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Amalia Luna, Maria Emilia Merino, Cecilio G Alberdi, Martin C Abba, Amada Segal-Eiras, Maria Virginia Croce Center of Basic and Applied Immunological Research, Faculty of Medical Sciences, National University of La Plata, Argentina Abstract: Breast cancer is the most common cause of cutaneous metastases from internal malignancies. Generally, the neoplastic cells are located in the dermis or hypodermis, while a finding of transepidermal elimination on cutaneous metastases is exceptional. In this report we present a patient with perforating cutaneous metastases from breast cancer with mucin 1 expression. Cutaneous, bone, lung, and hepatic lesions were detected two years after the diagnosis of the primary tumor. Keywords: breast cancer, cutaneous metastasis, transepidermal elimination, MUC1

Sandra O Demichelis, Marina T Isla-Larrain, Luciano Cermignani, Cecilio G Alberdi, Amada Segal-Eiras, María Virginia CroceCentre of Basic and Applied Immunological Research, Faculty of Medical Sciences, National University of La Plata, La Plata, ArgentinaObjective: In breast cancer, several tumor markers have been identified. The marker most extensively associated with breast cancer is MUC1. The objective of the study was to analyze prognostic and risk factors in relation to tumor markers in order to clarify breast cancer biology. A total of 349 primary tumor samples and lymph nodes from breast cancer patients were studied. Risk and prognostic factors were considered. An immunohistochemical approach was applied and an extensive statistical analysis was performed, including frequency analysis and analysis of variance. Correlation among variables was performed with principal component analysis.Results: All the antigens showed an increased expression according to tumor size increment; moreover, sialyl Lewis x expression showed a significant increase in relation to disease stage, whereas Tn and TF presented a positive tendency. Vascular invasion was related to sialyl Lewis x expression and number of metastatic lymph nodes. Taking into account risk factors, when a patient had at least one child, Lewis antigens diminished their expression. In relation to breastfeeding, sialyl Lewis x expression diminished, although its apical expression increased.Conclusion: Associations between MUC1 and carbohydrate antigens and risk and prognostic factors show the complexity of the cellular biological behavior that these antigens modulate in breast cancer.Keywords: breast cancer, Argentine women, risk factors, prognostic factors, antigenic expression

Lewis x functions as an adhesion molecule in glycolipids and glycoproteins since it mediates homophilic and heterophilic attachment of normal and tumoral cells. During malignancy, altered glycosylation is a frequent event; accumulating data support the expression of Lewis x in tumors although controversial results have been described including its relationship with patient survival. This report has been developed as an introduction to the relationship between Lewis x expression and breast cancer and head and neck squamous cell carcinoma (HNSCC). Results obtained in our laboratory are presented in the context of the literature.

Breast cancer spreading to different organs have been related to different molecules and mechanisms, but cutaneous metastasis remains unexplored. Increasing evidence showed that MUC1 and some of its carbohydrate associated antigens may be implicated in breast cancer metastasis. In this study we analyzed these tumor markers in order to identify breast cancer cutaneous metastatic profiles. A cohort of 26 primary tumors from breast cancer patients with cutaneous metastases were included; also, cutaneous and lymphatic node metastatic samples and primary tumors from breast cancer patients without metastases were analysed. Immunohistochemical (IHC) studies demonstrated that both underglycosylated MUC1 (uMUC1) and sialyl Lewis x (sLex) to be positively associated with cutaneous metastatic primary tumors (p < 0.05). Notably, a high percentage of tumors with cutaneous metastases were characterized as triple negative and Her2+ tumors (37.5 % and 29 %, respectively). Some discordant results were found between primary tumors and their matched cutaneous metastases. To determine if MUC1 variants may be carriers of carbohydrate antigens, subcellular fractions from a cutaneous metastatic lesion were obtained, immunoprecipitated and analyzed by Western blot. We found that the isolated uMUC1 with a molecular weight of>200 kDa was also the site for binding of anti-sLex MAb; in coincidence, a high correlation of positive IHC expression of both markers was observed. Our findings confirm that breast cancer cutaneous metastases were associated to highly malignant primary tumors and sustain the hypothesis that u-MUC1 and sLe x may drive breast cancer cutaneous metastases., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

