ARTICLE |
Correspondence to: Amada SegalEiras, CINIBA, Facultad de Ciencias Médicas, Calle 60 y 120, 1900 La Plata, Argentina. E-mail: as-eiras@netverk.com.ar
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Summary |
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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. (J Histochem Cytochem 51:781788, 2003)
Key Words: MUC1 cytoplasmic tail, breast cancer, immunohistochemical study
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Introduction |
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IMMUNOHISTOCHEMICAL STUDIES have indicated that MUC1 mucin is widely expressed by breast epithelium and that some of its changes have been associated with malignant transformation. MUC1 is expressed on the apical borders of normal secretory mammary epithelial cells, whereas carcinoma counterparts exhibit increased expression with different patterns, mainly over the entire surface (
MUC1 exists in a heterodimeric form with a large extracellular domain consisting predominantly of variable numbers of O-glycosylated 20-amino-acid tandem repeat peptides (VNTR) (mucin domain) bound via noncovalent forces (
MUC1 has been involved in cellcell (
The transmembrane and cytoplasmic domains of MUC1 are conserved among mammalian species with an amino acid identity of 8090% (
Immunohistochemical (IHC) MUC1 detection studies have included diverse anti-MUC1 monoclonal antibodies (MAbs) against the extracellular domain, mainly against different sequences from the VNTR (
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Materials and Methods |
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Tissue Specimens
A total of 98 pretreatment tumor samples were obtained from newly diagnosed breast cancer patients at different stages of disease; all cases were typed as invasive adenocarcinoma: not otherwise specified ductal type (NOS ductal) 56 (57%), papillary ductal carcinoma in situ (DCIS) with invasive component 8 (8%), lobular type (LC) 30 (31%). Four cases (4%) were medullary (MC). Disease staging was also established: 23 (23%) corresponded to stage I, 45 (46%) to stage II, 25 (25.5%) to stage III, and 5 (5%) to stage IV (Table 1). Tumors corresponding to NOS ductal type showed different grades of differentiation as follows: 7/56 (12%) were well differentiated, 11/56 (20%) moderately differentiated, and 38/56 (68%) were poorly differentiated tumors. Mean age was 56 with a range of 2586 years. The population under study included non-smoking women.
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Tissue samples from normal biopsy samples (n=7) obtained during breast cosmetic surgery as well as samples from six patients with benign breast disease (cystic changes, adenosis, and atypical hyperplasia) were also included as controls.
Procedures followed were in accordance with the Helsinki Declaration. Informed consent was obtained from all women included in this study.
Each tissue was studied using routine procedures. Specimens were sectioned in several parts to cover different programmed studies. A piece of tissue was fixed for histopathological diagnosis and IHC analysis. Another tissue sample was rinsed with sterile Hank's balanced solution and processed for preparation of subcellular fractions (see below).
Antibodies
Two monoclonal antibodies were assayed: MAb CT2, developed in Armenian hamster, directed against the last 17 amino-acids (SSLSYNTPAVAATSANL) of the cytoplasmic tail of MUC1 (
Methods
IHC Analysis.
The technique was performed according to standard procedures. The specimens were fixed in Methacarn (chloroform:methanol:acetic acid 60:30:10) for 2 hr and then transferred into 70% ethanol until processing in paraffin. Tissues were treated with 10 mM sodium citrate buffer at 100C for 5 min and incubated overnight at 4C with either CT2 or C595. In the first case, MAb CT2 diluted 1:500 was employed. After three washes with phosphate buffer, biotinSP-conjugated affinity-purified goat anti-Armenian hamster IgG (H+L) (Jackson ImmunoResearch; West Grove, PA) diluted 1:1000 was added and incubated for 1 hr. A final incubation with peroxidase-conjugated streptavidin (Jackson ImmunoResearch) was performed. MAb C595 diluted 1:1000 was used. After three washes with phosphate buffer, peroxidase-conjugated goat anti-mouse IgG (Dakopatts; Copenhagen, Denmark) diluted 1:400 was added and incubated for 60 min. The slides were counterstained with hematoxylin and coverslipped with mounting medium.
Negative controls were incubated with PBS instead of MAbs. Staining of cytoplasm and of plasma and nuclear membranes was evaluated. Cells were considered positive when at least one of these components was stained.
Sections were examined by light microscopy and the antibody staining patterns were scored in a semiquantitative manner. Staining intensity was graded as negative (-), low (+), moderate (++), or strong (+++) (
The pattern of reaction was classified according to other authors (
Preparation of Subcellular Fractions.
