An immunohistochemical panel consisting of EZH2, C-KIT,and CD205 is useful for distinguishing thymic squamous cell carcinoma from type B3 thymoma

Type B3 thymoma and thymic squamous cell carcinoma (SqCC) often cause a diagnostic problem due to their histological similarities. The aim of this study is to identify EZH2 as a novel and powerful biomarker that can effectively distinguish thymic SqCC from type B3 thymoma, and find optimal combinations among 11 markers. A total of 53 patients, comprising 26 with type B3 thymoma and 27 with thymic SqCC, were allocated to the discovery or validation cohorts, and immunohistochemical staining was performed and analyzed. The expression level of each marker was scored, and receiver-operator characteristic curve analysis was performed to evaluate their diagnostic accuracies. This analysis identified EZH2, C-KIT, and CD205 as useful markers for distinguishing thymic SqCC, and a combined panel approach using them further improved diagnostic accuracy in both the discovery and validation cohorts. In the combined cohorts analysis, EZH2 was the single best marker with 88.9% sensitivity and 100% specificity [area under the curve (AUC)?=?0.967]. The sensitivity and specificity were 85.2% and 100% (AUC?=?0.962) for C-KIT, and 100% and 73.1% (AUC?=?0.844) for CD205. The combined panel had the highest sensitivity and specificity at 96.3% and 100%, which was significantly or marginally higher than those of EZH2, C-KIT, and CD205 alone (P?=?0.071, 0.034, and 0.005, respectively). The present findings indicate that EZH2 is useful as a novel diagnostic marker for distinguishing thymic SqCC and that the panel approach can be used as an effective differential diagnostic tool in daily practice.     

The value of diffusion-weighted and dynamic contrast-enhanced imaging in the diagnosis of thymic epithelial tumors

Background: Thymic epithelial tumors (TETs) are clinically the most frequently encountered neoplasm of the prevascular mediastinum in adults. The role of chest magnetic resonance (MR) imaging has been increasingly stressed thanks to its excellent contrast resolution, freedom from ionizing radiation, and capability to provide additional information regarding tumors'' cellular structure and vascularity.Methods: This study aimed to establish the relationship between the MR findings and pathological classification of TETs, focusing on diffusion-weighted (DW) and dynamic contrast-enhanced (DCE) imaging. This retrospective cross-sectional study included 44 TET patients who underwent chest MR scanning. The tumors were classified into three groups according to the WHO classification: low-risk thymoma (LRT), high-risk thymoma (HRT), and non-thymoma (NT). Along with morphological characteristics, the apparent diffusion coefficient (ADC) value, time-intensity curve (TIC) pattern, and time to peak enhancement (TTP) of the tumors were recorded and compared between the three groups.Results: A smooth contour and complete or almost complete capsule were suggestive of LRTs. The median ADC value of the 44 tumors was 0.95 × 10^-3 mm²/sec. Among the three groups, LRTs had the highest ADC values, while NTs had the lowest. The differences between the ADC values of the three groups were statistically significant (p = 0.006). Using an ADC cutoff of 0.82 × 10^-3 mm²/sec to differentiate between LRTs and tumors of the two remaining groups, the area under the curve was 0.775, sensitivity was 100%, specificity was 50%, and accuracy was 65.91%. The washout (type 3) TIC pattern was the most prevalent, accounting for 45.45% of the population; this pattern was also predominantly observed in LRTs (71.43%). Although the median TTP of LRTs was lower than that of HRTs or NTs, no statistically significant differences were found between the TTPs of the three groups (p = 0.170).Conclusions: MR is a good imaging modality to preoperatively assess TETs. Morphological features, ADC value, TIC pattern, and TTP are helpful in preoperatively predicting TET pathology.     

