CEA Expression Research Articles

Added Value of Combined Gene and Protein Expression of CK20 and CEA in Non—macroscopically Involved Lymph Nodes of Colorectal Cancer

A methacarn fixation permits an approach that comprises multiple techniques. In this study the procedure is used to examine 100 mesenteric lymph nodes from patients with colon cancer by means of histology, immunohistochemistry (IHC), and reverse transcriptase polymerase chain reaction (RT-PCR). The evaluated nodes are found to be grossly free of metastases. The combined expression of both messenger RNA (mRNA) and protein is investigated to validate the presence of structural (cytokeratin 20, or CK20) and tumor-specific (carcinoembryonic antigen, or CEA) markers. Histological analysis shows micrometastases on 4 nodes. IHC analysis identifies isolated (CK20 and CEA positive) tumor cells on 14 other nodes. In this group, none of the nodes that are positive for CK20 IHC express the related mRNA. RT-PCR confirms the CEA IHC positivity in 50% of the cases. The double CEA IHC/RT-PCR positivity would have up-staged 33% of the pN0 cases to pN1. This approach offers a technological framework for further studies that aim to validate the clinical significance of protein/mRNA expression of tumor markers in colorectal cancer sentinel lymph nodes.

Fluorophore-conjugated anti-CEA Antibody for the Intraoperative Imaging of Pancreatic and Colorectal Cancer

IntroductionColorectal and pancreatic cancers together comprise the third and fourth most common causes of cancer-related death in the United States. In both of these cancers, complete detection of primary and metastatic lesions at the time of surgery is critical to optimal surgical resection and appropriate patient treatment. Materials and MethodsWe have investigated the use of fluorophore-labeled anti-carcinoembryonic antigen (CEA) monoclonal antibody to aid in cancer visualization in nude mouse models of human colorectal and pancreatic cancer. Anti-CEA was conjugated with a green fluorophore. Subcutaneous, orthotopic primary and metastatic human pancreatic and colorectal tumors were easily visualized with fluorescence imaging after administration of conjugated anti-CEA. The fluorescence signal was detectable 30 min after systemic antibody delivery and remained present for 2 weeks, with minimal in vivo photobleaching after exposure to standard operating room lighting. Tumor resection techniques revealed improved ability to resect labeled tumor tissue under fluorescence guidance. Comparison of two different fluorophores revealed differences in dose–response and photobleaching in vivo. ConclusionThese results indicate that fluorophore-labeled anti-CEA offers a novel intraoperative imaging technique for enhanced visualization of tumors in colorectal and pancreatic cancer when CEA expression is present, and that the choice of fluorophore significantly affects the signal intensity in the labeled tumor.

Prognostic value of immunohistochemical stain pattern for carcinoembryonic antigen in patients with completely resected pathologic stage I lung adenocarcinoma

7553 Background: Surgery alone remains the standard therapy for patients with stage I NSCLC based on the recent results of randomized control trials. There is, however, a subgroup of patients with stage I disease who have a poor prognosis, for whom adjuvant chemotherapy can be as effective as that for patients with more advanced disease. Preoperative serum CEA level has been reported to be an independent prognostic factor for stage I NSCLC. Nevertheless, adjuvant therapy is not performed based on the serum CEA level, because it can be influenced by smoking or other lung conditions. Therefore, a more definitive indicator is considered necessary. We hypothesized that immunohistochemical (IHC) CEA expression would be a more reliable and effective prognostic marker than serum CEA levels. Methods: Between 1986 and 2000, 219 patients who underwent complete resection at our hospital were diagnosed as having stage I lung adenocarcinoma. Immunohistochemical staining with the antibody for CEA was carried out on paraffin embedded sections of those tumors using the avidin-biotin- peroxidase complex method. Preoperative serum CEA levels and other clinicopathologic factors were also investigated by univariate and multivariate analysis. Results: IHC CEA was positive in 186 patients (85%). The membrane pattern, in which only the cell membrane was stained, was observed in 92 patients; the cytoplasmic pattern (CP), in which cytoplasm was diffusely stained, was observed in 94 patients; and a negative stain pattern was shown in 33 patients. We combined the cell membrane pattern and negative stain pattern as the non- cytoplasmic pattern (NCP) on the basis of survival analysis. Survival analysis demonstrated that CEA IHC patterns and serum CEA levels as well as age and blood vessels invasion were significant prognostic factors on both univariate and multivariate analysis. The 5-year survival for patients with a high serum CEA level and CP was only 38%, compared to 79% for normal serum CEA levels and CP, 78% for high serum CEA levels and NCP, and 93% for normal serum CEA levels and NCP. Conclusions: Both CEA IHC patterns and serum CEA levels were independent prognostic factors for patients with pathologic stage I lung adenocarcinoma. No significant financial relationships to disclose.

