SEREX技术筛选及鉴定食管癌肿瘤抗原

背景与目的:正常细胞向癌细胞转化过程中,突变的基因或各种异常表达的蛋白可以成为肿瘤抗原诱导机体的免疫反应,因此肿瘤患者的血清中存在着与肿瘤相关的自身抗体.重组cDNA表达文库血清学分析法(serological analysis of recombinant cDNA expression libraries,SEREX)是利用肿瘤患者血清中的自身抗体筛选、鉴定肿瘤抗原的技术.本研究拟采用SEREX的方法寻找食管癌自身抗体的相关肿瘤抗原,鉴定与食管癌发生、发展相关的基因和免疫治疗分子靶点,并为食管癌的诊断提供候选血清标志物.方法:用食管癌组织建立库容量达1.6×106 pfu的cDNA表达文库,SEREX筛选获得21个不同cDNA序列的阳性克隆,进一步使用SADA法分析其中4个抗原在10例食管癌及10例正常人血清中的反应.结果:在Homosapiens desmin(DES)等21个阳性克隆中,4个克隆与已知EST序列明显无同源性,另外17个克隆与已知基因高度同源.Ribosomal protein S4等4个抗原与食管癌患者和正常人血清反应阳性率分别为40%和0%、60%和10%、70%和20%、30%和20%.结论:Ribosomal protein S4等4个抗原普遍参与了食管癌患者的体液免疫反应,与食管癌患者血清的反应阳性率明显高于正常人的血清.本研究发现的21个食管癌抗原可作为食管癌治疗的潜在分子靶点和食管癌诊断新的候选血清学标志物.     

肝癌相关抗原calponin2的血清免疫反应及其意义

目的：检测肿瘤相关抗原calponin2对肝细胞癌(hepatocellular carcinoma, HCC)的特异性。方法：应用SEREX （serological identification of antigens by recombinant expression loning)技术检测calponin2抗原在肝癌、肺癌、胃癌、直肠癌、肝炎、肝硬化患者（各31例）及正常健康者（32人）血清的抗体阳性率。结果：相应抗体主要见于HCC患者血清,其血清阳性反应率为54.8% ;肝炎、肝硬化患者血清阳性反应率均为3.2%;肺癌、直肠癌、胃癌患者的血清阳性反应率分别是3.2%、97%和6.5%;正常人血清阳性反应率为3.1%。Calponin2抗体在肝癌血清中的阳性率最高（P<001）。Calponin2阳性率与AFP阳性率及含量、癌组织病理分级、患者年龄及γ谷氨酰转肽酶（γGT）水平无相关性。使用calponin2诊断肝癌的灵敏度、特异性和准确度分别为54.8%、95.2%和89.4%。结论: Calponin2在HCC中有一定的特异性,具有作为新的HCC血清学诊断潜在标志物的意义。     

血清抗端粒酶抗体测定的方法学建立及初步应用

目的 建立测定血清抗端粒酶抗体的Westerrl blot法.方法 应用免疫共沉淀法从转染了hTERT的AGS细胞株上清液,分离端粒酶催化亚单位,以此为抗原转至PVDF膜,建立测定血清抗端粒酶抗体的Western blot法并对胃癌和非胃癌患者血清进行端粒酶抗体检测.结果 31例胃癌组患者端粒酶抗体阳性率显著高于56例非胃癌组患者,胃癌组阳性率为29.0%（9/31）,非胃癌组为0（0/56）（P＜0.05）.测定血清端粒酶抗体对胃癌诊断敏感性为29.0%,特异性为100.0%.端粒酶自身抗体的半衰期较长,在术后3个月可仍为阳性.结论 建立的方法可用于检测血清端粒酶抗体,端粒酶抗体有望成为一个新的特异性肿瘤标志物.     

血清DNA—PRIA诊断大肠癌的价值

血清DNA－PRIA是一种新的恶性肿瘤的标志物，对诊断恶性肿瘤具有广谱性和诊断符合率高的特点，本研究测定49例大肠癌病人的血清DNA－PRIA，其阳性率达7347％（36／49），同时检测67例大肠良性疾病病人的血清DNA—PRIA，阳性率占4．48％（3／67）（P＜0．01），又检测100例正常人血清DNA—PRIA，其阳性率为0（P＜0．001），提示血清DNA－PRIA诊断大肠癌具有十分重要的价值，值得大力推广。     

血清DNA-P RIA对肺癌的诊断意义

血清DNA－PRIA是一种新的广谱的恶性肿瘤的标志物，对诊断恶性肿瘤具有广谱性和诊断符合率高的特点，本文测定肺癌病人血清58例，其阳性率占82．76％（48例），检测肺部良性疾病病人血清160例作对照，阳性率4．38％（7例），（p＜0．01），又测定100例正常人血清，阳性率为0（P＜0．01），提示血清DNA－PRLA对肺癌的诊断具有重要的临床意义。     