Effective use of bronchoscopy and sputa in the diagnosis of lung cancer

The records of 134 patients who underwent bronchoscopy at the Forbes Health System hospitals between January 1, 1982 and December 31, 1983 were reviewed. The number of pre- and postbronchoscopy sputa obtained on each patient, final diagnosis, and follow-up for 6 to 30 months were obtained. Tissue obtained at bronchoscopy yielded a diagnosis of malignancy in 71 of 84 (84.5%) patients who received that final diagnosis. Addition of postbronchoscopy sputa examination increased the yield to 73 of 84 (86.9%). Prebronchoscopy sputa contributed no diagnoses beyond those made by bronchoscopically obtained material. The cost to the patient of examining pre- and postbronchoscopy sputa in addition to bronchoscopically obtained cytologic and biopsy material is 70% higher than that of examining bronchoscopically obtained material alone. Indirect costs of increased hospital stay may also be significant. We propose early bronchoscopy without prebronchoscopy sputa in cases where there is clinical suspicion of lung cancer. Postbronchoscopy sputa should be obtained, but only processed for microscopic examination if the bronchoscopically obtained material does not yield a diagnosis.     

肺癌患者外周血T淋巴细胞及亚群CD28表达和活化的改变

目的检测共刺激分子CD28在肺癌患者外周血T淋巴细胞及亚群中的表达密度和细胞活化程度的变化,并探讨该变化与肺癌临床分期的关系.方法用三色荧光直标流式细胞法分别检测38例正常人与42例肺癌患者中血T淋巴细胞亚群中CD28的阳性率以及CD28+T淋巴细胞亚群活化率.结果与正常人相比,肺癌患者外周血中的CD3+CD28+,CD4+CD28+,CD8+CD28+淋巴细胞百分比均有下降(P＜0.05).淋巴细胞及其T亚群中的CD25+CD28+,CD38+CD28+淋巴细胞百分比亦有所下降(P＜0.05),但在不同临床分期的患者之间无显著性差异(P＞0.05).结论肺癌患者外周血T淋巴细胞的CD28表达密度及CD28+T细胞的活性均有降低,但下降幅度与临床分期无关.     

Killing effect of TNF-mediated by conditionally replicating adenovirus on esophageal cancer and lung cancer cell lines

Objective: The killing effect of TNF mediated by conditionally replicating adenovirus SG502 on human cancer cell lines was assessed by in vivo and in vitro experiments. Methods: The recombinant adenovirus SG502-TNF was used to infect human lung cancer cell line A549 and human esophageal cancer cell line TE-1. The expression of the exogenous gene and its inhibitory effect on the tumor cell lines were thus detected. Tumor transplantation experiment was performed in mice with the purpose of assessing the inhibitory effect of the adenovirus on tumor cells and tumor formation. The targeting of the adenovirus and the mechanism of tumor inhibition were discussed by in vivo imaging technology, HE staining and TUNEL assay. Results: Recombinant adenovirus SG502-TNF targeted the tumor cells specifically with stable expression of TNF, which produced a killing effect on tumor cells by regulating the apoptotic signaling pathway. Conclusion: Recombinant adenovirus SG502-TNF possessed significant killing effect on TE-1 cells either in vivo or in vitro. This finding demonstrated the potential clinical application of adenovirus SG502.     

Restricted expression of transgenic HLA-DRA gene in thymic epithelial cells and its role in acquisition of T cell tolerance to self-superantigens and processed DRα-derived peptide

We have established a set of transgenic mouse lines in which the HLA-DRA gene was expressed in different cell types. In one line (DRα-24), DRαEβ^b molecules were expressed on thymic medullary and cortical epithelial cells and all lineages of bone marrow-derived antigen-presenting cells (APC) except for thymic macrophages. By contrast, expression of the molecules in another line (DRα-30) was found on thymic medullary and cortical epithelial cells but not on bone marrow-derived APC in the thymus and periphery. To evaluate the role of thymic epithelial cells in acquisition of T cell tolerance, comparative analysis of DRα-24 and DRα-30 was performed. In DRα-30, T cells expressing TcR Vβ5 and Vβ11 were eliminated to comparable levels to those in DRα-24, suggesting that expression of the DRαEβ^b molecules on thymic epithelial cells are sufficient for clonal deletion of the self-superantigen-reactive T cells. In addition, CD4⁺ T cells from DRa-30 as well as those from DRα-24 were tolerant to DRα-derived peptide/I-A^b complex expressed on spleen cells from DRα-24 even in the presence of exogenous interleukin-2. These observations suggest that expression of the DRα chain in thymic epithelial cells could induce T cell tolerance directed toward naturally processed DRα-derived peptide bound to I-A^b molecules, probably via clonal deletion of the self-reactive T cells.     

