Presence Of Carcinoembryonic Antigen Research Articles

Naked eye detection of trace cancer biomarkers based on biobarcode and enzyme-assisted DNA recycling hybrid amplifications

Naked eye-based detection has received increasing research interest due to the simplicity nature of this type of assay. However, improving the sensitivity of the naked eye detection method for the monitoring of trace amount of target molecules remains a major challenge. Herein, we describe a biobarcode and an enzyme-assisted DNA recycling hybrid amplification strategy for naked eye detection of sub-picomolar carcinoembryonic antigen (CEA), a cancer biomarker. The presence of CEA and the corresponding antibodies results in the formation of immunocomplexes and the capture of the biobarcodes in a microplate. The massive barcode DNAs released from the biobarcodes hybridize with the G-quadruplex inactive hairpin DNA probes and form catalytic nicking sites for N.BstNBI endonuclease, which cleaves the barcode DNA/hairpin partial dsDNA, releases the G-quadruplex active sequences and recycles the barcode DNA. Due to the barcode DNA recycling process, numerous G-quadruplex active sequences are generated and associate with hemin to form peroxidase mimicking enzymes, which convert colorless ABTS2− to green color intensified ABTS•− to achieve naked eye detection of CEA down to 0.025ngmL−1 (0.14pM). The naked eye detection strategy reported herein can be applied also to complicated serum sample matrix, making this approach hold great promise for point-of-care diagnostic applications.

S03 * ALCOHOL, CANCER, HEPATITIS AND FATTY LIVER: INTERACTIONS AND CO-MORBIDITIES

S03 * ALCOHOL, CANCER, HEPATITIS AND FATTY LIVER: INTERACTIONS AND CO-MORBIDITIES

Ultrasensitive electrochemical immunoassay for carcinoembryonic antigen based on three-dimensional macroporous gold nanoparticles/graphene composite platform and multienzyme functionalized nanoporous silver label

Three-dimensional macroporous gold nanoparticles/graphene composites (3D-AuNPs/GN) were synthesized through a simple two-step process, and were used to modify working electrode sensing platform, based on which a facile electrochemical immunoassay for sensitive detection of carcinoembryonic antigen (CEA) in human serum was developed. In the proposed 3D-AuNPs/GN, AuNPs were distributed not just on the surface, but also on the inside of graphene. And this distribution property increased the area of sensing surface, resulting in capturing more primary antibodies as well as improving the electronic transmission rate. In the presence of CEA, a sandwich-type immune composite was formed on the sensing platform, and the horseradish peroxidase-labeled anti-CEA antibody (HRP-Ab2)/thionine/nanoporous silver (HRP-Ab2/TH/NPS) signal label was captured. Under optimal conditions, the electrochemical immunosensor exhibited excellent analytical performance: the detection range of CEA is from 0.001 to 10ngmL−1 with low detection limit of 0.35pgmL−1 and low limit of quantitation (LOQ) of 0.85pgmL−1. The electrochemical immunosensor showed good precision, acceptable stability and reproducibility, and could be used for the detection of CEA in real samples. The proposed method provides a promising platform of clinical immunoassay for other biomolecules

A high-throughput homogeneous immunoassay based on Förster resonance energy transfer between quantum dots and gold nanoparticles

A novel homogeneous immunoassay based on Förster resonance energy transfer for sensitive detection of tumor, e.g., marker with carcinoembryonic antigen (CEA), was proposed. The assay was consisted of polyclonal goat anti-CEA antibody labeled luminescent CdTe quantum dots (QDs) as donor and monoclonal goat anti-CEA antibody labeled gold nanoparticles (AuNPs) as acceptor. In presence of CEA, the bio-affinity between antigen and antibody made the QDs and AuNPs close enough, thus the photoluminescence (PL) quenching of CdTe QDs occurred. The PL properties could be transformed into the fluorometric variation, corresponding to the target antigen concentration, and could be easily monitored and analyzed with the home-made image analysis software. The fluorometric results indicated a linear detection range of 1–110ngmL−1 for CEA, with a detection limit of 0.3ngmL−1. The proposed assay configuration was attractive for carcinoma screening or single sample in point-of-care testing, and even field use. In spite of the limit of available model analyte, this approach could be easily extended to detection of a wide range of biomarkers.