Fractions were prepared from all breast tissues according to
Pellets, membrane as well as cytoplasmic fractions, were dialyzed against 1.41 M PBS at 4C for 48 hr, lyophilized, and stored at -20C. Samples were then analyzed by SDS-PAGE and immunoblotting.
SDS-PAGE and Immunoblotting.
The isolated fractions were analyzed under reducing conditions in SDS-PAGE in a discontinuous buffer system according to
When MAb C595 was used as second antibody, peroxidase-conjugated goat anti-mouse IgG (Dakopatts) diluted 1:800 was added. Nitrocellulose sheets were developed with 3,3'-diaminodiazobenzidine in 10 mM Tris-HCl (pH 7.4) containing 1 µg/ml 30% H2O2.
Statistical Analysis
Statistical study was performed employing different methods. A multivariate analysis was applied through principal component analysis (PCA) with Kendall correlation including all standardized data. Relationship among variables (metric and non-metric) was studied; a set of dichotomous variables (dummy) was used to represent each non-metric variable. Spearman rank correlation (p<0.05) was also run, including MUC1 detection through MAb CT2 and MAb C595 and disease stage, histological type, and histological grade. Comparison of percentage of positivity among groups was studied using 2 analysis.
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Results |
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Immunohistochemical Staining
Breast Carcinoma.
Employing MAb CT2, IHC analysis showed a high percentage of positive staining in 91/98 (93%) specimens. In 60/91 (66%) positive samples, the reaction comprised the entire specimen, while in 31/91 (34%) staining was restricted to a few areas of the sample. In most cases (82/91; 90%) the staining intensity was strong (+++).
Table 1 summarizes the percentage of positive results as well as the pattern of reaction according to the tumor type.
In NOS ductal carcinoma, considerable heterogeneity of staining at the cellular localization was found. In 44.2% of specimens, only plasma membranes reacted positively. Several tumors showed a continuous reaction, whereas others showed a discontinuous pattern. In several samples, luminal content was also stained. Frequently, cytoplasmic staining was observed together with a membrane reaction in a mixed pattern (Fig 1A and Fig 1B). In 17.3% of specimens, the reaction was restricted to the cytoplasm, either in a homogeneous pattern (Fig 1C) or with a granular aspect.
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A high percentage of LC (64%) was stained both in plasma membrane and in cytoplasm, with a mixed pattern (Fig 1D). In 6/28 specimens, reaction comprised only the cytoplasm, while staining restricted to plasma membrane was found in a few samples.
All papillary DCIS with an invasive component examined showed a positive reaction, mainly at the plasma membrane. Usually the reaction covered the papilla externally and internally lined the apical membrane (Fig 1E). Two specimens were also reactive at the cytoplasmic level (mixed pattern).
Cytoplasmic staining was predominant or exclusive in MC samples. As was expected, given that 93% of tumors were reactive, no statistically significant correlation was obtained among tumor stage, degree of differentiation, or histopathological tumor type and MUC1-CT expression detected with MAb CT2.
Immunohistochemical analysis was also performed employing an anti-MUC1 VNTR MAb (MAb C595). Intensity of reaction observed was as follows: 19/72 (26%) malignant samples showed strong intensity; 32/72 (44%) showed moderate staining; 21/72 (29%) showed low intensity, and 16/72 (22%) were negative.
Percentages of subcellular localization of MUC1 reactivity related to tumor type are summarized in Table 1. Similar percentages of membrane and cytoplasmic staining were found, mainly in a mixed pattern.
Statistical analysis did not show any correlation between MUC1 detection and disease stage, histological type, or histological grade. Multivariate analysis by PCA with Kendall correlation showed a significant correlation between MAb CT2 and C595 staining (=0.5147).
Control Samples. With the anti-cytoplasmic tail MAb (MAb CT2) all control specimens showed positive reactivity. MUC1 expression in samples of benign breast disease and normal tissues was similar. Some specimens showed a reaction restricted to a few areas, but others showed more extensive staining. The pattern of reaction was observed mainly at the apical level of plasma membranes (Fig 1F), although several samples also showed a cytoplasmic apical reaction.
With MAb C595, benign breast samples showed MUC1 tissue expression in two of six samples, while three of seven normal specimens were reactive. The staining pattern was mainly restricted to the apical plasma membrane.
