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Prognostic significance of muc1 epithelial mucin expression in breast cancer
Prognostic Significance of MUCI Epithelial Mucin Expression in Breast Cancer MICHAEL A. McGUCKIN, BSc, PHD, MICHAEL D. WALSH, BSc, BRENDAN G. HOHN, BAPPSCl, BRUCE G. WARD, FRCOG, PHD, AND R. GORDON WRIGHT, FRCPA The epithefial mucin produced by the MUCI gene is present in the apical cell membrane of normal breast epithelial cells and is highly expressed in many breast cancers. Several studies have provided conflicting evidence regarding the relationship between MUC1 expression and survival in breast cancer patients. In this study a detailed immunohistological analysis of MUC1 expression was performed using monoclonal antibody BC2 and was related to other tumor characteristics and patient sur~val. Patients whose tumors showed MUC1 expression in greater than 75% of tumor cells had significantly poorer dlsease-free and overall survival (P < .05). The proportion of cells showing cytoplasmic MUC1 expression was prog-
nostically significant, but the proportion of cells that lined gland spaces showing apical membrane staining was of no prognostic significance. A high level of MUC1 expression was significantly associated with the presence of axillary node metastases and estrogen receptors but not with other tumor characteristics. HUM PATHOL 26:432439. Copyright © 1995 by W.B. Saunders Company Key words: breast cancer, mucin, prognosis, survival, MUC1. Abbreviations: VNTR, variable number of tandem repeats; HMFGM, human milk fat globule membrane; PBS, phosphate-buffered saline.
Mucins are high molecular weight glycoproteins f o u n d in the cell m e m b r a n e s of h u m a n epithelial cells f r o m the m a m m a r y gland, salivary gland, digestive tract, respiratory tract, kidney, bladder, prostate, uterus, and rete testis, a During malignant transformation m e m b r a n e expression of these mucins often changes f r o m apical to circumferential coincident with loss of polarity of the epithelial cell, and synthesis increases markedly, often resulting in large amounts of mucin being either shed or secreted by t u m o r cells. Glycosylation of the peptide backbone also is altered, resulting in novel carbohydrate epitopes or exposure of the peptide backbone. 2 Recently, the genes o f seven of these mucins have b e e n fully or partially cloned. ~5 In all of these mucins a substantial portion of the core protein consists of sequences that are repeated a large and variable n u m b e r of times, known as the variable n u m b e r of tand e m repeats, or VNTR, region. Many antimucin m o n o c l o n a l antibodies have b e e n described that react with breast carcinomas. Some of these have b e e n raised using breast t u m o r cells as immunogens, but m a n y have b e e n raised against o t h e r types of tumors or the h u m a n milk fat globule m e m brane (HMFGM). Although we have previously d e m o n strated the presence of the MUC2 mucin and its association with p o o r survival in breast cancer, 6 the mucin f o u n d most c o m m o n l y in breast carcinoma is the product of the MUC1 gene, t e r m e d polymorphic epithelial
mucin. 7 Almost all anti-MUC1 core protein m o n o c l o n a l antibodies described react with the VNTR region. 8 However, m o n o c l o n a l antibodies with similar m i n i m u m epitopes (as assessed by binding to synthetic peptides) can show differing reactivities with breast carcinoma. T h e IgG1 mouse monoclonal antibody, BC2, used in this study was raised using HMFGM as the i m m u n o g e n , and reacts with very high affinity to the peptide epitope, APDTR, on the VNTR of MUC1 mucins. 9,a° Several studies have e x a m i n e d the relationship between expression o f MUC1 mucins and survival in breast cancer patients. 11-21 However, the results f r o m these studies are often contradictory, and m a n y are based on the analysis of relatively small n u m b e r s of patients with inadequate follow-up. MUC1 expression by breast tumors and mucin levels in serum also have b e e n related to estrogen receptor s t a t u s , 11'12'14-16'22-24 also with contradictory findings. T h e current study assesses MUC1 mucin expression in a retrospective series of breast carcinomas and compares the prognostic significance of this expression with that of other prognostic factors, such as the presence ofaxillary n o d e metastases, histological type and grade, t u m o r size, presence of coexistent carcinoma in situ, estrogen receptor status, and m e n o p a u s a l status.