Improved Activation toward Primary Colorectal Cancer Cells by Antigen-Specific Targeting Autologous Cytokine-Induced Killer Cells

Adoptive therapy of malignant diseases with cytokine-induced killer (CIK) cells showed promise in a number of trials; the activation of CIK cells from cancer patients towards their autologous cancer cells still needs to be improved. Here, we generated CIK cells ex vivo from blood lymphocytes of colorectal cancer patients and engineered those cells with a chimeric antigen receptor (CAR) with an antibody-defined specificity for carcinoembryonic antigen (CEA). CIK cells thereby gained a new specificity as defined by the CAR and showed increase in activation towards CEA+ colon carcinoma cells, but less in presence of CEA− cells, indicated by increased secretion of proinflammatory cytokines. Redirected CIK activation was superior by CAR-mediated CD28-CD3ζ than CD3ζ signaling only. CAR-engineered CIK cells from colon carcinoma patients showed improved activation against their autologous, primary carcinoma cells from biopsies resulting in more efficient tumour cell lysis. We assume that adoptive therapy with CAR-modified CIK cells shows improved selectivity in targeting autologous tumour lesions.

Activated macrophages containing tumor marker in colon carcinoma: immunohistochemical proof of a concept

The presence of carcinoembryonic antigen (CEA)-containing activated macrophages has been demonstrated in peripheral blood from patients with colorectal carcinoma. Macrophages migrate from the circulation into the tissue, phagocytose debris, and return to the bloodstream. Hence it seems likely that activated macrophages containing tumor debris, i.e., tumor marker, are present in the stroma of colorectal carcinoma. After phagocytosis, they could follow a hematogenic or lymphogenic route to the peripheral blood. The aim of this study is to assess the presence of tumor marker-containing activated macrophages in the stroma of colon carcinoma and in regional lymph nodes. From 10 cases of colon carcinoma, samples of tumor tissue and metastasis-free lymph nodes were cut in serial sections and stained for CD68 to identify macrophages and for CEA, cytokeratin, or M30 presence. Slides were digitalised and visually inspected using two monitors, comparing the CD68 stain to the tumor marker stain to evaluate the presence of tumor marker-positive macrophages. Macrophages containing tumor marker could be identified in tumor stroma and in metastasis-free regional lymph nodes. The distribution varied for the different markers, CEA-positive macrophages being most abundant. The presence of macrophages containing tumor marker in the tumor stroma and lymph nodes from patients with colon carcinoma could be confirmed in this series using serial immunohistochemistry. This finding supports the concept of activated macrophages, after phagocytosing cell debris, being transported or migrating through the lymphatic system. These results support the potential of tumor marker-containing macrophages to serve as a marker for diagnosis and follow-up of colon cancer patients.

A Novel Electrochemical Immunosensor Based on DNA/Fe<sub>3</sub>O<sub>4</sub> (Core) /ZrO<sub>2</sub>（Shell）one Dimension Magnetic Probes for Carcino-Embryonic Antigen

A novel and sensitive electrochemical enzyme-linked immunosorbent assay (ELISA) system of Hydroquinones (HQ)- peroxide hydantoin (CP) -horseradish-peroxidase (HRP) by using magnetic DNA linked core-shell Fe3O4@ZrO2（ZMPs）nanoparticles as labels and signal amplification was established. In the presence of carcinoembryonic antigen (CEA) analyte, the sandwich-type immunocomplex could be formed between HRP labeled CEA second antibody（CEA Ab2） modified DNA-ZMPs probes (DNA / (ZMPs-HRP-CEA Ab2) n) and nano-gold CEA modified glassy carbon electrode (GCE | CEA Ab1) as an immune electrode.The concentration of CEA was determined based on the current signal, which was generated in the reaction between HQ and CP catalyzed by HRP on the sandwich-type immunocomplex. ZMPs nanoparticles as label material can not only perform the rapid separation and purification of signal antibody on magnetic field, but also immensely enhance the labeled capacity of HRP-anti-CEA which can amplify the catalytic current signal. Furthermore, the electrode modification process was not required. The method provided a linear response range between 0.01 and 200 ng·mL-1with a detection limit of 4 pg·mL-1.Moreover, the proposed electrochemical ELISA method exhibited good precision, high sensitivity and acceptable reproducibility, and could be further developed for clinical detections of CEA and other biomarkers.

