ARTICLE |
Correspondence to: Georg F. Springer, Heather M. Bligh Cancer Research Laboratories, Chicago Medical School, 3333 Green Bay Rd., N. Chicago, IL 60064.
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Summary |
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The precursors of the blood group N and M-immunodominant structures, Tn and T (Thomsen-Friedenreich) epitopes (EPs) occur in 90% of carcinomas (CAs) but are masked in benign-diseased and healthy tissues. We determined quantitatively on 55 primary invasive ductal breast CAs, stages I to IV, the prognostic value of extent of Tn and T EP expression over an observation period exceeding 5 years postoperatively. Classical, established pathological and histological prognostic characteristic indicators associated with survival were subdivided by standard criteria into favorable and unfavorable categories. Tissue sections were reacted with monoclonal anti-Tn and -T antibodies, followed by the streptavidin-biotin-peroxidase-DAB procedure; counterstain was methyl green. Tn and T EPs were then quantitated by computerized image analysis. Of the 55 CAs, 51 clearly expressed Tn and T, and four had traces. Strong Tn EP expression was statistically significantly associated with shortened 5-year disease-free interval, increasing pTNM stages, positive lymph node status, and increasing combined histological grades. T EPs were usually well expressed but showed no significant association with prognostic factors. Our results suggest that quantitative immunohistochemistry-image analysis of Tn EPs of primary breast CAs may add new parameters to prognostication. (J Histochem Cytochem 45:1393-1400, 1997)
Key Words: invasive human ductal breast, carcinoma, Tn and T epitopes, immunohistochemistry, quantitative computerized, image analysis, prognostication of breast, carcinoma
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Introduction |
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Carbohydrate structures on cell surface glycoproteins play important roles in intercellular recognition, adhesion, and morphogenesis during ontogenesis. In malignant tumors, changes of CA cell surface glycoprotein structure have a major impact on progress of the tumor (1-O-Ser/Thr) and T (Galß1
3GalNAc-
1-O-Ser/Thr) are early stage-specific differentiation antigens (Ags) and carcinoma-associated glycopeptide EPs (
90% of all primary human CAs and in their metastases (
Established prognosticators of the clinical course of breast CA include the following: TNM stage, histological grade (tubule formation, nuclear pleomorphism and hyperchromasia, mitotic rate) and, to some degree, steroid receptor status. Ancillary prognosticators such as C-erb B-2, cathepsin D, Ki67, p53, aneuploidy, and angiogenesis, as well as carcinoembryonic antigens, vimentin and epithelial membrane antigen are being investigated (
Immunohistochemistry (IHC) is a powerful tool for detection of these well-characterized EPs and may thus aid in understanding of some pathogenetic aspects of CA. Conventional evaluation of immunostaining permits only semiquantitative interpretation of the density and distribution of Tn and T EPs. Computerized image analysis allows quantitation of immunostaining (
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Materials and Methods |
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Tissues
Paraffin blocks of primary breast CA archived in Evanston Hospital (Evanston, IL) were used in this project. We studied 55 primary infiltrating ductal breast CAs, stages I-IV. Ages at surgery ranged from 34 to 77 years (median 56 years). Twenty-five CA patients had lymph node metastases. Staging by the surgical pathologists was according to the pathological TNM classification (
The largest diameter of the primary CAs ranged from 0.5 to 10 cm (median 2.0 cm). All tissues were routinely processed by fixation in 0.1 M phosphate-buffered 10% aqueous formalin for 24-36 hr at 20-23C and then embedded in paraffin. Serial tissue sections 5 µm thick were cut from the blocks, placed on coated glass slides (SuperFrost/Plus; Fisher Scientific, Pittsburgh, PA), and dried overnight in an oven at 50C. One section from each block was stained with hematoxylin-eosin to authenticate the tissue and confirm the pathologist's diagnosis. Histopathological grades were assessed by a modification of the Bloom-Richardson method (
Antibodies
Two pools each of three rodent monoclonal antibodies (MAbs) were used for Tn and T EP demonstration, respectively. The fine specificities of these MAbs have been described previously, together with their other properties: mouse MAb BaGS-3 elicited by isolated O Tn Ag and BaGS-5 raised against O Tn RBCs (
Immunohistochemistry (IHC)
All procedures were performed at room temperature (20-23C) and all conditions remained uniform throughout this study. The sections were deparaffinized in xylene, rehydrated in decreasing concentrations of ethanol/water and rinsed in Dulbecco's PBS (DPBS), pH 7.4. Endogenous peroxidase of tissues was inactivated by 30-min incubation in 3% H2O2 in 80% methanol with 0.1 M sodium azide. The sections were then rinsed with DPBS, treated with 0.3% BSA in DPBS for 30 min, and incubated with primary Ab. The fluids in the anti-Tn MAb pool were prediluted: BaGS-3 1:80; BaGS-5 1:80; HBTn-1 1:80. Similarly, the anti-T pool consisted of 3 MAbs diluted as follows: RS1-114 1:100; HT-8 1:200; HBT1 1:60. Incubation with primary Abs was 60 min, followed by washing in DPBS. Specimens were then incubated for 30 min with a 1:600 dilution of biotinylated goat anti-mouse Igs (DAKO). The slides were then washed in DPBS, incubated with a 1:300 dilution of streptavidin and biotinylated horseradish peroxidase complex (SABC) (DAKO) for 30 min, washed again, and reacted for 7 min in the dark with 0.05% 3-3', 4-4'-diaminobenzidine tetrahydrochloride (DAB; Sigma, St Louis, MO) in DPBS containing 0.01% H2O2 (
Quantitative Image Analysis
Computerized image analysis (
Quantitative image analysis was essentially by the methods described by 33% was considered as low vs high EP expression. To prove the reproducibility of the computerized image analysis system, the same CA area (e.g., the largest CA area of one section) was selected and measured by the same observer on different days or by different observers to compare the results. In addition, duplicate sections were stained on different days and measured to evaluate the technical reproducibility.