RHBDD2 is an intramembrane pseudoprotease member of the Rhomboid superfamily. Our previous studies in breast and colorectal cancer indicate an association between RHBDD2 overexpression and advanced tumor stages. Two alternative transcriptional variants have been described for RHBDD2, which would be encoding for different RHBDD2 protein isoforms. The expression of these RHBDD2 variants/isoforms and its association with breast cancer was the focus of this study. First, expression of RHBDD2 splicing variants was evaluated in normal and breast tumor samples. RHBDD2 variant 2 overexpression was detected in tumors in respect to normal breast tissues at the mRNA and protein levels (P<0.05). Moreover, RHBDD2 variant 2 expression was associated with poor prognostic factors such as basal‑like intrinsic subtype (P<0.05), high proliferation (P<0.01) and long‑term risk‑of‑recurrence (P<0.01) scores. Second, the expression of both variants was evaluated under nutritional‑deprived conditions in breast cancer cell lines. Results demonstrated that RHBDD2 splicing was switched from mRNA variant 1 to variant 2 in association with a significant increment of protein isoform B in response to glucose starvation treatment. Therefore, we propose that the switch from the RHBDD2 variant 1, expressed in normal epithelial cells, to variant 2 occurs as an adaptive phenotype to bypass the stressful tumor microenvironment and promote tumor progression. Finally, the RHBDD2 subcellular localization was corroborated at the Golgi apparatus and their associated v‑SNARE transport vesicles, suggesting a putative new role for RHBDD2 in the protein trafficking of human breast cancer cells.

Head and neck squamous cell carcinoma (HNSCC) is an aggressive disease with poor prognosis without appropriate prognostic markers. Previous research shows that Lewis antigens have been involved in carcinoma dissemination and patients´ survival. Fucosyl and sialyltransferases are the enzymes implicated in the Lewis antigens synthesis. The purpose of this study was to evaluate the prognostic utility of Lewis antigens in HNSCC. We conducted a prospective research including histological samples from 79 patients with primary HNSCC. Lewis x and sialyl Lewis x expression were detected by immunohistochemistry; patient's data, progression free, and overall survival were documented. A statistical correlation study of antigenic expression and patients´ histopathological variables was performed. Cox regression models with internal validation procedures were employed to analyze survival data. By immunohistochemistry, Lewis x was detected in 34/79 (43%) tumor samples, while sialyl Lewis x only in 11/79 (14%). Lewis x expression showed a positive correlation with tumor differentiation and a better overall survival for Lewis x + patients was detected. Moreover, multivariate Cox's regression analysis showed that Lewis x is an independent predictor of better overall survival. The in silico analysis supported the presence of deregulated fucosyl (FUT4) and sialyltransferase (ST3GAL4) in the Lewis synthetic pathway related to patient survival. These results suggest that Lewis x expression is associated with a better outcome in patients with HNSCC.

During the past years, molecular studies through high-throughput technologies have led to the confirmation of critical alterations in colorectal cancer (CRC) and the discovery of some new ones, including mutations, DNA methylations, and structural chromosomal changes. These genomic alterations might act in concert to dysregulate specific signaling pathways that normally exert their functions on critical cell phenotypes, including the regulation of cellular metabolism, proliferation, differentiation, and survival. Targeted therapy against key components of altered signaling pathways has allowed an improvement in CRC treatment. However, a significant percentage of patients with CRC and metastatic CRC will not benefit from these targeted therapies and will be restricted to systemic chemotherapy. Mechanisms of resistance have been associated with specific gene alterations. To fully understand the nature and significance of the genetic and epigenetic defects in CRC that might favor a tumor evading a given therapy, much work remains. Therefore, a dynamic link between basic molecular research and preclinical studies, which ultimately constitute the prelude to standardized therapies, is very important to provide better and more effective treatments against CRC. We present an updated revision of the main molecular features of CRC and their associated therapies currently under study in clinical trials. Moreover, we performed an unsupervised classification of CRC clinical trials with the aim of obtaining an overview of the future perspectives of preclinical studies., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Secreted mucins constitute a crucial part of the gel that protects respiratory and digestive epithelia, being MUC2/Muc2 the predominant gel-forming mucin of the intestine while MUC5AC/Muc5ac is one of the gel-forming mucins most expressed at the airways. In this study, we have analyzed Muc2 and Muc5ac during rat development by using immunohistochemistry, Western blotting and RT-PCR. We demonstrated that rat Muc2 was expressed in fetal intestinal goblet cells of surface epithelium of villi and developing Lieberkühn crypts. In neonates and adults, Muc2 was expressed at luminal goblet cells of small and large intestine and at gastric mucous and glandular cells. Muc5ac protein was observed in embryonic gastric and lung samples; expression increased during development and postnatal and adult life. After birth, a low reaction was detected at the tracheal surface epithelium and glands, which increased in adults., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Over the last few years rhomboid genes have gained interest because of its association with cancer and neurodegenerative diseases. In previous studies, we demonstrated that human RHBDD2 is over-expressed in the advanced stages of breast and colorectal cancers, suggesting a favorable role in cell proliferation. So far little is known about the expression of RHBDD2 in other tissues and other species, and because of similarities between cancer and embryonic cells, this study focused on the evaluation of Rhbdd2 expression in embryonic and adult rat tissues. By IHC and RT-PCR, Rhbdd2 was identified in early stages of most tissues analyzed, with high expression in brain, spinal cord, kidney and embryonic skin. In adult tissues, the expression remained elevated while salivary glands became positive. Furthermore, Rhbdd2 showed a high expression in the most proliferative stages of the rat mammary gland. Indeed, similar findings were observed in the mouse mammary epithelial cell line HC11, in which Rhbdd2 resides in the Golgi apparatus, and at different stages of mouse mammary gland development. Therefore, Rhbdd2 would be implicated in embryonic and adult tissue proliferation., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

