correlation with the biological behavior of ESCC. Methods： The protein expres- sions of p53, p57（Kip2） and CD68 were detected in 51 cases of ESCC with S-P immunohistochemical method. Results： The total positive rate of those proteins was p53 64.71%, CD68 58.82% and p57（Kip2） 45.09% respectively in ESCC. The positive expression rate of p57（Kip2） was significantly lower in the positive p53 of ESCC than in the negative p53 （P 〈 0.05）. The positive expression rate of p57（Kip2） was significantly lower in the positive CD68 of esophageal squamous cell carcinoma than in the negative （P 〈 0.05）. The positive expression of p53 and CD68 were related to differentiate and TNM of ESCC, but p57（Kip2） was not related to TNM （P 〉 0.05）. Conclusion： There are significant negative correlations between p57（Kip2） and p53, CD68 protein expression and related to biological behavior. Multy predictors are better guide to patients than single predictor.

Dendritic cell co-stimulatory and co-inhibitory markers in chronic HCV: An Egyptian study

AIM:To assess co-stimulatory and co-inhibitory markers of dendritic cells(DCs)in hepatitis C virus(HCV)infected subjects with and without uremia.METHODS:Three subject groups were included in the study:group 1 involved 50 control subjects,group2 involved 50 patients with chronic HCV infection and group 3 involved 50 HCV uremic subjects undergoing hemodialysis.CD83,CD86 and CD40 as co-stimulatory markers and PD-L1 as a co-inhibitory marker were assessed in peripheral blood mononuclear cells by realtime polymerase chain reaction.Interleukin-10(IL-10)and hyaluronic acid(HA)levels were also assessed.All findings were correlated with disease activity,viral load and fibrogenesis.RESULTS:There was a significant decrease in costimulatory markers;CD83,CD86 and CD40 in groups2 and 3 vs the control group.Co-stimulatory markers were significantly higher in group 3 vs group 2.There was a significant elevation in PD-L1 in both HCV groups vs the control group.PD-L1 was significantly lower in group 3 vs group 2.There was a significant elevation in IL-10 and HA levels in groups 2 and 3,where IL-10was higher in group 3 and HA was lower in group 3 vs group 2.HA level was significantly correlated with disease activity and fibrosis grade in group 2.IL-10 was significantly correlated with fibrosis grade in group 2.There were significant negative correlations between co-stimulatory markers and viral load in groups 2 and3,except CD83 in dialysis patients.There was a significant positive correlation between PD-L1 and viral load in both HCV groups.CONCLUSION:A significant decrease in DC co-stimulatory markers and a significant increase in a DC coinhibitory marker were observed in HCV subjects and to a lesser extent in dialysis patients.

THE OVEREXPRESSION AND SIGNIFICANCE OF CYCLIN D1 AND P53 IN CERVICAL SQUAMOUS CELL CARCINOMAS

Objective To investigate the significance of overexpresson of cyclin D1 and P53 protein in cervical squamous cell carcinomas.Methods Fifty cases of invasive cervical squamous cell carcinomas and 10 cases of normal cervical squamous epithelia were investigated with immunihistochemical technique. Results The overexpression of cyclin D1 and P53 in invasive cervical carcinomas was 70% and 50%, respectively. There was no overexpression of them in the control group. The overexpression of cyclin D1 in grade Ⅱ and Ⅲ was much higher than that in gradeⅠ(P<0.05). The overexpresson of cyclin D1 in stage Ⅲ of cervical carcinoma was significantly higher than that in stage Ⅱ (P<0.05). The overexpression of P53 in grade Ⅱ and grade Ⅲ of cervical carcinoma was remarkably higher than that in grade Ⅰ (P<0.05).Conclusion The action point of both cyclin D1 and P53 may be at G1/S transition. The overexpression of them was associated with development and progression of cervical carcinoma probably in different mechanisms and different pathways.