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Statistical Analyses
Two basic parameters were assessed for their statistic significance: (a) association of prognostic factors, defined by pathological TNM stage (2 cm), lymph node status (negative vs positive), combined histological grade (I and II vs III), and disease-free interval (Table 2).
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Fisher's exact test (3 or
0.4 (
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Results |
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Immunohistochemistry
Breast CA cells usually have large quantities of Tn and T EPs in cytoplasm and on cell membranes, demonstrable specifically with the anti-Tn and anti-T MAb pools used. Of the 55 primary invasive ductal CA studied, 51 expressed Tn and T EPs abundantly and four had only traces (1-5%). In the majority of cases, both Tn and T EPs were well expressed, a finding characteristic for CAs in contrast to normal and benign-diseased tissues (see Discussion). However, T EPs were mainly seen in lumina of well-differentiated CA and adjacent CA cells (Figure 1A), and T EP staining showed a wide variability in both well- and poorly-differentiated CAs, whereas staining for Tn EPs was in general more extensive in cytoplasm and on cell membranes than that of T EPs (Figure 1B-D). Compared to primary CAs, metastatic CAs in lymph nodes usually showed increased Tn and decreased T EP expression (Figure 1E and Figure 1F). Normal breast tissues and benign breast lesions did not bind anti-T and -Tn MAbs (see Discussion) and were found to be negative, even on replicate sections from the same paraffin block, except occasionally in the glandular lumina (Figure 2A) or in apocrine metaplasia, which frequently showed some staining in cytoplasm for Tn or T EP (Figure 2B).
Histologically normal and benign glandular epithelial cells in the vicinity of the CA served as built-in controls (Figure 1C, lefthand side, lower quarter, and 1D, right side of photo). None of the negative controls (omission of primary Ab or its preabsorption) showed any staining for Tn and T EPs. Intra- and interobserver measurements of the same CA area on different days under constant conditions gave nearly the same results; the mean run-to-run variations were less than 8%.
Statistical Analyses
The results of statistical analyses of disease-free interval of our cohort in relation to established prognostic indicators of breast CA are listed in Table 1. The most highly significant prognostic parameters related to the disease-free interval are pTNM stage (p=0.002), and combined histological grade (p=0.002), followed by lymph node status (p=0.016) and primary tumor size (p=0.032). Degree of association for all parameters is strong.
Table 2 shows that percentage of Tn-immunostained CA area has a strong association with disease-free interval (p=0.006) and combined histological grade (p=0.001). High Tn EP expression is also associated with pTNM stage (p=0.027), and lymph node metastasis (p=0.013), but tumor size is without significant p value (p=0.109) and only weak association (0.39). Therefore, in all instances, degree of association is strong except with tumor size. It is notable that there was no association of T EP expression with any of the prognostic indicators; therefore, the data are not shown in Table 2.
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Discussion |
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We and others have previously shown that both immunoreactive T and Tn EPs are almost always found in breast CA, as determined with human monospecific, polyclonal antibodies, and only rarely in small amounts in normal and benign tissues (
Recent advances in studies of mucin structure and glycosylation revealed that in breast and other CA cells the product of the MUC1 gene is aberrantly glycosylated and that Tn, T EPs, and core peptides are exposed (
Here, we employed MAb pools directed against uncovered Tn and T EPs in breast CA and demonstrated that the extent of Tn and T EP expression in CA can be quantitatively analyzed immunohistochemically, which may add to prognostication. The expression of Tn EPs predominated in invasive CAs over T EPs and the increasing expression of Tn EPs was highly significantly associated with established, prognostically unfavorable parameters of breast CA. Quantitative measurements of Tn EP expression provide important information on the metastatic potential of a CA and on patients' disease-free interval, provided that strict reproducibility of results is ascertained by the stringent controls as described in the Materials and Methods. Strict quantitation of biological phenomena is an overriding goal to comprehend and control pathophysiological phenoma (cf. Gordon-Taylor 1948).
In addition to aberrant glycoslation, there is another way, that may lead to the exposure of Tn and T EPs on cells. Previous studies have documented that sialic acid may be released from major sialoglycoproteins of erythrocyte membranes as the result of cell aging (
Our studies concerned overall densities of Tn and T EPs of carcinoma structures; single cells were not investigated. Therefore, we have no spatial resolution and no data on subpopulations. We plan to obtain information on the absolute concentration of both of these EPs per particular cell using a quantitative standard such as that described by
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Acknowledgments |
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Supported by US National Cancer Institute grant no. CA 22540 and by the Heather M. Bligh Cancer Research Fund.
We are grateful to M. Kaufman, MD for making the paraffin blocks of breast tissue available, to W. Rheault, PhD, PT, for guiding us in the statistical work and performing most of it, to M. Ghazizadeh, MD, for some preliminary investigations, and to J. Prasad and S. Kutsch for their excellent technical assistance.
Received for publication February 11, 1997; accepted May 20, 1997.
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