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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 <55 years old. AP stage III was observed at X = 49 years old versus DP at X = 54 years old; cutaneous and/or thoracic wall invasion was found at diagnosis in 15.4% of DP, while it was absent in all APs. Both groups showed advanced stages in younger patients. In general, tumor size versus age showed a negative relationship (p < 0.04); this observation was only significant for AP (p < 0.02). ER, PR expression, and Her2neu status presented differences between the two populations. Percentage of AP patients with positive ER were 78.8%, positive PR: 79.5%; while Her2-neu positive status was found in 5.3%. In the case of DP patients, positive percentages were: ER: 66.3%, PR: 51.2%; and finally Her2-neu: 38.8%. Histological and nuclear grade DP compared to AP were significantly higher, while vascular and lymphatic invasion showed the inverse DP: 30% versus AP: 18%, p < 0.01. Taking into account the number of children, significant differences between groups were found: DP, 2.8+/-0.11 while AP, 1.9+/-0.13 (media +/-standard error). In general, it was observed that patients who had breastfed presented lower number of metastatic lymph nodes than those who had not. 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. [ABSTRACT FROM AUTHOR]

The purposes of this research were 1- to study the expression of MUCI peptidic and carbohydrate epitopes in normal, benign and malignant breast epithelia and 2- to correlate this expression with breast cancer disease stage. An immunohistochemical approach was performed in 81 tissues obtained from normal, benign and malignant sections; studies were performed following standard procedures. Three anti-MUC1 peptide core monoclonal antibodies (MAbs), C595, HMFGI and SM3 were used and 4 anti carbohydrate antigens MAbs: sLewis x (KM93), Lewis x (KM380), Lewis y (C 14) and Tn. Statistical analysis was done employing a Principal Component Analysis with Kendall correlations. Malignant tissues expressed peptidic and carbohydrate MUC1 epitopes with high intensity and elevated percentages of positivity; MUC1 protein core was reactive in 89% in stage I, 87% in stage II, 75% in stage III and 83% in stage IV. SM3 MAb showed its highest staining (83%) in tumors belonging to stage IV patients. Lewis x and sLewis x were found coexpressed with a correlation value of 0.5487; Lewis y diminished its expression from 67% in stage I tumors to 50% in stage IV while Tn showed the lowest expression respect to the other antigens in any of the stages. Reactivity of benign and normal samples was restricted to the apical cell surface. Statistical analysis showed a separation between normal and dysplasia versus cancer samples (p<0.05). Conclusion: carbohydrate antigens are expressed in breast tissue under different patterns, intensity and extension being predominant in cancer cells. [ABSTRACT FROM AUTHOR]

MUC I is a highly glycosylated mucin with a large extracellular domain, a transmembrane fraction as well as a 72-aminoacid cytoplasmic tail (CT). The present research was developed to study the pattern of MUC 1 mucin expression in breast carcinoma with an anti MUCI cytoplasmic tail (CT) MAb since it is a reliable indicator of the abundance of cell-associated MUC1. A total of 98 pretreatment tumor samples were obtained from breast cancer patients; all cases were typed as invasive adenocarcinoma; disease staging, histopathological type and grade of differentiation were established. Samples from normal and benign conditions were also included. Immunohistochemistry (IHC) was performed following standard procedures. The pattern of reaction was classified as lineal when the plasmatic membrane was reactive, cytoplasmic and mixed. Subcellular fractions were prepared followed by SDS-PAGE and Western blots analysis. The antibody employed was CT2 monoclonal antibody (MAb) directed against the last 17 aminoacids of the MUCI-CT. IHC showed a high percentage of positive staining in about 90% specimens; frequently, 50% or more tumor cells stained either with a homogeneous distribution, or restricted to some areas of the sample. Benign and normal samples showed a more restrictive pattern usually confined to the plasmatic membrane. By Western blot, subcellular fractions derived from malignant samples showed different bands of reaction at approximately 30 kDa (the most intense), at 60-50 and at 180 kDa. Conclusions: the pattern of expression of MUC I CT in breast epithelium may constitute a better indicator for MUC 1 production since it does not depend on glycosylation. [ABSTRACT FROM AUTHOR]

The present study was undertaken to study the relationship of tumor-associated antigens with colorectal carcinoma by means of immunohistochemistry (IHC) with a panel of monoclonal antibodies (MAbs). Materials and Methods: Tumor tissue samples were studied from 59 patients with colorectal cancer (Dukes stages B, C and D), 24 benign colorectal conditions and 24 colorectal normal mucosa. Immunohistochemistry was performed following standard procedures; MAbs against the following tumor associated antigens were included: MUC2 mucin (PMHl), MUCl (C595) carcinoembryonic antigen (CEA, C365), colonic glycolipid (C505), normal colorectal antigen (NCA, C198), Lewis x (KM380), sialyl Lewis x (KM93), Lewis y (C 14 MAb) and Tn hapten. Immune reaction was graded according to positive staining, intensity and distribution. Results: in colorectal cancer tissue sections, a high expression of tumor antigens associated with metastatic dissemination was found: Lewis x, 62%; MUC 1 and MUC2, 47%; Lewis y, 46% and slaty] Lewis x, 44%; Tn was positive in 36%, CEA in 32%, NCA, 44% and colonic glycolipid in 82%. Benign samples showed a remarkable positive expression of glycolipid antigen, WC 1, MUC2, NCA and Lewis x; an interesting feature was the expression of MUC 1 in invasive areas of several specimens. Normal tissue samples mainly reacted with glycolipid antigen, NCA and Lewis x. Conclusion: Colorectal cancer expresses strongly a variety of tumor antigens, which are involved in recurrence and metastatic processes. [ABSTRACT FROM AUTHOR]

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.)