MATERIALS AND METHODS Patients
From the Department of Obstetrics and Gynecology and the Department of Surgery, University of Queensland, Royal Brisbane Hospital; the Department of Pathology, Royal Brisbane Hospital; and the Department of Gynecologic Oncology, Royal Women's Hospital, Herston, Australia. Accepted for publication August 8, 1994. Supported by a grant from the Queensland Cancer Fund and Australian National Health and Medical Research Council Project Grant No. 92-1140. Address correspondence and reprint requests to Michael A. McGuckin, BSc, PhD, Department of Obstetrics and Gynecology, University of Queensland, Clinical Sciences Building, Royal Brisbane Hospital, Herston, 4029, Australia. Copyright © 1995 by W.B. Saunders Company 0046-8177/95/2604-001355.00/0
432
One hundred seventy-one cases of invasive breast carcinoma diagnosed at the Department of Pathology of the Royal Brisbane Hospital between 1971 and 1986 were selected for this study. The cases were sequential and unselected other than being excluded if there was insufficient histological material (including axillary node biopsy) or inadequate followup information. Histological classification of these invasive cancers was performed by one investigator (RGW). The results of the clinical follow-up and immunohistochemistry study were independently collected. These cases included 140 ductal, 20 lobular, five medullary, one mucinous, two papillary, and one each of apocrine, tubulolobular, and squamous
MUC1 MUCIN IN BREASTCANCER (McGuckin et al) TABLE 1. Grouping of Tumor and Patient Characteristic Variables Used for Statistical Analyses Variable Number of axillary node metastases Tumor diameter Tumor grade Overall proportion of tumor cells stained by BC2 (%) Intensity of BC2 staining Proportion of tumor cells with cytoplasmic MUC1 (%) Proportion of tumor cells with apical membrane MUC1 (%) Menopausal status Histological type Estrogen receptor status Microvascular invasion Coexistent carcinoma in situ Age (yr)
Groupings 0, 1-3, >3 ~<10 mm, 11-20 mm, 21-50 nlm, >50 mlrl 1,2,3 <75, >75
of invasive tumor cells and in situ tumor cells showing MUC1 expression were classified separately into five groups: no staining, 0% to 25%, 26% to 50%, 51% to 75%, and 76% to 100% of cells positive. The proportion of cells showing cytoplasmic staining and the proportion of cells exposed to glandular spaces showing apical membrane staining were classified similarly. The intensity of staining for each of these classifications also was assessed using a five-point scale.
Statistical Analyses Chi-squared tests were used to investigate associations between MUC1 expression and other tumor characteristics. Univariate differences between recurrence-free and overall survival estimates for the variables listed in Table 1 were assessed using actuarial survival analyses and log-rank t e s t s . 26 Those parameters showing significant univariate differences in survival were included in multivariate Cox proportional hazard regression models. 27 Data were stored using the Paradox 3.0 relational database (Borland International, Scotts Valley, CA). All statistical analyses were performed using the SAS/STAT 6.04 statistical program (SAS Institute Inc, Cary, NC) on a microcomputer.
scores 0-2, scores 34 <50, >50 <50, >50 Premenopausal and perimenopausal, postmenopausal Ductal, lobular, other Positive, negative Present, absent Present, absent <50, />50
RESULTS cell carcinomas. Coexistent ductal carcinoma in situ was present in 54 of these cases and lobular carcinoma was present in six. Histological grading was performed using the Nottingham modification of the Bloom and Richardson system. 25 Peritumoral microvascular invasion was recorded where present. Data regarding tumor size, estrogen receptor status, menopausal status, and presence of axillary node metastases were obtained from clinical charts and pathology records. Estrogen receptor data, derived from a biochemical dextran-coated charcoal method, were available in only 100 of the cases.
