Carcinoembryonic Antigen Gene Research Articles

Pathological classification of malignant peritoneal mesothelioma and comparative analysis with peritoneal carcinomatosis

Objective: To analyze the pathological classification of malignant peritoneal mesothelioma (MPeM) and screen the immunohistochemical markers that can distinguish MPeM from peritoneal metastatic carcinoma (PC) . Methods: In June 2020, the pathological results of peritoneal biopsy of 158 MPeM and 138 PC patients from Cangzhou Central Hospital, Cangzhou People's Hospital, and Cangzhou Hospital of Integrated Traditional Chinese and Western Medicine from May 2011 to July 2019 were retrospectively analyzed, and the pathological classifications of MPeM in Cangzhou were summarized. Immunohistochemical markers of MPeM and PC patients were analyzed, and receiver operating characteristic curve (ROC curve) was drawn for differential diagnosis of MPeM and PC. Results: There were 55 male and 103 female MPeM patients in Cangzhou, with an average age of 57.1 years old. The asbestos exposure rate was 91.14% (144/158). The most common pathological classifications were cutaneous type, accounting for 90.51% (143/158). There were significant differences in the expression of calreticulum protein, CK5/6, vimentin, D2-40, carcinoembryonic antigen (CEA) and tail type homologous nuclear gene transcription factor 2 (CDX-2) between MPeM and PC (P<0.05). Among the 6 positive markers, the sensitivity of calreticulum protein was the highest (0.905) and CEA was the lowest (0.428) . Conclusion: Calreticulum protein, CK5/6, vimentin, D2-40, CEA and CDX-2 may be used as specific markers to distinguish the diagnosis of MPeM from PC.

Comparison of Three Different Methods of Transfection for the Production of Recombinant Adenovirus Expressing Human Carcinoembryonic Antigen Gene.

Adenoviral vectors (AdVs) are widely used as a gene delivery vehicle and vaccine design due to their genetic stability, transfer capacity of large genes, production at high titers, and remarkable efficacy of transduction. One of the most important applications of AdVs is in cancer immunotherapy. Tumor-associated antigens are overexpressed in cancer cells; however, they cannot induce immune responses sufficiently. Therefore, the immune system must be stimulated against these antigens to kill the cancer cells. This study described the construction steps of a recombinant AdV expressing human carcinoembryonic antigen (CEA) gene. Furthermore, in order to achieve a high titer of the virus, an efficient transfection was required. Three various transfection reagents were compared to achieve the best method of transfection. Carcinoembryonic antigen was cloned into the pAdV and transfected into the A293 cells using three different reagents, including polyethylenimine (PEI), calcium phosphate, and DMRIE-C. The PEI had the highest transfection efficiency, which was selected for the transfection of the recombinant plasmid. It has low toxicity for cells and is suitable for large-scale transfection. The virus produced in this study can be applied as a vaccine in cancer immunotherapy for stimulating the immune system against CEA-expressing tumors.

Effect of extracellular vesicles of Lactobacillus rhamnosus GG on the expression of CEA gene and protein released by colorectal cancer cells.

Background and Objectives:Almost all living cells secret nano-sized structures enclosed by the lipid bilayer called extracellular vesicles (EVs) into their extracellular milieu. These EVs play important roles in several physiological processes as a cargo delivery system. In probiotics, EVs are the main communication tool with the host. The present study aimed to assess the effect of EVs originated from Lactobacillus rhamnosus GG on the Carcinoembryonic antigen (cea) gene expression and protein (CEA) synthesis in the SW480 and HT-29 cell lines.Materials and Methods:Different concentrations of Lactobacillus rhamnosus GG EVs were applied on the SW480 and HT-29 cell lines. The MTT assay, Real-Time PCR, and ELISA analysis methods were exploited to explore the cell viability and the expression level of the cea gene in comparison with the β-actin gene as the control.Results:The two concentrations of 80 and 100 μg/ml of Lactobacillus rhamnosus GG EVs considerably affected the anti-proliferation and increased the amount of both CEA mRNA and protein (p < 0.05).Conclusion:Our findings showed that EVs of Lactobacillus rhamnosus GG could induce the gene expression and protein synthesis of CEA. Also, they reduced the cell proliferation of HT29 and SW480. Thus, probiotics such as EVs of Lactobacillus rhamnosus GG could be useful for preventing colorectal cancer.

