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Aim Colorectal cancer (CRC) is one of the most prevalent malignancies in Argentina with 11,043 new cases and 6,596 deaths estimated to have occurred in 2008. The present study was developed to clarify the differential expression of MUC1, MUC2, sLex, and sLea in colorectal cancer patients and their relationship with survival and clinical and histological features. Methods Ninety primary tumor samples and 43 metastatic lymph nodes from CRC patients were studied; follow-up was documented. Twenty-six adenoma and 68 histological normal mucosa specimens were analyzed. An immunohistochemical approach was applied and statistical analysis was performed. Results In tumor samples, MUC1, sLea, and sLex were highly expressed (94%, 67%, and 91%, respectively); also, we found a significantly increased expression of the 3 antigens in primary tumors and metastatic lymph nodes compared with normal mucosa and adenomas. MUC2 was expressed in 52% of both normal mucosa and CRC samples; this reactivity significantly decreased in metastatic lymph nodes (pConclusions The correlation of sLex overexpression in primary tumors and metastatic lymph nodes, the discrimination among the normal, adenoma, and CRC groups based on sLex expression, as well as its association with recurrence and survival, all suggest a prognostic role of sLex in Argentinian CRC patients.

The aim of this study was to elucidate whether the IgG humoral immune response to breast cancer cells is directed to the aberrant mucin-1 (MUC1) associated to this type of cancer. To this aim, an adaptation of immunohistochemistry (IHC) was performed on samples of 45 breast cancer tissues, 12 benign disease tissues, and 31 normal tissues, incubated with matched serum samples from the same patients. Each serum sample was also incubated, with a modified immunocytochemistry (ICC), with MCF7 cells. In both techniques, serum was employed instead of the primary antibody. In the case of IHC, the reactivity with sera diminished when added after previous incubation of the tumor/tissue with an anti-MUC1 mAb; the reduction in reactivity was: from 93% to 44% in breast cancer tissues, and from 100% to 67% in benign disease tissues. The reactivity of normal samples (36%) remained unchanged. In the case of ICC, the reactivity with sera decreased after incubation with anti-MUC1 mAb from 71% to 16% in breast cancer tissues, from 83% to 0% in benign disease tissues, and from 52% to 10% in normal serum samples. These results were confirmed employing siRNA MUC1 transient gene knockdown. By Western blot analysis – after immunoprecipitation (IP) of the circulating MUC1– and ELISA, the TF antigen was detected in circulating MUC1 in all breast cancer and benign samples while Tn was detected in 38% of the samples. The existence of IgG autoantibodies against aberrantly glycosylated MUC1 may have a protective role and may contribute to a better prognosis in some patients. Enhancement of this natural immune response may constitute an alternative therapeutic strategy.

In Argentina, there are no studies evaluating neither breast cancer screening nor risk and prognostic factors in relation to socioeconomic status among women in metropolitan areas. Taking into account that Argentina presents social and economical disparities and that there is a mixture of features of both developed and developing societies, it is interesting to compare prognostic and risk factors in disadvantaged and advantaged women as it would clarify the influence of socioeconomic factors in breast cancer biology. The purpose of this study was to compare risk and prognostic factors of invasive breast cancer in two different Argentine populations. Study participants and data collection. A total of 625 women who had a histologically confirmed diagnosis of invasive primary breast cancer were included; 270 patients belonged to a private clinic of the city of La Plata (province of Buenos Aires) belonging to an Advantaged Population (AP) and 355 patients belonged to a public hospital of the city of Neuquén (province of Neuquén, Patagonia) belonging to a Disadvantaged Population (DP). Women of these geographical regions and first diagnosed with invasive primary breast carcinoma from 2002 until 2007 were eligible as cases. There were no racial or ethnic differences between the two groups of women; all of them were born in Argentina. Risk factors included age at diagnosis, menarche and menopause status, breastfeeding and parity, while prognostic factors were: disease stage, number of metastatic lymph nodes, tumor size, histological and nuclear grade, vascular invasion, ER, PR, and Her2neu statuses. Methods: Statistical analysis included frequency analysis and ANOVA (p < 0.05). Results: A remarkable difference between the two populations was found: the age at diagnosis was significantly lower in DP than in AP: 63% of DP versus 44% of AP was Conclusions: Patients belonging to these two different geographical regions constitute two different populations. Breastfeeding and number of children, considered in relation to socio-economic features, are important risk factors of invasive breast cancer. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-07-13.

