Amada Segal-Eiras

E14129 Background: Different morfologic features (MF) are associated with a poor prognosis. The aim of this research was to relate MF with antigenic expression. Methods: 90 primary CCR tumor samples (69% were colonic and 31% rectal cancer), 64 lymphatic nodes (LN) and 22 control samples (adenomas and normal colorectal mucosa). MF were: localization, type, extent of local invasion, histologic and nuclear grade, vascular and lymphatic invasion and metastatic nodes (N+). Antigens studied were: MUC2, MUC1, MUC5AC, CEA, beta-catenin; carbohydrate antigens: Lewis x (Lex), sialyl Lewis x (sLex), Lewis y (Ley), sialyl Lewis a (sLea) and Tn hapten. Standard haematoxilyn/eosin and immunohistochemistry (IH) was performed. Positive response was evaluated by means of frequency analysis (p < 0.05). A reactivity index (RI) was calculated as intensity (I) x percentage of positive area (A); RI values varied from 0-12; low (0-6) and high (7-12). Intensity, percentage of positive area, IH pattern and RI were analyzed employing Chi-Square test and ANOVA (p < 0.05). Results: 65% corresponded to pure adenocarcinoma, 23% with mucinous component, 12% pure mucinous. Local invasion was detected: muscular in 96%, subserosal in 16%, serosal in 7% and fat in 37% while 31/64 (48%) LN showed metastatic invasion. Antigenic expression: CCR samples expressed MUC2 in 52% of cases; MUC1, 94%; MUC5AC, 14%; CEA, 93%; beta-catenin, 98 %; Lex, 74%; sLe x, 66%, Ley; 91%; sLea, 90% and Tn, 41%. N+ expressed MUC2, 30%; MUC1, 89%; MUC5AC, 19%; CEA, 80%; beta-catenin, 83%; Lex, 69%; sLex, 72%; Ley, 75%; sLea, 86% while Tn, 44%. A positive significant correlation was found between tumor type and antigenic expression: pure adenocarcinoma and CEA, adenocarcinoma with mucinous component and sLea and beta-catenin, pure mucinous carcinoma and Tn hapten and MUC2. Moreover, a positive tendency was found between muscular invasion and Ley and sLex while a positive significant correlation was found between well differentiated tumors and CEA and beta-catenin; a negative significant correlation between nuclear grade and beta-catenin. Conclusions: This study revealed correlations among antigenic expression and MF that may be important considering CCR patient prognosis. No significant financial relationships to disclose.

E14060 Background: In CCR, antigenic expression in primary tumors and also in metastatic lymphatic nodes (N+) may be useful for early detection of disease progression. The purpose of the present research was to establish the possible relationship among antigenic expression in tumor and N+ in order to disease free survival (DFS) and overall survival (OS) in CCR patients. Methods: A total of 90 CCR patients were studied: 90 primary CCR tumor samples and 64 lymphatic nodes; 22 samples from adenomas and normal colorectal mucosa specimens were employed as controls. Antigens studied were: MUC2, MUC1, MUC5AC, CEA, beta-catenin; carbohydrate antigens such as Lewis x (Lex), sialyl Lewis x (sLex), Lewis y (Ley), sialyl Lewis a (sLea) and Tn hapten. Immunohistochemistry was performed following standard procedures with antigenic retrieval. Positive response was evaluated by means of frequency analysis (p < 0.05). A reactivity index (RI) was calculated as intensity (I) x percentage of positive area (A); RI values varied from 0-12; low (0-6) and high (7-12). Intensity, percentage of positive area, immunohistochemical pattern and RI were analyzed employing Chi-Square test and ANOVA (p < 0.05), after data standardization. Results: Primary malignant samples expressed MUC2 in 52% of cases; MUC1, 94%; MUC5AC, 14%; CEA, 93%; beta-catenin, 98 %; Lex, 74%; sLe x, 66%, Ley; 91%; sLea, 90% and Tn, 41%. N+ expressed MUC2, 30%; MUC1, 89%; MUC5AC, 19%; CEA, 80%; beta-catenin, 83%; Lex, 69%; sLex, 72%; Ley, 75%; sLea, 86% while Tn, 44%. In general, CCR showed a higher RI than control samples. Patients with a low Lex RI in primary tumor presented a longer DFS and OS than patients with a high Lex RI (44 vs. 26 months and 4.1 vs. 2.5 years, respectively). In N+ patients, Lex and sLex were also studied in lymphatic node; a low sLex RI was correlated with a longer DFS and OS than patients with a high sLex RI (44 vs. 14 months and 3.7 vs. 1.5 years, respectively), (p 0.07). In N+ with a low Lex RI presented a longer DFS than patients with a high Lex RI (24 vs. 18 months). Conclusions: This study showed that, in CCR patients, carbohydrate antigenic expression may indicate a difference in DFS and OS. No significant financial relationships to disclose.