The immune-modulating pregnancy-specific glycoproteins evolve rapidly and their presence correlates with hemochorial placentation in primates

BackgroundPregnancy-specific glycoprotein (PSG) genes belong to the carcinoembryonic antigen (CEA) gene family, within the immunoglobulin gene superfamily. In humans, 10 PSG genes encode closely related secreted glycoproteins. They are exclusively expressed in fetal syncytiotrophoblast cells and represent the most abundant fetal proteins in the maternal blood. In recent years, a role in modulation of the maternal immune system possibly to avoid rejection of the semiallogeneic fetus and to facilitate access of trophoblast cells to maternal resources via the blood system has been suggested. Alternatively, they could serve as soluble pathogen decoy receptors like other members of the CEA family. Despite their clearly different domain organization, similar functional properties have also been observed for murine and bat PSG. As these species share a hemochorial type of placentation and a seemingly convergent formation of PSG genes during evolution, we hypothesized that hemochorial placentae support the evolution of PSG gene families.ResultsTo strengthen this hypothesis, we have analyzed PSG genes in 57 primate species which exhibit hemochorial or epitheliochorial placentation. In nearly all analyzed apes some 10 PSG genes each could be retrieved from genomic databases, while 6 to 24 PSG genes were found in Old World monkey genomes. Surprisingly, only 1 to 7 PSG genes could be identified in New World monkeys. Interestingly, no PSG genes were found in more distantly related primates with epitheliochorial placentae like lemurs and lorises. The exons encoding the putative receptor-binding domains exhibit strong selection for diversification in most primate PSG as revealed by rapid loss of orthologous relationship during evolution and high ratios of nonsynonymous and synonymous mutations.ConclusionThe distribution of trophoblast-specific PSGs in primates and their pattern of selection supports the hypothesis that PSG are still evolving to optimize fetal-maternal or putative pathogen interactions in mammals with intimate contact of fetal cells with the immune system of the mother like in hemochorial placentation.

Abstract 606: Circulating proteins associated with ovarian cancer risk and survival are enriched for immune-related pathways

Abstract Most women with epithelial ovarian cancer (EOC) are diagnosed after the disease has metastasized and five-year survival rates in this group continue to remain very poor. Circulating proteins may illuminate EOC etiology and have the potential to serve as biomarkers for early detection and diagnosis. We performed a two-sample Mendelian randomization (MR) study with circulating protein levels as exposures and susceptibility to EOC and its histological subtypes as outcomes. We used previously identified quantitative trait loci (QTLs) robustly associated with plasma levels of 1,478 proteins as genetic instruments and corresponding summary genetic association statistics for these instruments derived from 22,406 EOC cases and 40,941 controls. Among proteins that had only a single QTL variant available as a genetic instrument, we identified 12 proteins whose circulating levels were associated with overall EOC risk at a False Discovery Rate (FDR) of &amp;lt; 0.1. For nine of the 12 proteins, the association was driven by variants near ABO, which is both a genome-wide significant high grade serous histotype risk locus and a hub QTL for several proteins. Three of these 12 proteins and their QTLs were, however, not linked to the ABO locus and included the products of the NOTCH signaling genes MFAP2 and DLK1. Among proteins that had multiple QTL variants available for the creation of more robust multi-variant genetic instruments, we identified 33 protein level-EOC risk associations at FDR &amp;lt; 0.1. Once again, we observed that the majority of associations (19/33) were driven by variants at the ABO locus. Nine of the 14 remaining associations were for overall and high grade serous EOC risk and included predominantly immune system proteins: ARTS1 (HLA class I), CPNE1 (involved in innate immunity), Macrophage mannose receptor (HLA class I), DQA2 (HLA class II), RGAP1 (innate immunity), Cathepsin H (innate immunity), and FCG2B (the low affinity receptor for the Fc region of immunoglobulin gamma). We observed one mucinous histotype-specific association (complement C4) and two clear cell histotype-specific associations (KI2S2 and PSG4). KI2S2 is a Killer cell immunoglobulin-like receptor expressed by natural killer cells and PSG4 is a member of the carcinoembryonic antigen gene family that also includes the gene encoding the tumor marker for colorectal cancer. Finally, we also performed two-sample MR analyses using survival as the outcome and identified two proteins whose plasma levels were associated with overall EOC survival at FDR &amp;lt; 0.1: SULT1E, a phenol sulfotransferase that conjugates sulfates to estrogens, and MA2B2. Thus, we identified circulating proteins that are candidate biomarkers for EOC risk and survival. They represent promising starting points for follow-up studies and the measurement of plasma levels of these proteins in EOC cohorts is required to validate these associations. Citation Format: Daniel P. Considine. Circulating proteins associated with ovarian cancer risk and survival are enriched for immune-related pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 606.