Detection of hTERT mRNA, CEA, CA19-9 in pleural effusion and its clinical significance

OBJECTIVE To explore the clinical significance of the human telomerase reverse transcriptase (hTERT) mRNA, CEA and CA19-9 in differential diagnosis of benign and malignant pleural effusions. METHODS Concentrations of CEA and CA19-9 in pleural effusions were assayed using automated chemiluminescence, and expression of hTERT mRNA was detected by RT-PCR. RESULTS The positive rates of hTERT mRNA, CEA and CA19-9 expression in the group with malignant effusions were significantly higher compared to the group with benign effusion ( P < 0.05). The sensitivity (%), specificity (%) and diagnostic accordance rates (%) of the 3 tumor markers were as follows: i ) hTERT mRNA: 81.8/90.5/86.1; ii ) CEA: 52.3/92.9/72.1; iii ) CA19-9: 34.1/90.5/61.6. The positive rates of hTERT mRNA + CEA (%) expression in the pleural effusions were 97.7. CONCLUSION All of these tumor markers can be helpful for differential diagnosis of pleural effusions. hTERT mRNA had more clinical value in differentiation of the pleural efffusions. CA19-9 is unfit to be as an optimal index. The combined assay of hTERT mRNA and CEA in pleural eff usions can further raise the positive detection rate of the tumor markers and can be helpful in producing a diagnosis.

Histopathological characterization of a syngeneic orthotopic murine bladder cancer model

We developed and characterized by histopathology and immunohistochemistry a syngeneic murine bladder tumor model derived from the MB49 tumor cell line. Bladder tumor implantation was achieved by intravesical instillation of 5 x 105 MB49 tumor cells in C57BL/6 mice. A chemical lesion of the bladder was performed in order to promote intravesical tumor implantation. The bladder wall lesion was accomplished by transurethral instillation of silver nitrate (AgNO3). After 15 days, the animals were sacrificed, examined macroscopically for intravesical tumor and bladder weight. Histology and immunohistochemistry were performed using cytokeratin 7 (CK7), carcinoembrionic antigen (Dako-CEA), p53 and c-erbB2 oncoprotein (Her2/neu). Twenty-nine out of 30 animals (96.7%) developed intravesical tumors in a 15-day period. Macroscopically, the mean bladder weight was 0.196g (0.069-0.538g), 10 to 15 times the normal bladder weight. The immunohistochemical analysis showed significant membrane expression of CEA and CK7: a similar finding for human urothelial cancer. We also characterized absence of expression of p53 and anti-Her2/neu in the murine model. High tumor take rates were achieved by using the chemical induction of the bladder tumor. Although electric cauterization is widely described in the literature for syngeneic orthotopic animal models, the technique described in this study represents an alternative for intravesical bladder tumor implantation. Moreover, the histopathology and immunohistochemical analysis of the murine bladder tumor model derived from the MB49 cell line showed a resemblance to human infiltrating urothelial carcinoma, allowing clinical inference from experimental immunotherapy testing.

Biological characterization of two xenografts derived from human CUPs (carcinomas of unknown primary)

BackgroundCarcinomas of unknown primary site (CUP) are epithelial malignancies revealed by metastatic lesions in the absence of any detectable primary tumor. Although they often adopt an aggressive clinical pattern, their basic biology remains poorly understood. Laboratory research on their biology have been hampered so far by the absence of cell lines representative of CUPs.MethodsWe attempted xenografts of CUP clinical specimens in immunodeficient mice and subsequent in vitro culture of transplanted malignant cells. Whenever possible, malignant xenografted or cultured cells were characterized by microsatellite genotyping, immunohistology, electron microscopy, multifish chromosome analysis and search of TP 53 gene mutations.ResultsSuccessful xenografts were achieved in 2 cases out of 4. One of them (Capi1) was lost after 3 passages whereas the other one (Capi3) has been adapted to in vitro culture and is currently available to the scientific community with reliable identification based on microsatellite genotyping. Both Capi1 and Capi3 have histological characteristics of adenocarcinomas and display intense expression of EMA, CEA and cytokeratin 7. Multifish chromosome analysis demonstrated a translocation involving chromosomes 4 and 21 in both specimens. Distinct rare missense mutations of the TP53 gene were detected in Capi1 (codon 312) and Capi3 (codon 181); the codon 181 mutation is consistent with a previously reported similar finding in a small series of CUP specimens. Finally, intense membrane expression of c-kit was recorded in Capi3.ConclusionOur data suggest that xenografted tumors can be obtained from a substantial fraction of CUP clinical specimens. The hypothesis of a preferential association of CUPs with TP 53 mutations of codon 181 deserves further investigations. The Capi3 cell line will be a useful tool for assessment of novel c-kit inhibitors.

