Translation initiation factor eIF-4G is immunogenic, overexpressed, and amplified in patients with squamous cell lung carcinoma

BACKGROUND: Recently, the authors reported identification and cloning of several novel immunogenic antigens in squamous cell lung carcinoma. Of 14 corresponding genes, 9 mapped in an amplified chromosomal region with the gene for eIF-4G that was amplified most frequently. METHODS: Recombinant eIF-4G was expressed in E. coli and screened with sera from patients with squamous cell carcinoma of the lung and of the head and neck. Protein extracts from squamous cell carcinoma tissues were analyzed for eIF-4G expression by Western blot analysis. Mutation analysis was performed using an automatic DNA sequencer. RESULTS: The authors screened a spectrum of 33 heterologous sera from lung carcinoma patients and detected antibodies against eIF-4G in 5 of these sera (15%). They found no immune response to eIF-4G in 17 sera from squamous cell carcinoma tissues derived from the head and neck. In addition, no antibodies were found to eIF-4G in a group of 17 control sera from individuals without known tumor formation. Sequence analysis of the eIF-4G gave no indication of mutations. To analyze the expression of eIF-4G, a monoclonal antibody was used. Western blot analysis clearly showed overexpression in the tumor tissue compared with the corresponding normal lung tissues. CONCLUSIONS: The translation initiation factor eIF-4G is the first protein in which the gene shows amplification, has increased expression in a human tumor, and induces an immune response in patients. eIF-4G lends itself as a marker for the diagnosis of and possibly as a future therapeutic target in patients with squamous cell lung carcinoma.     

Serological analysis of human renal cell carcinoma

Serological analysis of cDNA expression libraries (SEREX) has proven to be a useful technique in the quest to elucidate the repertoire of immunogenic gene products in human cancer. We have applied the SEREX method to human renal cell carcinoma (RCC) in order to identify associated immunogenic gene products. cDNA expression libraries were prepared from a RCC tumor, a RCC cell line and human testis. The 3 libraries were screened with sera from 35 RCC patients and 15 healthy controls. Approximately 4.5 x 10(6) phage plaques were screened resulting in 234 positive clones, which corresponded to 74 different gene products. The seroreactivity toward 49 of these antigens was assessed. Seroreactivity to 21 (43%) of the antigens was similar in RCC patients and healthy controls, 9 antigens (18%) elicited antibodies more frequently and 19 antigens (39%) solely in RCC patients. In the reverse setting, reactivity of RCC patients' sera was tested against a panel of 44 previously identified "tumor-associated" antigens via the SADA (serum antibody detection array) method; 6 antigens reacted with RCC patients' and healthy donors' sera, 8 were reactive only with RCC patients' sera. From the 27 antigens identified by SEREX and SADA, which did not react with sera from healthy controls, 10 antigens reacted with a significant proportion of RCC patients' sera and 77% of RCC patients' sera reacted at least with one of these antigens. Sera from patients with non-malignant renal diseases or an autoimmune disease did not react with these 10 antigens.     

Identification of tumor-associated antigens by screening phage-displayed human cDNA libraries with sera from tumor patients

Screening cDNA libraries from solid human tumors with sera of autologous patients (SEREX) has proven to be a powerful approach to identifying tumor antigens recognized by the humoral arm of the immune system. In many cases, application of this methodology has led to the discovery of novel tumor antigens as unknown gene products. We tried to improve the potency of the SEREX approach by combining it with phage-display technology. We designed a new lambda vector to express protein fragments as N-terminal fusions to the D capsid protein and generated high-complexity cDNA libraries from human breast carcinoma cell lines and solid tumors. Screening these phage-displayed libraries required limited amounts of sera from patients and efficiently identified several tumor antigens specifically reacting with sera from breast cancer patients.     

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.     

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.     

Antitumor effectiveness of different amounts of electrical charge in Ehrlich and fibrosarcoma Sa-37 tumors

Background

Tumor-associated antigens recognized by humoral effectors of the immune system are a very attractive target for human cancer diagnostics and therapy. Recent advances in molecular techniques have led to molecular definition of immunogenic tumor proteins based on their reactivity with autologous patient sera (SEREX).

Methods

Several high complexity phage-displayed cDNA libraries from breast carcinomas, human testis and breast carcinoma cell lines MCF-7, MDA-MB-468 were constructed. The cDNAs were expressed in the libraries as fusion to bacteriophage lambda protein D. Lambda-displayed libraries were efficiently screened with sera from patients with breast cancer.

Results

A panel of 21 clones representing 18 different antigens, including eight proteins of unknown function, was identified. Three of these antigens (T7-1, T11-3 and T11-9) were found to be overexpressed in tumors as compared to normal breast. A serological analysis of the 21 different antigens revealed a strong cancer-related profile for at least five clones (T6-2, T6-7, T7-1, T9-21 and T9-27).