Expression of KAI1/CD82 and MRP-1/CD9 in Transitional Cell Carcinoma of Bladder

The expression of KAI1/CD82 and MRP-1/CD9 in transitional cell carcinoma of bladder （TCCB） and its clinical significance were investigated. Immunohistochemistry was used to detect KAI1/CD82 and MRP-1/CD9 protein expression in 52 TCCB specimens. Correlation between the expression of KAI1/CD82 and MRP-1/CD9 to clinicopathologic factors was statistically analyzed. The results showed that the positive rate of KAI1/CD82 and MRP-1/CD9 in TCCB was 50% and 61.5%, respectively. The MRP-1/CD9 and KAI1/CD82 expression was significantly associated with grade of TCCB （P〈0.05）, but no correlation was found between MRP-1/CD9 or KAI1/CD82 expression and clinical stage of TCCB （P〉0.05）. The expression level of MRP-1/CD9 and KAI1/CD82 in recurrent TCCB samples was lower than that in non-recurrent samples （P〈0.05）. Meanwhile, the correlation between the KAI1/CD82 expression and MRP-1/CD9 expression was statistically significant （r=0.316, P〈0.05）. It was concluded that KAI1/CD82 and MRP-1/CD9 expression may be important prognostic indicators and potentially useful for assessing the biological behavior of TCCB.

Expression of Nerve Growth Factor and Hypoxia Inducible Factor-1α and Its Correlation with Angiogenesis in Non-Small Cell Lung Cancer

Summary： In order to investigate the expression of nerve growth factor （NGF） and hypoxia inducible factor-1α （HIF-1α） and its correlation with angiogenesis in non-small cell lung cancer （NSCLC）, paraffin-embedded tissue blocks from 20 patients with NSCLC were examined. Twenty corresponding para-cancerous lung tissue specimens were obtained to serve as a control. The expression of NGF, HIF-1α, and vascular endothelial growth factor （VEGF） in the NSCLC tissues was detected by using immunohistochemistry. The microvascular density （MVD） was determined by CD31 staining. The resuits showed that the expression levels ofNGF, HIF-1α and VEGF in the NSCLC tissues were remarkably higher than those in the para-cancerous lung tissues （P〈0.05）. There was significant difference in the MVD between the NSCLC tissues （9.19±1.43） and para-cancerous lung tissues （2.23±1.19） （P〈0.05）. There were positive correlations between NGF and VEGF, between HIF-1α and VEGF, and between NGF and HIF-1α in NSCLC tissues, with the spearman correlation coefficient being 0.588, 0.519 and 0.588, respectively. In NSCLC tissues, the MVD had a positive correlation with the three factors （P〈0.05）. Theses results suggest that NGF and HIF-1α are synergically involved in the angiogenesis of NSCLC.

香叶基香叶基焦磷酸合成酶在肺鳞状细胞癌中的表达及临床意义

目的·利用生物信息学和免疫组织化学法探究在肺鳞状细胞癌(lung squamous cell carcinoma,LUSC)组织中香叶基香叶基焦磷酸合成酶(geranylgeranyl diphosphate synthase 1,GGPS1)的表达及其临床意义。方法·首先从UCSC Xena平台下载LUSC组织与配对正常组织的转录组数据,使用R语言进行数据标准化和差异表达分析,并利用UALCAN数据库进行验证;采用UALCAN和LinkedOmics数据库分析LUSC患者中GGPS1表达与临床病理特征关系;利用Kaplan-Meier Plotter数据库探究LUSC患者GGPS1表达对预后的影响。应用最小绝对收缩和选择算子(least absolute shrinkage and selection operator,LASSO)回归分析筛选基因相关系数及风险评分。通过列线图和校正曲线评价GGPS1对LUSC的诊断价值。采用STRING、GeneMANIA数据库构建GGPS1蛋白质相互作用(protein-protein interaction,PPI)网络。运用R语言挑选与GGPS1相关差异基因,并进行基因本体论(Gene Ontology,GO)和京都基因和基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)富集分析。采用免疫组织化学法检测LUSC患者GGPS1表达情况,并分析其与临床病理特征和预后的相关性。结果·通过TIMER2.0数据库检索到GGPS1在多数肿瘤中表达均升高,且在LUSC中呈高表达。UCSC Xena、UALCAN数据库中GGPS1在LUSC的表达高于癌旁组织(均P<0.05)。UALCAN和LinkedOmics数据库发现GGPS1在指标分期较晚患者中的表达水平更高,且Kaplan-Meier Plotter数据库显示LUSC患者GGPS1高表达总生存期(overall survival,OS)较短(P<0.05)。基于LASSO回归评估LUSC患者有较好的风险预测效能。构建LUSC患者的个体化预测模型具有最佳预测准确度。GO、KEGG结果显示,GGPS1相关基因主要与蛋白质代谢、调节脂质和胆固醇代谢过程、尼古丁成瘾、磷脂酰肌醇3激酶(phosphatidylinositol3-kinase,PI3K)/蛋白激酶B(protein kinase B,AKT)/哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)、过氧化物酶体增殖物激活受体(peroxisome proliferatoractivated receptors,PPAR)信号通路等有关。GGPS1的代谢功能可能促进肿瘤发生。免疫组化结果提示GGPS1主要定位于细胞质中,且LUSC组织中表达高于癌旁组织(P<0.05),GGPS1高表达与LUSC患者肿瘤大小、淋巴结转移和TNM分期有关(均P<0.05),且GGPS1表达高的患者OS明显短于低表达者(P=0.000)。多因素Cox回归分析提示GGPS1可作为LUSC的独立预后因素。结论·相较于正常肺组织,GGPS1在LUSC中表达显著升高,尤其在肿瘤体积较大、淋巴结转移阳性及晚期的患者中表达升高更明显;且GGPS1过表达是LUSC患者预后差的独立预测因子。GGPS1有望成为新的LUSC诊治和预防的分子靶点。