D e t e c t i o n of MUC1 M u c i n by Immunohistochemistry All paraffin blocks containing tumor from each primary breast carcinoma were sectioned at 3 #m. Sections were stained with the BC2 monoclonal antibody using an avidinbiotin complex immunoperoxidase technique. Sections were dewaxed and rehydrated, treated for 10 minutes with 3% (v/ V) H202 in 18% (v/v) methanol in 0.1 m o l / L phosphate buffer containing 0.9% (w/v) NaC1, pH 7.4 (phosphate-buffered saline [PBS]), to limit endogenous peroxidase activity, washed three times in PBS, and then nonspecific binding blocked in 4% skim milk powder in PBS for 15 minutes. After brief washing sections were placed flat in a humidified box and incubated with BC2 (diluted 1:5,000 in PBS) monoclonal antibody mouse ascites for 45 minutes. The sections were washed thoroughly in PBS after each antibody incubation. Sections were subsequently incubated with 1:150 biotinylated sheep antimouse immunoglobulins (Amersham Australia Pty Ltd, Castle Hill, Australia) for 45 minutes and then with 1:150 streptavidin-horseradish peroxidase (Amersham Australia Pty Ltd) for 30 minutes. After washing peroxidase activity was detected using 3,3'-diaminobenzidine as a chromogen with H2O 2 as a substrate. The sections were counterstained with hematoxylin and mounted. As negative controls, serial sections were stained as above but with PBS substituted for the primary antibody. Immunoperoxidase-stained tumors were examined by two investigators (MAM and MDW). The overall proportions
433
Expression of MUC1 in Normal Breast Epithelium MUC1 was e x p r e s s e d by m o s t b r e a s t e p i t h e l i a l cells f o u n d a d j a c e n t to c a r c i n o m a s . E x p r e s s i o n was usually l i m i t e d to t h e apical m e m b r a n e o f t h e s e cells (Fig 1).
Expression of MUC1 in C a r c i n o m a In Situ BC2 s t a i n i n g was d e t e c t e d in 40 o f 60 cases (67%) o f c o e x i s t e n t c a r c i n o m a in situ o f e i t h e r d u c t a l o r l o b u lar type. I n c r i b r i f o r m a n d m i c r o p a p i l l a r y cases a p i c a l m e m b r a n e s t a i n i n g was c h a r a c t e r i s t i c (Fig 2), w h e r e a s in c o m e d o a n d solid h i s t o t y p e s i n t e n s e diffuse cytop l a s m i c s t a i n i n g also was o f t e n s e e n (Fig 3). I n s o m e cases MUC1 was d e t e c t e d in a l a r g e p e r c e n t a g e o f cells in in situ lesions w h e n t h e invasive t u m o r cells s h o w e d o n l y a low level o f e x p r e s s i o n .
FIGURE 1. Apical staining of normal breast duct epithelium. (Immunoperoxidase, BC2 antibody; original magnification × 100.)
HUMAN PATHOLOGY
Volume 26, No. 4 (April 1995)
TABLE 2. Percentage of Invasive Breast Cancers Showing MUC1 Expression, Cytoplasmic Expression, and Apical and Circumferential M e m b r a n e Expression Percentage of Cells Positive (%) Classification
0
1-25
26-50
51-75
76-100
Overall Cytoplasmic Apical membrane
1 8 13
12 14 22
8 20 21
19 29 13
60 29 30
A s s o c i a t i o n s B e t w e e n MUC1 Expression a n d O t h e r Tumor C h a r a c t e r i s t i c s
MUC1, as detected by antibody BC2, was expressed in all but two of the 171 invasive carcinomas. However, the pattern of expression and the p r o p o r t i o n of positive t u m o r cells varied greatly between tumors. Table 2 shows the p r o p o r t i o n of t u m o r cells showing MUC1 expression and the p r o p o r t i o n showing expression in the cytoplasm and in the apical m e m b r a n e . In some tumors expression was restricted to the apical m e m b r a n e of t u m o r cells lining gland spaces (Fig 4). In other tumors strong diffuse cytoplasmic staining was p r o m i n e n t in the presence (Fig 5) or absence of m e m b r a n e staining. Twenty-five tumors showed apical staining in the absence of cytoplasmic staining, whereas only 11 tumors showed cytoplasmic staining in the absence of m e m b r a n e staining. Cytoplasmic MUC1 expression was intense in most lobular carcinomas, and in these tumors intracytoplasmic vacuoles also were often present (Fig 6).