原发性双侧肺癌癌巢、癌旁、淋巴结和外周血免疫功能的探讨

目的:探讨原发性双侧肺癌患者不同病灶的免疫功能及其治疗方式的选择。方法:收集广东祈福医院双侧肺癌患者两次手术切除样本,应用HE染色和肿瘤相关突变基因二代测序技术确定患者双肺肿瘤为原发性双侧肺癌;分别分离左肺和右肺肿瘤切除术时癌巢、癌旁、淋巴结和外周血单个核细胞,流式细胞术检测T细胞和自然杀伤(natural kille...     

精氨酸酶1在食管癌患者外周血的表达及其临床意义

目的检测精氨酸酶1(arginase1,Arg1)在食管癌中的表达,分析其与髓源性抑制细胞(myeloid-derived suppressorcells,MDSCs)消长的关系,探讨癌症患者机体免疫抑制状态的可能机制。方法应用流式细胞术检测30例食管癌患者PBMC中的MDSCs比例;实时荧光定量PCR检测PBMC中Arg1、IL-6和TNF-αmRNA的表达水平;酶联免疫吸附试验(ELISA)检测血浆Arg1,IL-6和TNF-α含量。结果食管癌患者外周血PBMC中MDSCs比例及Arg1的mRNA水平均明显高于健康对照组(P<0.05),而IL-6和TNF-α的mRNA水平均低于健康对照组(P<0.05)。食管癌患者血浆Arg1含量高于健康对照组(P<0.05),而IL-6和TNF-a含量与健康对照组相比无明显差异(P>0.05)。此外,食管癌患者Arg1 mRNA水平和蛋白水平均与MDSC含量呈正相关(P<0.01)。结论 Arg1在食管癌患者外周血中的高表达与MDSCs水平和食管癌的发生、发展有着密切的关系,其检测将有助于临床食管癌的辅助诊断和预后判断。     

人上皮性卵巢癌细胞系侧群细胞的肿瘤干细胞样细胞生物学特性及膜蛋白差异表达的鉴定

目的 鉴定上皮性卵巢癌细胞侧群细胞(SP细胞)肿瘤干细胞样细胞生物学特性,并检测侧群细胞内差异蛋白质的表达.方法 流式分选上皮性卵巢癌细胞系SKOV3和A2780具有外泌Hochest33342染料生物学特性的侧群细胞,鉴定其肿瘤干细胞样细胞相关生物学特性.运用细胞培养稳定同位素标记(SILAC)的定量蛋白质组学技术,...     

人肺癌相关单克隆抗体的制备及其抗原的纯化

目的：研制抗人肺癌细胞（A549）单克隆抗体，并对其所识别的抗原进行免疫亲和层析纯化。方法：以人肺癌细胞系A549免疫BALB／c小鼠，常规融合，以间接ELISA筛选，免疫组化研究单克隆抗体的特性，通过免疫亲和层析纯化所识别相关的抗原。结果：成功获得了一株能够稳定分泌抗人肺癌相关抗原单克隆抗体的杂交瘤细胞株289，并提取了其识别的相关抗原。结论：2139单克隆抗体及其所识别抗原有可能进一步用于肺癌的实验室诊断。     

Immunodetection of neuron-specific enolase and keratin in cytological preparations as an aid in the differential diagnosis of lung cancer

The results of neuron-specific enolase (NSE) and keratin immunodetection in cytological specimens of sputum secured from 41 patients with lung cancer are presented. All 19 cases of small-cell carcinoma showed intense immunoreactivity for NSE. No such immunoreactivity was found in 21 of 22 cases of non-small-cell carcinoma. The single positive result was from a case of large-cell undifferentiated carcinoma. All 10 cases of squamous-cell carcinoma showed immunoreactivity for keratin. The 19 cases of small-cell carcinoma showed no such reactivity. Our findings indicate that immunostaining for NSE and keratin is a valuable aid when a definite diagnosis of small-cell carcinoma of the lung can not be made on the basis of conventional cytologic features.     

