乳腺癌组织中ER、PR和CEA相关性研究

应用免疫组化技术亲和组化法和ＡＢＣ法,检测了１０６例乳腺癌组织中ＥＲ、ＰＲ和ＣＥＡ水平。其阳性率依次为８３％、８１．１％和８８．７％。其中７９．２％的乳腺癌ＥＲ和ＰＲ表达一致。在９８例ＥＲ和／或ＰＲ阳性乳腺癌中有９２例（９３．９％）呈ＣＥＡ阳性,８例ＥＲ和ＰＲ阴性乳腺癌中６例（７５％）为ＣＥＡ阴性,８９％乳腺癌ＣＥＡ与ＥＲ和ＰＲ表达一致。在癌的分级表达中,随着癌的组织学分级增高ＣＥＡ阳性率增高,而ＥＲ和ＰＲ阳性率减低。结果表明,乳腺癌ＥＲ、ＰＲ和ＣｈＡ表达可反映肿瘤的不同生物学特征。同时检测三者对选择内分泌治疗及判断预后更有意义。     

Carcinoembryonic antigen, ferritin, tissue polypeptide antigen, and CA15/3 in breast cancer: relationship between carcinoma and normal breast tissue

The study of tumor markers in breast cancer tissue may supply information on the tumor's biological features and its clinical behaviour. Forty-nine primary breast cancer patients are evaluable to date. CEA, ferritin, TPA and CA15/3 were measured with radioimmunometric methods in the cytosol of carcinoma and normal tissue from the same breast. The concentrations of the four markers were higher in the tumor than in normal tissue in 42/49 cases for CEA, 47/49 for ferritin, 42/49 for TPA and in 24/29 for CA15/3. However, an overlap was found between carcinoma and normal tissue levels, particularly for CEA and TPA. We can conclude that the four substances studied may be markers of malignancy in breast carcinoma when non-malignant breast tissue from the same patient is determined at the same time, whereas assays within a single, unknown breast tissue sample may be useful only in the case of ferritin and, partly, CA15/3.     

DNA analysis of malignant effusions. Comparison with cytologic diagnosis and carcinoembryonic antigen content.

Twenty-three consecutive malignant effusions from 19 patients submitted for cytologic examination were analyzed for carcinoembryonic antigen (CEA) content and for DNA analysis by flow cytometry. The study was undertaken to determine if the addition of DNA analysis would improve the sensitivity of cytologic diagnosis and CEA assay. CEA examination was performed on Papanicolaou-stained smears and hematoxylin-and-eosin-stained cell blocks. Final diagnoses were correlated with histologic examination (four patients), clinical and radiologic studies, and follow-up. The malignant effusions in 19 patients were secondary to carcinoma of the breast (5), lung (5), ovary (1), endometrium (1), mucinous carcinoma of the colon (1), unknown primary (1), extraovarian papillary carcinoma (1), mesothelioma (2) and large cell lymphoma (2). The sensitivity of cytologic diagnosis was 100% and specificity 100%. DNA aneuploidy, defined as the presence of two separate peaks in the histogram, was present in 7 of 23 fluids (sensitivity, 30%). Four fluids had insufficient cells for analysis, and one histogram showed debris (following chemotherapy). DNA aneuploidy was detected in effusions secondary to carcinoma of the breast (4), lung (1) and lymphoma (2). Using 5 ng/mL as the cutoff, the sensitivity of CEA was 68%. DNA analysis of cells in malignant effusions is less sensitive than cytologic diagnosis, and CEA assay and is not recommended for routine use in the diagnosis of malignant effusions.     

乳腺癌AgNOR计数与癌胚抗原的免疫组化研究

应用核仁组成区嗜银蛋白（ＡｇＮＯＲ）技术及癌胚抗原（ＣＥＡ）免疫组化染色对９８例乳腺良、恶性病变进行对比研究。结果表明：ＡｇＮＯＲ计数与肿瘤增殖活跃程度及生物学行为是一致的。乳腺癌中ＡｇＮＯＲ计数显著高于良性病变（Ｐ＜０．０１）。浸润癌ＡｇＮＯＲ计数比其他类型乳腺癌高。ＣＥＡ染色在乳腺良性病变中基本阴性,恶性病变中阳性率８０．８％。乳腺癌中ＡｇＮＯＲ计数与ＣＥＡ分布之间呈线性相关（ｒ＝０．８２,Ｐ＜０．０５）。ＣＥＡ阳性乳腺癌组与ＣＥＡ阴性组ＡｇＮＯＲ计数差异显著（Ｐ＜０．０５）。提示：ＡｇＮＯＲ定量研究和ＣＥＡ分布在乳腺良、恶性病变的鉴别及肿瘤恶性程度的研究中具有相似的参考价值。     