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.)

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.)

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.

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.

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.

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.

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.

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: 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.

This paper is a review on immune complexes in malignant tumours. High levels of immune complexes have been detected in the serum of patients with malignant solid tumours, leukemias and lymphomas. These studies were performed using non-specific methods. Results obtained by different laboratories are in general coincident; elevated levels of immune complexes in pre-treatment samples and in recurrent disease and, normal values in post-treatment studies indicating a disease free state. Nevertheless, there are certain variations among the results obtained by different techniques suggesting the necessity to check various methods for each tumour type. Although there is no clear evidence that immune complexes in cancer patients contain tumor related products, the current status of clinical research suggests the potential of circulating immune complexes assays for monitoring malignant diseases.

We are dedicated to the study of circulating immune complexes (CIC) associated with different diseases: malignant tumors, leprosy and rheumatoid arthritis. Immune complexes were evaluated by various methods: 125I-Clq binding assay, 125I-IgG binding test, 125I-bovine conglutinin binding assay and polyethylene glycol precipitation test (3.5% and 2.5%). Techniques for the isolation and splitting of CIC in their components were performed in sera from patients with tumors and with leprosy. These methods consisted in the combination of CIC with protein A followed by elution with different buffers. CIC splitting techniques were first applied on immune complexes formed in vitro (BSA-aBSA, OVA-aOVA). The analysis of CIC fractions was done by SDS-PAGE, immunoelectrophoresis and immunoblotting techniques. Results were as follows: CIC levels correlated with active stages of disease, decreasing during remission so that CIC detection can be useful to evaluate response to treatment. The isolation and splitting of immune complexes into their components resulted in the obtention of immunologically active fractions, especially in sera from patients with gastrointestinal and breast cancer and with leprosy.

The object of the present work was to investigate: a) The cytotoxic activity of cytophilic antibody in sera from guinea pigs sensitized with the testicular antigen; b) The presence of immune complexes. In vitro cytotoxicity induced by hyperimmune sera or by the cytophilic antibodies eluted from the corresponding macrophages, was demonstrated after incubation with either germinal cells or spermatozoa. Marked cytotoxicity was also observed in vivo when sera or antibodies were injected into the tests, as shown by sequential histologic studies. Circulating immune complexes, as tested by the 125I-C1q binding assay, were not detected in these sera. On the contrary, high levels of circulating immune complexes were found when the polyethyleneglycol precipitation test was used. It can be concluded that the cytotoxic effect induced by cytophilic antibodies of IgG2 nature, may be associated with polyethyleneglycol precipitating immune complexes.

A total of 122 sera from acute lymphoblastic leukemia (ALL) patients were analyzed for circulating immune complexes (CIC) by two methods: the 125I-Clq binding assay and the polyethylene glycol precipitation test (PEG). The results were correlated with induction, remission and relapse stages of the disease. Using the first method the levels of CIC in induction were 15.18 +/- 9.15, with 19/29 positive cases (65.50%), P less than 0.001 compared with controls. In the remission phase the levels were 9.02 +/- 5.62, 11/45 (24.49%) nonsignificant P value, and in relapse they were 16.14 +/- 11.17 28/48 (58.33%) P less than 0.001. The PEG precipitation test results were: 0.33 +/- 0.10, 8/22 (36.36%); 0.24 +/- 0.11, 10/48 (20.83%) and 0.28 +/- 0.10, 6/28 (21.42%), respectively. Thus the values of CIC as measured by PEG in the three clinical of phases ALL did not differ significantly from controls. This contrasts with results obtained by the radioiodinated C1q binding assay, where the incidence of positive values was significantly higher in induction and in relapse and lower in the remission phase. These observations were extended in sequential vertical studies performed in a group of patients. These results suggest that raised CIC detected by the 125I-C1q method may reflect a progressive state in ALL and that quantitation of these immune complexes may provide an adequate biochemical marker for prognosis.

In the mouse, retrovirus B and C are causal agents of mammary cancer and leukemia, respectively. In previous work it was demonstrated that sera of leukemia-lymphoma patients possess antibodies which react with antigenic determinants of Type C virus present on AKR-lymphoma and AKR-thymus targets. The object of this paper was to determine whether these antibodies also reacted with Type B viral antigens present on a virus-induced BALB-mammary carcinoma; at the same time a comparative study was carried out with sera of breast cancer patients. A total of 325 sera were obtained from 277 leukemia-lymphoma cases under protocol treatment: 232 acute lymphoid leukemia, 23 acute myeloid leukemia, 15 chronic myeloid leukemia and 55 Hodgkin lymphoma sera. A total of 240 sera were obtained from breast cancer patients at the time of diagnosis and 196 sera from normal blood donors served as controls. Using indirect immunofluorescence with labeled anti-human IgG and the murine targets, antibodies were encountered in a high percentage of cancer cases and were consistently absent in normal sera. The results confirm the presence of antibodies reacting with murine Type C virus in leukemia-lymphoma cases and indicate the presence of antibodies reacting to both Type B and C retroviruses in the sera of breast cancer patients.