Data for the overall p r o p o r t i o n o f cells stained with BC2 were divided into two classes (tumors showing staining in less than 75% and in greater than 75% of cells) on the basis of survival analysis results (see Results). Associations between overall MUC1 expression and other t u m o r characteristics are shown in Table 3. Weak significant associations were f o u n d between a high level of MUC] expression and both estrogen receptor and axillary n o d e positivity. MUC] expression was high in 76% of estrogen receptor-positive tumors c o m p a r e d with 54% of estrogen receptor-negative tumors. MUC1 expression was high in 76% of tumors f r o m patients with m o r e than three nodes containing metastases c o m p a r e d with 64% of patients with one to three nodes involved and 51% of patients with no nodal metastases. Although there were variations in MUC1 expression in different t u m o r size groups, these did not follow a consistent pattern. No significant associations were f o u n d between overall MUC1 expression and histological type, t u m o r grade, m e n o p a u s a l status, the presence of microvascular invasion, or coexistent carcin o m a in situ. T h e p r o p o r t i o n of cells showing cytoplasmic MUC1 expression was not significantly associated with any other t u m o r characteristics. T h e p r o p o r t i o n of cells showing staining of apical m e m branes also was not associated with any characteristics
FIGURE 3. Comedo ductal carcinoma in situ of the breast showing cytoplasmic and luminal membrane expression of MUC1 mucin. (Immunoperoxidase, BC2 antibody; original magnification ×200.)
FIGURE 4. Infiltrating ductal carcinoma of the breast with MUC 1 mucin expression limited to apical staining of tumor cells lining gland spaces. (Immunoperoxidase, BC2 antibody; original magnification x 160.)
FIGURE 2. Micropapillary ductal carcinoma in situ of the breast showing apical membrane expression of MUC1 mucin. (Immunoperoxidase, BC2 antibody; original magnification x160.)
Expression o f MUC1 in I n v a s i v e C a n c e r
434
MUC1 MUCIN IN BREASTCANCER (McGuckin et al)
TABLE 3.
Associations Between the Proportion of Breast Tumor Cells Showing MUC1 Expression as Defined by Monoclonal Antibody BC2 and Other Tumor Characteristics (the Number of Tumors in Each Classification Is Shown) Parameter Estrogen receptors No. of axillary n o d e metastases T u m o r size (mm)
FIGURE 5. Infiltrating ductal carcinoma of the breast showing diffuse cytoplasmic and circumferential membrane expression of MUC1 mucin. (Immunoperoxidase, BC2 antibody; original
magnification × 160.)
T u m o r grade
Histological type
Menopausal status
with the exception of tumor grade. Grade 1 tumors showed a higher proportion of cells with apical membrane staining than grade 2 and 3 tumors. MUC1 Expression R e l a t e d to Survival
The results of univariate analyses of recurrencefree and overall survival are presented in Tables 4 and 5, respectively. The number of lymph nodes containing metastases was the strongest predictor of both recurrence-free and overall survival. Other significant variables were tumor size, grade, and the overall proportion of cells showing MUC1 expression for overall survival, and both the overall proportion of cells showing MUC1 expression and the proportion showing cytoplasmic MUC1 for recurrence-free survival. Overall MUC1 expression was classified into tumors showing less than and greater than 75% of cells positive after consideration of actuarial survival curves for the four quartiles. The poorer overall and recurrence-free survivals (P <
FIGURE6. Lobular carcinoma of the breast showing both diffuse and vacuolar cytoplasmic expressionof MUC1 mucin. (Im-
munoperoxidase, BC2 antibody; original magnification × 160.)
435
Carcinoma in situ Microvascular invasion
Classification Negative Positive 0 1-3 >3 1-10 11-20 21-50 >50 1 2 3 Ductal Lobular Other Pre/periPostPresent Absent Present Absent
BC2 <75%
BC2 >75%
25 11 41 17 10 1 37 17 13 17 29 22 57 4 7 17 51 25 43 21 47
30 34 42 30 31 9 39 41 14 31 52 20 83 16 4 16 87 43 60 19 84
X2, P 4.74, .029 7.52, .023
9.66, .022
3.70, NS
5.93, NS
2.36, NS 0.43, NS 3.54, NS
NOTE. Chi-squared tests were used to test for significant differences in the proportions of MUCl-positive t u m o r s within the classifications of o t h e r t u m o r characteristics.