DNA methylation of HDAC4 gene affects the trans-differentiation process of hMSCs into sweat gland like cells

To investigate the changesof DNA methylation in histone deacetylases 4 gene (HDAC4) and its effectduring the trans-differentiation process of human mesenchymal stem cells (hMSCs) into sweat gland like cells (SGLCs). Selected cell lines of human mesenchymal stem cells (hMSCs) were cultured and expended in vitro, the third generation ofhMSCs and heat-shocked sweat gland cells were picked up, and were co-culturedwith adding inducible factor in the transwell chamber. The sweat gland like cells (SGLCs)in experiment group and the hMSCs in control group were collected, the changes of DNA methylation degree of CpG dinucleotide sitesin histone deacetylases 4 gene (HDAC4) promotor were detected by methylation specific PCR (MSP)andMaldi-TOF Mass Array. And then, the hMSCs in experiment group were treated with 5-aza-CdR (5-aza-2-deoxycytidine, 10 μmol/L), while the hMSCsin control group were culturedwith PBS at the same time. ThemRNA expressions of HDAC4 gene and carcino-embryonic antigen (CEA)gene in the two groups were measured by RT-PCR. The methylation of HDAC4gene in hMSCs was in high level before induction, the methylation degreeof CpG dinucleotide sites located in cg2463009 was 0.901, and the methylation degree of HDAC4gene in SGLCs was markedly decreased by 37% after induction, which was 0.531. The methylationlevel of CpG dinucleotide sites located in cg14823429was changed from 0.687to 0.386 after induction. The mRNA expression of HDAC4 gene was upregulated in test group after treated with 5-aza-CdR for 48 hours, the mRNA expression of CEA gene related with transdifferentiation was enhanced too at the same term, there was significantly statistic difference compared with control group (P<0.05). Methylation of HDAC4 gene participates in the regulation of the trans-differentiation of hMSCs into sweet gland like cells.

Recent expansion and adaptive evolution of the carcinoembryonic antigen family in bats of the Yangochiroptera subgroup

BackgroundExpansions of gene families are predictive for ongoing genetic adaptation to environmental cues. We describe such an expansion of the carcinoembryonic antigen (CEA) gene family in certain bat families. Members of the CEA family in humans and mice are exploited as cellular receptors by a number of pathogens, possibly due to their function in immunity and reproduction. The CEA family is composed of CEA-related cell adhesion molecules (CEACAMs) and secreted pregnancy-specific glycoproteins (PSGs). PSGs are almost exclusively expressed by trophoblast cells at the maternal-fetal interface. The reason why PSGs exist only in a minority of mammals is still unknown.ResultsAnalysis of the CEA gene family in bats revealed that in certain bat families, belonging to the subgroup Yangochiroptera but not the Yinpterochiroptera subgroup an expansion of the CEA gene family took place, resulting in approximately one hundred CEA family genes in some species of the Vespertilionidae. The majority of these genes encode secreted PSG-like proteins (further referred to as PSG). Remarkably, we found strong evidence that the ligand-binding domain (IgV-like domain) of PSG is under diversifying positive selection indicating that bat PSGs may interact with structurally highly variable ligands. Such ligands might represent bacterial or viral pathogen adhesins. We have identified two distinct clusters of PSGs in three Myotis species. The two PSG cluster differ in the amino acids under positive selection. One cluster was only expanded in members of the Vespertilionidae while the other was found to be expanded in addition in members of the Miniopteridae and Mormoopidae. Thus one round of PSG expansion may have occurred in an ancestry of all three families and a second only in Vespertilionidae. Although maternal ligands of PSGs may exist selective challenges by two distinct pathogens seem to be likely responsible for the expansion of PSGs in Vespertilionidae.ConclusionsThe rapid expansion of PSGs in certain bat species together with selection for diversification suggest that bat PSGs could be part of a pathogen defense system by serving as decoy receptors and/or regulators of feto-maternal interactions.