Combination of Measles Virus Virotherapy and Radiation Therapy Has Synergistic Activity in the Treatment of Glioblastoma Multiforme

Glioblastoma multiforme is the most frequent primary brain tumor in adults and represents one of the most lethal malignancies with a median survival of 12-16 months. We have previously shown that an oncolytic measles virus derivative expressing soluble human carcinoembryonic antigen (MV-CEA) has significant antitumor activity against glioblastoma multiforme cell lines and xenografts. Radiation therapy (RT) represents one of the mainstays of glioma treatment. Here we tested the hypothesis that the combination of RT with MV-CEA would have synergistic activity against gliomas. 3-(4,5-Dimethyl-thiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and clonogenic assays were used to test cytoxicity of the combination treatment in vivo. To examine the mechanism of synergy, one-step viral growth curves, terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays, and Western blot analyses were performed. In vivo assessment of synergistic antitumor activity was conducted in a U87 glioma model. MTS and clonogenic assays showed a strong synergistic interaction between MV-CEA and RT in glioblastoma multiforme cells including both primary and established glioma lines. Furthermore, significant antitumor efficacy was observed in vivo in a subcuteneous U87 xenograph model. There was significant prolongation of survival (P = 0.001) in the combination treatment group as compared with single modality- or control-treated animals. One-step viral growth curves showed increased viral burst size by up to 2 log in MV/RT combination-treated cells, as compared with single agent MV-CEA-treated glioma cells. Changes in CEA levels and expression of viral N and H protein were also consistent with increased viral production. Furthermore, TUNEL assays and Western blot analysis showed increase in apoptosis in MV/RT combination-treated cells. The pan-caspase inhibitor Z-VAD-FMK and the caspase-8 inhibitor Z-IETD-FMK, but not the caspase-9 inhibitor Z-IEHD-FMK, protected glioma cells from MV-CEA/RT-induced cleavage of poly(ADP-ribose) polymerase (PARP), indicating that the apoptotic death in combination-treated cells is mostly mediated via the extrinsic caspase pathway. The Fas/Fas ligand interaction blocking antibody NOK-1 blocked MV/RT-induced PARP cleavage whereas the Fas agonistic antibody CH11 increased PARP cleavage in MV/RT combination-treated cells. Reverse transcription-PCR, fluorescence-activated cell sorting analysis and immunohistochemistry showed up-regulation of Fas in combination-treated tumor in vitro and in vivo cells. There is synergy between MV-CEA and RT in vitro and in vivo. The synergistic effect of the combination seems to be due to increase in viral burst size and increase in apoptotic cell death. This latter effect is mostly mediated via the extrinsic caspase-8 pathway, activated via increased signaling through the Fas death receptor pathway. These results could have translational implications in glioma therapy.

Clinical Relevance of the Expression of mRNA of MMP-7, MMP-9, TIMP-1, TIMP-2 and CEA Tissue Samples from Colorectal Liver Metastases

Background: Nowadays we know that survival rates do not differ between repeated and single liver resections for colorectal liver metastases (CLM). To be able to determine patients prone to early recurrence, the use of different markers with a better prognostic value than the routinely employed tumor markers is required. Aim of Study: The aim of our study was to assess mRNA expression of MMP-7, MMP-9, TIMP-1, TIMP-2 and CEA in tissue samples from CLM and their relationship to disease-free interval (DFI) and overall survival (OS). Patients and Methods: The liver tumor biopsies were obtained from 40 patients suffering from CLM treated with radical surgery. mRNA expression levels of CEA, MMPs and TIMPs and a housekeeping gene (GAPDH) were quantified using RT-PCR. Results: The increased expression of CEA, MMP-9 and TIMP-1 in CLM was associated with a short DFI and a high tendency to early CLM recurrence. Statistical analysis confirmed CEA, MMP-9 and TIMP-1 expression as prognostic factors of survival. Conclusion: This study demonstrated the importance of CEA, MMP-9 and TIMP-1 in the prognostication of DFI and OS.