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.

Unlabelled: Some mucin genes have been detected during human embryonic and fetal organ development; however, little is known about mucin expression in epidermal development, neither in humans nor in other species. The present research was developed to explore Muc5ac skin expression during prenatal and postnatal rat development. Immunohistochemistry (IHC), Western blotting (WB) and RT-PCR were employed. By IHC, Muc5ac protein was found early in embryonic epidermis from day 13 of gestation until seven days after birth when the surface epidermis became negative and the reaction was restricted to secreting sebum cells. In coincidence with IHC findings, WB analysis showed a band at approximately 200KDa at the same periods of development. Results were also confirmed by RT-PCR. Muc5ac expression in rat embryonic epidermis suggests that Muc5ac may play a protective role in embryonic skin previous to birth which may be replaced by pile covering. To our knowledge, this is the first report which confirmed Muc5ac expression during skin development., Conclusion:   Muc5ac expression in rat embryonic epidermis suggests that Muc5ac may play a protective role in embryonic skin previous to birth which may be replaced by pile covering. To our knowledge, this is the first report which confirmed Muc5ac expression during skin development.

Aim: A descriptive study was developed in an entire Argentine rural community considering breast cancer risk factors, preventive strategies and breast cancer incidence., Patients and Methods: the study comprised of 83 women. A questionnaire of 34 items was employed; a mammogram and a breast ultrasound were performed. ANOVA and Pearson correlation were employed., Results: Mean age was 54.5 years; 69% of women were postmenopausal; 96% had children; breastfeeding was X=10 months/child; Body Mass Index (BMI) was X=27.8 kg/m(2); 13% had first-degree relatives with breast cancer; 90% of women considered mammographic screening a necessary study. One woman had presented breast cancer. Argentine screening guidelines were not followed and an inverse relationship between education level and age of first mammogram was found (p<0.05). Mammographic and ultrasound studies did not reveal potential abnormalities., Conclusion: Peculiar social and cultural characteristics may be relevant to evaluate breast cancer risk factors in Argentina., (Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Indoleamine-2,3-dioxygenase (IDO) has been established as a normal mechanism of peripheral tolerance and immunosuppression. Besides, malignant tumors release microvesicles (MV) related with tumor dissemination. The aims of this study were to determine the expression of IDO in breast cancer and circulating microvesicles from breast cancer patients and to perform an in silico analysis to find genes co-expressed to IDO. One hundred and twenty-two tissue and serum breast samples (91 malignant, 21 benign, and 10 normal), and MCF7, MDA-MB-231, and T47D breast cancer cell lines were included. Standard immunohistochemistry (IHC), immunocytochemistry (ICC), Western blot (WB), and RT-PCR were employed. Microvesicle isolation from plasma samples was obtained by serial centrifugation and ultracentrifugation. By IHC, 60 % breast cancer, 43 % benign, and 20 % normal samples were positive. Significant differences were found among normal, benign, and malignant samples. Breast cancer stages I, II, and III expressed IDO in 42, 66, and 71 % of samples, respectively, while breast cancer cell lines also reacted; by WB, 9/25 microvesicles fractions showed bands at 42 kD. In silico analysis of IDO 1 gene expression in breast cancer showed its association with several genes related to immune response and apoptosis. Moreover, IDO and co-expressed genes were found predominately in basal and erbB2 subtypes. The cumulative data indicate a high expression of IDO in breast cancer which increased with higher stages. Furthermore, IDO was found in association with circulating breast cancer MV, while experimental and in silico gene expression revealed that IDO was mainly expressed in a triple-negative subgroup.