High expression of CEACAM19, a new member of carcinoembryonic antigen gene family, in patients with breast cancer.

Carcinoembryonic antigen (CEA) family members play important roles in malignancies and are introduced as biomarkers in different types of cancers. Among them CEACAM19 (CEAL1) gene, a new member of the CEA family, remains to be fully elucidated. The aim of this study was investigating the mRNA expression level of CEACAM19 in tumor samples of breast cancer patients compared to breast tissue of normal individuals. We evaluated the expression level of this gene in 75 breast tumors by using real-time quantitative PCR. Also, we studied the correlation between CEACAM19 expression and clinicopathological features and hormone receptors status, including estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 of patients. Out of the enrolled patients, six of them (7.9%) showed low expression, ten (13.2%) showed normal expression and 59 (77.6%) showed high expression of CEACAM19. There was a significant correlation between high expression of CEACAM19 gene in tumor samples compared to normal tissues (P=0.039). No significant correlation was seen between clinicopathological factors and disease-free survival with mRNA levels of CEACAM19 in tumor samples, while the difference between the expression of CEACAM19 in ER/PR-positive and ER/PR-negative breast cancer patients was statistically significant (P=0.046). In conclusion, CEACAM19 showed high expression in tumor samples compared to normal mammary tissue. In addition, CEACAM19 may represent as a novel therapeutic target in certain subgroups of breast cancer patients such as ER/PR-negative. Critical roles of CEA proteins in tumor progression may nominate them as robust potential targets for therapeutic intervention in near future.

Phase I study of alphaviral vector (AVX701) in colorectal cancer patients: comparison of immune responses in stage III and stage IV patients

Meeting abstracts AVX701 is an alphaviral replicon particle vaccine expressing a modified carcinoembryonic antigen gene (CEA(6D)). We previously reported the preliminary results of a Phase I/II study of AVX701 in metastatic colorectal cancer patients (Morse, J Clin Invest. 2010; 120: 3234–3241).

Prognostic value of genetic detection using CEA and MAGE in peritoneal washes with gastric carcinoma after curative resection: result of a 3-year follow-up.

Peritoneal metastasis is the most frequent cause of death in patients with gastric cancer. Reverse transcriptase-polymerase chain reaction (RT-PCR) assay of peritoneal washes has been used to predict peritoneal metastasis of gastric carcinoma. We applied carcinoembryonic antigen (CEA) and melanoma-associated gene (MAGE) RT-PCR for the detection of peritoneal metastasis of gastric carcinoma after curative surgery and evaluated its clinical significance.Peritoneal washes were obtained from 117 patients with gastric carcinoma. MAGE A1–A6 and CEA RT-PCR were performed, and the results were evaluated according to their clinicopathologic characteristics. Three-year follow-up clinical studies were periodically performed, and disease-free survival rates were retrospectively investigated using the medical records.Among 117 peritoneal fluids, 11 cases (9.4%) revealed MAGE expression and 38 cases (32.5%) revealed CEA expression. When focusing on recurrence rates, RT-PCR-positive had much higher recurrence rates than RT-PCR-negative cases (32.5% vs 5.2%, P < 0.01). Univariate analysis revealed that depth of invasion, lymph node metastasis, tumor node metastasis (TNM) stage, Lauren classification, and MAGE and CEA expressions were independent prognostic factors for recurrence. In a multivariate analysis, MAGE expression and TNM stage were significantly and independently related to recurrence in patients who underwent curative resection. MAGE expression was determined to be the most important prognostic factor for recurrence (hazard ratio: 12.487, P < 0.01).It is feasible to identify free cancer cells in peritoneal lavage by using a MAGE A1–A6 and CEA RT-PCR. MAGE RT-PCR results disclosed significant associations with peritoneal recurrence and proved to be the most important factor for the recurrence rate in patients with gastric carcinoma who had undergone radical resection.