An immunohistochemical analysis was employed to determine the expression of carbohydrate antigens associated to mucins in normal epithelia. Tissue samples were obtained as biopsies from normal breast (18), colon (35) and oral cavity mucosa (8). The following carbohydrate epitopes were studied: sialyl-Lewis x, Lewis x, Lewis y, Tn hapten, sialyl-Tn and Thomsen-Friedenreich antigen. Mucins were also studied employing antibodies against MUC1, MUC2, MUC4, MUC5AC, MUC6 and also normal colonic glycolipid. Statistical analysis was performed and Kendall correlations were obtained. Lewis x showed an apical pattern mainly at plasma membrane, although cytoplasmic staining was also found in most samples. TF, Tn and sTn haptens were detected in few specimens, while sLewis x was found in oral mucosa and breast tissue. Also, normal breast expressed MUC1 at a high percentage, whereas MUC4 was observed in a small number of samples. Colon specimens mainly expressed MUC2 and MUC1, while most oral mucosa samples expressed MUC4 and MUC1. A positive correlation between MUC1VNTR and TF epitope (r=0.396) was found in breast samples, while in colon specimens MUC2 and colonic glycolipid versus Lewis x were statistically significantly correlated (r=0.28 and r=0.29, respectively). As a conclusion, a defined carbohydrate epitope expression is not exclusive of normal tissue or a determined localization, and it is possible to assume that different glycoproteins and glycolipids may be carriers of carbohydrate antigens depending on the tissue localization considered.

Background: The glycoprotein MUC1 is overexpressed and underglycosylated in cancer cells. MUC1 is translated as a single polypeptide that undergoes autocleavage into 2 subunits (the extracellular domain and the cytoplasmic tail), and forms a stable heterodimer at the apical membrane of normal epithelial cells. The MUC1 cytoplasmic tail localizes to the cytoplasm of transformed cells and is targeted to the nucleus. Aims: To study the expression of the MUC1 extracellular subunit in cell nuclei of neoplastic breast, head and neck, and colon samples. Materials and methods: 330 primary tumor samples were analyzed: 166 invasive breast carcinomas, 127 head and neck tumors, and 47 colon tumors; 10 benign breast disease (BBD) and 40 normal specimens were also included. A standard immunohistochemical method with antigen retrieval was performed. Nuclear fractions from tissue homogenates and breast cancer cell lines (ZR-75, MDA-MB-231, MCF7, and T47D) were obtained and analyzed by Western blotting (WB). The anti-MUC1 extracellular subunit monoclonal antibody HMFG1 was used for immunohistochemistry. Results: 37/166 breast cancer specimens, 5/127 head and neck cancer specimens, 2/47 colon cancer samples, and 3/10 BBD samples showed immunohistochemical staining at the nuclear level. No nuclear reaction was detected in normal samples. By WB, breast and colon cancer purified nuclear fractions showed reactivity at 200 kDa in 3/30 breast and 3/20 colon cancer samples as well as purified nuclear fractions obtained from breast cancer cell lines. Conclusions: This study shows that the MUC1 extracellular domain might be translocated to the cell nucleus in breast, head and neck, and colon cancer as well as BBD., Facultad de Ciencias Médicas

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.

Serum from patients with different malignancies contain an abnormal concentration of a a1-acidic-glycoprotein (AAG) and also, increased levels of AAG are associated with the presence of tumor mass. In the present report, serum levels of AAG were measured by radial immunodiffusion in squamous cell carcinoma of the head and neck (SCCHN) patients taking into account disease status parameters such as tumor localization, stage and extension of disease. Immunohistochemical methods, SDS-PAGE and Western-blotting were employed to study the expression of AAG and a carbohydrate related antigen (sialyl Lewis x) in tumor tissues and derived fractions. AAG showed abnormal levels in 7/15 oral cavity tumor patients sera, 2/5 oropharynx and 5/10 larynx tumors; increased AAG serum levels belonged to patients with disseminated disease. On the other hand, the presence of AAG and sialyl Lewis x were demonstrated in carcinoma cells and in derived fractions from tumor tissues belonging to patients with elevated AAG serum levels. In the present study, we have found elevated levels of AAG in serum samples from SCCHN patients; these neoplastic cells are capable to express AAG.