CD1a标记树突细胞在声门型喉鳞癌中的表达及其临床意义

背景与目的:树突细胞(dendriticcell,DC)是最重要的抗原呈递细胞(antigenpresentingcell,APC),而CD1a作为未成熟DC的标记物,在肿瘤的发生及发展过程中有一定的影响。本文旨在探讨CD1a+DC与声门型喉癌病理分级、T分期及局部复发的关系,从而间接了解它与预后的关系。方法:收集111例有完整病理资料及临床随诊资料的声门型喉鳞癌病例,并取17例非肿瘤组织做对照。用免疫组化技术检测CD1a+DC在癌组织中的表达,分析癌组织中CD1a+DC在不同病理分级、T分期及局部复发组织中的表达,间接了解其与声门型喉鳞癌预后的关系。结果:111例声门型喉鳞癌病例中,CD1a阳性率为59.46%(66/111);高分化组阳性率为71.43%(55/77),中低分化组阳性率为28.57%(11/34);T1+T2组阳性率为67.16%(45/67),T3+T4组阳性率为47.73%(21/44)。局部复发组阳性率为42.86%(12/28),局部无复发组阳性率为65.06%(54/83)。17例非肿瘤组织中CD1a均为阴性。结论:声门型喉鳞癌中,肿瘤细胞分化越差,T分期越高,则CD1a标记树突细胞阳性率越低。局部复发者CD1a+DC阳性率低。CD1a可作为预测声门型喉鳞癌预后的参考指标之一。

口腔颊粘膜癌组织中CD1a^+的树突状细胞的免疫组化分析

目的通过免疫组化技术分析树突状细胞(dendritic cell,DC)在口腔颊粘膜鳞状细胞癌(oralsquamous cell carcinoma,OSCC)中的表达,探讨其在肿瘤组织中的功能状态。方法随机选取22例临床收集的未经任何其他非手术治疗的颊粘膜OSCC患者标本,以10例正常口腔粘膜(normal mucosa,NM)和10例口腔非特异性炎症上皮(inflammatory mucosa,IM)为对照,采用CD1a单抗通过免疫组化技术标记上皮组织中的DCs,结果进行统计学分析。结果 NM组中有3例未见CD1a+DC表达,OSCC组及IM组中各有1例未见CD1a+DC表达,OSCC组与NM组、OSCC组与IM组之间CD1a+DC的计数均有显著性差异(P<0.05);肿瘤不同病理分级中,癌旁组织(ESCC)中CD1a+DC的数量在OSCCⅠ级与Ⅱ级间有显著性差异(P<0.05);ESCC中CD1a+DC在有或无淋巴结转移中的差别有显著意义(P<0.05)。结论 ESCC中CD1a+DC与肿瘤分级呈显著负相关,与颊癌的预后相关。