Rhomboid domain containing 2 (RHBDD2) was previously observed overexpressed and amplified in breast cancer samples. In order to identify biological pathways modulated by RHBDD2, gene expression profiles of RHBDD2 silenced breast cancer cells were analyzed using whole genome human microarray. Among the statistically significant overrepresented biological processes, we found protein metabolism—with the associated ontological terms folding , ubiquitination, and proteosomal degradation—cell death, cell cycle, and oxidative phosphorylation. In addition, we performed an in silico analysis searching for RHBDD2 co-expressed genes in several human tissues. Interestingly, the functional analysis of these genes showed similar results to those obtained with the microarray data, with negative regulation of protein metabolism and oxidative phosphorylation as the most enriched gene ontology terms. These data led us to hypothesize that RHBDD2 might be involved in endoplasmic reticulum (ER) stress response. Thus, we specifically analyzed the unfolding protein response (UPR) of the ER stress process. We used a lentivirus-based approach for stable silencing of RHBDD2 mRNA in the T47D breast cancer cell line, and we examined the transcriptional consequences on UPR genes as well as the phenotypic effects on migration and proliferation processes. By employing dithiothreitol as an UPR inducer, we observed that cells with silenced RHBDD2 showed increased expression of ATF6, IRE1, PERK, CRT, BiP, ATF4, and CHOP (p <0.01). We also observed that RHBDD2 silencing inhibited colony formation and decreased cell migration. Based on these studies, we hypothesize that RHBDD2 overexpression in breast cancer could represent an adaptive phenotype to the stressful tumor microenvironment by modulating the ER stress response.

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 (p<0.05). A multiple comparison analysis showed that MUC1 and sLex discriminated among 3 groups: normal, adenoma, and CRC tissues. The increase of sLex expression showed an association with recurrence, and survival analysis showed that a high sLex staining was significantly associated with a poor survival. By multivariate analysis MUC1 inmunoreactivity correlated positively and significantly with tumor size, while MUC2 expression showed the opposite correlation., Conclusions: 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.

Rhomboid is an evolutionary conserved and functionally diversified group of proteins composed of proteolytically active and inactive members that are involved in the modulation of multiple biological processes such as epidermal growth factor receptor signaling pathway, endoplasmic reticulum-associated degradation, cell death, and proliferation. Recently, several human rhomboid genes have been associated with the development of chronic myeloid leukemia and pituitary, colorectal, ovarian, and breast cancers. In this study, we evaluated the mRNA and protein expression profiles of rhomboid genes in cancer cell lines and breast tissue/tumor samples. In silico analysis of publicly available gene expression datasets showed that different rhomboid genes are specifically expressed according to the breast cancer intrinsic subtypes. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis showed a significant RHBDD2 mRNA overexpression in advanced breast cancer compared with normal tissue samples (p = 0.012). In addition, we found that RHBDL2 and PARL mRNA expression was associated with a low/intermediate histologic tumor grade (p = 0.024 and p = 0.015, respectively). Immunohistochemistry analysis showed a significant increase of RHBDD2 protein expression in association with breast cancer samples negative for progesterone receptor (p = 0.015). Moreover, protein expression analysis corroborated the quantitative RT-PCR results, indicating that breast primary tumors belonging to patients with a more disseminated disease expressed significantly increased levels of RHBDD2 protein compared with less disseminated tumors (p = 0.01).

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 previous studies, we identified rhomboid domain containing 2 (RHBDD2) gene to be markedly overexpressed in breast cancer patients that developed recurrence of the disease. In this study, we evaluated for the first time RHBDD2 gene expression in colorectal cancer (CRC). Five public available DNA microarray studies were compiled in a homogeneous dataset of 906 colorectal samples. The statistical analysis of these data showed a significant increase of RHBDD2 expression in the advanced stages of CRC (p < 0.01). We validated these findings by immunohistochemistry on 130 colorectal tissue samples; RHBDD2 protein overexpression was also observed in the advanced stages of the disease (p < 0.001). In addition, we investigated RHBDD2 expression in response to the chemotherapy agent 5-fluorouracile (5FU). We detected a significant increase of RHBDD2 mRNA and protein after 5FU treatment (20-40 μM; p < 0.001). Overall, these results showed that RHBDD2 overexpression might play a role in colorectal cancer progression.