转录因子Foxp3在肺癌细胞的表达及其意义

目的研究转录因子Foxp3在肺癌细胞中的表达及其对肿瘤免疫的影响。方法用常规RT-PCR检测Foxp3在肺癌细胞系中的表达,进一步用Western blot验证其蛋白水平的表达;将表达Foxp3的肿瘤细胞及用Foxp3 siRNA沉默后的肿瘤细胞分别与CD4+CD25-T细胞共培养,CFSE评价免疫效应细胞增殖情况。结果 Foxp3在肺癌细胞系中出现表达;肺癌细胞与CD4+T细胞进行共培养,可显著抑制共培养体系中CD4+T细胞增殖;肺癌细胞Foxp3表达沉默后,可显著改善共培养体系中肺癌细胞对CD4+T细胞增殖的抑制作用。结论在肺细胞癌细胞系发现Foxp3的表达,肺癌细胞Foxp3的表达参与对肿瘤特异性T细胞增殖的抑制。     

miRNAs 在不同级别宫颈癌中的差异性表达分析及其临床意义

宫颈癌是女性常见的恶性肿瘤之一,虽然高危型人乳头瘤病毒持续性感染是导致宫颈癌发生的最主要因素,但是表观遗传学调控在其发展过程中也具有重要作用。研究发现,微小核糖核酸（ microRNAs , miRNAs ）在不同级别宫颈癌中有明显差异性表达,并广泛参与了宫颈癌发生的各个生物学过程。本文系统分析了85篇实验性文章中报道的不同阶段宫颈癌组织的 miRNAs 差异性表达谱,并将其分别归类到宫颈上皮内瘤样病变1-3级（ cervical intraepithelial neoplasia , CIN 1-3）和宫颈癌4个病理分级中,为评价 miRNAs 在宫颈癌早期诊断和预后监测中的临床应用价值提供了科学的理论依据。     

苦豆子生物碱对体外培养的肺癌细胞的抑制作用

目的 对4种苦豆子生物碱的体外抗肺癌活性及机制进行研究,并评价其对正常细胞的毒性,以期筛选出具有高效低毒抗肿瘤作用的药物.方法 用噻唑蓝（MTT）法测定4种苦豆子生物碱对人肺癌细胞和仓鼠肺细胞增殖活性的影响;用激光共聚焦显微镜观察Hochest33342染色后肺癌细胞凋亡的形态学变化;用流式细胞仪检测异硫氰酸荧光素标记的膜联蛋白Ⅴ/碘化丙啶（Annexin Ⅴ-FITC/PI）双染后肺癌细胞的凋亡率.结果 4种苦豆子生物碱对肺癌细胞的生长具有不同程度的抑制作用,呈剂量依赖关系,以氧化槐定碱的抗肺癌活性最强,且对正常肺细胞的毒性较小;Hochest33342染色和流式细胞术显示氧化槐定碱可以明显诱导人非小细胞肺癌细胞A549发生凋亡和坏死.结论 4种苦豆子生物碱均具有一定的抗肺癌作用,以氧化槐定碱的活性最强,具有高效低毒的特点,适合作为抗肿瘤候选药物进一步开发.     