CD1a和CD83在舌鳞癌中的表达及其意义

目的通过对树突状细胞(dendritic cells,DC)表面标志物CD1a和CD83在舌鳞癌、癌前病变及正常组织中表达状况的研究,探讨其在舌鳞癌中的功能状态及其与舌鳞癌患者临床病理学特征之间的关系。方法随机选取手术切除的舌鳞癌、舌癌前病变及舌正常组织标本共50例,应用免疫组织化学法检测DC表面标志物CD1a、CD83的表达情况。结果 CD1a在舌鳞癌、癌前病变和舌正常组织中的表达率分别为50%、0%和0%,差异有统计学意义(P=0.001)。CD83在舌鳞癌、癌前病变和舌正常组织中的表达率分别为33%、0%和0%,差异有统计学意义(P=0.017)。在舌鳞癌组织中,CD83的表达水平在不同临床分期中有差异(P=0.014)。结论舌鳞癌患者局部微环境中存在一定的免疫反应,CD83+DC数量多的患者可能有较好的预后。

Expression of CD44V6 and PCNA in squamous cell carcinomas

OBJECTIVE: To investigate the expression of cluster of differentiation 44 variant 6 (CD(44V6)) and proliferating cell nuclear antigen (PCNA) in ocular squamous cell carcinomas. METHODS: Streptavidin-biotin complex (SABC) immunohistochemistry was used to explore the expression of CD(44V6) and PCNA in 35 cases of ocular squamous cell carcinomas, 20 cases of papillomas, and 11 cases of normal eyelid tissue. RESULTS: The CD(44V6) positive rate was 62.9% (22/35) in ocular squamous cell carcinomas, 15.0% (3/20) in papillomas, but not detectable in the 11 cases of normal eyelid tissue. The positive expression rates of CD(44V6) in ocular squamous cell carcinomas were significantly higher than in benign tumors (chi(2) = 11.57, P

免疫组化法与RNAscope原位杂交法检测宫颈鳞状细胞癌组织中PD⁃1和PD⁃L1表达一致性研究

目的研究免疫组化法与RNAscope原位杂交法检测宫颈鳞状细胞癌组织中程序性死亡分子1(PD⁃1)和程序性死亡分子1配体(PD⁃L1)表达一致性。方法回顾性分析2019年3月至2021年2月被江阴市中医院病理科诊断为宫颈鳞状细胞癌的80例患者石蜡组织标本。采用免疫组化法与RNAscope原位杂交法测定宫颈鳞状细胞癌组织中PD⁃1和PD⁃L1表达情况,分析此2种方法检测癌组织中PD⁃1和PD⁃L1表达一致性。分析RNAscope原位杂交法检测癌组织中PD⁃1mRNA、PD⁃L1mRNA表达与其临床病理特征的关系。随访1年,记录所有患者总生存时间。结果免疫组化法结果显示,80例宫颈鳞状细胞癌组织标本中PD⁃1蛋白阳性52例,阳性率为65.00%;PD⁃L1蛋白阳性60例,阳性率为75.00%。RNAscope原位杂交法结果显示,80例宫颈鳞状细胞癌组织标本中PD⁃1蛋白阳性49例,阳性率为61.25%;PD⁃L1蛋白阳性57例,阳性率为71.25%。免疫组化法与RNAscope原位杂交法检测PD⁃1表达一致性良好(Kappa值=0.847,P>0.05);免疫组化法与RNA⁃scope原位杂交法检测PD⁃L1表达一致性良好(Kappa值=0.861,P>0.05)。PD⁃1mRNA、PD⁃L1mRNA表达与患者年龄、脉管侵犯、淋巴结转移、神经侵犯、肿瘤分期(TNM)、肿瘤直径无关(P>0.05)。随访1年,PD⁃1mRNA阳性表达与PD⁃1mRNA阴性表达的总生存曲线比较差异无统计学意义(P>0.05);PD⁃L1mRNA阳性表达与PD⁃L1mRNA阴性表达的总生存曲线比较差异无统计学意义(P>0.05)。结论免疫组化法与RNAscope原位杂交法检测宫颈鳞状细胞癌组织中PD⁃1和PD⁃L1表达一致性较好,且PD⁃1mRNA、PD⁃L1mRNA表达与患者总生存时间无关。