Objective: In breast cancer, several tumor markers have been identified. The marker most extensively associated with breast cancer is MUC1. The objective of the study was to analyze prognostic and risk factors in relation to tumor markers in order to clarify breast cancer biology. A total of 349 primary tumor samples and lymph nodes from breast cancer patients were studied. Risk and prognostic factors were considered. An immunohistochemical approach was applied and an extensive statistical analysis was performed, including frequency analysis and analysis of variance. Correlation among variables was performed with principal component analysis., Results: All the antigens showed an increased expression according to tumor size increment; moreover, sialyl Lewis x expression showed a significant increase in relation to disease stage, whereas Tn and TF presented a positive tendency. Vascular invasion was related to sialyl Lewis x expression and number of metastatic lymph nodes. Taking into account risk factors, when a patient had at least one child, Lewis antigens diminished their expression. In relation to breastfeeding, sialyl Lewis x expression diminished, although its apical expression increased., Conclusion: Associations between MUC1 and carbohydrate antigens and risk and prognostic factors show the complexity of the cellular biological behavior that these antigens modulate in breast cancer.

During embryonic development, studies on mouse and human embryos have established that Muc1/MUC1 expression coincides with the onset of epithelial sheet and glandular formation. This study aimed therefore at evaluating the temporal and spatial expression of Muc1 at different stages of rat development. In this experiment, 80 animals were included: 64 rat foetuses at 13, 14, 15, 16, 17, 18, 19 and 20 days of gestation from pregnant females (WKAH/Hok), 8 embryos each stage. Standard immunohistochemistry was performed using anti-MUC1 cytoplasmic tail polyclonal antibody (CT33). The reaction was considered positive when more than 5% of the cells were stained; reaction patterns were: L = linear, membrane, C = cytoplasmic and M = mixed; nuclear staining was also recorded. Intensity was graded as negative (-), low (+), moderate (++) and strong (+++). Muc1 expression was observed with a low intensity on 13th day (13 d) in the stomach, lung and kidney; at 14 d, small intestine and pancreas were also reactive; at 16 d, liver and esophagus and at 18 d, trachea and salivary glands. During the development, intensity increased while the pattern of expression changed: at the first days of gestation, it was predominantly linear and apical while during further development an increase in cytoplasmic expression was observed. Trachea, stomach, kidney and lung epithelia were the more reactive tissues. In specimens belonging to neonates and adults, all tissues analyzed showed similar Muc1 expression. The findings of this study assess that Muc1 is highly expressed in the epithelial rat embryonic development.

The MUC1 gene is aberrantly overexpressed in approximately 90% of human breast cancers. Several studies have shown that MUC1 overexpression is due to transcriptional regulatory events. However, the importance of gene amplification as a mechanism leading to the increase of MUC1 expression in breast cancer has been poorly characterized. The aim of this study was to evaluate the role of MUC1 gene amplification and protein expression in human breast cancer development. By means of real-time quantitative polymerase chain reaction and immunohistochemical methods, 83 breast tissue samples were analyzed for MUC1 gene amplification and protein expression. This analysis showed MUC1 genomic amplification and a positive association with the histopathological group in 12% (1 out of 8) of benign lesions and 38% (23 out of 60) of primary invasive breast carcinoma samples (P = 0.004). Array-comparative genomic hybridization meta-analysis of 886 primary invasive breast carcinomas obtained from 22 studies showed MUC1 genomic gain in 43.7% (387 out of 886) of the samples. Moreover, we identified a highly statistical significant association between MUC1 gene amplification and MUC1 protein expression assessed by immunohistochemistry and Western blot test (P < 0.0001). In conclusion, this study demonstrated that MUC1 copy number increases from normal breast tissue to primary invasive breast carcinomas in correlation with MUC1 protein expression., (2010 Elsevier Inc. All rights reserved.)