胃癌相关抗原MGd1-Ag在消化道肿瘤血清诊断中的初步应用

 本文对386例消化系统肿瘤、消化系统良性疾病、正常人血清中一种新的胃癌相关抗原表达情况进行检测,以探讨其在消化道肿瘤诊断中的意义。结果如以正常人组检测OD值的均数加二个标准差之和作为判断肿瘤的阴性阈值。阳性率分别为,食道癌14.3%,胃癌45%,结肠癌50%,直肠癌18.6%,肝癌、胰腺癌则为阴性。166例良性消化系疾病总假阳性率为18.7%,41例正常人仅1例阳性。实验结果提示血清中MGdl Ag的检测对胃癌结肠癌的诊断具有一定价值。     

应用SEREX技术筛选和鉴定非小细胞肺癌肿瘤相关抗原

背景与目的 目前用于非小细胞肺癌诊断的标志物为数不多,为寻找更多的早期诊断和靶向治疗相关的标志物,本研究应用SEREX技术筛选与鉴定非小细胞肺癌肿瘤相关抗原.方法 使用非小细胞肺癌患者血清对人肺鳞癌和腺癌噬菌体展示文库进行生物淘选和SEREX筛选.PCR扩增阳性克隆插入片段后测序,结果 与GeneBank数据库中已知基因进行同源性比较,分析其生物学特性.结果用非小细胞肺癌血清对噬菌体展示文库进行筛选,共获得31个阳性克隆,测序后发现其代表15个基因,其中12个与已知cDNA序列同源,与肺癌或其它肿瘤的发生、发展关系密切;另外3个未发现有同源基因,可能是新基因.结论 应用SEREX技术发现15个肺癌相关抗原和3个肺癌相关抗原的候选基因,为进一步的深入研究打下良好的基础.     

β-HCG与肝细胞癌的相关性研究

目的评估人绒毛膜促性腺激素（β-HCG）检测在肝细胞癌（HCC）诊断和预后中的价值。方法用微粒子酶免发光技术（MEIA）测定83例HCC患者，25例肝硬化患者和62名正常人血清的5种HCC血清标志物，包括β-HCG、甲胎蛋白（AFP）、糖链抗原19—9（CA19—9）、糖链抗原125（CA125）、癌胚抗原（CEA）。结果通过5种血清学指标检测，HCC患者5种血清学指标与肝硬化组和正常人组比较差异均有显著性（P〈0．01），β-HCG和AFP在Ⅰ期HCC和Ⅰ、Ⅲ期HCC中表达差异无显著性。部分AFP阴性患者β-HCG可阳性。结论β-HCG检测可提高HCC早期诊断率，同时，也可能为HCC生物治疗提供一个新的靶点。     

肿瘤相关抗原的抗体检测在原发性肝癌早期诊断中的价值

目的: 对10种肿瘤相关抗原(tumor-associated antigen,TAA)的自身抗体检测在原发性肝癌早期诊断中的价值进行评价,从而寻找一种真实可靠的肝癌早期诊断方法。 方法: 应用间接酶联免疫吸附试验(ELISA)检测原发性肝癌患者血清和正常人血清中的10种TAA(针对10个抗原Calnuc、CyclinE、CDK2、CIAP、RalA、p62、p53、Cy-clinB1、Koc、Imp1)的自身抗体,利用流行病学方法对检测结果的真实性进行评价。 结果: 每种TAA单独检测时,大多数指标的灵敏度偏低,但是肝癌患者抗体阳性率明显高于正常人;10种TAA两两联合起来时检测抗体阳性率,除了Calnuc抗体和CDK2抗体联合时阳性率为18.0%,其余联合均大于26.0%,明显高于单个抗体的检测结果,抗体阳性率最高达到50.0%,其中CyclinE和CIAP、CyclinE和Koc、CyclinE和Imp1等抗体阳性率均为50.0%;对10种TAA进行不同的组合,逐渐增加抗原数目进行检测,结果随着检测抗体的增多,诊断的灵敏度随之增加,10种抗体联合检测的灵敏度达到了88.0%,特异度也达到了86.2%,阳性预测值为84.6%,阴性预测值为89.3%。阳性似然比为6.25,阴性似然比为0.14,说明10种TAA检测肝癌的临床价值较高,Kappa值为0.74,提示该实验诊断结果与真实值之间高度一致。 结论: 利用10种TAA抗体组合检测肝癌具有较高的真实性,可作为现场高危人群筛检和临床中肝癌早期诊断的一种方法。     