口腔鳞癌VEGF诱导树突状细胞致免疫耐受的机制研究

目的探讨口腔鳞状细胞癌(oral squamous cell carcinoma,OSCC)中血管内皮细胞生长因子(vascular endothelial growth factor,VEGF)诱导树突状细胞(dendritic cells,DCs)向免疫耐受型转化的作用及机制。方法将树突状细胞分为4组:对照组(DC)、VEGF组(DC中加入外源性VEGF)、共培养组(DC与SCC7细胞共培养)及抗VEGF组(DC与SCC7细胞共培养后加入VEGF抗体),采用流式细胞术(flow cytometry,FCM)检测DC表面标记分子的表达情况;为了检测DC对T细胞增殖的影响,将实验分为5组:空白组(T细胞)、对照组(T细胞+DC)、VEGF组(T细胞+DC+VEGF)、共培养组(T细胞+DC+SCC7细胞上清)及抗VEGF组(T细胞+DC+SCC7上清+VEGF抗体),采用混合淋巴细胞反应(mixed lymphocyte reaction,MLR)检测;分别应用Western blot、real time PCR及FCM检测各组DC中吲哚胺-2,3-双加氧酶(indoleamine⁃2,3⁃dioxygenase,IDO)和程序性死亡受体配体1(programmed cell death l ligand 1,PD⁃L1)的表达情况;收集肿瘤细胞抗原致敏的DC诱导形成的细胞毒性T细胞(cytotoxic lymphocyte,CTL)与肿瘤细胞共培养,检测其对肿瘤细胞的杀伤活性,实验分为4组:对照组(T细胞+DC)、IDO抑制剂组(T细胞+DC+IDO抑制剂)、抗PD⁃L1组(T细胞+DC+PD⁃L1抗体)及联合用药组(T细胞+DC+IDO抑制剂+PD⁃L1抗体);小鼠荷瘤模型中观察IDO抑制剂及抗PD⁃L1抗体对瘤体大小及脾脏指数的影响。结果与对照组比较,VEGF组、共培养组和抗VEGF组DC表面标记分子的表达均有所降低;但是,抗VEGF组DC表面标记分子的表达稍高于VEGF组及共培养组。VEGF组及共培养组DC刺激T细胞增殖显著弱于对照组,而抗VEGF组DC刺激T细胞增殖能力有所恢复。与对照组相比,VEGF组、共培养组DC中IDO和PD⁃L1的表达均显著升高,而抗VEGF组DC中IDO和PD⁃L1的表达均有所下调。IDO抑制剂组、抗PD⁃L1组和联合用药组DC表面CD86、CD11C表达、刺激T细胞的增殖能力及对肿瘤细胞的杀伤活性均显著高于对照组,其中联合用药组升高最明显。小鼠体内荷瘤实验结果显示,IDO抑制剂组、抗PD⁃L1组和联合用药组肿瘤体积均明显缩小,其中联合用药组瘤体缩小最为显著;3组小鼠的脾脏指数较对照组均有所升高。结论OSCC微环境中的VEGF通过刺激IDO和PD⁃L1的表达,抑制DC的成熟分化,使其向免疫耐受型DC转化,从而促进肿瘤局部免疫耐受微环境的形成。