The aim was to compare the expression of MUC1 and carbohydrate antigens in 124 tissue samples; 42 fibroadenoma (FA), 23 nonproliferative benign diseases (NPF), 25 usual epithelial hyperplasia (UEH), 7 atypical ductal hyperplasia (ADH), and 27 breast normal tissues. An immunohistochemical approach was adopted, using the following antibodies: reactive with MUC1 variable number of tandem repeats (C595, HMFG2, and SM3 monoclonal antibodies), anti-MUC1-cytoplasmic tail polyclonal antibody (CT33), and anti-carbohydrate antigens (sialyl Lewis x, Lewis x, Lewis y, Tn, and Thomsen-Friedenreich epitopes). Positive area of reaction, intensity, and pattern of expression were considered. A reactivity index was calculated as intensity (I) x 100+percentage of positive area (A). Statistical analysis comprised frequency analysis, P < 0.05, analysis of variance, and multiple correlation with principal component analysis. All samples expressed MUC1, detected by at least one anti-MUC1 antibody whereas Lewis x was the carbohydrate antigen most frequently found in all groups whereas variable number of tandem repeats MUC1 and Lewis x showed the highest correlation: 93% of normal samples, 62.5% of NPF, 87% of FA, 85% of UEH, and finally 80% of ADH. Although principal component analysis using reactivity indexes explained only 39% of data variability, normal samples appeared grouped and separated from benign breast diseases, which remained spread. Thomsen-Friedenreich was the only antigen that showed an increased tendency for positive expression and intensity from NPF through FA, UEH to ADH, whereas it was not detected in normals. With respect to the pattern of expression, an apical pattern was predominantly found in all the groups.

In the course of breast cancer global gene expression studies, we identified an uncharacterized gene known as RHBDD2 (Rhomboid domain containing 2) to be markedly over-expressed in primary tumors from patients with recurrent disease. In this study, we identified RHBDD2 mRNA and protein expression significantly elevated in breast carcinomas compared with normal breast samples as analyzed by SAGE (n=46) and immunohistochemistry (n=213). Interestingly, specimens displaying RHBDD2 over-expression were predominantly advanced stage III breast carcinomas (p=0.001). Western-blot, RT-PCR and cDNA sequencing analyses allowed us to identify two RHBDD2 alternatively spliced mRNA isoforms expressed in breast cancer cell lines. We further investigated the occurrence and frequency of gene amplification and over-expression affecting RHBDD2 in 131 breast samples. RHBDD2 gene amplification was detected in 21% of 98 invasive breast carcinomas analyzed. However, no RHBDD2 amplification was detected in normal breast tissues (n=17) or breast benign lesions (n=16) (p=0.014). Interestingly, siRNA-mediated silencing of RHBDD2 expression results in a decrease of MCF7 breast cancer cells proliferation compared with the corresponding controls (p=0.001). In addition, analysis of publicly available gene expression data showed a strong association between high RHBDD2 expression and decreased overall survival (p=0.0023), relapse-free survival (p=0.0013), and metastasis-free interval (p=0.006) in patients with primary ER-negative breast carcinomas. In conclusion, our findings suggest that RHBDD2 over-expression behaves as an indicator of poor prognosis and may play a role facilitating breast cancer progression.

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.

Many pathogens require direct binding to, or penetration of, mucosal cells to cause pathology. Cell surface mucins are critical components of mucosal defense. Mucin 1, named MUC1 in humans and Muc1 in non-human species, is a transmembrane glycoprotein expressed in apical mammalian epithelial tissues. The aim of this study was to determine the Muc1 profile expression in healthy cat epithelial tissues. An extensive analysis of Muc1 expression was performed by immunohistochemistry (IHC), Western blot (WB) and RT-PCR. By IHC, the presence of Muc1 protein was observed in the epithelial cells of the esophagus, stomach, trachea, lung, small and large intestine, liver, pancreas, salivary glands, lactating mammary glands and bladder. The predominantly linear patterns of reaction as well as the ubiquitous expression of feline Muc1 were consistent with normal human tissues. By WB, a band of 35kDa, corresponding to that predicted for the Muc1 cytoplasmic tail, was detected. The RT-PCR analysis showed a fragment of 115bp, consistent with that found in MCF7 and T47D human cell lines. The results showed that the widest distribution of feline Muc1 expression is in the mucosal tissues most at risk of infections such as the gastrointestinal tract, respiratory tract and lactating mammary gland. This study provides a normal model of cat Muc1 pattern expression as a starting point to evaluate and compare the expression of this epithelial mucin in pathological feline tissues. We believe that the CT33 antibody and the universal primers designed could be valuable tools for veterinary pathologists involved in the diagnostic interpretation of alterations in Muc1 expression of infected tissues in cats.