There is convincing epidemiological evidence that multiparity provides protection against the development of breast cancer. In the present study we evaluated the levels of MUC1 and MUC1 circulating immune complexes (MUC1-CIC) in 135 serum samples obtained from healthy women. The study population included 13 women who had never been pregnant, 31 primiparous pregnant women, 36 multiparous pregnant women who had lactated, 5 multiparous pregnant women who had never lactated, 24 multiparous non-pregnant women who were lactating at the time of the study, 24 multiparous non-pregnant women who had lactated, and 2 multiparous non-pregnant women who had never lactated. The purpose of this work was to detect MUC1 variations during pregnancy and lactation as well as to study the possible induction of a humoral immune response against MUC1 in these conditions. We employed ELISA techniques to measure MUC1 (CASA test) and MUC1-CIC (IgM and IgG) using two anti-MUC1 monoclonal antibodies (MAbs): C595 and SM3. Statistical analysis was performed using the ANOVA test. The pooled results pertaining to pregnant versus non-pregnant women were compared and significant differences were observed in MUC1 and MUC1-CIC-IgM levels detected with both MAbs; the MUC1-CIC-IgG levels detected with C595 were increased in the pregnant group while the MUC1-CIC-IgG levels detected with SM3 did not show any significant differences. When the results were compared between lactating and non-lactating women, no significant differences were found. In conclusion, MUC1 and MUC1-CIC-IgM, detected with both MAbs, and MUC1-CIC-IgG levels detected with the MAb C595 are apparently induced by pregnancy.

The most common clinical form of lung cancer is a disseminated disease with distant metastases; several years of cancer progression precede presentation, and this ultimately limits the efficacy of curative therapy. In this immunohistochemical study, we examined a mucinous adenocarcinoma cell line, maintained by xenogeneic transplantation, and a spontaneous metastatic variant which produces distant tumors (in liver, spleen and kidney). The aim was to investigate possible parameters which characterize the metastatic process. Histopathological comparison between the two subcutaneous transplanted tumor lines showed that both lines presented a similar cellular morphology, a different pattern of cellular growth and an increased vascularization in the metastatic line with respect to its parent. All the tumor sections expressed differential immune reactivity with monoclonal antibodies against Lewis y (MAb C14), sialyl-Lewis x (MAb SNH3) and Lewis x (MAb FH2) determinants. Neither expressed MUC 1 mucins detectable with monoclonal antibodies reactive with the mucin protein core (MAbs C595 and SM3) nor was carcinoembryonic antigen (MAb C365) expressed. Neoplastic cells were reactive with an anti-pan cytokeratin monoclonal antibody confirming their epithelial histogenesis. Our findings have been evaluated with respect to defining metastatic phenotypes in lung cancer by examination of distinct histopathological and immunological parameters.

BackgroundHNSCC 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 methodsFifty 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.ResultsMUC1 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.ConclusionPossibly, 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.

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 may be implicated in tumour spreading which may be also influenced by an anti-MUC1 immune response. In this pilot study, we report the pattern of carbohydrate and peptidic MUC1-associated epitopes on carcinoma cells isolated from bone marrow (BM), taking into account primary tumour histopathologic features. We also bring information about the anti-MUC1 humoral response in these patients. Seventeen patients with invasive breast carcinoma were included. A sample of the primary tumour, a serum sample and a BM aspirate were obtained from each patient. Clinical features studied were tumour size, number of metastatic nodes, histological type and disease stage. Standard immunohistochemistry was performed with antigenic retrieval using different monoclonal antibodies (MAbs): anti carbohydrate antigens: Lewis x (KM380), sLewis x (KM93), Lewis y (C14) and Tn, anti-MUC1 peptide core MAbs: C595, HMFG2 and SM3, anti-cytokeratins, anti-protoncogenes ErbB2 and ErbB3 (IgG) MAbs and also anti-CD34 and anti-CD45 MAbs. ELISA techniques were employed to study circulating MUC1 as well as free and complexed anti-MUC1 antibodies. Immunohistochemical results showed that carbohydrate antigenic expression increases in BM neoplastic cells compared to the original tumours. However, we were not able to demonstrate that a humoral immune response to MUC1 has been induced in these patients. Finally, the employed procedures allow the selective immortalisation of micrometastatic carcinoma cells since short-term cell lines were established.