Carcinoembryonic antigen in effusions. A diagnostic adjunct to cytology

A total of 189 effusion specimens (100 benign and 89 malignant) submitted for cytologic examination were assayed for carcinoembryonic antigen (CEA) by an enzyme immunoassay to determine whether the addition of CEA evaluation to cytologic study would improve the diagnostic accuracy for the detection of malignancy. The sensitivity and specificity were 78% and 90%, respectively, for a cytologic diagnosis of malignancy and 68% and 99%, respectively, for a positive CEA (greater than 5 ng/mL). CEA assay was negative in the most common epithelial malignancies of the female genital tract (15 of 17 cases), mesotheliomas (5), lymphomas (7) and alveolar-cell carcinoma of lung (1). CEA assay was positive in 55 of 89 cases of malignancy, including 14 cases with cytologically negative malignant effusions. The CEA assay sensitivity for lung carcinoma (95% for adenocarcinoma, 100% for oat-cell carcinoma and 100% for carcinosarcoma), breast carcinoma (95%), and gastrointestinal carcinoma (100%) were all over 90%. No significant difference in the levels of CEA was noted between gastrointestinal and lung adenocarcinomas. Oat-cell carcinomas and squamous-cell carcinomas had lower values. In cases of an effusion with an unknown primary, an elevated CEA in the fluid is diagnostic of metastatic carcinoma arising from the breast, lung or gastrointestinal tract.     

CEA is the major PHA-L-reactive glycoprotein in colon carcinoma cell lines and tumors: relationship between K-ras activation and beta1-6 branching of N-linked carbohydrate on CEA

Previously we have shown that a positive correlation existed between the presence of beta1-6 branching of N-linked carbohydrate (detected as PHA-L reactivity) and the level of Ras activation in colon carcinoma cell lines. In these cell lines the major PHA-L-reactive species was found to be 180 kDa. Here we identified this species to be carcinoembryonic antigen (CEA) by demonstrating that: (a) CEA immunoreactivity and PHA-L reactivity colocalized on blots of crude cellular membranes from these cell lines, and that (b) immunoprecipitation of CEA resulted in quantitative coprecipitation of PHA-L reactivity at 180 kDa. Metabolic labeling of cell line HTB39 with [(3)H]mannose revealed that CEA was the predominantly labeled glycoprotein. This indicated that CEA was the major PHA-L-reactive species due its high level of expression. The amount of PHA-L reactivity present on CEA, expressed as the PHA-L/CEA ratio, was found to vary between cell lines. This ratio was found to correlate closely with the level of Ras activation in these cells. In cellular membrane isolated from primary colon carcinoma, the major PHA-L-reactive species was also 180 kDa. This reactivity colocalized with CEA immunoreactivity, indicating that the major beta1-6-branching glycoprotein in membranes from primary colon carcinoma was CEA. Similar to that seen in cell lines, the amount of PHA-L reactivity on CEA in human tumor samples varied, suggesting that a similar paradigm of Ras-induced expression of beta1-6 branching may occur in human colon carcinoma.     

Immunoperoxidase detection of carcinoembryonic antigen in fine needle aspirates of breast carcinoma. Correlation with studies in tissue sections

The peroxidase-antiperoxidase technique was used to demonstrate the presence of carcinoembryonic antigen (CEA) in fine needle aspirates and the corresponding tissue sections in 50 cases of carcinoma of the breast. The incidence of CEA positivity in the aspiration smears was 76%. A good correlation was observed between the results in the aspiration smears and in the tissue sections: 82% in the initial correlation and 90% when additional tissue sections from four of nine discordant cases proved to be positive for CEA. Five cases remained positive for CEA in the aspiration smears and negative for CEA in the tissue sections. This preliminary data indicates that aspiration smears can be used to demonstrate the CEA status in mammary carcinomas.     