Hypoxia upregulates carcinoembryonic antigen expression in cancer cells

Carcinoembryonic antigen (CEA, ceacam5) is an important tumor-associated antigen with reported roles, e.g., in immunological defense, cell adhesion, cell survival and metastasis. Its overexpression in cancer cells is known to involve transcriptional activation of the CEA gene, but the underlying molecular details remain unclear. Here, we show that hypoxia and intracellular alkalinization, 2 factors commonly found in solid tumors, increase CEA protein expression in breast (MCF-7) and colorectal (CaCo-2 and HT-29) cancer cells. The increase was comparable (2-3-fold) to that observed in colorectal carcinomas in vivo. CEA promoter analyses further revealed that this upregulation involves a known binding site for HIF-1 transcription factor (5'-ACGTG-3') within one of the CEA promoter's positive regulatory elements (the FP1 site; the E-box). Accordingly, deletion or targeted mutagenesis of this motif rendered the CEA promoter unresponsive to hypoxia. Our chromatin immunoprecipitation data confirmed that endogenous HIF-1alpha binds to the CEA promoter in hypoxic cells but not in normoxic cells. Moreover, overexpression of the hypoxia-inducible factor (HIF-1alpha) was sufficient to increase CEA protein expression in the cells. In contrast, c-Myc, which is known to bind to the overlapping E-box, did not potentiate HIF-1alpha-induced CEA expression. CEA overexpression in vivo was also found to coincide with the expression of carbonic anhydrase IX, a well-known hypoxia marker. Collectively, these results define CEA as a hypoxia-inducible protein and suggest an important role for the tumor microenvironmental factors in CEA overexpression during tumorigenesis.

Molecular markers expression in mediastinal nodes from resected stage I non-small cell lung cancer (NSCLC): Prognostic impact and potential role as markers of occult micrometastasis

21016 Background: 5-y surv in surgically resected St I NSCLC is 60–70% whereas in cases with affected lymph nodes (LN) it is &lt; 50%. The main risk factor for recurrence is nodal disease. Current histopathologic analysis can miss occult micrometastases in nodal tissues at initial diagnosis. Detection of micromets with a more sensitive technique would be useful to define a high-risk population selecting patients (p) for postop treatment. We assessed the role of several genes mRNA expression in pathological negative LN from resected St I NSCLC p as markers of occult micromets and correlated the results with relapse, and survival. Methods: Paired tumor and histological negative LN (n=344) obtained by systematic mediastinal lymphadenectomy from 38 surgically resected St I NSCLC p were analyzed for the presence of 12 genes mRNA expression using RT-Q-PCR in an ABIPRISM 7500. RNA was extracted using ABIPRISM 6100. Specifically designed primers and probes were purchased from Applied Biosystems as Assay-on-demand; GADPH was used as an endogenous control. Samples were also analyzed by ICH for LN staging. Results: 38 NSCLC p; 12 adenoca, 16 squamous cell, 10 undifferentiated . From the 12 tested genes CEA and PLUNC were found with high expression in lung tissue and low or null expression in normal LN. We consider molecular + LN those in which expression of CEA or PLUNC was detected. In the 344 pathological negative LN, 13% (44/344) were positive for CEA, 16% (54/344) for PLUNC. The expression patterns were similar for both markers. At a median follow-up of 24 mo (9–46) 11 p had died from NSCLC and 1 had died without recurrence. None of the living p had tumor recurrence. For the prognostic assessment, molecular + LN were classified as N1 and N2. Median DFS was 15 ± 11.74 mo in p with N2 molecular + nodes was and has not yet reached in cases of molecular (-) LN (p=0.028). Median survival of p with N2 molecular positive nodes was 17.3 ± 5.7 mo and has not yet reached (p= 0.0083) for molecular (-) LN. Conclusions: CEA and PLUNC mRNA expression could be used as molecular markers of occult micrometastases in mediastinal LN showing a prognostic effect . CEA and PLUNC expression provides a tool for selecting high-risk p considered for adjuvant therapies No significant financial relationships to disclose.

Expression of Tumor-Associated Differentiation Antigens, MUC1 Glycoforms and CEA, in Human Thymic Epithelial Cells: Implications for Self-Tolerance and Tumor Therapy

Expression of tissue-restricted self-antigens in the thymus, termed promiscuous gene expression, imposes T cell tolerance and protects from autoimmune diseases. This antigen pool also includes various types of tumor-associated antigens (TAA) previously thought to be secluded from the immune system. The scope of promiscuous gene expression has been defined by mRNA analysis at the global level of isolated medullary thymic epithelial cells (mTECs). Information at the protein level on the frequency of mTECs expressing a given antigen, on coexpression patterns, and post-translational modifications is largely missing. We report here promiscuous expression at the protein level of two TAA, MUC1 and CEA, in situ and in purified human mTECs. Both antigens are expressed in 1% to 3% of mTECs, either individually or coexpressed in the same cell. Using a panel of anti-MUC1 monoclonal antibodies recognizing different post-translational modifications, i.e., glycoforms of MUC1, we show that only fully glycosylated forms of MUC1 and the differentiation-dependent glycoforms were detected on mTECs, but not the cancer-associated glycoforms. Our findings imply that MUC1 and CEA are amenable to central tolerance induction, which might, however, be incomplete in case of tumor cell-restricted MUC1 glycoforms. Knowledge of these subtleties in promiscuous gene expression may, in the future, assist the selection of T cell tumor vaccines for clinical trials.