Tumor MUC1 expression as well as levels of MUC1, MUC1 circulating immune complexes (MUC1-CIC) and free antibodies against MUC1 (IgG and IgM-MUC1) were evaluated in 70 breast cancer patients with different stages of disease. Controls included: 135 serum samples from healthy women, normal mammary tissue samples (n = 7) and benign breast disease specimens (n = 6). In all assays, pre- and post-vaccination serum samples from breast cancer patients belonging to a vaccination protocol developed at the Memorial Sloan Kettering Cancer Center (New York, USA) were included as controls. Serum MUC1 was measured through Cancer Associated Serum Antigen test and CA15-3 test. Employing ELISA, MUC1-CIC-IgG/M were measured with either C595 or SM3 monoclonal antibodies (MAb) as catchers and also free antibodies against MUC1 (IgG and IgM) using 100mer peptide as catcher. Employing multivariate statistical analysis, results were correlated with age, tumor type, stage of disease and grade of differentiation. By quantitative immunohistochemistry using three anti-MUC1 core protein MAbs (C595, HMFG2 and SM3), tumor MUC1 was detected in 60/70 (86%) breast cancer specimens which reacted with at least one of these MAbs. High MUCI serum levels were detected in 14/67 (21%); IgG and IgM anti-MUC1 antibodies were found elevated in 32 and 14%, respectively, while IgG-MUC1-CIC-measured with C595 in 42% and IgM-MUC1-CIC in 54%; finally, SM3 was positive in 43 and 18%, respectively. Results of these studies demonstrate that in a group of breast cancer patients, MUC1 was detected both in tissue specimens as well as free in serum samples; furthermore, MUC1 can also circulate complexed with IgG and IgM antibodies; thus an accurate measurement should include free and complexed forms. On the other hand, immunohistochemical studies on breast cancer tissues may contribute to reveal different MUC1 glycoforms.

Serum levels of MUC1 and antibodies (Abs) against MUC1 (IgG and IgM-MUC1) were evaluated in healthy women related to pregnancy and lactation status. A total of 149 serum samples were obtained from: nulliparous, primiparous pregnant, multiparous pregnant that have lactated, multiparous pregnant without lactation, multiparous non-pregnant actual lactating, multiparous non-pregnant that have lactated and finally, multiparous non-pregnant women without lactation. In all assays, we included pre- and post-serum samples belonging to a breast cancer patient vaccinated with a MUC1 derived peptide. CASA test was employed to measure MUC1 while IgG- and IgM-MUC1 serum Abs were evaluated with an ELISA using a 100 mer peptide as catcher. In all groups, mean IgM levels were higher than IgG mean values; when samples were grouped in pregnants versus non-pregnants, a significant difference was detected with both Abs, being raised in non-pregnants. When samples were grouped in lactating versus non-lactating a significant difference was detected with IgG-MUC1, being raised in lactating women while no significant difference was found with IgM-MUC1. The evaluation of serum MUC1 levels confirmed previous results since a significant difference between pregnant versus non-pregnant groups was found while lactating versus non-lactating samples did not. Conclusions: (i) Increased MUC1 serum levels are apparently associated with pregnancy but not with lactation; (ii) MUC1 Abs are mainly associated with lactation and with non-pregnant status. These results may be considered a contribution on studies about protection against breast cancer induced by pregnancy and lactation.

The purpose of this report was the initiation and further maintenance of tumor cells from a primary larynx squamous cell carcinoma. A tumor fragment was mechanically dissociated, the cells were grown in RPMI medium, being the primary culture dependent on the presence of epidermal growth factor and insulin; during subsequent passages the adaptation to conventional growth conditions was obtained. Cells grew in monolayer with an epitheliod shape, showing a pavement-like arrangement; at confluence, cells piled up without contact inhibition maintaining the same morphology. Population doubling time was about 48 h with a colony-forming efficiency of 10%. Immunocytochemical characterization was performed with a panel of monoclonal antibodies reactive against tumor associated antigens, including mucin glycoproteins and related carbohydrate antigens, carcinoembryonic antigen (CEA), p53 as well as cytokeratins, vimentin and desmin. T201 expressed CEA, sialyl Lewis x, Lewis x, Lewis y, MUC1 mucin, Tn hapten, p53, vimentin and cytokeratins. On the other hand, a modal chromosome diploid number of 46 occurring in 74% of cells was detected. Present data confirmed that the methodology employed was adequate for the establishment and characterization of a new cell line which can provide a useful model to study biological and immunological aspects of larynx squamous cell carcinoma.