CEA levels in gastric juice in precancerous conditions and cancer

First described in 1965 as a specific antigen for cancer of the colon, CEA is now considered to be an antigen associated with many types of malignant neoplasia, although the CEA-Test's role in clinical routine has yet to be clearly defined. In the present study CEA levels in gastric juice were measured in subjects with gastric carcinoma (n = 25) and with benign gastric lesions (n = 171). CEA was significantly (p less than 0.05) higher in patients with gastric carcinoma (GC) than in subjects with benign gastric lesions, other than chronic atrophic gastritis (CAG) associated with intestinal metaplasia (IM). In this latter condition CEA levels were similar to those in patients with GC. These results suggest that the assay of CEA in gastric juice could be included in the diagnostic program for gastric cancer and its precursors with the aim of assessing its utility as risk indicator in the management of precancerous conditions and lesion.     

乳腺癌ConA、LCA、PSA免疫组化研究

运用ＡＢＣ法研究５０例乳腺癌三种凝集素受体（Ｌｅｃｔｉｎ－Ｒ）的表达,并以乳腺纤维瘤和乳腺小叶增生症各１０例作为对照。结果表明：该三种凝集素受体在乳腺良、恶性病变中的表达率无显著性差异。ＣＯｎＡ－Ｒ在乳腺癌以胞浆型定位为主,在纤维腺瘤则以胞膜型多见（Ｐ＜０．０５）。ＬＣＡ－Ｒ和ＰＳＡ－Ｒ在乳腺三种病变中均以胞浆型为主。ＣｏｎＡ－Ｒ与乳腺癌分化程度具良好的相关性;ＬＣＡ－Ｒ与乳腺癌瘤体大小有关。该三种凝集素受体与ＥＲ、ＰＲ及ＣＥＡ的表达未显示密切的关联。     

OVX1 and CEA in patients with colon carcinoma, colon polyps and benign colon disorders

The OVX1 tumor marker promises to complement CA125 for detection of early stage ovarian carcinoma. OVX1 has also been shown to be elevated in colon cancer patients. This study is designed to assess serum OVX1 levels in patients with specific stages of colon cancer, colon polyps or other GI disorders. Serum OVX1 and CEA were measured by radioimmunoassay or enzyme immunoassay for 206 patients at the time of colonoscopy or staging for colon carcinoma. In patients with stage I, II, III, or IV colon carcinoma, serum OVX1 was positive in 37%, 48%, 74% and 63%, respectively. Fifty-three percent of patients with colon polyps had elevated OVX1 levels, while OVX1 levels were positive in only 7% of healthy controls. If both OVX1 and CEA were considered, at least one of these markers was elevated in 36%, 60%, 79% or 89% of patients with stage I, II, III or IV colon carcinoma, respectively. The majority of patients with inflammatory bowel disease or diverticulosis also had elevated OVX1 levels. Both markers were positive in 27% of patients with colon carcinoma, and not in any patients with a normal colonoscopy or with a diagnosis of diverticulosis or hemorrhoids. In conclusion, serum OVX1 improves the sensitivity of CEA for detecting colon polyps and colon cancer; however, the use of OVX1 in this setting is hindered by its elevation in non-malignant colonic processes.     

Carcinoembryonic antigen and CD44 variant isoforms cooperate to mediate colon carcinoma cell adhesion to E- and L-selectin in shear flow

Selectin-mediated adhesion of tumor cells to platelets, leukocytes, and endothelial cells may regulate their hematogenous dissemination in the microvasculature. We recently identified CD44 variant isoforms (CD44v) as functional P-, but not E- or L-, selectin ligands on colon carcinoma cells. Moreover, an approximately 180-kDa sialofucosylated glycoprotein(s) mediated selectin binding in CD44-knockdown cells. Using immunoaffinity chromatography and tandem mass spectrometry, we identify this glycoprotein as the carcinoembryonic antigen (CEA). Blot rolling assays and flow-based adhesion assays using microbeads coated with CEA immunopurified from LS174T colon carcinoma cells and selectins as substrate reveal that CEA possesses E- and L-, but not P-, selectin ligand activity. CEA on CD44-knockdown LS174T cells exhibits higher HECA-452 immunoreactivity than CEA on wild-type cells, suggesting that CEA functions as an alternative acceptor for selectin-binding glycans. The enhanced expression of HECA-452 reactive epitopes on CEA from CD44-knockdown cells correlates with the increased CEA avidity for E- but not L-selectin. Through the generation of stable knockdown cell lines, we demonstrate that CEA serves as an auxiliary L-selectin ligand, which stabilizes L-selectin-dependent cell rolling against fluid shear. Moreover, CEA and CD44v cooperate to mediate colon carcinoma cell adhesion to E- and L-selectin at elevated shear stresses. The novel finding that CEA is an E- and L-selectin ligand may explain the enhanced metastatic potential associated with tumor cell CEA overexpression and the supportive role of selectins in metastasis.     