Background: In cancer patients, MUC1 glycoprotein may carry Lewis y which could be involved in immune response. Purposes: 1- to evaluate the presence of Lewis y and MUC1 in circulating immune complexes (Lewis y/CIC and MUC1/CIC, respectively) and their correlation; 2- to analyze the possible presence of Lewis y in carbohydrate chains of tumoral MUC1 glycoprotein and 3- to correlate serum and tissue parameters considered.Methods: Pretreatment serum and tissue breast samples from 76 adenocarcinoma, 34 benign and 36 normal specimens were analyzed. Anti-MUC1 and anti-Lewis y MAbs were employed. To detect Lewis y/CIC and MUC1/CIC, ELISA tests were developed; serum samples containing MUC1 were previously selected by Cancer Associated Serum Antigen (CASA). Immunoprecipitation (IP) was performed in 9 malignant, benign and normal samples and analyzed by SDS-PAGE and Western blot. Lewis y and MUC1 expression was studied by immunohistochemistry (IHC). Statistical analysis was performed employing principal component analysis (PCA), ANOVA, Tukey HSD, Chi square test and classical correlation (p < 0.05).Results: By ELISA, Lewis y/IgM/CIC levels showed statistically significant differences between breast cancer versus benign and normal samples; mean ± SD values expressed in OD units were: 0.525 ± 0.304; 0.968 ± 0.482 and 0.928 ± 0.447, for breast cancer, benign disease and normal samples, respectively, p < 0.05. Lewis y/IgG/CIC did not show any statistically significant difference. MUC1/IgM/CIC correlated with Lewis y/IgM/CIC. By CASA, 9 samples with MUC1 values above the cut off were selected and IP was performed, followed by SDS-PAGE and Western blot; bands at 200 kDa were obtained with each MAb in all the samples. By IHC, with C14 MAb, 47.5%, 31% and 35% of malignant, benign and normal samples, respectively, showed positive reaction while all the samples were positive with anti-MUC1 MAb; in both cases, with a different pattern of expression between malignant and non malignant samples.Conclusion: Our findings support that in breast cancer there was a limited humoral immune response through Lewis y/IgM/CIC levels detection which correlated with MUC1/IgM/CIC. We also found that Lewis y might be part of circulating MUC1 glycoform structure and also that Lewis y/CIC did not correlate with Lewis y expression.