A comprehensive phylogenetic and structural analysis of the carcinoembryonic antigen (CEA) gene family.

The carcinoembryonic antigen (CEA) gene family belongs to the immunoglobulin (Ig) superfamily and codes for a vast number of glycoproteins that differ greatly both in amino acid composition and function. The CEA family is divided into two groups, the carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) and the pregnancy-specific glycoproteins. The CEA family members are implicated in pleiotropic (patho)physiological functions including cell–cell adhesion, pregnancy, immunity, neovascularization, regulation of insulin homeostasis, and carcinogenesis. In general, the CEA-encoded proteins are composed of an extracellular region with Ig variable and constant-like domains and a cytoplasmic region containing signaling motifs. Of particular interest, the well-studied human and mouse CEA genes are arranged in clusters in a single chromosome. Taking into account this characteristic, we made an effort to reconstruct the evolutionary history of the CEA gene family. Toward this end, the publicly available genomes were searched extensively for CEA homologs. The domain organization of the retrieved protein sequences was analyzed, and, subsequently, comprehensive phylogenetic analyses of the entire length CEA homologous proteins were performed. A series of evolutionarily conserved amino acid residues, functionally important, were identified. The relative positioning of these residues on the modeled tertiary structure of novel CEA protein domains revealed that they are, also, spatially conserved. Furthermore, the chromosomal arrangement of CEA genes was examined, and it was found that the CEA genes are preserved in terms of position, transcriptional orientation, and number in all species under investigation.

Microfluidic bead-based multienzyme-nanoparticle amplification for detection of circulating tumor cells in the blood using quantum dots labels

This study reports the development of a microfluidic bead-based nucleic acid sensor for sensitive detection of circulating tumor cells in blood samples using multienzyme-nanoparticle amplification and quantum dot labels. In this method, the microbeads functionalized with the capture probes and modified electron rich proteins were arrayed within a microfluidic channel as sensing elements, and the gold nanoparticles (AuNPs) functionalized with the horseradish peroxidases (HRP) and DNA probes were used as labels. Hence, two signal amplification approaches are integrated for enhancing the detection sensitivity of circulating tumor cells. First, the large surface area of Au nanoparticle carrier allows several binding events of HRP on each nanosphere. Second, enhanced mass transport capability inherent from microfluidics leads to higher capture efficiency of targets because continuous flow within micro-channel delivers fresh analyte solution to the reaction site which maintains a high concentration gradient differential to enhance mass transport. Based on the dual signal amplification strategy, the developed microfluidic bead-based nucleic acid sensor could discriminate as low as 5fM (signal-to-noise (S/N)3) of synthesized carcinoembryonic antigen (CEA) gene fragments and showed a 1000-fold increase in detection limit compared to the off-chip test. In addition, using spiked colorectal cancer cell lines (HT29) in the blood as a model system, the detection limit of this chip-based approach was found to be as low as 1 HT29 in 1mL blood sample. This microfluidic bead-based nucleic acid sensor is a promising platform for disease-related nucleic acid molecules at the lowest level at their earliest incidence.