The progression from uncontrolled cell proliferation to invasion and metastasis of epithelial tumors is partially understood. Alteration of epithelial mucin expression have been described in different malignant localizations but only few attempts have been made to identify mucin expression in malignant laryngeal tumors. In the present report, results are shown of studies on the expression of mucins and carbohydrate related antigens in laryngeal cancer and on the isolation of MUC1 mucin from this tumor tissue. Malignant laryngeal specimens were processed for immunohistochemical analysis and for extranuclear membrane fractions (ENM) which were obtained by ultracentrifugation. Subsequently, ENM samples were centrifuged in density-gradient; the analysis of fractions was performed by means of SDS-PAGE and Western-blotting. The panel of monoclonal antibodies (MAbs) included anti MUC1 mucin, anti Lewis x, anti sialyl Lewis x, anti Lewis y, anti MUC-5B, anti oral mucin (gp230), anti Tn hapten, anti p53 and anti cytokeratins. By immunohistochemistry, it was possible to detect MUC1 mucin, Lewis x and Lewis y showing strong reactions while sialy1-Lewis x and Tn antigen only reacted weakly in a few cells; cytokeratins were detected in all samples. In ENM derived fractions obtained by CsC1 centrifugation, MUC1 was demonstrated by Western blotting. Conclusions: (1) laryngeal cancer antigenic expression comprises mostly MUC1 mucin, Lewis x, Lewis y as well as Tn antigen and (2) the methodology here employed is useful to isolate MUC1 from tumor samples.

The morphology, cell growth, antigenic expression and tumorigenicity of cell subpopulations from the A549 lung adenocarcinoma isolated by Percoll gradient separation have been analysed. Four subpopulations were obtained (subpopulations A, B, C and D). Immunocytochemical analysis of several antigens was performed with monoclonal antibodies (MAbs): MUC1 mucin (C595, HMFG1 and HMFG2), MUC5B (PANH2); gp230 (PANH4); carbohydrate antigens including sialyl Lewis x (KM93), Tn antigen (83D4), Lewis y (C14); 5, 6, 8, 17 and 19 cytokeratins and p53. The cell population D tended to form cell aggregates that piled up on the monolayer similar to overgrowth cultures of the A549 parental cell line, whereas A, B and C cell subpopulations formed well spread monolayers. Both parental A549 and subpopulation D secreted abundant mucus. The topographic distribution and secretion production were correlated with tumorigenic assays since only subpopulation D grew in nude mice exhibiting reduced latency period; these characteristics correlated with the fast growth of the subpopulation D in vitro. Immunocytochemical analysis demonstrated that subpopulation D showed greater expression of MUC1 mucin and carbohydrate antigens such as Tn antigen, sialyl Lewis x and Lewis y and less expression of cytokeratins, p53, MUC5B and gp230; conversely, subpopulations A, B and C showed the opposite antigenic profile. Our results illustrate heterogeneity in the A549 cell line; subpopulations A, B and C retained characteristics of more differentiated adenocarcinoma while subpopulation D displayed features of a less differentiated tumor line.

The occurrence of increased circulating immune complexes (CIC) in sera of patients with esophageal cancer and their usefulness for diagnosis and prognosis have not been demonstrated. Circulating acute-phase proteins (APP) related to esophageal cancer have been described but without any association with CIC. This is a study to measure CIC, C-reactive protein (CRP), and alpha 1-acidic glycoprotein (AAG) in pretreatment esophageal cancer sera and to analyze the presence of both APP associated with these CIC. Increased CIC levels were found in 57% of sera from esophageal cancer patients; elevated CRP was detected in 87% and AAG in 47%. Western blot analysis showed the presence of CRP and AAG in CIC-derived fractions. We conclude that: (1) CIC, CRP, and AAG are elevated in esophageal cancer sera; (2) they may be considered possible useful clinical parameters in pretreatment esophageal cancer patients; (3) these APPs appear in CIC precipitates and may possibly be involved in their composition.