Introduction: Recent studies have demonstrated that members of the GATA-binding protein (GATA) family (GATA4 and GATA5) might have pivotal roles in the transcriptional upregulation of mucin genes (MUC2, MUC3 and MUC4) in gastrointestinal epithelium. The zinc-finger GATA3 transcription factor has been reported to be involved in the growth control and differentiation of breast epithelial cells. In SAGE (serial analysis of gene expression) studies we observed an intriguing significant correlation between GATA3 and MUC1 mRNA expression in breast carcinomas. We therefore designed the present study to elucidate whether MUC1 expression is regulated by GATA3 in breast cancer cells. Methods: Promoter sequence analysis of the MUC1 gene identified six GATA cis consensus elements in the 5' flanking region (GATA1, GATA3 and four GATA-like sequences). Chromatin immunoprecipitation and electrophoretic mobility-shift assays were employed to study the presence of a functional GATA3-binding site. GATA3 and MUC1 expression was analyzed in vitro with a GATA3 knockdown assay. Furthermore, expression of GATA3 and MUC1 genes was analyzed by real-time RT-PCR and immunohistochemistry on breast cancer-specific tissue microarrays. Results: We confirmed the presence of a functional GATA3-binding site on the MUC1 promoter region in the MCF7 cell line. We determined that GATA3 knockdown assays led to a decrease in MUC1 protein expression in MCF7 and T47D cells. In addition, we detected a statistically significant correlation in expression between GATA3 and MUC1 genes at the mRNA and protein levels both in normal breast epithelium and in breast carcinomas (p = 0.01). GATA3 expression was also highly associated with estrogen receptor and progesterone receptor status (p = 0.0001) and tumor grade (p = 0.004) in breast carcinomas. Conclusion: Our study provides evidence indicating that GATA3 is probably a mediator for the transcriptional upregulation of MUC1 expression in some breast cancers.

Background: HNSCC progression to adjacent tissue and nodes may be mediated by altered glycoproteins and glycolipids such as MUC1 mucin. This report constitutes a detailed statistical study about MUC1 expression and anti-MUC1 immune responses in relation to different clinical and pathological parameters which may be useful to develop new anti HNSCC therapeutic strategies.Patients and methods: Fifty three pre treatment HNSCC patients were included: 26 (49.1%) bearing oral cavity tumors, 17 (32.1%) localized in the larynx and 10 (18.8%) in the pharynx. Three patients (5.7%) were at stage I, 5 (9.4%) stage II, 15 (28.3%) stage III and 30 (56.6%) at stage IV. MUC1 tumor expression was studied by immunohistochemistry employing two anti-MUC1 antibodies: CT33, anti cytoplasmic tail MUC1 polyclonal antibody (Ab) and C595 anti-peptidic core MUC1 monoclonal antibody. Serum levels of MUC1 and free anti-MUC1 antibodies were detected by ELISA and circulating immune complexes (CIC) by precipitation in polyethylene glycol (PEG) 3.5%; MUC1 isolation from circulating immune complexes was performed by protein A-sepharose CL-4B affinity chromatography followed by SDS-PAGE and Western blot. Statistical analysis consisted in Multivariate Principal Component Analysis (PCA); ANOVA test (Tukey's test) was employed to find differences among groups; nonparametrical correlations (Kendall's Tau) were applied when necessary. Statistical significance was set to p < 0.05 in all cases.Results: MUC1 cytoplasmic tail was detected in 40/50 (80%) and MUC1 protein core in 9/50 (18%) samples while serum MUC1 levels were elevated in 8/53 (15%) patients. A significant statistical correlation was found between MUC1 serum levels and anti-MUC1 IgG free antibodies, while a negative correlation between MUC1 serum levels and anti-MUC1 IgM free antibodies was found. Circulating immune complexes were elevated in 16/53 (30%) samples and were also statistically associated with advanced tumor stage. MUC1 was identified as an antigenic component of IgG circulating immune complexes. Moreover, poorly differentiated tumors were inversely correlated with tumor and serum MUC1 detection and positively correlated with node involvement and tumor mass.Conclusion: Possibly, tumor cells produce MUC1 mucin which is liberated to the circulation and captured by IgG antibodies forming MUC1-IgG-CIC. Another interesting conclusion is that poorly differentiated tumors are inversely correlated with tumor and serum MUC1 detection.