Redirected perforin-dependent lysis of colon carcinoma by ex vivo genetically engineered CTL

The redirection of autologous lymphocytes to predefined tumor target Ags has considerable potential for the immunotherapeutic treatment of cancer; however, robust experimental systems for comparing various approaches have not been developed. Herein, we have generated a single chain variable domain anti-carcinoembryonic Ag (CEA) Fcepsilon receptor I gamma-chain fusion (scFv anti-CEA) receptor and demonstrated high-level expression of this chimeric receptor in naive mouse T lymphocytes by retroviral gene transduction. These gene-modified CTL were able to lyse CEA+ targets and secrete high levels of IFN-gamma following Ag stimulation. Depletion studies demonstrated that specific tumor cell cytotoxicity was mediated by gene-modified CD8+ T cells. Importantly, in increasingly stringent tests of efficacy in vivo, transduced CTL were sequentially shown to reject CEA+ colon carcinoma cells in a Winn assay and then reject established s.c. colon carcinoma in scid or syngeneic mice. Furthermore, using gene-targeted and scFv anti-CEA receptor-transduced donor CTL, perforin and IFN-gamma were demonstrated to be absolutely critical for the eradication of colon carcinoma in mice. In summary, we have developed a highly efficient gene transfer system for evaluating chimeric receptor expression in cytotoxic lymphocytes. This series of experiments has revealed the utility of scFv anti-CEA chimeras in providing mouse T cells the capacity to reject colon carcinoma in an Ag- and perforin-specific manner.     

The usefulness of a panel of immunostains in the diagnosis and differentiation of metastatic malignancies in pericardial effusions

Pericardial effusions are not uncommon in patients with an advanced malignancy Rarely malignancies may present initially with a pericardial effusion. Cytological examination of pericardial fluid may be valuable in differentiation of these cases. However, a metastatic tumour in serous effusion may not always show the functional differentiation of the primary tumour. In such a situation, although a wide range of special studies have been suggested for the diagnosis of malignancy we have found the use of a panel of a few common immunostains to be useful in confirming or suggesting the site of a primary tumour. The material for this study consisted of 76 pericardial fluids obtained between January 1991 and October 1998 from 46 males (mean age 59 years) and 30 females (mean age 52 years). Metastatic malignancy was diagnosed in 22 of the 76 patients and in 7/22 cases pericardial effusions were the initial presentation. The subsequent follow-up in the seven cases revealed adenocarcinoma of lung (n = 2), small cell anaplastic carcinoma of lung (n = 1), squamous cell carcinoma lung (n = 1), melanoma leg (n = 1), non-Hodgkin's lymphoma retroperitoneal lymph nodes (n = 1) and carcinoma of the breast (n = 1). Of the remaining 15 cases with a known history of malignancy, eight had cancers (three adeno; two small cell; one poorly differentiated, and two squamous cell types) of the lung; breast (n = 3); colon (n = 1); melanoma (n = 2) and non Hodgkin's lymphoma (n = 1). Immunostains which were useful in the diagnosis were EMA, CEA, cytokeratin, B72.3, HMB45, vimentin, S100, LCA, L26 and kappa and lambda light chains.     

Cellular growth modulation. I. Effects of extracellular matrix and tumor cell conditioned medium

Earlier studies reported the enzymatic modulation of the cell surface in malignant transformation of human normal mammary epithelial cells and in conversion of mammary carcinoma. Carcinoembryonic antigen (CEA) is a neoplasm-associated antigen, its production and release is used to monitor changes in cell phenotype. The present study shows that CEA production and release by human colon carcinoma (CCC), and by colon cells from patients with familial polyposia coli (FPC) and ulcerative colitis (UCC) is inhibited when the cells are cultured in contact with confluent normal colon epithelial (HNCEC) cell monolayer. Footprints left behind and/or conditioned media from HNCEC cells inhibited, whereas footprints left behind and/or conditioned media from CCC, FPC or Ucc enhanced CEA release. During sequential passages of HNCEC cells grown on footprints and/or in spent media from CCC cultures, HNCEC cells acquire the ability to produce and release CEA, and to develop tumors in athymic Nu/Nu mice. On the other hand, during sequential passages, CCC, FPC or UCC grown in spent media, or on footprints left behind HNCEC cells, showed significant decrease in CEA production and release, and in oncologic ability in athymic mice. It is concluded that both the extracellular matrix, and a growth-regulating factor(s) in the spent medium modulate cellular transformation. Quantitative data on CEA-release indicate that FPC and UCC represent an intermediary stage between normal colon epithelial cells and colon carcinoma cells, i.e. a preneoplastic stage.     