.05) for patients with greater than 75% of tumor cells showing MUC1 expression are demonstrated by the survival curves in Figs 7 and 8. Cytoplasmic and apical MUC1 expression was classified into tumors showing less than and greater than 50% of cells positive after consideration of actuarial survival curves for the four quartiles. The poorer recurrence-free survival (P < .05) and the trend for poorer overall survival (not significant, P = .076) for patients showing high levels of cytoplasmic MUC1 expression are demonstrated in Figs 9 and 10. The proportion of cells showing apical membrane staining showed no prognostic significance (Figs 11 and 12). Intensity of BC2 staining, menopausal status, histological type, estrogen receptor status, and the presence of microvascular invasion and carcinoma in situ had no significant effect on recurrence-free or overall survival in univariate analyses. The number of lymph nodes with metastatic carcinoma, tumor size and grade, the overall proportion of cells showing MUC1 expression, and the proportion showing cytoplasmic MUC1 expression were combined in proportional hazards multivariate regression analyses for both overall and recurrence-free survivals (Table 6). In these analyses the only variable to show prognostic significance independent from the highly significant nodal status was histological grade in the overall survival model only. Tumor size and the proportion of cells showing overall or cytoplasmic MUC1 expression were not significant prognostic indicators independent of nodal status.
HUMAN PATHOLOGY
Volume 26, No. 4 (April 1995)
TABLE 4. Univariate Analyses of the Effect of 13 Variables on Recurrence-Free Survival (5-Year and 10-Year Actuarial Survival Estimates [Mean + SEM] Are Shown)
Variable N u m b e r o f axillary node metastases P r o p o r t i o n of cells with MUC1 e x p r e s s i o n (%) P r o p o r t i o n o f cells with c y t o p l a s m i c MUC1 e x p r e s s i o n (%) T u m o r size Tumor grade I n t e n s i t y o f BC2 stain Microvascular invasion
Age P r o p o r t i o n o f cells showing apical membrane MUC1 expression Presence of c a r c i n o m a in situ Estrogen receptors M e n o p a u s a l status H i s t o l o g i c a l type
5-Year Survival
10-Year Survival
(%)
(%)
X 2, P
TABLE S. Univariate Analyses of the Effect of 13 Variables on Overall Survival (5-Year and 10-Year Actuarial Survival Estimates [Mean _+ SEM] Are Shown) 5-Year Survival (%)
10-Year Survival (%)
0 1-3 >3 1 2 3 <75 >75
94 ± 5 82 -+ 12 51 -+ 15 92 ± 8 8 O _+ 9 69 ± 15 86 _+ 8 77 _+ 8
78 _+ 13 54 + 22 1 7 ± 13 73 _+ 18 49+14 60_+ 18 68 + 15 50 _+ 15
59.85, <.0001
~10 10-20 21-50 >50
90 ± 19 82± 8 85 -+ 10 62 + 18
72 + 35 59_+ 15 59 _+ 19 49 _+ 20
3.85, .0499
Variable
0 1-3 >3 <75 >75
85 + 63 _+ 34_+ 76 _+ 61 -+
7 14 15 10 10
72 -+ 12 44 _+ 21 13_+ 12 65 + 14 41 _+ 14
54.78, < . 0 0 0 1
<50 >50
74 -+ 10 62 _+ 10
59 -4- 16 46 _+ 12
4.82, .028
Number of axillary node metastases Tumor grade
6.91, .009 Proportion of cells with MUC1 expression (%) Tumor size (ram)
Proportion of cells with cytoplasmic MUC1 expression Menopausal status
2.00, NS 1.79, NS 1.39, NS 1.28, NS
0.43, NS 0.38, NS 0.002, NS
4.31, .038
1.83, NS 1.75, NS 0.65, NS
Coexistent carcinoma in situ Estrogen receptors Histological type Microvascular invasion Intensity of BC2 stain Proportion of cells showing apical membrane MUC1 expression
0.65, NS
4.50, .034
3.16, NS
Age
0.96, NS 0.95, NS
X2, P
0.62, NS 0.39, NS 0.26, NS 0.02, NS 0.004, NS
N O T E . L o g - r a n k tests w e r e u s e d to test for d i f f e r e n c e s in survival; X 2 a n d p r o b a b i l i t y are shown.
NOTE. Log-rank tests were used to test for differences in survival; X2 and probability are shown.
DISCUSSION
T h e staining patterns observed using the m o n o clonal antibody BC2 in this study a p p e a r similar to those obtained in previous studies using different m o n o clonal antibodies reactive with the MUC1 core protein.