GPI‐anchored CEA family glycoproteins CEA and CEACAM6 mediate their biological effects through enhanced integrin α5β1‐fibronectin interaction&

Carcinoembryonic antigen (CEA) and CEA family member CEACAM6 are glycophosphatidyl inositol (GPI)-anchored, intercellular adhesion molecules that are up-regulated in a wide variety of human cancers, including colon, breast, and lung. When over-expressed in a number of cellular systems, these molecules are capable of inhibiting cellular differentiation and anoikis, as well as disrupting cell polarization and tissue architecture, thus increasing tumorigenicity. The present study shows that perturbation of the major fibronectin receptor, integrin alpha5beta1, underlies some of these biological effects. Using confocal microscopy and specific antibodies, CEA and CEACAM6 were demonstrated to co-cluster with integrin alpha5beta1 on the cell surface. The presence of CEA and CEACAM6 was shown to lead to an increase in the binding of the integrin alpha5beta1 receptor to its ligand fibronectin, without changing its cell surface levels, resulting in increased adhesion of CEA/CEACAM6-expressing cells to fibronectin. More tenacious binding of free fibronectin to cells led to enhanced fibronectin matrix assembly and the formation of a polymerized fibronectin "cocoon" around the cells. Disruption of this process with specific monoclonal antibodies against either fibronectin or integrin alpha5beta1 led to the restoration of cellular differentiation and anoikis in CEA/CEACAM6 producing cells.

Value of carcinoembryonic antigen and cytokeratins for the detection of recurrent disease following curative resection of colorectal cancer

To evaluate the efficacy of postoperative serial assay of carcinoembryonic antigen (CEA) and cytokeratins for the detection of recurrent disease in patients with colorectal adenocarcinoma after radical surgery. Between 1993 and 2000, 120 patients with colorectal adenocarcinoma underwent radical surgery in the Department of Surgical Gastroenterology, Federal University of Sao Paulo-Escola Paulista de Medicina, Sao Paulo, Brazil. Periodic postoperative evaluation was performed by assaying markers in peripheral serum, colonoscopy and imaging examination. Presence of CEA was detected using the Delfia method with 5 microg/L threshold, and cytokeratins using the LIA-mat TPA-M Prolifigen method with 72 U/L threshold. In the first postoperative year, patients without recurrent disease had normal levels of CEA (1.5 +/- 0.9 microg/L) and monoclonal tissue polypeptide antigen-M (TPA-M, 64.4 +/- 47.8 U/L), while patients with recurrences had high levels of CEA (6.9 +/- 9.8 microg/L, P < 0.01) and TPA-M (192.2 +/- 328.8 U/L, P < 0.05). During the second postoperative year, patients without tumor recurrence had normal levels of CEA (2.0 +/- 1.8 microg/L) and TPA-M (50.8 +/- 38.4 U/L), while patients with recurrence had high levels of CEA (66.3 +/- 130.8 microg/L, P < 0.01) and TPA-M (442.7 +/- 652.8 U/L, P < 0.05). The mean follow-up time was 22.3 mo. There was recurrence in 23 cases. Five reoperations were performed without achieving radical excision. Rises in tumor marker levels preceded identification of recurrences: CEA in seven (30%) and TPA-M in eleven individuals (48%). Intensive follow-up by serial assay of CEA and cytokeratins allows early detection of colorectal neoplasm recurrence.

Detection of epithelial tumour RNA in the plasma of colon cancer patients is associated with advanced stages and circulating tumour cells.