Utility of p16INK4a, CEA, Ki67, P53 and ER/PR in the Differential Diagnosis of Benign, Premalignant, and Malignant Glandular Lesions of the Uterine Cervix and Their Relationship with Silverberg Scoring System for Endocervical Glandular Lesions

Early detection of premalignant and malignant glandular lesions of the uterine cervix and their distinction from benign mimics is crucial but sometimes difficult. In this study, we investigated utility of expression of p16, CEA, Ki67, p53 and ER/PR in evaluating the benign, premalignant, and malignant glandular lesions of the uterine cervix. A total of 35 cervical cone or LEEP cases were collected including 14 adenocarcinoma in situ (AIS), 7 endocervical glandular dysplasia (EGD), and 14 benign mimics (BM). A histological score for each case according to the criteria proposed by Silverberg group was assigned independently by 4 pathologists. Formalin-fixed, paraffin-embedded sections were immunostained with p16, CEA, p53, Ki67, and ER/PR. Immunoreactivity was scored based on intensity (0 = none, 1 = mild, 2 = moderate, 3 = marked) and percentage of cells staining (0 = <1%,1 = 2-10%, 2 = 11-40%, 3 = >40%). A comparison of histological scores and immunoscores in 3 diagnostic categories was analysed. The histological scores assigned independently by 4 pathologists were all equal or above 6 for AIS, between 3 and 5 for EGD, and equal or below 3 for BM. There was increased expression of p16 and CEA in EGD compared with BM (P < 0.05), with further increase in expression of these markers in AIS compared with EGD (P < 0.05). Ki67 expression was significantly increased in AIS compared to EGD (P < 0.05) as well as compared to BM (P < 0.05). Ki67 expression was only slightly increased in EGD as compared to BM. There was a loss of ER/PR in cervical AIS, but not in EGD. Our results indicate that the Silverberg scoring system is a useful tool in differential diagnosis of cervical glandular lesions for increased diagnostic accuracy and interobserver agreement. Most cervical glandular lesions can be differentiated by using a combination of histological scores with a panel of immunomarkers.

Expression of liver-enriched nuclear factors and their isoforms in α-fetoprotein-producing gastric carcinoma cells

Alpha-fetoprotein (AFP)-producing gastric cancer (AFP-GC) is a highly malignant variant of adenocarcinoma with aberrant hepatocellular phenotype. A detailed understanding of the regulation of its liver phenotype is lacking. Liver-enriched nuclear factors (LENFs) are implicated in the transcriptional regulation of AFP in the fetal liver. To investigate the regulatory role of LENFs in AFP-GCs, the expression of LENFs including CCAAT/enhancer binding protein (C/EBP)-β, C/EBP-α, hepatocyte nuclear factor (HNF)-1α, HNF-1β and HNF-4α was investigated in 3 cell lines of AFP-GC and 7 cell lines of control GC. The liver activating protein (LAP), an activating isoform of C/EBP-β, was predominantly expressed in AFP-GCs, whereas the liver inhibitory protein (LIP), an inhibitory isoform of C/EBP-β, predominated in the control GCs. HNF-1α was relatively suppressed in AFP-GCs. HNF-4α was expressed in one of three AFP-GC cell lines. C/EBP-α and HNF-1β were expressed at the same levels in both cell types of GC. AFP-GCs expressed a set of hepatocyte-related proteins (e.g., transferrin and albumin) while they still retained the several glandular cell-related proteins (e.g., MUC2). The induction of LIP reduced transferrin expression and induced CEA expression in an AFP-GC line. Collecting these results, it was suggested that the contribution of LENFs, especially isoforms of C/EBP-β, is possibly important in phenotypic regulation of AFP-GCs.

Endocervical glandular lesions: A diagnostic approach combining a semi-quantitative scoring method to the expression of CEA, MIB-1 and p16

To investigate whether combining a semi-quantitative scoring method with the immunohistochemical expression of CEA, MIB-1 and p16, would improve the diagnostic accuracy of endocervical glandular lesions. The hematoxylin and eosin-stained sections of 95 cervical biopsies were examined by 4 different observers and were grouped into three categories, benign, dysplasia and adenocarcinoma in situ, depending on the degree of nuclear stratification, nuclear atypia and the number of mitosis and apoptotic figures. Each case was also stained immunohistochemically with antibodies against CEA, Ki-67 (MIB-1) and p16. Staining was graded as negative, weak and positive. The accuracy of the scoring method alone was compared to the accuracy of combining the score with the immunostaining results. Using the semi-quantitative scoring system, most of the cases that were initially diagnosed as atypical hyperplasia or tuboendometrial metaplasia fell into the benign category. This scoring system discriminates effectively (Kruskal-Wallis, p<0.001) between the three categories (benign, endocervical glandular dysplasia and adenocarcinoma in situ). When analyzing the immunohistochemical score, only Ki-67 staining seems to be effective mostly in discriminating between normal glands or glands with atypical hyperplasia and epithelial glandular dysplasia. Ki-67, CEA and p16 failed to discriminate between tuboendometrial metaplasia and epithelial glandular dysplasia. Combining the semi-quantitative scoring system with the immunohistochemical results discriminates between the three categories equally well as the semi-quantitative scoring system alone (Kruskal-Wallis, p<0.001). Nevertheless, the proportion of cases that were classified similarly to the prestudy diagnosis was higher when the combined score was used. Combining a semi-quantitative scoring scheme with the immunohistochemical expression of CEA, MIB-1 and p16 seems to be of value in classifying some endocervical glandular lesions.