1- to detect alpha 1-acid glycoprotein (AGP) and sialyl Lewis x (sLex) in colorectal malignant, benign and normal samples; 2- to isolate AGP from colorectal cancer and 3- to study its immunoreactivity with an anti-sLex monoclonal antibody (MAb). tissue and serum samples from 88 patients with colorectal cancer, 22 adenomas and 23 normal were included. Expression of AGP and sLex was studied by immunohistochemistry (IHC); isolation approach: AGP was precipitated with ammonium sulphate and immunoprecipitated with anti-AGP MAb. The immune complex formed was isolated by protein A-Sepharose CL-4B affinity chromatography and further eluted; fractions were analysed by SDS-PAGE and Western-blot. Statistical analysis was performed by means of Principal Component Analysis. By Western blot employing anti-AGP MAb and sLex MAbs, isolated fractions from malignant samples showed a band at about 45 kD. IHC revealed that AGP was expressed in 70% of colorectal carcinoma samples, 50% of benign and 35% of normals. SLex was detected in 31% of malignant samples, 41% of benign and in one normal sample. In malignant samples, AGP reaction comprised the whole specimen with a strong and homogeneous staining while normal and benign samples showed a restricted reaction. In cancer, sLex expression consisted in an intense reactivity in membrane, cellular debris and some cytoplasmic foci while normal and benign samples were occasionally stained. A statistically significant positive correlation was found between AGP and sLex expression. Serum AGP levels were measured by radial immunodiffusion and statistical comparative analysis with tissue expression did not show a correlation between both parameters. AGP may constitute a carrier of sLex in colorectal cancer.

The dissemination of a malignant neoplasia is a complex process, which requires a set of molecules that remains unknown. It has been suggested that mucins and their carbohydrate-associated antigens...

Our aim was to determine the pattern of expression of MUC1 mucin cytoplasmic tail (MUC1 CT) in breast carcinoma. A total of 98 invasive breast adenocarcinoma tumor samples were assayed by immunohistochemical (IHC) analysis. The pattern of reaction was classified as membrane, cytoplasmic, or mixed. Subcellular fractions were prepared after SDS-PAGE and Western blotting. The antibodies employed were anti-MUC1 CT (CT2 monoclonal antibody, MAb) and C595 MAb against the extracellular MUC1 core protein. With the CT2 MAb, IHC showed a high percentage of positive staining in 93% of specimens, with membrane staining the most common pattern observed. C595 MAb was reactive in 73% of specimens. Similar percentages of membrane and cytoplasmic staining were found, mainly in a mixed pattern. Western blotting showed different bands. With the CT2 MAb, the membrane fraction showed the most intense reaction; a strong band of reaction was detected at approximately <30 kD. With the C595 MAb, in most cases a double band at 200 kD was found. In breast epithelium, the pattern of MUC1 CT expression may constitute an indicator of MUC1 production because it does not depend on glycosylation. The pattern and extension of MUC1 CT positivity do not vary according to the histopathological subtype of the tumor.

An immunological analysis to study MUC1 mucin core protein and carbohydrate associated antigens as tissue tumor markers in head and neck carcinoma was performed. Twenty nine patients with the following tumor localizations were included: tongue (n=10), larynx (n=8), oral cavity (n=4), maxillary sinus (n=3), tonsillar ring (n=3) and pharynx (n=1); seven samples of epithelium obtained from normal organs at the same localizations were studied as controls. Immunohistochemical analysis was performed following standard procedures and reaction was graded according to staining intensity and distribution. From each tissue section, membrane, cytoplasmic and nuclear moieties were obtained by differential centrifugation with subsequent fractionation by density gradient centrifugation (6M guanidium chloride-CsCl); subcellular moieties and CsCl derived fractions were analyzed by immunoblotting. Monoclonal antibodies (MAbs) reacting with the core protein of MUCI (C595) and associated carbohydrate antigens were: Tn, 83D4 MAb; Lewis y antigen (Le y), C14 MAb; Lewis x antigen (Le x), KM380 MAb and sialyl Lewis x (sLe x), KM93 MAb. Statistical analysis was undertaken by Spearman rank correlation. In tumor samples, the immunohis-tochemical identification of MUCl core protein and associated antigens was extended; differences were found in the pattern and intensity of expression; results were corroborated by immunoblotting although in a few samples there was not coincidence between both methods. Localization, tumor mass or node involvement did not show significant differences for any of the antigens studied. Conclusions: 1) head and neck carcinoma expressed MUCI and associated carbohydrate antigens in high levels; 2) no relationship between antigenic expression and tumor status was found.