Discovering novel lung cancer associated antigens and the utilization of their autoantibodies in detection of lung cancer

ObjectiveThe identification of tumor-associated antigens (TAAs) and their corresponding autoantibodies in lung cancer (LC) may expand our vision of cancer immunity. This study aims to screen novel TAAs to distinguish LC from the healthy population.MethodsIn our previous study, 35 genes encoding LC-associated TAAs were identified from the serological analysis of recombinant cDNA expression libraries (SEREX), and Oncomine database was further used to identify potential genes in cancer progression. Autoantibody to TAAs were tested by enzyme-linked immunosorbent assay (ELISA) in sera from 1379 participants in validation set and verification set.FindingsBased on analysis of three independent microarrays in Oncomine, ten genes were consistently dysregulated in LC. The sera level and positive frequency of the anti-TOP2A, anti-ACTR3, anti-RPS6KA5 and anti-PSIP1 from LC patients were higher than normal control in validation set. The area under curve (AUC) of anti-TOP2A, anti-ACTR3, anti-RPS6KA5 and anti-PSIP1 was respectively 0.758, 0.787, 0.707, 0.668. The sensitivity of these four autoantibodies for LC detection ranged from 26.63 % to 32.07 % with the specificity over 90 %. Data from the verification set confirmed the results. Except that, the frequency of serum autoantibody against TOP2A (43.3 %) and ACTR3 (50.0 %) was significantly higher in early stage LC than late stage (23.6 % and 22.3 %, respectively).ConclusionTOP2A, ACTR3, RPS6KA5 and PSIP1 can elicit humoral immune response in LC and their autoantibodies have relationship with the tumorigenesis of LC. Anti-TOP2A and anti-ACTR3 have the potential to serve as a serological biomarkers in early stage LC.     

食管癌高发区人胚胎发育过程中食管上皮组织P16、PCNA蛋白的表达及其与形态学变化关系

目的初步探讨食管癌高发区人胎儿食管上皮组织抑癌基因p16和增殖细胞核抗原(proliferation cell nuclear antigen,PCNA)与人胚胎发育过程中食管上皮细胞增殖特性的关系。方法采用免疫组化ABC法检测3～10月份胎儿食管上皮组织中p16和PCNA的表达状况。结果在26例食管癌高发区人胎儿食管上皮组织中,p16蛋白总阳性率61.5%(16/26),p16蛋白阳性率在低月龄组中明显低于高月龄组(P<0.05),PCNA单位面积平均阳性细胞数为(450±156)/mm2,PCNA单位面积阳性细胞数在低月龄组中明显高于高月龄组(P<0.05)。结论食管癌高发区人胎儿食管上皮组织中p16基因的表达和PCNA单位面积阳性细胞数与胎龄密切相关,可以客观反映食管癌高发区人胎儿食管上皮的组织分化状态。     

联合检测血清中CYFRA21-1、NSE对肺癌诊断的价值

目的探讨联合检测血清中肿瘤标记物CYFRA21-1、NSE对肺癌早期诊断、病情监测及疗效判定的价值。方法将肺癌患者分为非小细胞肺癌组(NSCLC)和小细胞肺癌组(SCLC),以健康人及肺部良性病变患者(BLD)为对照组。静脉取血,采用免疫放射法(IRMA)、放射免疫法(RIA)分别检测血清CYFRA21-1和NSE。结果血清CYFRA21-1水平在NSCLC组明显高于SCLC和BLD组(P<0.01),其对NSCLC诊断的阳性率显著高于SCLC组(58.1%vs16.7%,P<0.01)。NSE则在SCLC组明显高于NSCLC和BLD组(P<0.01),其对SCLC诊断的阳性率为66.7%,显著高于NSCLC组24.3%(P<0.01)。血清CYFRA21-1、NSE联合检测与各单项指标相比,可将肺癌诊断的敏感度、特异度和阳性率分别提高到79.1%、91.7%和85.0%(P<0.01)。此外,在NSCLC组,血清CYFRA21-1含量变化与肿瘤分期呈正相关,且与患者病情的变化有相关性。结论联合检测血清中CYFRA21-1、NSE水平可明显提高肺癌早期诊断的阳性率,从而提高肺癌的早期诊断。临床上动态监测血清CYFRA21-1水平对NSCLC的进程及疗效判断有一定的价值。     

Prognostic value of FHIT, CTNNB1, and MUC1 expression in non-small cell lung cancer