Carcinoembryonic antigen content in fine needle aspirates of the lung. A diagnostic adjunct to cytology.

Carcinoembryonic Antigen (CEA) was measured in 59 consecutive fine needle aspirates (FNAs) of the lung from 58 patients to determine if the CEA content would enhance the sensitivity of the cytologic diagnosis. Twenty-eight males and 30 females with tumors 1-40 cm in diameter were studied. Final diagnoses were correlated with the clinical history, radiologic studies, tissue (when available) and follow-up. Image-guided FNAs were performed by radiologists using a 22-gauge Chiba needle and 20-mL syringe with one to four passes per specimen. Cytologic examination included rapid assessment in the radiology suite and a final diagnosis in 24 hours. CEA was measured by enzyme immunoassay using monoclonal antibody. Nine benign aspirates and 50 malignant aspirates were diagnosed. The sensitivity of cytology was 86% and specificity, 100%. Using 5 ng/mL as the cutoff, the sensitivity of CEA for malignant aspirates was 50% and specificity, 90%. The combined sensitivity of CEA and cytology was 95%. The mean CEA in malignant aspirates was 131 ng/mL and in benign aspirates, 2.41. The highest mean CEA was seen in adenocarcinoma, 402.6 ng/mL. Lower CEA content was seen in epidermoid carcinoma (58.6 ng/mL), large cell carcinoma (8.09), oat cell carcinoma, metastatic carcinoma of the kidney and breast, thymoma and lymphoma (each less than 1 ng/mL). Elevated CEA alone was diagnostic in two aspirates of bronchioloalveolar carcinoma; carcinoma with an unknown primary source, three; and large cell carcinoma, one. The adjunctive use of CEA in FNAs of the lung enhances the sensitivity of the cytologic diagnosis.     

Dendritic cell-mediated cross-presentation of antigens derived from colon carcinoma cells exposed to a highly cytotoxic multidrug regimen with gemcitabine, oxaliplatin, 5-fluorouracil, and leucovorin, elicits a powerful human antigen-specific CTL response with antitumor activity in vitro

Gemcitabine, oxaliplatin, leucovorin, and 5-fluorouracil (GOLF) is a novel multidrug regimen inducing high levels of necrosis and apoptosis in colon carcinoma cells. This regimen is also able to promote a process of Ag remodeling including up-regulation of immunotherapy targets like carcinoembryonic Ag (CEA), thymidylate synthase (TS). We have conducted a preclinical study aimed to investigate whether these drug-induced modifications would also enhance colon cancer cell immunogenicity. Several CTL lines were thus generated by in vitro stimulating human HLA-A(*)02.01(+) PBMCs, from normal donors and colon cancer patients, with autologous dendritic cells cross-primed with cell lysates of colon cancer cells untreated, irradiated, or previously exposed to different drug treatments including the GOLF regimen. Class I HLA-restricted cytolytic activity of these CTL lines was tested against colon cancer cells and CEA and TS gene transfected target cells. These experiments revealed that CTLs sensitized with GOLF-treated cancer cells were much more effective than those sensitized with the untreated colon carcinoma cells or those exposed to the other treatments. CTL lines sensitized against the GOLF-treated colon cancer cells, also expressed a greater percentage of T-lymphocyte precursors able to recognize TS- and CEA-derived peptides. These results suggest that GOLF regimen is a powerful antitumor and immunomodulating regimen that can make the tumor cells a suitable means to induce an Ag-specific CTL response. These results suggest that a rationale combination of GOLF chemotherapy with cytokine-based immunotherapy could generate a chemotherapy-modulated Ag-specific T-lymphocyte response in cancer patients able to destroy the residual disease survived to the cytotoxic drugs.     