Conclusions

Preliminary results indicate that patient serum reactivity against five of the antigens is associated with tumor disease. The novel T7-1 antigen, which is overexpressed in breast tumors and recognized specifically by breast cancer patient sera, is potentially useful in cancer diagnosis.     

Cancer Testis Antigen,NOL4, Is an Immunogenic Antigen Specifically Expressed in Small-Cell Lung Cancer

To identify cancer/testis (CT) antigens and immunogenic proteins, immunoscreening of testicular and small-cell lung cancer cell line NCI-H889 cDNA libraries was performed using serum obtained from a small-cell lung cancer (SCLC) patient. We obtained 113 positive cDNA clones comprised of 74 different genes, designated KP-SCLC-1 through KP-SCLC-74. Of these genes, 59 genes were found to be related to cancers by EMBASE analysis. Three of these antigens, including KP-SCLC-29 (NOL4), KP-SCLC-59 (CCDC83), and KP-SCLC-69 (KIF20B), were CT antigens. RT-PCR and western blot analysis showed that NOL4 was frequently present in small-cell lung cancer cell lines (8/9, 8/9). In addition, NOL4 mRNA was weakly, or at a low frequency, or not detected in various cancer cell lines. Our results reveal that NOL4 was expressed at protein levels in small-cell lung cancer tissues (10/10) but not detected in lung adenocarcinoma and squamous cell carcinoma by immunohistochemical analysis. Serological response to NOL4 was also evaluated by western blot assay using NOL4 recombinant protein. A humoral response against NOL4 proteins was detected in 75% (33/44) of small-cell lung cancer patients and in 65% (13/20) of healthy donors by a serological western blot assay. These data suggest that NOL4 is a specific target that may be useful for diagnosis and immunotherapy in SCLC.     

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.     

Identification of the antigens predominantly reacted with serum from patients with hepatocellular carcinoma

BACKGROUND: To identify antigens specifically recognized by the immune surveillance system in patients with hepatocellular carcinoma (HCC), the authors examined two complementary DNA (cDNA) libraries of moderately differentiated HCC by serologic analysis of recombinant cDNA expression libraries (SEREX). METHODS: The libraries were screened with autologous patients' sera, and sequences of the reacted clones were determined. To study the immunoreactivity of the antigens, sera from 20 patients with HCC, from 20 healthy volunteers, and from 16 patients with chronic viral hepatitis were examined. RESULTS: Twenty-seven antigens were identified. They included SART1, p57Kip2, ROCK-1, gamma-catenin, and heat shock proteins, which are classified as tumor-associated genes. Three of 27 antigens-Tat-binding protein-1 (TBP-1), beta4 integrin-binding protein (p27[BBP]), and ribosomal protein L30 (rpL30)-were reacted predominantly with sera from patients with HCC (55% of patients, 45% of patients, and 20% of patients, respectively). Patients in the control group had no antibodies against these three antigens. Seventy percent of patients with HCC had the antibody against at least one of these antigens. CONCLUSIONS: Disease specific humoral immune response against TBP-1, p27(BBP), and rpL30 was induced in patients with HCC, and the antibodies against these antigens also may be used as tumor markers.     

Antigens recognized by autologous antibody in patients with renal-cell carcinoma.

The screening of cDNA expression libraries derived from human tumors with autologous antibody (SEREX) is a powerful method for defining the structure of tumor antigens recognized by the humoral immune system. Sixty-five distinct antigens (NY-REN-1 to NY-REN-65) reactive with autologous IgG were identified by SEREX analysis of 4 renal cancer patients and were characterized in terms of cDNA sequence, mRNA expression pattern, and reactivity with allogeneic sera. REN-9, -10, -19, and -26 have a known association with human cancer. REN-9 (LUCA-15) and REN-10 (gene 21) map to the small cell lung cancer tumor suppressor gene locus on chromosome 3p21.3. REN-19 is equivalent to LKB1/STK11, a gene that is defective in Peutz-Jeghers syndrome and cancer. REN-26 is encoded by the bcr gene involved in the [t(9:22)] bcr/abl translocation. Genes encoding 3 of the antigens in the series showed differential mRNA expression; REN-3 displays a pattern of tissue-specific isoforms, and REN-21 and REN-43 are expressed at a high level in testis in comparison to 15 other normal tissues. The other 62 antigens were broadly expressed in normal tissues. With regard to immunogenicity, 20 of the 65 antigens reacted only with autologous sera. Thirty-three antigens reacted with sera from normal donors, indicating that their immunogenicity is not restricted to cancer. The remaining 12 antigens reacted with sera from 5-25% of the cancer patients but not with sera from normal donors. Seventy percent of the renal cancer patients had antibodies directed against one or more of these 12 antigens. Our results demonstrate the potential of the SEREX approach for the analysis of the humoral immune response against human cancer.     