Unlabelled: Mucins are related to infectious and non-infectious diseases in Veterinary and Human Medicine. MUC1 mucin is a transmembrane glycoprotein expressed on the apical surface of human epithelia while MUC5AC is the predominant secreted mucin expressed in human gastric epithelium and goblet cells of lung and eyes. MUC5AC C-terminus cysteine rich regions and the cytoplasmic tail of MUC1 domains are conserved among several mammalian species., Objective: to compare the expression of MUC1 and MUC5AC mucins in mammalian epithelia. CT33 anti-MUC1 cytoplasmic tail (MUC1CT) polyclonal antibody and 45M1 anti-MUC5AC monoclonal antibody were employed. By immunohistochemistry, MUC1CT was expressed in most tissues while MUC5AC was restricted to gastric surface epithelium and goblet cells from trachea and lung. By western blot, MUC1CT showed a band at approximately 35 kDa in most tissues; MUC5AC revealed bands at >180 kDa in stomach and lung secretions from rat, cat, pig and cow. When rat MUC5AC was immunoprecipitated, a band at about 180 kDa was obtained.

Background: In head and neck squamous cell carcinoma (HNSCC), tumor markers may be helpful to evaluate prognosis accurately as well as to improve therapy selection. Detection of human MUC1 has been widely employed for the evaluation of carcinoma patients. This article aims to study MUC1, Tn, sTn, and Lewis antigenic expression in primary HNSCC, lymph node metastasis, and local recurrences., Methods: We used immunohistochemistry, tissue homogenization and differential centrifugation, isopycnic density gradient centrifugation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blot., Results: In primary tumors, MUC1 was detected in 80.0% of the samples; sLewis x in 23.2%, Lewis x in 45.6%, and Lewis y in 40.8%. Tn and sTn were found in 4.0% and 6.4% of samples, respectively. In metastatic lymph nodes, MUC1 showed a similar positive reaction as in primary tumors. Lewis y was detected in 20% lymph nodes whereas Lewis x, sLewis x, Tn, and sTn did not show differences. Some recurrences expressed MUC1 and only a few Lewis antigens, whereas Tn and sTn were not detected., Conclusion: In primary HNSCC and metastatic nodes, a high expression of MUC1 and Lewis antigens was detected that diminished in local recurrences. We also found that differentiated tumors mainly expressed a linear pattern of MUC1CT and Lewis x.

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: 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.

Unlabelled: The immunohistochemical detection (IHC) of MUC1-CT employing a polyclonal antibody (CT33) in relation to CT2 monoclonal antibody (MAb) was analyzed. Western blot (WB) was used to determine the molecular mass of CT., Materials and Methods: We studied 163 breast and 89 colorectal cancer specimens, 10 breast and 14 colorectal benign conditions, and 12 breast and 20 colorectal normal samples. From each tumor sample, subcellular fractions were obtained and analyzed by SDS-PAGE and WB. A nonparametric statistical analysis was employed; data were standardized and a Kendall-Tau correlation was applied., Results: By IHC, 146/163 (90%) and 151/163 (93%) of breast cancer were positive with CT33 and CT2, respectively; a statistically significant correlation was obtained (t=0.5199). Seven out of ten (70%) benign breast specimens were positive with CT33 while all samples stained with CT2; in normal breast sample tissues, all were positive with both Abs. In colorectal cancer samples, both antibodies stained 47/89 (53%) samples; CT2 reacted in 13/14 (93%) of benign samples while CT33 showed a positive reaction in 9/14 (64%) of benign specimens. In normal samples, CT2 showed staining in 17/20 (85%) of samples and CT33 was reactive in 12/20 (60%). By WB, in breast and colorectal cancer samples, similar results were obtained with both antibodies: a main band at about 30kDa which represents the smaller subunit., Conclusion: CT33 polyclonal antibody has demonstrated its efficacy to detect MUC1 in breast and colorectal cancer tissues with similar reactivity to CT2. It is worthwhile to affirm that CT33 is a good indicator of MUC1 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.

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 anti-Tn 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.

Objectives: 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)., Materials and Methods: 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., Results: 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., Conclusion: 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 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.

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.