A comparative analysis of K-ras mutation and carcinoembryonic antigen in pancreatic cyst fluid

Background/aimsAnalysis of cystic fluid may be useful in distinguishing between benign and malignant cysts which has significant impact on their management. The aim of our study was to assess the diagnostic utility of carcinoembryonic antigen (CEA) and K-ras gene mutation in pancreatic cysts fluid. MethodsThe study included 56 patients with pancreatic cystic fluid collected for analysis. The cysts were classified as benign (simple cysts, pseudocysts, serous cystadenoma) - 39 patients or premalignant/malignant (mucinous cystadenoma, IPMN, cystadenocarcinoma) - 17 patients. The patients history, CEA fluid concentrations and presence of K-ras mutation were analyzed. ResultsCEA were higher in patients with malignant cysts (mean levels 238 ± 12.5 ng/ml; range 32.8–4985 ng/ml) compared to benign lesions (mean levels 34.5 ± 3.7 ng/ml; range 3.9–693 ng/ml; p < 0.001). K-ras mutation correctly classified 11 of 17 patients with premalignant/malignant lesions. It was also detected in 1 patient with final diagnosis of benign cyst (the sensitivity 64.7% and the specificity 97.4%; p < 0.01). If CEA and molecular analysis were combined in that cysts with either CEA level>45 ng/ml or presence of K-ras mutation, than 16 of 17 premalignant/malignant cysts were correctly identified (94.1%). ConclusionMolecular analysis of pancreatic cyst fluid adds diagnostic value to the preoperative diagnosis and should be considered when cyst cytologic examination is negative for malignancy.

Loss of Mammal-specific Tectorial Membrane Component Carcinoembryonic Antigen Cell Adhesion Molecule 16 (CEACAM16) Leads to Hearing Impairment at Low and High Frequencies

The vertebrate-restricted carcinoembryonic antigen gene family evolves extremely rapidly. Among their widely expressed members, the mammal-specific, secreted CEACAM16 is exceptionally well conserved and specifically expressed in the inner ear. To elucidate a potential auditory function, we inactivated murine Ceacam16 by homologous recombination. In young Ceacam16(-/-) mice the hearing threshold for frequencies below 10 kHz and above 22 kHz was raised. This hearing impairment progressed with age. A similar phenotype is observed in hearing-impaired members of Family 1070 with non-syndromic autosomal dominant hearing loss (DFNA4) who carry a missense mutation in CEACAM16. CEACAM16 was found in interdental and Deiters cells and was deposited in the tectorial membrane of the cochlea between postnatal days 12 and 15, when hearing starts in mice. In cochlear sections of Ceacam16(-/-) mice tectorial membranes were significantly more often stretched out as compared with wild-type mice where they were mostly contracted and detached from the outer hair cells. Homotypic cell sorting observed after ectopic cell surface expression of the carboxyl-terminal immunoglobulin variable-like N2 domain of CEACAM16 indicated that CEACAM16 can interact in trans. Furthermore, Western blot analyses of CEACAM16 under reducing and non-reducing conditions demonstrated oligomerization via unpaired cysteines. Taken together, CEACAM16 can probably form higher order structures with other tectorial membrane proteins such as α-tectorin and β-tectorin and influences the physical properties of the tectorial membrane. Evolution of CEACAM16 might have been an important step for the specialization of the mammalian cochlea, allowing hearing over an extended frequency range.

Recent Advances in Active Specific Cancer Vaccine Treatment for Colorectal Cancer

Cloning techniques to identify genes and peptides of tumor-associated antigens have created new possibilities for the immunotherapy of patients with advanced cancer. Here, we review recent clinical trials of specific cancer vaccines, mainly HLA-restricted peptides, and epitope-encoding vectors for advanced colorectal cancer (CRC). Many researchers initially focused on carcinoembryonic antigen (CEA) as an immunologic target antigen that is overexpressed on virtually all CRCs. A recombinant vaccine containing the CEA gene and dendritic cells (DCs) loaded with CEA peptide was administered to patients with CEA-elevated CRC. Although CEA-specific responses were detected, the clinical responses were limited. Recently, new types of clinical trials--namely, a personalized protocol to take into account the immunological diversity of cytotoxic T cell responses among patients and a novel cancer-testis antigen protocol that uses multiple peptides derived from genes identified by the cDNA array method--have been introduced. The personalized protocol seemed to be better than the classical (non-personalized) protocol in terms of clinical response and survival. Novel cancer-testis antigen protocols that use multiple CRC-derived peptides were recently conducted in patients with advanced CRC. The preliminary study yielded promising results regarding specific T cell responses to peptides and survival benefits. In this review, we summarize these results and discuss future perspectives.