Comprehensive expression analysis using microarrays has identified a number of differentially expressed genes in smoke-exposed bronchial epithelium and non-small cell lung cancers (NSCLCs). To evaluate the prognostic relevance of these proteins in NSCLCs, we used immunohistochemistry to investigate the expression of beta-catenin (CTNNB1), dickkopf, Xenopus, homolog of 3 (DKK3 gene), fibroblast growth factor receptor 3 (FGFR3), fragile histidine triad (FHIT), tumor protein p53 (TP53), mucin1 (MUC1), topoisomerase II alpha (TOP2A), and glutathione S-transferase-Pi (GST) in a cohort of patients (n = 125). We correlated the expression data with clinicopathologic features and clinical outcome. In addition, SNaPshot multiplex assays (Applied Biosystems, Darmstadt, Germany) and restriction fragment length polymorphism analysis were used to screen for activating point mutations at the hot spots of FGFR3 in a cohort of 30 samples of NSCLC. Using Kaplan-Meier analysis, we observed significantly better overall survival in adenocarcinomas compared with squamous cell cancers (P = .049). Loss of FHIT expression showed a strong association with shorter overall survival in both histologic types of NSCLC (squamous cell cancers, P < .001; adenocarcinomas, P = .001). In adenocarcinomas, the cytoplasmic expression of beta-catenin was associated with shorter survival (P = .012); MUC1 expression was associated with worse prognosis in patients with squamous cell cancers (P = .002). The nuclear staining of TP53 (P = .008) and TOP2A (P = .059) was associated with cancers without lymphonodal metastases. A correlation with positive staining of TOP2A (P = .03) and FGFR3 positivity (P = .057) was found in adenocarcinomas of male patients. Positive MUC1 stainings were associated with squamous cell cancers of male patients (P = .03). DKK3 expression did not show any significant association with clinical outcome or pathologic features. The screening of the FGFR3 sequence in lung cancers showed only wild-type sequences and did not detect mutations in the known hot spots for FGFR3 mutations. We conclude that the immunohistochemical loss of FHIT expression and the positivity for beta-catenin and MUC1 in NSCLC are useful prognostic markers, whereas the variable expression of TP53, TOP2A, and FGFR3 in relation to the different histologic types of NSCLC and sex of the patients is suggestive for different underlying molecular pathways.     

Alcohol dehydrogenase isoenzymes and aldehyde dehydrogenase activity in the sera of patients with esophageal cancer

Various alcohol dehydrogenase (ADH) isoenzymes and aldehyde dehydrogenase (ALDH) exist in the human esophageal mucosa. In our last experiments we have shown that ADH and ALDH are present also in the esophageal cancer cells. Moreover, the activities of total ADH and class IV isoenzymes were significantly higher in cancer tissue than in healthy mucosa, which suggests that these changes may be reflected by enzyme activity in the serum. Therefore, we measured the activity of total alcohol dehydrogenase, and classes I–IV of this enzyme and aldehyde dehydrogenase in the sera of patients with this cancer. Serum samples were taken for routine biochemical investigation from 67 patients with esophageal cancer before treatment. Total ADH activity was measured by photometric method with p-nitrosodimethylaniline (NDMA) as a substrate and ALDH activity by the fluorometric method with 6-methoxy-2-naphtaldehyde as a substrate. For the measurement of the activity of class I and II isoenzymes, we employed the fluorometric methods, with class-specific fluorogenic substrates. The activity of class III alcohol dehydrogenase was measured by the photometric method with formaldehyde and class IV with m-nitrobenzaldehyde as a substrate. A statistically significant increase of class IV alcohol dehydrogenase isoenzymes was found in the sera of cancer patients. The median activity of this class isoenzyme in the total cancer group increased by about 26.5% (7.42 mU/l) in comparison to the control level (5.46 mU/l). The total alcohol dehydrogenase activity was significantly higher (30%) among patients with cancer. The activities of other tested ADH isoenzymes and total ALDH were unchanged. The activity of the class I ADH isoenzyme was significantly higher in the sera of drinkers with esophageal cancer than non-drinking patients. The increased total activity of alcohol dehydrogenase and class IV isoenzyme in the sera of patients with esophageal cancer probably can be caused by release of this isoenzyme from cancer cells or might be stimulated by alcohol drinking.