Although circulating tumour DNA has been detected in patients with different types of cancer, little is known of free RNA in cancer patients. We investigated the presence of RNA from epithelial tumours in plasma from patients with colorectal carcinomas, and its correlation with tumour characteristics and circulating tumour cells. beta-actin mRNA was analysed to assess the viability of plasma RNA in samples from 53 patients with colonic cancer and 25 controls. Subsequently, nested primers were used to detect the presence of cytokeratin 19 (CK19) and carcinoembryonic antigen (CEA) RNA in the same samples. Nine clinicopathological parameters were studied to correlate the molecular and clinical parameters. Additionally, we investigated for micrometastases in blood in 18 of these patients and in 10 of the controls samples. All samples had detectable quantities of beta-actin RNA. In the controls, one case (4%) was positive for CEA and five (20%) for CK19 RNA; of the 53 patients, 17 cases (32%) were positive for CEA and 39 (73.6%) for CK19 RNA. This was statistically significant (p=0.000001). Advanced stages (p=0.03) and soluble CEA status (p=0.03) were associated with the presence of CEA, CK19, or both RNAs in plasma. Lymph node metastases (p=0.06) and vascular invasion (p=0.07) were almost significant. On the basis of these results, we examined the possible presence of micrometastases in blood in several of these patients. The presence of plasma tumour RNA was found to be associated with circulating tumour cells in blood (p=0.04). Epithelial tumour RNA is detectable in plasma from colon cancer patients. This molecular event is associated with advanced stages and circulating tumour cells. Our results could offer new approaches in the diagnosis and monitoring of colon cancer.

Large Cell Undifferentiated Carcinoma of the Parotid Gland Containing Malignant Oncocytic Change

The light microscopic, electron microscopic and immunohistochemical features of a large cell undifferentiated parotid gland carcinoma are presented. The tumor was composed predominantly of undifferentiated, polygonal large cells with focally admixed malignant oncocytic cells. The presence of carcinoembryonic antigen, lactoferrin, lysozyme and secretory component in the oncocytic tumor cells was found immunohistochemically, whearas no immunoreactivity with the tested biological markers was observed in undifferentiated polygonal large cells. Ultrastructurally, numerous mitochondria with crescent-shaped cristae were packed in the tumor cells within the oncocytic cell areas, and junctional complexes between the neighboring cells represented contiguous tight junctions and distinct desmosomes in the area of undifferentiated cells.

An immunohistochemical study of the incidence and significance of carcinoembryonic antigen in malignant endometrium.

In an immunohistochemical study of the presence of carcinoembryonic antigen (CEA) in endometrial carcinomas, it was found that CEA was detectable in 92 of 171 neoplasms, an incidence of 53.8%. The antigen was unrelated to important prognostic variables, both clinical and histopathologic, and overall there was no difference in survival rates between CEA-positive and CEA-negative endometrial neoplasms during the 10-year follow-up period. CEA-negative carcinomas did, however, confer longer survival in certain subgroups of patients: those with adenocarcinomas arising from a hyperplastic endometrium, those with neoplasms of poor differentiation, and those who, in addition to surgery, received irradiation treatment. It is suggested that immunoperoxidase staining for CEA may provide an independent method for predicting radiosensitivity in endometrial carcinomas, and also may be indicative of prognosis within some forms of this malignancy.

Presence of carcinoembryonic antigen in hepatic neoplasms of Rivulus ocellatus marmoratus

Carcinoembryonic antigen (CEA), a glycoprotein, is found in the embryonic gut and liver, and is in malignant neoplasms of the intestine and liver of humans. CEA is used as a diagnostic marker for liver and gut neoplasms. The objective of this preliminary study was to determine the usefulness of CEA as a diagnostic marker in liver neoplasms of the mangrove rivulus, Rivulus ocellatus marmoratus, and to compare the results to those reported in mammals. A commercially-available polyclonal antibody kit, Autoprobe III (Fisher Scientific, Houston, Texas), which utilizes avidin-biotinperoxidase complex was used to stain CEA. Diethylnitrosamine-induced neoplasms and control livers from a previous study were used. These paraffin sections were stored at room temperature for at least six years. Both Bouin's-fixed and formalin-fixed tissues were used. A positive control (1-day-old mangrove rivulus larvae), and a negative control (omission of CEA antibody) were also used. CEA was found as red deposits in the gut mucosa, epidermis, and the brush boarder and tubular epithelium of the kidney of the 1-day-old-fish, control fish and the fish that had liver neoplasms. No CEA was found in the negative controls, the livers of postive controls and the normal adult livers. The liver tumors that had characteristics of biliary neoplasms stained positively for CEA, but the hepatocellular neoplasms were negative. The presence of CEA in the gut mucosa and the biliary neoplasms of fish is similar to the results of similar studies of human tissues.