应用多肽芯片研究非小细胞肺癌患者血清中的EGFR自身抗体

背景与目的 自身抗体作为新的肿瘤标志物在肺癌的早期诊断和预后评价中可能发挥重要作用,本研究利用多肽芯片检测非小细胞肺癌患者血清中表皮生长因子受体(epidermal growth factor receptor,EGFR)的自身抗体,并筛选自身抗体识别的抗原表位.方法 使用Intavis公司ASPSL多肽芯片合成仪合成EGFR多肽芯片,利用多肽芯片检测非小细胞肺癌患者血清中EGFR自身抗体,并筛选自身抗体识别的抗原表位.结果 使用EGFR多肽芯片检测了20例非小细胞肺癌患者,结果有6例阳性,阳性率为30%,在该6例阳性患者中发现了9个高频位点,并且有8个高频位点集中在EGFR胞外段的第Ⅲ和第Ⅳ结构域.结论 本研究为我们进一步研究EGF咖EGFR自身抗体的功能提供了新的线索.     

Serological identification of tumor antigens of esophageal squamous cell carcinoma

Autoantibodies are often detected in the patients with esophageal cancer. We applied serological analysis of recombinant cDNA expression libraries (SEREX) to a case of esophageal squamous cell carcinoma in order to identify tumor antigens. A cDNA library derived from an esophageal cancer cell line was bacterially expressed and screened for interaction with antibodies in five allogeneic sera of patients with esophageal squamous cell carcinoma. To examine the specific immunoreactivity of the antigens, sera from 16 more patients with esophageal squamous cell carcinoma, 16 patients with gastric cancer, 16 patients with colon cancer, 16 patients with breast cancer and 37 healthy volunteers were screened. We identified 11 independent cDNA clones that potentially encoded esophageal cancer tumor antigens. The identified cDNA clones were SURF1, HOOK2, CENP-F, ZIC2, hCLA-iso, Ki-1/57, enigma, HCA25a, SPK and two EST clones named LOC146223 and AGENCOURT_7565913. The sero-positive rates of antibodies against SURF1 (48%), LOC146223 (38%), HOOK2 (14%) and AGENCOURT_7565913 (14%) were significantly higher in esophageal cancer patients than in healthy controls. At least one of these antibodies was detected in 18 (86%) of 21 sera from esophageal cancer patients. A disease-specific humoral immune response against SURF1, LOC146223, HOOK2 or AGENCOURT_7565913 was observed in most patients with esophageal squamous cell carcinoma. Antibodies against these SEREX antigens may represent a pool of candidates for serum tumor markers of esophageal squamous cell carcinoma.     

Profiling tumor-associated autoantibodies for the detection of colon cancer.

PURPOSE: The purpose of the present study was to screen the autoantibody signature of colon cancers to develop serum markers for colon cancer detection. EXPERIMENTAL DESIGN: A phage cDNA expression library of colon cancer was built. The library was sequentially screened by a pool of 10 colon cancer sera, goat antihuman IgG, and a pool of two healthy sera to identify phage-expressed antigens recognized by tumor-associated antibodies. The clones picked out by these screening were subjected to a training set with 24 colon cancer sera and 24 healthy sera. The antigen combination, which got the most satisfactory classification, was tested by an independent set of 24 colon cancer sera with equal number of sera from normal donors. The carcinoembryonic antigen (CEA) level of these sera was detected for the additional classification analysis with or without the antigen combination. RESULTS: A cDNA expression library consisting of 2 x 10(6) primary clones was prepared. After three turns of screening, 24 antigens recognized by tumor-associated antibodies were picked out for serum marker identification. The training set showed that a six-marker combination got the most satisfactory classification in a logistic regression model; leave-one-out validation achieved 91.7% sensitivity and 91.7% specificity. In a testing set with this marker panel, we correctly predicted 85% of the samples. Although according to CEA level alone, we correctly predicted 75% of the samples with 42% of cancer patients misclassified. When CEA was combined with the six markers, the sensitivity and specificity increased to 91.7% and 95.8%, respectively. The six antigen sequences in the phage display system are relatively short peptides. Only two of them showed homology to known protein sequences. CONCLUSIONS: Autoantibodies against phage-expressed antigens derived from colon cancer tissues could be used as serum markers for the detection of colon cancer.     