Serological identification of immunogenic antigens in acute monocytic leukemia

In order to improve disease-free survival and potentially a cure, it is necessary to identify more potent leukemia antigen. Here, we defined the acute monocytic leukemia-associated antigen (LAA) recognized by the humoral immune system for the first time. We have applied the method of serologic analysis of recombinant cDNA expression library (SEREX) on acute monocytic leukemia (FAB M5), followed by DNA sequencing and analyzing of positive clones. Then, the reactivity of normal and other leukemia sera with positive clones were performed. Thirty-five distinct novel antigens reactive with autologous IgG were identified by SEREX analysis on an acute monocytic leukemia patient and were characterized according to cDNA sequence and the reactivity with allogeneic sera. Twenty of the 35 antigens identified in this study were recognized by IgG antibodies in normal sera, and the remaining 15 were recognized exclusively by sera from allogeneic leukemia patients but not by normal donor sera, suggested that the immune response to these 15 antigens are leukemia related. The 15 immunogenic antigens detected by immune responses in the autologous host facilitate the identification of epitopes recognized by antigen-specific cytotoxic T lymphocytes (CTL) and are potential candidates for diagnosis and immunotherapy in acute myeloid leukemia (AML).     

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.     

A Study of the p53 Tumor Suppressor Gene in Yunnan Tin Miner''''s Lung Carcinoma

目的：为深入探讨云锡矿工肺癌高发的发病机理,找寻早期诊断的方法和理论依据。方法：应用免疫组化方法对云锡矿工肺癌41例（M组）、非云锡地区非矿工人群肺癌25例（NM组）和正常肺组织15例（C组）进行p53检测比较研究。数据用四格表确切概率法进行组间显著性测验。结果：发现p53阳性率M组为60．98％,NM组为36％,C组为几三组间差异有高度显著性（P＜0．0）,M组与NM组亦有显著差异（P＜0．05）,且M组与NM组间鳞癌的p53阳性存在显著差异（0．05＜P＜0．10）。结论：说明突变型p53在肺癌中发生频率高,云锡矿粉特殊性致癌因素可较多地导致p53突变,这也是云锡矿工肺癌中鳞癌多见的可能原因之一。肺癌周围增生支气管粘膜上皮和11型肺泡上皮出现p53阳性,提示增生上皮已有p53突变存在,值得引起注意。     

Toward an early diagnosis of lung cancer: an autoantibody signature for squamous cell lung carcinoma

Serum‐based diagnosis offers the prospect of early lung carcinoma detection and of differentiation between benign and malignant nodules identified by CT. One major challenge toward a future blood‐based diagnostic consists in showing that seroreactivity patterns allow for discriminating lung cancer patients not only from normal controls but also from patients with non‐tumor lung pathologies. We addressed this question for squamous cell lung cancer, one of the most common lung tumor types. Using a panel of 82 phage‐peptide clones, which express potential autoantigens, we performed serological spot assay. We screened 108 sera, including 39 sera from squamous cell lung cancer patients, 29 sera from patients with other non‐tumor lung pathologies, and 40 sera from volunteers without known disease. To classify the serum groups, we employed the standard Naïve Bayesian method combined with a subset selection approach. We were able to separate squamous cell lung carcinoma and normal sera with an accuracy of 93%. Low‐grade squamous cell lung carcinoma were separated from normal sera with an accuracy of 92.9%. We were able to distinguish squamous cell lung carcinoma from non‐tumor lung pathologies with an accuracy of 83%. Three phage‐peptide clones with sequence homology to ROCK1, PRKCB1 and KIAA0376 reacted with more than 15% of the cancer sera, but neither with normal nor with non‐tumor lung pathology sera. Our study demonstrates that seroreactivity profiles combined with statistical classification methods have great potential for discriminating patients with squamous cell lung carcinoma not only from normal controls but also from patients with non‐tumor lung pathologies. © 2008 Wiley‐Liss, Inc.     