The aim of this study was to examine tissue from patients with breast carcinoma or benign breast disease for the presence of monoclonal-antibody-defined antigens, including the MUC1 mucin and carcinoembryonic antigen CEA. The tests were performed by sodium dodecyl sulphate/polyacrylamide gel electrophoretic separation of proteins, electrophoretic transfer to nitrocellulose membranes and immunostaining with the monoclonal antibodies. Some of the antigens identified are known to circulate at high levels in some but not necessarily all, breast carcinoma patients. Serum from a panel of ten breast cancer patients was subjected to a fractionation procedure designed to release antigen from immune complexes, and again these samples were analysed for the presence of monoclonal-antibody-defined antigens. A high frequency of positive reactions was detected by the anti-MUC1 monoclonal antibody C595 with both breast carcinoma subcellular membrane fractions as well as antigen fractions eluted from circulating immune complexes. No reactions were observed with equivalent materials from benign breast disease samples. The findings illustrate the variability in antigen expression between breast tumours. The data also indicate that a proportion of patients respond to their tumour by the production of antibodies that recognise the MUC1 antigen in their circulation.

Introduction Despite of an excellent oncologic surgery with broad normal tissue margins, the mucosa next to the bed anastomosed is a frequent place of tumoral recurrence on colorectal cancer. Objective: This study was performed to evaluate the expression of mucins and carbohydrates associated antigens in normal mucosa colorectal specimens (NMC), mucosa adjacent to colorectal cancer (MACCR) and malignant colorectal tumour samples (CCR). Methods Ninety colorectal cancer tumour samples and their MACCR, and also 68 NMC were included. Monoclonal antibodies (MAbs) against the following antigens were employed: anti-MUC1 (HMFG1), MUC2 (H-300) and MUC5AC (45M1), anti-Tn (HB-Tn1), Lex (KM380), sLex (KM93), Ley (C70) and sLea (1116-N5-19-9). An immunohistochemical approach following standard procedures was performed. Statistical analysis: an univariate statistical analysis with Chi2 was applied. Results Malignant samples expressed MUC1 in 94% of cases, MUC2 in 52.4%, MUC5AC in 14.3%, Tn in 41%, Lex in 74.4%, sLex in 66.7%, Ley in 91.8% and sLea in 90.7%. Taking into account MUC2 and Ley expression, a positive correlation was found between MACCR and CCR: MUC2, P = .0006 and Ley, P = .0008. In malignant samples, MUC1 expression was increased compared to NMC (P = .04), also this mucin was 22.2% higher in MACCR than NMC. The expression of sLex and Ley was higher in CCR than NMC (179% and 33.6%, respectively). Expression of most antigens showed an increased tendency from NMC to MACCR and CCR: MUC1 (27.9%, 34.1% and 94%, respectively), Lex (60.9%, 30.5% and 74.4%, respectively), sLex (23.9%, 16.9% and 66.7%, respectively), Ley (68.7%, 78.3% and 91.8%, respectively) and sLea (62.1%, 45.6% and 90.7%, respectively). On the other hand, MUC5AC expression decreased in the mentioned sequence (51.5%, 20.7% and 14.3, respectively). Conclusion Our results may support the hypothesis that the MACCR could be an intermediate point or “transitional zone” between NMC and CCR. Further studies are needed in order to correlate these findings with clinical outcomes. Disclosure All authors have declared no conflicts of interest.

Colorectal carcinoma (CRC) is the second leading cause of death among malignancies in the world. Postoperative chemotherapy is widely accepted as the standard modality for the treatment of this disease; however 50% of patients within an oncology protocol will develop tumor metastatic dissemination. The drug 5-fluorouracil (5-FU) is one of the most commonly used agents for the treatment of CRC at early and advanced tumor stages, but clinical resistance is a major limitation. The identification of novel predictive biomarkers of resistance to 5-FU treatment would allow developing new therapies and improving the quality of life for CRC patients. The aim of this study was to evaluate the role of RHBDD2 gene expression in human colorectal carcinogenesis and its effect on treatment with chemotherapeutic agent 5-FU. Firstly, we analyzed a dataset of 430 colorectal samples (32 healthy control, 355 primary CRC and 43 metastatic CRC) derived from two independent public available gene expression studies: Bittner et al., 2005 (GEO Acc.#GSE2109), and Sabates-Bellver et al., 2007 (GEO Acc.#GSE8671). A statistical significant increase in RHBDD2 expression was detected between normal colorectal samples and CRC specimens with and without metastasis (p In conclusion, our findings suggest that RHBDD2 over-expression might play a role in colorectal neoplastic progression, modulating the response of colon cancer cells to 5-FU treatment. Further studies are needed to evaluate the relevance of RHBDD2 gene expression as a predictive biomarker as well as a therapeutic target to enhance drug efficacy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3801. doi:10.1158/1538-7445.AM2011-3801

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 re...