Enhancement of anti-murine colon cancer immunity by fusion of a SARS fragment to a low-immunogenic carcinoembryonic antigen

BackgroundIt is widely understood that tumor cells express tumor-associated antigens (TAAs), of which many are usually in low immunogenicity; for example, carcinoembryonic antigen (CEA) is specifically expressed on human colon cancer cells and is viewed as a low-immunogenic TAA. How to activate host immunity against specific TAAs and to suppress tumor growth therefore becomes important in cancer therapy development.ResultsTo enhance the immune efficiency of CEA in mice that received, we fused a partial CEA gene with exogenous SARS-CoV fragments. Oral vaccination of an attenuated Salmonella typhimurium strain transformed with plasmids encoding CEA-SARS-CoV fusion gene into BALB/c mice elicited significant increases in TNF-α and IL-10 in the serum. In addition, a smaller tumor volume was observed in CT26/CEA-bearing mice who received CEA-SARS-CoV gene therapy in comparison with those administered CEA alone.ConclusionThe administration of fusing CEA-SARS-CoV fragments may provide a promising strategy for strengthening the anti-tumor efficacy against low-immunogenic endogenous tumor antigens.

Biology of Krüppel‐like factor 6 transcriptional regulator in cell life and death

An essential role for the Krüppel-like transcription factor family has been determined in the regulation of remarkable processes including cell proliferation, differentiation, signal transduction, oncogenesis, and cell death. A member of this group, Krüppel-like factor 6 (KLF6), identified on the basis of its ability to regulate a group of genes belonging to the carcinoembryonic antigen gene family, has been involved in human carcinogenesis. Early studies proposed a tumor suppressor function for KLF6 because of its ability to reduce cell proliferation through several biochemical mechanisms including regulation of cell cycle components, oncogene products, and apoptosis. Mutations within the klf6 gene, decreased expression and/or loss-of-heterozygosity were associated with the development of different human malignancies, and, hence, further supporting the tumor suppressor function of KLF6. This view has been challenged by other studies in distinct types of human cancers describing infrequent genetic alterations of klf6 gene or even enhanced expression in some tumors. The scenario about KLF6 function became still more complex as the description of oncogenic KLF6 splice variant 1 (SV1) with dominant negative activity against the wild type KLF6 (wtKLF6) protein. Additionally, increased evidence is suggesting that KLF6 is a bonafide target of several signaling cascades, which ultimate regulatory effect on this protein could drive decisions of cell life and death, facing the dilemma about how wtKLF6 could be involved in both processes. These apparently conflicting situations, emerged by apparently opposite effects mediated by wtKLF6, may be related, at least in part, to the biological cross-talk with the c-Jun oncoprotein. Depending on the stimulus received by the cell, wtKLF6 interaction with c-Jun determines different cell outcomes such as proliferation control or apoptosis. Thus, KLF6 responsiveness represents a kind of cell warning signal on receiving different stimuli, including oncogenic activation and microbial infections, orchestrating the implementation of proliferation and apoptotic programs.

DNA vaccine expressing repeated carcinoembryonic antigen (CEA) 625–667 induces strong immunity in mice

The efficacy of immunization with DNA plasmids for single truncated carcinoembryonic antigen (CEA) peptide or triple repeated CEA peptides in mice was evaluated. A DNA fragment the truncated CEA gene (nucleotide 625–667) encoding two helper T lymphocyte (HTL) epitopes was amplified by PCR and cloned for generating recombinant plasmids for single CEA 625–667 (pcDNA-CEA 625–667) or triple CEA 625–667 (pcDNA-triCEA 625–667), respectively. Subsequently, groups of BALB/c female mice were intramuscularly injected with pcDNA-CEA 625–667, pcDNA-triCEA 625–667 or control pcDNA3.0 vector, respectively. Ten days after the last immunization, the frequency of splenic CD4 + and CD8 + T cells in the mice was determined by flow cytometry. The antigen-specific proliferation of splenic T cells and cytokine production ex vivo were analyzed by 3H-TdR uptake and cytokine ELISA, respectively. The levels of serum antibodies against CEA in the mice were detected by Western blot and ELISA. Although immunization with plasmid for the CEA 625–667 peptide(s) did not alter the frequency of CD4 + and CD8 + T cells in mice, vaccination with plasmid for CEA peptide induced strong antigen-specific T cell proliferation, particularly for the plasmid encoding the triple-repeated CEA peptides, accompanied by significantly elevated levels of IFN-γ secreted by T cells from the mice immunized with triple-repeated peptides. Furthermore, immunization with the plasmid for CEA peptide stimulated higher levels of antigen-specific antibody responses in mice. Vaccination with the plasmid for the triple repeated CEA peptides induced stronger Th1 responses. Our findings may be useful for the development of effective DNA vaccine for the immunotherapy of cancer.