Distribution of lung adenocarcinoma-associated antigens in human tissues and sera defined by monoclonal antibodies KM-52 and KM-93

Two monoclonal antibodies to human lung adenocarcinoma, KM-52 and KM-93, were generated by the novel immunizing procedure using mice rendered tolerant to the normal human lung (N. Hanai et al., Cancer Res., 46: 4438-4443, 1986). KM-93 recognized sialylated carbohydrate epitope on the antigen different from CA19-9 and DU-PAN-2, while KM-52 recognized the protein antigen. Both antigens were different from carcinoembryonic antigen, alpha-fetoprotein, and beta 2-microglobulin. Distribution of KA-52 and KA-93, the antigens recognized by KM-52 and KM-93, respectively, in various tissues and sera was investigated. In immunoperoxidase staining, KM-93 reacted strongly and frequently with tumor cells of lung adenocarcinoma and partially with those of lung squamous cell carcinoma, large cell carcinoma, and small cell carcinoma. In normal adult and fetal tissues, KA-93 was expressed on the surface of a small number of cells of the lung, pancreas, liver, kidney, and bone marrow. KM-52 reacted selectively with tumor cells of adenocarcinoma among four different histological types of lung carcinoma. In normal adult and fetal tissues, KA-52 was distributed on a small number of cells of the lung, stomach, intestine, and pancreas. Of the two monoclonal antibodies, KM-93 could be used in detecting the antigen in sera of patients with lung cancer. The KA-93 level in sera was determined by the sandwich-type enzyme-linked immunosorbent assay. Serum with a high KA-93 level was found in 34 of 70 patients with lung adenocarcinoma (48.6%), one of 67 healthy adults (1.5%), and none of 32 patients with benign diseases (0%). Combined detection by KA-93 with KA-32, a new tumor marker of lung squamous cell carcinoma (N. Hanai et al., Cancer Res., 46: 5206-5210, 1986), elevated the positive percentage in patients with lung squamous cell carcinoma (52.7%) and with lung adenocarcinoma (59.5%). These results suggested that KM-52 and KM-93 would be potential monoclonal antibodies in immunohistological diagnosis and serum diagnosis of lung adenocarcinoma, respectively.     

p53 antibodies in the sera of lung cancer patients: Comparison with p53 mutation in the tumour tissue

This study examined the sensitivity and specificity of serum auto-antibodies to p53 protein as a non-invasive marker of p53 genetic alterations or protein accumulation in lung cancer cases. A sensitive ELISA to detect serum p53 antibodies was developed and used to examine sera from 186 patients undergoing pulmonary surgery for a suspected lung cancer. Target antigens in ELISA were wild-type p53 protein and 5 peptides covering the N- and C-terminal parts of the protein. Sixteen sera were positive for serum p53 antibodies in both ELISAs and all were among the 136 patients with confirmed primary lung carcinoma. Of 50 patients with other pulmonary diseases, none had p53 antibodies. In 92 cancer patients exons 5 to 8 of the p53 gene were examined for mutations by denaturing gradient gel electrophoresis and direct sequencing of PCR products. Forty-seven tumours had a p53 mutation and 7 (15.2%) of these were positive for p53 antibodies. Two patients had serum antibodies but no detectable mutation in exons 5 to 8. Frequencies of p53 mutations and serum antibodies were higher in squamous cell carcinoma patients than in adenocarcinoma. Accumulation of p53 protein in tumour tissue was observed in 32 patients, but only 5 were positive for p53 antibodies. In conclusion, serum p53 antibodies were detected only in a proportion of lung cancer cases, but the majority were specifically associated with a detectable p53 mutation in the tumour. © 1995 Wiley-Liss, Inc.     

The tumor antigen repertoire identified in tumor-bearing neu transgenic mice predicts human tumor antigens

FVB/N mice transgenic for nontransforming rat neu develop spontaneous breast cancers that are neu positive and estrogen receptor negative, mimicking premenopausal human breast cancer. These animals have been widely used as a model for immunobased therapies targeting HER-2/neu. In this study, we used serological analysis of recombinant cDNA expression libraries to characterize the antigenic repertoire of neu transgenic (neu-tg) mice and questioned the ability of this murine model to predict potential human tumor antigens. After screening 3 x 10(6) clones from 3 different cDNA libraries, 15 tumor antigens were identified, including cytokeratin 2-8, glutamyl-prolyl-tRNA synthetase, complement C3, galectin 8, and serine/threonine-rich protein kinase 1. Multiple proteins involved in the Rho/Rho-associated, coiled coil-containing protein kinase (Rock) signal transduction pathway were found to be immunogenic, including Rock1, Rho/Rac guanine nucleotide exchange factor 2, and schistosoma mansoni adult worm antigen preparation 70. All of the identified antigens are self-proteins that are expressed in normal tissues in addition to breast tumors and the majority of the antigens are intracellular proteins. More than half of the mouse tumor antigens have human homologues that have been reported previously as tumor antigens. Finally, the tumor-specific antibody immunity and marked immune cell infiltration that was observed in mice with spontaneous tumors were not observed in mice with transplanted tumors. Our results indicate that neu-tg mice bearing spontaneous tumors develop humoral immunity to their tumors similar to cancer patients and that tumor antigens identified in transgenic mouse may predict immunogenic human homologues.