结肠腺瘤的免疫组织化学及组织化学研究

本文应用免疫组织化学CEA、MC_5(PAP法)和组织化学AB(PH2.5)/PAS、HID/AB(PH2.5)染色方法,对43例结肠腺瘤、52例结肠腺瘤伴中、重度不典型增生,13例结肠腺瘤伴癌变,15例结肠腺癌和10例正常结肠组织进行了相关抗原及大肠粘蛋白的研究探讨。结果发现五类组织之间其着色含量都呈现一定的差异,尤其是随着腺瘤的异型程度增大,这种差异更为明显。本实验结果提示,上述染色法对结肠腺瘤癌变的逐渐演变过程的变化具有一定的参考价值。     

Ulex europeus type I agglutinin detects carcinoembryonic antigen in extracts of human colorectal carcinoma

Recent interest has focused on fucosylated epitopes expressed on human neoplasms. The plant lectin Ulex europus agglutinin, Type I (UEA) binds fucosylated oligosaccharides, while UEA-reactive substances have a tissue distribution similar to carcinoembryonic antigen (CEA). We sought to determine if UEA reacted with CEA in extracts of fresh primary and metastatic colorectal carcinomas and paired normal tissues. The extracts were electrophoretically transferred to nitrocellulose membranes after the proteins were separated by SDS-PAGE in 10% polyacrylamide gels. The transfer membranes were then stained with peroxidase-conjugated UEA (UEA-P) or antibody to CEA (CEA-P). UEA-P reacted with a 170-190-kDa band in extracts of 22 of 30 primary tumors, 10 of 12 metastases, but only 1 of 5 villous adenomas. UEA-P generally did not react with normal colon or liver extracts. UEA-P also did not bind to 170-190-kDa molecules in Western transfers of a breast carcinoma metastatic to bowel and a focal nodular hyperplasia of liver. CEA-P displayed similar reactivity and detected CEA in a tumor extract negative for UEA. Fucose blocked binding of UEA-P to Western transfers of tumor extracts. CEA-P reacted with a 170-190-kDa substance in tumor extracts eluted with fucose from a column of immobilized UEA. Thus, UEA reacts with fucosylated oligosaccharides on most, but not all, species of CEA and may be a useful adjunct to anti-CEA immunohistochemistry.     

Immunohistochemical analysis of p53 and HER-2/neu proteins in human tumors.

We examined samples of tumors of human breast, ovary, and colon of various degrees of malignancy for the expression of p53 protein, using a panel of anti-p53 antibodies and peroxidase immunohistochemistry. Of 66 tumor cases (24 cases of ovarian carcinoma, 23 cases of colon adenocarcinoma, and 19 cases of breast carcinoma), 36 (53%) showed high levels of expression of p53 using a human-specific antibody, and 16 (24%) showed high expression of a mutant form of p53. In the mutant p53-positive breast tumor samples, six (86%) were positive for HER-2/neu reactivity, compared with colon (0/4) and ovarian tumors (1/5). The pattern of p53 intracellular localization and tissue distribution, and the relationship between the expression of mutant p53 and cell differentiation, were also examined; poorly differentiated cells showed either overexpression of p53 or higher levels of mutant p53 in comparison with more normal cells.     

A dual-function DNA vaccine encoding carcinoembryonic antigen and CD40 ligand trimer induces T cell-mediated protective immunity against colon cancer in carcinoembryonic antigen-transgenic mice

A carcinoembryonic Ag (CEA)-based DNA vaccine encoding both CEA and CD40 ligand trimer achieved effective tumor-protective immunity against murine colon carcinoma in CEA-transgenic mice by activating both naive T cells and dendritic cells. Peripheral T cell tolerance to CEA was broken in a prophylactic model by this novel, dual-function DNA vaccine, whose efficacy was further enhanced by boosts with a recombinant Ab-IL-2 fusion protein (huKS1/4-IL-2). These conclusions are supported by four lines of evidence. First, a lethal challenge of MC38-CEA-KS Ag murine colon carcinoma cells was for the first time completely rejected in 100% of experimental animals treated by oral gavage of this DNA vaccine carried by attenuated Salmonella typhimurium, followed by five boosts with huKS1/4-IL-2. Second, specific activation of dendritic cells was indicated by their marked up-regulation in expression of costimulatory molecules B7.1 (CD80), B7.2 (CD86), and ICAM-1. Third, a decisive increase over control values was observed in both MHC class I Ag-restricted cytotoxicity of CTLs from successfully vaccinated mice and secretion of proinflammatory cytokines IFN-gamma and IL-12. Fourth, activation of CTLs was augmented, as indicated by up-regulation of activity markers LFA-1, CD25, CD28, and CD69. Taken together, these results suggest that a dual-function DNA vaccine encoding CEA and CD40 ligand trimer combined with tumor-targeted IL-2 may be a promising strategy for the rational development of DNA-based cancer vaccines for future clinical applications.