食管鳞状细胞癌PD-L1不同抗体免疫组织化学检测的一致性分析

背景与目的:程序性死亡[蛋白]配体-1(programmed death ligand-1,PD-L1)是食管癌免疫治疗应用最广泛的疗效预测生物标志物,准确可靠的PD-L1检测对于筛查免疫治疗的潜在受益者至关重要。本研究旨在探讨食管鳞状细胞癌(esophageal squamous cell carcinoma,ESCC)患者PD-L1的表达情况以及7种不同克隆号PD-L1抗体免疫组织化学(immunohistochemistry,IHC)检测的一致性,为规范食管癌PD-L1 IHC检测及后续临床研究提供参考。方法:收集2020年1月—2021年12月复旦大学附属肿瘤医院的146例ESCC手术切除标本,用PD-L122C3、SP263、28-8、E1L3N XP、CST E1L3N、BP6099和MXR0067种克隆号抗体进行染色,并采用联合阳性评分(comprehensive positive score,CPS)、肿瘤细胞比例评分(tumor proportion score,TPS)和免疫细胞评分(immune cell proportion score,IPS)3种评分标准,在不同临界值下对抗体的一致性进行统计学分析。结果:PD-L122C3与两种伴随诊断抗体(SP263、28-8)对比结果显示:CPS临界值为10时,3种抗体之间均一致性较好(Kappa:0.66~0.80);CPS临界值为1时,28-8与22C3、SP263抗体一致性较好(Kappa=0.80、0.65),22C3与SP263一致性一般(Kappa=0.49);TPS临界值为10%和1%时,3种抗体之间均具有很高的一致性(Kappa:0.79~0.89,0.71~0.91)。PD-L122C3与4种国产抗体(E1L3N XP、CST E1L3N、BP6099、MXR006)对比结果显示:CPS临界值为10和TPS临界值为10%时,5种抗体之间均一致性较好(Kappa:0.67~0.88,0.65~0.79);CPS临界值为1时,5种抗体之间一致性较好或一般(Kappa:0.58~0.88);TPS临界值为1%时,5种抗体之间均一致性很好(Kappa:0.85~0.92);IPS临界值为1%和10%时,5种抗体之间一致性一般或较差(Kappa:0.46~0.66,0.30~0.62)。结论:PD-L122C3与两种伴随诊断抗体(SP263、28-8)在CPS和TPS中一致性较好;PD-L122C3与4种国产抗体(E1L3N XP、CST E1L3N、BP6099、MXR006)在CPS和TPS中一致性较好,而采用IPS时不同克隆号抗体的一致性较差。这些抗体之间具有一定的可互换性,可为食管鳞状细胞癌PD-L1临床检测提供更多选择。

口腔鳞状细胞癌中IGF1的表达及其临床意义

目的探讨口腔鳞状细胞癌(OSCC)组织及细胞中胰岛素样生长因子1(IGF1)的表达水平及其临床意义。方法免疫组织化学技术检测IGF1在115例OSCC组织(OSCC组)及74例邻近正常组织(正常组)中的蛋白表达,分析与OSCC临床病理因素及预后之间的关系。利用蛋白免疫印迹(Western blot)和实时荧光定量PCR(qRT-PCR)检测口腔正常上皮细胞系HOK和OSCC细胞系CAL-27、TCA-8113、SCC-15和SCC-25中IGF1蛋白和mRNA水平。结果IGF1在OSCC组中的高表达率为72.17%,明显高于正常组(2.70%)(P<0.001)。IGF1用于诊断OSCC的ROC曲线下面积(AUC)为0.81,灵敏度为0.73,特异性为0.82。IGF1的表达与OSCC分化程度、T分期和浸润深浅有关(P=0.03,P=0.02,P=0.02),与性别、年龄、N分期、TNM分期、吸烟、饮酒、HPV感染无关(P>0.05)。Kaplan-Meier和COX回归分析显示,IGF1高表达、分化程度、T分期和浸润深浅是影响患者预后的相关因素(P<0.01,P=0.04,P=0.03,P=0.04)。COX多因素表明,IGF1高表达是影响患者预后的独立因素(P=0.01)。Western blot和qRT-PCR结果显示,IGF1在OSCC细胞系中的蛋白及mRNA表达水平高于正常口腔上皮细胞(P<0.05)。结论IGF1在OSCC中高表达,是OSCC潜在的诊断及预后不良标记物。

Fine-Tuned Expression of Programmed Death 1 Ligands in Mature Dendritic Cells Stimulated by CD40 Ligand is Critical for the Induction of an Efficient Tumor Specific Immune Response

During maturation, murine myeloid dendritic cells （DCs） upregulated the expressions of CDllc, CD25, CD40, CD80, CD86, MHC Ⅱ and programmed death 1 ligands 1 and 2 （PD-L1 and PD-L2）. Differential expression patterns of PD-L1 and PD-L2 were found when DCs were triggered by CD40 ligand and TNF-α. PD-L1 expression was repressed and PD-L2 expression remained unchanged in mature CD40-ligated DCs, whereas TNF-α stimulated DCs kept high expression of PD-L1 and significantly enhanced PD-L2 expression on DCs. Proliferations of T lymphocytes stimulated by immature DCs were enhanced by blockade of the PD-1 and PD-1 ligand interaction. But inhibitive effects were found in T lymphocytes stimulated by CD40-ligated DCs. With the fine-tuned expressions of PD-L1 and PD-L2, CD40-1igated DCs could sustain a longer activation period and elicit a more efficient T lymphocyte activation. Cellular ＆ Molecular Immunology.