Subcellular Fractions
All tissue samples were subjected to subcellular fractionation followed by SDS-PAGE and Western blotting analysis of their derived fractions. In malignant tissue fractions, this analysis demonstrated the presence of several bands at different molecular weights (MW). With MAb CT2, the fraction showing the most intense reaction was the membrane moiety. Under denatured conditions, a smear reaction was usually obtained from 20 kD up to <200 kD, although some bands were impossible to identify. An example is shown in Fig 2A, where a strong band of reaction was found at approximately <30 kD and other minor bands were found at 60 kD. The cytoplasmic and nuclear fractions showed the same reaction, with a minor intensity of staining. In some other samples, the reaction at <30 kD was split into two different bands (data not shown).
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The incubation with MAb C595 showed a double band of reaction at approximately 180200 kD in all malignant tissue-derived fractions (Fig 2B). In one third of samples, bands of MW >200 kD were also found in pellet, membrane, and cytoplasmic fractions. A smear reaction from >50 kD up to >200 kD was frequently observed in all fractions. In pellet fractions, some samples (32%) showed additional bands at 70, 60, and 50 kD. In 29% of membrane fractions, bands of about 100, 70, 60, 50, 40, and 25 kD were found. Finally, in 50% of cytoplasmic fractions, bands of 80, 70, 60, 50, 40, 25 kD were observed.
Control Samples
By employing MAb CT2, pellet, membrane, and cytoplasmic fractions belonging to control samples showed bands at 30 kD and also at about 60 kD. In some samples, a double band at >180 kD, corresponding to membrane fractions, was identified.
With MAb C595, a double band at 180 kD was found in all fractions. A smear reaction from 5060 kD up to >200 kD was frequently observed in pellet, nuclear, and membrane fractions. In pellet fractions derived from three normal and two benign breast disease samples, bands at 70 kD and at 80 kD were also found.
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Discussion |
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To our knowledge, this is the first extended IHC study developed in mammary gland tissues by employing an anti-MUC1 CT MAb (MAb CT2). Results were compared with an MUC1 tandem reactive MAb (MAb C595) which may place our findings in the context with the previously published literature. In breast cancer, MAb CT2 reacted with 93% of samples, and MAb C595 stained 73.5%. All benign and normal samples were reactive with CT2 but C595 failed to react with some control specimens.
Breast carcinoma tumors are frequently reactive with anti-VNTR MUC1 MAbs. Differences in percentages of reactive tumors depend on the MAb employed (
MAb C595 binds the RPAP sequence (
On the other hand, MAb SM3 (
Different authors (reviewed by
With respect to subcellular localization of MUC1, we have performed a detailed study of samples. In breast cancer, MAb CT2 showed reactivity mainly at the plasma membrane. The cytoplasmic staining was more frequently found in a mixed pattern. MAb C595 showed a similar percentage of cytoplasmic and membrane staining, mainly in a mixed pattern. Most samples did not show any polarization of the reaction which, on the other hand, constituted the hallmark of normal and benign tissue sample staining. In 330 breast carcinomas,
The use of an anti-MUC1-CT may constitute an accurate marker for MUC1 because it has not been described whether this MUC1 fraction can be liberated by the cell, although extracellular MUC1 may be secreted and released to serum as well as to ascites fluid (
It is possible that the 60-kD band is an alternative splice variant of MUC1, such as MUC1/Y (
In a percentage of subcellular fractions, different bands of low MW were also found, which may be due either to different steps of cellular metabolic processing or to generation by sample treatment (
Many carcinoma-associated markers are glycoconjugates whose expression undergoes temporal or spatial regulation. MUC1 mucin is a well-documented example of such a molecule. In this sense, an anti-cytoplasmic tail MAb that recognizes the molecule, regardless of the presence of the VNTR sequence, may be useful to target intracellular MUC1 processing, and may also be an accurate marker at the membrane level. It may detect heterodimeric MUC1, MUC1/A, and MUC1/B variants as well as MUC/Y, MUC/X, and MUC1/Z isoforms. Alternatively, reactivity with MAb C595 may be explained by the fact that other spliced forms, such as MUC1/SEC, may also be expressed.
Our study indicates that the pattern of expression of MUC1 CT in breast epithelium may constitute a better indicator of MUC1 production because it does not depend on glycosylation. It also shows that the pattern and extent of MUC1 CT positivity do not vary according to the histopathological subtype of breast cancer.
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Acknowledgments |
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Supported by CONICET (BID-CONICET: PICT05-06544 and PIP: 4718/96) and by the University of La Plata. This research project is part of the Program PRECANMA (Program for Breast Cancer Prevention) CICPBA-CINIBA, La Plata, Prov. Buenos Aires, Argentina).
We thank Dr Clemente Sala and Dr Jorge Gori for providing patient samples and Lic. Sandra Demichelis for statistical analysis.
Received for publication October 21, 2002; accepted January 22, 2003.
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