Evaluation of mRNA markers for micrometastases detection by RT-PCR in lymph nodes (LN), peripheral blood (PB) and bone marrow (BM) of breast cancer patients (pts)

20068 Background: Previous RT-PCR study showed that hMAM and CEA mRNA could be detected in 95% and 78% breast carcinomas, respectively. Analyzed in normal LN, these two markers were the only ones not expressed, compared to other mRNA markers which could be detected in a variable number of cases (Marchetti 2001). We evaluate the expression of CEA and hMAM mRNA in breast cancer pts in order to investigate if the identification of these transcripts could be correlated to in the LN, PB and BM. Methods: Tumor, normal gland, LN and PB were obtained from 49 breast cancer pts who have been referred to surgery during the period of Jun/2000 to Jan/2004.BM could be obtained from 21 of these cases. Before the manipulation of the tumor, 5 mL of PB was collected and 5 mL of BM was aspirated from the iliac crest. First level axillary LN were sectioned in two. One of the halves was immediately frozen in liquid nitrogen. CEA and hMAM transcripts have been analysed by RT-PCR. Results: Tumors were positive for CEA in 38 (77.6%) cases and LN were positive in 15 cases (36.7%). 6/49 pts expressed CEA in normal breast (12.2%). Five of these, tumor also expressed CEA. Two pts (4.1%) showed CEA expression in PB and in BM. One patient expressed CEA in BM but not in PB. All pts that showed expression in PB, BM and LN, also showed transcript of CEA in tumor. hMAM transcripts were found in 39 (79.6%) normal gland tissue and in 41 of tumor samples (85.7%). LN were positive to hMAM in 12 cases (24.5%). 5 of these (10.2%) were also positive in PB and BM samples. The pts showing hMAM expression in PB, BM and LN also showed its expression in neoplasic tissue. Analyzing both markers, two pts showed the expression of CEA and hMAM in the PB and BM samples. In 21 of the 49 cases the comparison of hMAM and CEA was possible to be performed in all tissues. CEA and hMAM expression associated to larger number of LN affected. Conclusions: CEA and hMAM transcripts can be detected in majority of breast cancer tissue. Specificity of hMAM seems to be higher than CEA, which can be detected in more than 10% of non-tumor breast tissue. Synchronicity of CEA or hMAM expression in the tumor and in extra-mammary sites was high, suggesting that these markers may provide an interesting strategy for follow-up micrometastatic disease. No significant financial relationships to disclose.

Survival Stratification Pattern of Colorectal Carcinoma with CEA MMP-2 and p27kip1 Expression

The prognosis varies greatly in colorectal caner (CRC) patients, even in the same stage. We examined the correlation between the expression of a reportedly wide range of biomarkers (MMP-2, CEA, p27kip1, β-catenin, CDC25B, Mucin-1, VEGF-D, c-erbB-2/neu, TGF-α and C-met) and clinicopathological features in CRC patients in order to identify a panel of biomarkers predictive of CRC prognosis. The expressions of these ten individual biomarkers in 127 CRC were analyzed by immunohistochemistry method. Univariate and multivariate analysis were done to analyze these biomarkers expression with the disease free survival time in CRC. Among markers studied, high expression of MMP-2, CEA and low expression of p27kip1 are significantly related to poor prognosis in univariate analysis. The expression of MMP-2, CEA, p27kip1, TNM staging, and differentiation are independent prognostic factors for survival by Cox regression analysis. The coexpression pattern of MMP-2, CEA and p27kip1 MCP has significant prognostic value in all patients. Cox regression reveals that coexpression pattern of MMP-2, p27kip1 and CEA, TNM stage and differentiation are independent prognostic factors in CRC. Though prognosis in patients with CRC is difficult to predict, the results of this study provide further evidence that the combination of biomarkers of MMP-2, CEA and p27kip1, or MCP, is more informative than any single tumor biomarker alone for the survival stratification of CRC. MCP may be a useful marker for clinical management and may provide multiple targets for therapeuticintervention.