Immunotherapy of human breast cancer is a rapidly growing experimental area based on peptidic as well as carbohydrate tumor-associated antigens. Cell surface carbohydrates are characteristic of different stages of normal development and differentiation; distinct carbohydrates are expressed in tissue- and cell-specific manners during those processes. Under pathological conditions such as neoplasia, changes in carbohydrate structures can almost always be present. In the successful development of carbohydrate anticancer vaccines, adjuvant and carrier molecules that promote the presentation of an antigen to the immune effector cells are prominent factors. At present, keyhole limpet hemocyanin (KLH) is the most widely used carrier protein in cancer immunotherapy, and QS-21 is the adjuvant most widely employed. In breast cancer, the immunogenicity of TF, Tn, STn and globo H antigens has been explored in different clinical trials. Approaches including vaccines against STn and globo H are presently being assayed with expectancy. From the experience obtained, it is possible to conclude that: 1) the employment of glycolipopeptide with clustered epitopes seems to be more effective than that of related structures with single epitopes in inducing antitumor cell antibodies; 2) the conjugation to carriers is best achieved using KLH; 3) totally synthetic constructs can be better immunogens in conjunction with a suitable adjuvant such as QS-21; in some cases, this adjuvant leads to a bypass in the need for specific T-cell help to stimulate IgG as well as IgM antibodies. In other cases, a T-cell mediated immune response is obtained, and (4) the development of a totally synthetic vaccine would greatly facilitate the production of the vaccine for large scale clinical trials with the attainment of regulatory approval., ((c) 2002 Prous Science. All rights reserved.)

Unlabelled: 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.

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-lgM levels detected with both MAbs; the MUC1-CIC-lgG levels detected with C595 were increased in the pregnant group while the MUC1-CIC-lgG 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-lgM, detected with both MAbs, and MUC1-CIC-4gG levels detected with the MAb C595 are apparently induced by pregnancy.

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.

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.

Unlabelled: 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 immunohistochemical 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.

Unlabelled: 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 sialyl-Lewis x and Tn antigen only reacted weakly in a few cells; cytokeratins were detected in all samples. In ENM derived fractions obtained by CsCl 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.

A serie of 29 human normal, benign and malignant breast tissues were cultivated in an attempt to isolate and propagate primary breast epithelial cells in vitro. Explants methodology as well as disaggregation techniques (enzymatic and mechanical) were employed to obtain better culture conditions. Cells derived from breast malignant tissue were propagated in an appropriate and survived in culture for at least 6 months, exhibiting a marked preference to grow in suspension (independent anchorage). Tumorigenicity assays in nude mice were performed with malignant cells obtained from primary culture cells as well as with cells achieved from successive passages. The rate of long time cell survival from malignant and non-malignant tissues demonstrated the accuracy of the methodology but it also emphasised the need for improving technology to obtain cell lines with long survival.

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.

Breast carcinoma cells may express a variety of clinically relevant epitopes, some of which are associated with aberrant glycosylation of MUC1 mucin molecules, as well as determinants which are commonly expressed on their normal molecular counterparts. The present investigation is primarily an immunochemical analysis of MUC1 epitopes and other tumour associated antigenic determinants, as defined by their reaction with monoclonal antibodies and expressed in normal, benign and malignant epithelia. It was determined that malignant tissues of the breast expressed MUC1 mucin, as well as the Le(y) hapten and CEA, at different intensities, cellular distribution and patterns and percentages of positively stained cells. Conversely, benign tissues expressed a low intensity of MUC1 which was restricted to apical cell surface membranes and lumen debris; a similar pattern was found in some normal breast sections. It was concluded that MUC1 mucin exhibits heterogeneous antigenicity (as defined by its reactivity with a panel of related anti-MUC1 monoclonal antibodies) which is predominantly related to the progression of malignant disease. Le(y) is a marker of breast neoplasia, while CEA was found on only a small proportion of tumours. These immunohistochemical findings are considered in the context of improving breast cancer diagnosis and therapy.

Breast mucins are expressed by malignant epithelial cells and they elicit an immune reaction. The up-regulation of mucin expression is association with tumour invasion, this mucin called MUC-1 reduces the cell-cell interaction facilitating cell detachment. The MUC-1 gene product, known as polymorphic epithelial mucin is a transmembrane high molecular weight glycoprotein. The molecule of MUC-1 has a central polypeptidic core with a carbohydrate linked in O-linkage to serines and threonines. The carbohydrate side chain epitope of MUC-1 molecule produced by breast cancer cells is heavily sialylated, giving their physical properties and increasing their immunogenicity. The development of monoclonal antibodies (MAb) has led to study the MUC-1 in subcellular extracts, tissues and culture supernatants from breast cancer and also colorectal carcinoma. The pattern of tumour cell staining with labeled MAb varies according with the grade of malignancy; these MAb bind either to peptide sequence and/or to the glycosylated epitopes. MUC-1 has a clinical relevance because serum concentrations may be useful for monitoring the response to therapy and progress of disease. MUC-1 epitope masking has been described since specific antibodies can combine with them forming immune complexes. Finally, mucins have been considered to develop vaccines against cancer, targeting specific carbohydrate and mucin epitopes.

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.