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 employ...

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. 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). 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. 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., Facultad de Ciencias Médicas

Sera from 62 patients with osteogenic sarcoma and 12 with malignant giant-cell tumour were tested for the presence of immune complexes by the 125I-Clq binding assay. Elevated serum Clq binding activity was found in 67.7% of the osteogenic sarcoma patients and in 75% of the giant-cell tumour patients. These results were compared with those obtained with five sera from patients with benign bone tumours and 20 sera from normal young donors. In the last two groups, the incidence of elevated Clq-binding activity was 0% and 5%, respectively. In some patients with giant-cell tumours, pre- and post-operative serum samples were studied, showing a decrease in test values after tumour resection. Preliminary sequential studies of individual patients indicate that the 125I-Clq binding assay may be useful for monitoring patients with bone tumours.

CANCERHASbecomean increasingly important disease incanine populations, partly asaresult ofadvances inveterinary medicine, whereelimination of many infectious diseases by vaccination has considerably extended theaverage lifespanofdogs.Deathsarenow more frequently attributed todiseases ofoldage, ofwhichcancer isanimporant example (Hannant etal., 1978). Manydiseases ofdogspresent asimilar clinico-pathological pictureto their counterparts inhumans,andthereare several comparative studies relating clinicalandmorphological aspects ofcanine tumours. Thereare,however, fewreports existing on theadaptability ofclassic markersof disease. A studyof the comparative pathology betweenhuman andcanineosteosarcoma (Owen,1969) showedthatthebiological behaviours of this tumourissimilar inthetwospecies. Otherneoplasms ofcomparative medical andveterinary interest include spontaneousmammarycarcinoma (Owen, 1979) wherecirculating immunecomplexes have beendemonstrated. Immunecomplexes havebeenstudied in relation tothepathogenesis ofhumanand animaldisorders including malignant diseases (Hoffken etal., 1978b; Baldwin etal., 1979; Termanetal., 1980). Inrecent years sensitive techniques havebeenapplied to detect immunecomplexes inserafrom patients withmany typesoftumour (reviewed by Baldwin& Robins, 1980). Circulating immunecomplexes inhuman osteosarcoma havebeenstudied using the Clqbinding testbyTsangetal.(1979) and inthislaboratory by Segal-Eiras etal. (1980). Thepossibility ofinvestigating alarge population ofdogswitho.s.allowed the development ofthepresent study,in whichtheincidence ofserawithraised Clq bindingcouldbe determined, and a preliminary investigation ofchanges inClq binding withdisease progress couldbe made. Theseinvestigations wereperformed in parallel with human bone-tumour immune-complex studies, andusedhuman Clq,whichhaspreviously beenshownto bindrat(Hoffken etal., 1978a) anddog (Terman etal., 1979)immunecomplexes. Following themethodofYonemasu& Stroud(1971)Clqwas prepared and purified frompooled normalhumansera and its puritywas checkedby immunoelectrophoretic

To investigate the influence of sialic acid removal on MUC1 peptidic and carbohydrate epitope reactivity in head and neck squamous cell carcinoma (HNSCC), tumor samples belonging to 24 HNSCC patients were studied by standard immunohistochemistry (IHC) with and without desialylation with 0.1 U/ml neuraminidase. From each tumor sample, subcellular fractions were obtained and analyzed by SDS-PAGE and Western blotting (WB). Three monoclonal antibodies (MAbs) were used: C595 MAb directed to MUC1 protein core, an antiTn hapten MAb, and an anti-sTn hapten MAb; a comparative analysis between desialylated and sialylated samples was performed. By IHC without neuraminidase treatment, 19 of 24 samples reacted with anti-MUC1 peptidic epitope, while Tn hapten was not detected and sTn was found in 1 of 24 cases. Desialylation increased either the number of reacting cells or the intensity of the reaction with C595 and anti-Tn MAbs, and some negative samples became positive. On the other hand, sTn expression decreased with desialylation. By WB, several bands from >200 to 25 kDa were found; desialylation increased high-molecular-weight bands, diminishing the detection of low-molecular-weight ones. The use of desialylation is a suitable treatment that contributes to the exposure of MUC1-associated epitopes, which may be related to the spreading of HNSCC. (Pathology Oncology Research Vol 11, No 2, 74–81)