Abstract 842: The detection of circulating tumor cells is a negative prognostic factor in colorectal cancer

Abstract Purpose: We tested the hypothesis that the presence of circulating tumor cells (CTCs) is a negative prognostic factor in patients with stage I-III colorectal cancer (CRC). Patients and methods: This was a prospective study to test for the presence of CTCs in peripheral blood and bone marrow in 182 colorectal cancer patients at the time of surgery and one month after surgery using real-time RT-PCR for carcinoembryonic antigen (CEA) and cytokeratin 20 (CK20). Results: Overall, 24 of 147 (16.3%) stage I-III CRC patients developed recurrence/metastasis postoperatively in a mean follow-up period of 22.9 months. There was no correlation between CTCs and conventional clinical pathological features. In total, 51.7% of patients were CTC positive detected in peripheral blood and/or bone marrow at the time of surgery using CEA and/or CK20 and 47% of patients were CTC positive one month after surgery. At surgery, CTC positive patients measured as CEA mRNA copy number in peripheral blood showed significantly poorer disease-free survival (DFS) (p=0.05). One month after surgery, both increased above threshold CEA and/or CK20 mRNA levels in bone marrow and CK20 mRNA level in blood correlated with shorter DFS (p=0.005, resp. p=0.0035). In addition, systemic increase in CEA gene expression, even if the values remained below cut-off threshold also correlated with poorer DFS (p=0.005). Conclusion: Detection of CTCs at surgery and/or one month after surgery is an independent negative prognostic factor for relapsing-free survival in stage I-III colorectal cancer. Acknowledgments of research support: This study was supported by grants MSM 151100001, IGA MZCR NR/7804-5 and IGA MZCR NR/9076. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 842.

Anti-tumor immunotherapy despite immunity to adenovirus using a novel adenoviral vector Ad5 [E1-, E2b-]-CEA.

Adenovirus serotype 5 (Ad5) has been widely used in clinical trials because it expresses inserted transgenes robustly and augments the innate immune response. Strategies to improve Ad5 vectors that can circumvent Ad5 immunity have become a critical issue, especially for use as a cancer immunotherapeutic in which repeated immunization is required. In this study, we constructed a novel Ad5 vector with unique deletions of the viral DNA polymerase and the pre-terminal protein region (Ad5 [E1-, E2b-]). This vector contains the carcinoembryonic antigen (CEA) gene insert and is designed to induce cell-mediated immunity (CMI) against the tumor-associated target. The CEA immunogenicity and in vivo anti-tumor effects of repeated immunizations with Ad5 [E1-, E2b-]-CEA compared with those observed with current generation Ad5 [E1-]-CEA were tested in Ad5 pre-immunized mice. We report that Ad5-immune mice immunized multiple times with Ad5 [E1-, E2b-]-CEA induced CEA-specific CMI responses that were significantly increased over those detected in Ad5-immune mice immunized multiple times with a current generation Ad5 [E1-]-CEA. Ad5 immune mice bearing CEA-expressing tumors that were treated with Ad5 [E1-, E2b-]-CEA had increased anti-tumor response as compared with Ad5 [E1-]-CEA treated mice. These results demonstrate that Ad5 [E1-, E2b-]-CEA can induce CMI immune responses which result in tumor growth inhibition despite the presence of pre-existing Ad5 immunity. Multiple re-immunizations using the same vector platform are now possible with the novel Ad5 [E1-, E2b-] platform.