44. Combination of Oncolytic Measles Virus Strains and Radiation Therapy Has Synergistic Activity in the Treatment of Glioblastoma Multiforme

Background: Glioblastoma Multiforme (GBM), the most frequent primary brain tumor in adults, is one of the most lethal malignancies with a median survival of 12-16 months. We have previously shown that oncolytic measles virus derivatives expressing soluble human carcinoembryonic antigen (MV-CEA) have significant antitumor activity against GBM cell lines and xenografts. Here we tested the hypothesis that the combination of RT with MV-CEA would have synergistic activity against gliomas.Methods: MTS (Promega) and clonogenic assays were performed in U87 and U251glioma cell lines (ATCC) to assess the impact of the different treatment modalities on cell proliferation and survival. One-step viral growth curves, Western blots to assess expression of viral proteins, TUNEL assays, immunostaining for Fas/caspase-8 and western immunobloting for caspase-8, caspase-9, caspase-3 and PARP were employed to investigate the mechanism of the synergistic interaction between MV-CEA and RT in GBM cell lines.Results: MTS and clonogenic assays revealed a synergistic interaction between MV-CEA and RT in GBM cells. The optimal combination index for the RT/MV-CEA combination was 0.524 for U87 (0.01MOI/10GY) and 0.529 for U251 (0.05MOI/7.5GY), indicating strong synergy. Morphologic changes of U251 and U87 cells in response to the combination treatment demonstrated significant augmentation of cytopathic effect. In clonogenic assays, colony formation was significantly decreased in the MV-CEA/RT group as compared to either MV-CEA or RT alone. One-step viral growth curves demonstrated increased MV-CEA propagation by at least 2 logs in MV-CEA/RT combination treated cells, as compared to glioma cells treated with single agent MV-CEA. Changes in CEA levels and expression of viral N protein were also consistent with viral titer changes. TUNEL staining showed increased positivity in RT/MV-CEA group as compared to MV-CEA or RT alone. Furthermore, Western blot analysis demonstrated an increase in activated caspase-8, caspase-3 and PARP expression levels after combination treatment. The pan-caspase inhibitor Z-VAD-FMK completely protected cells from MV/RT induced cleavage of PARP, while the caspase-8 inhibitor (Z-IETD-FMK) partially protected glioma cells from MV-CEA/RT induced cleavage of PARP, and the caspase-9 inhibitor Z-IEHD-FMK showed no protection.Conclusions: Our results indicate that there is significant synergy between MV-CEA and RT in vitro. The synergistic effect of the combination appears to be due to increased viral bust size and increase in apoptotic death. This latter effect is mostly mediated via the extrinsic caspase 8 pathway. Experiments in primary glioma lines and animal experiments are ongoing. These results could have immediate translational implications in glioma therapy. Background: Glioblastoma Multiforme (GBM), the most frequent primary brain tumor in adults, is one of the most lethal malignancies with a median survival of 12-16 months. We have previously shown that oncolytic measles virus derivatives expressing soluble human carcinoembryonic antigen (MV-CEA) have significant antitumor activity against GBM cell lines and xenografts. Here we tested the hypothesis that the combination of RT with MV-CEA would have synergistic activity against gliomas. Methods: MTS (Promega) and clonogenic assays were performed in U87 and U251glioma cell lines (ATCC) to assess the impact of the different treatment modalities on cell proliferation and survival. One-step viral growth curves, Western blots to assess expression of viral proteins, TUNEL assays, immunostaining for Fas/caspase-8 and western immunobloting for caspase-8, caspase-9, caspase-3 and PARP were employed to investigate the mechanism of the synergistic interaction between MV-CEA and RT in GBM cell lines. Results: MTS and clonogenic assays revealed a synergistic interaction between MV-CEA and RT in GBM cells. The optimal combination index for the RT/MV-CEA combination was 0.524 for U87 (0.01MOI/10GY) and 0.529 for U251 (0.05MOI/7.5GY), indicating strong synergy. Morphologic changes of U251 and U87 cells in response to the combination treatment demonstrated significant augmentation of cytopathic effect. In clonogenic assays, colony formation was significantly decreased in the MV-CEA/RT group as compared to either MV-CEA or RT alone. One-step viral growth curves demonstrated increased MV-CEA propagation by at least 2 logs in MV-CEA/RT combination treated cells, as compared to glioma cells treated with single agent MV-CEA. Changes in CEA levels and expression of viral N protein were also consistent with viral titer changes. TUNEL staining showed increased positivity in RT/MV-CEA group as compared to MV-CEA or RT alone. Furthermore, Western blot analysis demonstrated an increase in activated caspase-8, caspase-3 and PARP expression levels after combination treatment. The pan-caspase inhibitor Z-VAD-FMK completely protected cells from MV/RT induced cleavage of PARP, while the caspase-8 inhibitor (Z-IETD-FMK) partially protected glioma cells from MV-CEA/RT induced cleavage of PARP, and the caspase-9 inhibitor Z-IEHD-FMK showed no protection. Conclusions: Our results indicate that there is significant synergy between MV-CEA and RT in vitro. The synergistic effect of the combination appears to be due to increased viral bust size and increase in apoptotic death. This latter effect is mostly mediated via the extrinsic caspase 8 pathway. Experiments in primary glioma lines and animal experiments are ongoing. These results could have immediate translational implications in glioma therapy.