Serum and tissue levels of CEA, TPA, CA 125 and CA 15.3 in patients with lung cancer.

Forty-nine healthy subjects (Group I), 24 patients with benign lung diseases (Group II) and 48 patients surgically treated for lung cancer (Group III): 28 with squamous cell carcinoma (SCC) and 20 with adenocarcinoma (adenoca), were tested for the presence of carcinoembryonic antigen (CEA), tissue polypeptide antigen (TPA), cancer antigen CA 125 and antigen CA 15.3. The four markers were measured in the serum of the patients of the three groups and in the cytosol extract of tumoral and peritumoral tissues of Group III subjects. The mean levels of serum CEA and TPA were significantly higher in squamous cell carcinoma and in adenocarcinoma patients than in normal subjects. In benign lung disease serum CEA was equal and TPA slightly higher than in normal subjects. CA 125 was higher in the serum of patients with malignant diseases compared to normal or benign lung diseases but this difference was not statistically significant. Serum CA 15.3 levels were similar in all subjects studied. CA 125 in squamous cell carcinoma cytosol was much higher than in peritumoral cytosol whereas the other three markers were not significantly different in tumor cytosol or peritumoral cytosol. A direct correlation between serum and cytosol values was observed for CEA, but not for the other markers. The levels of the four markers in serum and cytosol did not correlate with the stage or grade of the tumors.

An Immunohistological Comparison of Primary Lung Carcinosarcoma and Sarcoma

We wished to assess the antigenic expression of primary lung tumors diagnosed as either carcinosarcoma or sarcoma in order to determine whether this information would be useful in distinguishing the two. We therefore immunohistochemically analyzed six pulmonary carcinosarcomas and five primary lung sarcomas for the presence of carcinoembryonic antigen (CEA), S100 protein, cytokeratin and vimentin using commercially available monoclonal and polyclonal antibodies on formalin fixed tissues. Six of six carcinosarcomas stained positively for cytokeratin while none of the sarcomas stained. In three carcinosarcomas both the carcinomatous and sarcomatous areas were positive while in three only the carcinomatous areas were positive. CEA staining was present in five carcinosarcomas and absent in all the sarcomas. CEA positivity was strong and not confined to those tumors with obvious gland formation. Staining for S100 protein was positive in two carcinosarcomas but only in those areas showing chondroid differentiation. Immunohistochemical staining for vimentin using two different monoclonal antibodies gave inconsistent results. We conclude that in differentiating between a carcinosarcoma and a sarcoma of the lung, immunohistochemical staining for both cytokeratin and CEA are useful with cytokeratin marginally preferable. The data indicate that carcinosarcoma of the lung, like that of the upper aerodigestive tract, expresses antigens suggesting both epithelial and mesenchymal differentiation.

Immunocytochemical Demonstration of Intermediate Filament Proteins, S‐100 Protein and CEA in Apocrine Sweat Glands and Apocrine Gland Derived Lesions of the Dog

The presence of carcinoembryonic antigen (CEA), intermediate filament proteins and S-100 protein in normal and pathological canine apocrine sweat glands was investigated, using a standard immunoperoxidase technique. The normal apocrine sweat glands showed a constant immunoreactivity in all the cases studied. The cells of the acini and of the ducts only reacted with the antikeratin antibody. The myoepithelial cells reacted positively with the antisera antikeratin and anti protein S-100. Epithelial cells of apocrine cysts, sweat gland adenomas and sweat gland carcinomas showed the same immunoreaction than normal apocrine cells. Proliferating myoepithelial cells were also positive for vimentin. In two out of three adenocarcinomas a positive reaction with the anti CEA could be detected in the glandular cells. This can be due to the presence in glandular cells of CEA or of Nonspecific Crossreacting Antigen (NCA). These findings indicate that demonstration of keratin is a useful aid in the detection of apocrine gland derived lesions in the dog. Similarly, S-100 protein is a marker for myoepithelial cells. Further research is necessary to investigate the expression of CEA in canine tumours.