Isolation and characterization of human lung cancer antigens by serological screening with autologous antibodies

Serological analysis of a recombinant cDNA expression library (SEREX) derived from two lung adenocarcinoma cancer cell lines using autologous sera led to the isolation of 41 positive cDNA clones comprising 28 different antigens. They coded for a variety of nuclear and cytoplasmic proteins. Among the antigens, nucleoporin 107 (NUP107) was isolated most frequently (5 of 41 clones). The second most frequently isolated antigen was coded for by C21orf58 (4 of 41 clones). During serological analysis of selected antigens based on their reactivity to sera from normal individuals and lung cancer patients, none of the antigens showed a cancer-restricted recognition pattern. However, five genes including NUP107 showed higher expression when we examined the changes in gene expression in five different adenocarcinoma cell lines, including those used in SEREX, compared with their levels in normal lung tissues by cDNA microarray analysis. On the other hand, the expression levels of five genes including C21orf58 were down regulated in all adenocarcinoma cell lines. This SEREX study combining comprehensive gene expression assays has added to the growing list of lung cancer antigens, which may aid the development of diagnostic and immunotherapeutic reagents for patients with lung cancer.     

Novel tankyrase-related gene detected with meningioma-specific sera.

In many meningiomas, alterations of chromosome 22 can be found, and the NF2 (neurofibromatosis type 2) gene, in particular, is of great interest as a putative gene involved in meningioma. Because the NF2 gene is not mutated in all meningiomas, additional genes may be involved. Instead of looking for alterations directly at the DNA level, we used the immune response of meningioma patients to identify immunogenic antigens that may be associated with the disease. We screened a fetal brain cDNA expression library with sera pools from different patients bearing meningioma classified according to the three WHO grades, using the serological identification of antigens by recombinant expression cloning immunological screening method. Here, we report the finding of a new tankyrase-related protein. We found 16 overlapping clones with homologies to tankyrase when we screened the library with the common-type meningioma sera pool and 2 such clones when we screened the library with the atypical meningioma sera. The anaplastic meningioma sera did not identify any tankyrase-related clones. We tested some of the newly identified clones with 13 single sera, 6 of which (37.5%) reacted positively with the tankyrase-related clones. In addition, we screened the tankyrase-related clone with six sera pools from individuals without obvious disease. Although 1 of 24 (4.2%) normal sera reacted with the tankyrase-related clone, we found a striking difference in the frequency of reactivity to this clone by sera from patients bearing tumors corresponding to the three WHO meningioma grades; common-type sera was the most frequently reactive. Northern blot analysis demonstrates expression of the novel tankyrase gene in two common-type meningiomas from patients with immune response.     

Characterization of human colon cancer antigens recognized by autologous antibodies

The screening of cDNA expression libraries derived from human tumors with autologous antibody (SEREX) has proven to be a powerful method for defining the structure of tumor antigens recognized by the humoral immune system. In the present study, 48 distinct antigens (NY-CO-1–NY-CO-48) reactive with autologous IgG were identified by SEREX analysis in 4 patients with colon cancer. Sequencing analysis showed that 17 of the cDNA clones were previously uncharacterized molecules and 31 represented known gene products. The individual cDNA clones were analyzed in the following manner: a search for mutations or other structural changes; an analysis of mRNA expression in a panel of normal tissues; and a frequency analysis of the antibody response to the expressed product in the sera of colon cancer patients and normal individuals. The initial analysis showed NY-CO-13 to be a mutated version of the p53 tumor suppressor gene. Three of the 48 antigens showed a differential pattern of mRNA expression, with NY-CO-27 (galectin-4) expressed primarily in gastrointestinal tract, and NY-CO-37 and -38 showing a pattern of tissue-specific isoforms. With regard to immunogenicity, 20 of the 48 antigens were detected by allogeneic sera; 14 of these were reactive with sera from both normal donors and cancer patients, and 6 other clones (NY-CO-8, -9, -13, -16, -20 and -38) reacted exclusively with sera from colon cancer patients (ranging from 14% to 27%). Our results on colon cancer illustrate both the complexity and the potential of the SEREX approach for analysis of the humoral immune response against human cancer. Int. J. Cancer76:652–658, 1998.© 1998 Wiley-Liss, Inc.     

Identification of CLUAP1 as a human osteosarcoma tumor-associated antigen recognized by the humoral immune system

Since the prognosis of human osteosarcoma in advanced stage remains poor, the development of new and effective therapies including immunotherapy is required. To identify tumor-associated antigens of osteosarcoma applicable to the immunotherapy of this malignancy, we employed the serological analysis of recombinant cDNA expression library (SEREX) technique that defines tumor antigens recognized by the humoral immune system. Screening a cDNA library derived from an osteosarcoma cell line MG63 with sera from osteosarcoma patients identified 43 positive clones, representing 14 distinct antigens. Among them, CLUAP1 (clusterin-associated protein 1) was highly expressed in osteosarcoma tissue samples and cell lines. Overexpression of CLUAP1 was observed in other malignancies including ovarian, colon, and lung cancers. Our results suggest that CLUAP1 may be useful as a prognostic/diagnostic marker and/or for a target of immunotherapy of osteosarcoma.