Optimizing the methods of whole lung cancer RNA-loaded dendritic cells

Dendritic cells (DCs) are the unique antigen-presenting cells that can activate naive T lymphocytes. This function is critical for inducing specific immune response. DCs-based vaccines have been used broadly in immunotherapy for many carcinomas. Constructing vaccines by transfecting total tumor RNA into DCs can be done with a few tumor tissues and need not to identify tumor antigens, so it is especially suitable for lung cancer which lacks tumor-specific antigens but has great heterogenicity and weak immunogenicity. Currently, the best transfection stage and method are still indefinite. So, the objective of this study is to explore the best condition of transfecting total RNA extracted from lung cancer tissues into DCs. Ten patients with lung cancer were enrolled whose tumor tissues were CEA and MUC1 positive in immunohistochemical staining. Total tumor RNA were extracted by one-step method. Then DCs and T cells were separated and cultured from peripheral blood monocytes and the RNA was transfected into the DCs in different stages with different methods. CEA and MUC1 expression in the transfected DCs were measured by flow cytometry analysis and T cells' proliferation was examined by mixed lymphocyte reaction (MLR). The expression of CEA and MUC1 protein in immature DCs (11.33±2.64, 39.68±7.25) was remarkably higher than that in mature DCs (5.46±1.63, 27.17±4.16) after transfection with total RNA of lung cancer tissues (P < 0.01), and the DCs presented more powerful effects on T cell proliferation. The CEA and MUC1 expression on DCs were significantly higher in electroporation transfection group (20.53±3.64, 65.39± 9.33) than that in lipofection group (11.33±2.64, 39.68±7.25) and passive pulsing transfection group ( 0.91±0.27,18.53±3.26)(P < 0.01), and the DCs in electroporation transfection group presented more powerful effects on stimulating T cell proliferation than the other two groups did. Transfecting total tumor RNA into immature DCs by using electroporation is a good way to construct DCs-based vaccines for lung cancer and to achieve a higher activity to stimulate T cell proliferation.

Genetic and Pathologic Changes associated with Lymphovascular Invasion of Colorectal Adenocarcinoma

Lymphovascular invasion (LVI) is a biological manifestation of aggressive behavior in colorectal cancer. This study sought to identify and examine the association between genetic and pathologic alterations implicated in this invasive tumor progression. We consecutively recruited 81 and 79 colorectal cancer patients with and without LVI, respectively. Biological changes were evaluated by clinicopathological parameters together with CEA and E-cadherin expressions using immune staining. Allelic loss or MSI was examined using 10 microsatellite markers on chromosomes 10, 16, 18, and TGFbetaRII, possibly associated with colorectal cancer. The germline mutation of BMPR1A and SMAD4 was also sought. Tumor stage and lymph node metastasis were significantly greater in patients with LVI tumor than without it (P < 0.001). Decreased CEA expression was closely correlated with allelic loss or MSI at D16S421, D18S46, and D18S474 (P = 0.004-0.047). Allelic loss at D10S14 was specific to LVI tumors (P = 0.007). Using multivariate analysis, allelic loss at D18S46 significantly correlated with histological differentiation (P = 0.02). In addition, allelic loss and MSI at D18S474, histological differentiation, and expression of CEA and E-cadherin were closely associated with the progression of LVI (P = 0.005-0.049). However, no germline mutation in BMPR1A or SMAD4 was detected in all patients regardless of LVI status. In summary, in a subset of colorectal cancers, histological differentiation and expression of CEA or E-cadherin appear to determine aggressive behavior such as LVI. These changes are closely associated with chromosomal alterations at 10q22-23, 16q22 and 18q21, which carry several tumor suppressor genes.

P-649 Role of CEA, PLUNC and CK19 mRNA expression in lymph nodes from resected stage I non-small cell lung cancer (NSCLC) patients as markers of occult micrometastasis: A pilot study

P-649 Role of CEA, PLUNC and CK19 mRNA expression in lymph nodes from resected stage I non-small cell lung cancer (NSCLC) patients as markers of occult micrometastasis: A pilot study