Clear-Cell Syringoma

Clear-cell syringoma is a histologic variant of syringoma that is otherwise clinically indistinguishable from ordinary syringoma. This variant is formed by cells that have pale or clear cytoplasm as a result of glycogen accumulation. There is a high association of clear-cell syringoma and diabetes mellitus. A case of clear-cell syringoma associated with diabetes mellitus is described. Electron microscopic examination revealed that periluminal cells showed intra- and extracytoplasmic multivesicular bodies that may be characteristic of the clear-cell variant. Immunohistochemistry for carcinoembryonic antigen (CEA) showed ordinary syringomas as in the presence of CEA within and surrounding duct-like spaces.

CEA immunoreactivity in odontogenic tumors and keratocysts

Forty-five oral tumors and cysts were stained immunohistochemically for the presence of carcinoembryonic antigen (CEA). CEA, or a CEA-like antigen that is not nonspecific cross-reacting antigen (NCA), was demonstrated in the majority of aggressive or malignant tumors showing squamous differentiation, including cases of ameloblastoma, odontogenic carcinoma, and squamous carcinoma. CEA immunoreactivity was also found in cases of odontogenic keratocyst and focally in squamous odontogenic tumors but was not found in any of the ameloblastic fibromas, myxofibromas, odontogenic adenomatoid tumors, malignant melanomas, or apical cysts.

Prognostic value of immunohistochemically identified CEA, SC, AFP, and S‐100 protein‐positive cells in gastric carcinoma

Gastric tissues from 85 patients with advanced carcinoma were studied immunohistochemically for localization of carcinoembryonic antigen (CEA), secretory component (SC) and alpha-fetoprotein (AFP), and for S-100 protein-positive cells. Our objective was to determine if these factors could serve as prognostic indicators. Forty patients died of recurrence or metastases within 2 years (Group I), and the remaining 45 survived 10 years or longer (Group II) after resection. CEA-positive cells were present in 93% and 89% of Groups I and II, respectively. In case of staining for CEA, positive results were seen not only in the malignant cells but also in the stroma in 35% of Group I but none in Group II. The immunohistochemical status of SC was not essentially different between Groups I and II; the positive staining was recognized in 50% and in 57%, respectively. Gastric carcinomas rarely reacted to AFP. S-100 protein-positive cells, which play an important role in local immunity, were often present in Group II (47%) but rarely in Group I (10%). Thus, in addition to the various prognostic factors available to the surgical pathologist, the presence of CEA and the appearance of S-100 protein-positive cells in tumor tissue will aid in predicting the prognosis of patients with advanced gastric carcinoma.

CEA and HMFG in Hyperplastic and Malignant Lesions of the Breast

In order to evaluate a possible transition from proliferative lesions of the breast to ductal carcinoma in situ (DCIS), an immunohistochemical retrospective analysis was carried out. Twelve patients with hyperplasia without atypia, 11 patients with hyperplastic lesions with atypia, 21 patients with DCIS and 24 patients with invasive carcinoma were studied. The expression of carcino-embryonic antigen (CEA), a dedifferentiation marker, was investigated, applying one monoclonal antibody (Amersham). The expression of human milk fat globulin (HMFG), a differentiation marker, was studied by means of three monoclonal antibodies. The results reveal that no CEA activity can be demonstrated in normal and hyperplastic breast tissue, either with or without atypia. The monoclonal antibody was positive in 48% of DCIS and 50% of the invasive carcinomas. We failed to observe a correlation between the presence of CEA and the differentiation of the tumor. The application of this antiserum adds no substantial information about the phenotypic alterations during the progression from atypical hyperplasia to DCIS. HMFG was present in benign as well as in malignant breast lesions. Therefore, we conclude that HMFG is not useful for the evaluation of the phenotypic change from atypical hyperplasia to malignancy. However, as pointed out by others, it can be used in a diagnostic panel of different antibodies in the distinction between epithelial and non-epithelial lesions.