T h e MUC1 epithelial mucin, detected by the antibody BC2, was expressed in n o r m a l breast, carcinoma in situ, and almost all invasive breast cancers. A high level of expression by invasive cancers was associated with p o o r e r overall and disease-free survivals. Much is yet to be understood regarding the roles of this cornplex molecule in both normal and malignant epithelial cells, and arguments can be p r o p o s e d classifying the presence of mucins as b o t h g o o d and bad prognostic indicators. Association with good prognosis is consistent with observations that expression of this mucin may be a m a r k e r of differentiation in breast cancer a6 as well as with the demonstration of b o t h T- and B-cell i m m u n e responses to MUC1 peptide epitopes in breast and ovarian cancer. 28'29 Conversely, these molecules may act to protect t u m o r cells f r o m adverse physicochemical conditions (such as low p H ) 3° may be involved in cellular adhesion i m p o r t a n t in invasion and metastasis, 31'3z and also could act as i m m u n e blocking factors. 31'33 In addition to identification of the mucin core proteins, the detection of specific carbohydrate structures, which themselves may have biological importance, expressed on MUC1 and other mucins using carbohydrate-specific antibodies and lectins adds complexity to these considerations. 436
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MONTHS F I G U R E 7. Disease-free survival in breast cancer patients with greater than a n d less than 75% of tumor cells showing MUC1 expression.
MUC1 MUCIN IN BREAST CANCER (McGuckin et al)
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FIGURE 10. Overall survival in breast cancer patients with
MUC1 localization varying from a restricted presence on apical cell membranes to strong diffuse cytoplasmic staining, in addition to heterogeneity between tumors in the proportion of tumor cells showing MUC1 expression, also was reported in studies using antibodies HMFG1 and HMFG2, u'lz'lyAs NCRC11,1~-163s DF3, t9'21 F36/22,19 115D8 and 139H2, as and BrE-3. 21 These antibodies have all been shown to react with synthetic peptides based on the VNTR sequence of MUC1.34-~6 However, in immunohistochemical studies combining more than one antibody, significant differences in reactivity between antibodies within individual breast cancer sections have been described. 16'a7 Differences in antibody reactivity also have been demonstrated using enzymelinked i m m u n o s o r b e n t assay and Western blotting techniques on tumor-derived MUC1 mucins, sT'3s Despite
reacting with similar m i n i m u m epitopes in peptide studies, these antibodies show differences in reactivity with native mucin, probably caused by the conformational presentation of the peptide epitopes or the influence of adjacent oligosaccharides. Only studies using antibody NCRC11 have consistently shown any association between MUC1 expression and tumor grade. A high proportion of tumor cells staining with this antibody was associated with low grade in several studies, la16,ls In one of these studies NCRC11 staining was highly significantly associated with grade, but staining with HMFG1, HMFG2, 115D8, and 139H2 were not, further highlighting differences in reactivity with these antibodies. 18 In the current study the overall proportion of cells staining with BC2 was not associated with tumor grade, but the proportion of cells showing
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FIGURE 11. Disease-free survival in breast cancer patients with greater than and less than 50% of tumor cells lining gland spaces showing apical membrane MUC1 mucin expression.
FIGURE 9. Disease-free survival in breast cancer patients with greater than and less than 50% of tumor cells showing cytoplasmic MUC1 mucin expression.
437
HUMAN PATHOLOGY
Volume26, No. 4 (April 1995)
apical m e m b r a n e staining was higher in grade 1 than in higher grade tumors. This observation in well-differentiated tumors is consistent with the pattern of MUC1 expression in normal breast epithelial cells. In the current study estrogen receptor positivity was associated with a higher proportion of tumor cells showing BC2 staining. Several studies have addressed expression of MUC1 in relation to expression of estrogen receptors with conflicting results. Large studies using antibody N C R C l l found a positive association between a high level of NCRC11 positivity and estrogen receptor positivity, 14'16 although other studies with smaller numbers of patients failed to demonstrate this association) s'~5 The associations, if any, between tumor differentiation, estrogen receptor expression, and MUC1 production remain indefinite. In the current study a high proportion of cells positive using BC2 also was associated with the presence of nodal metastases, whereas studies using NCRC11 and other MUC1 antibodies showed no association with nodal metastases. ~6'18 The association with nodal metastases is consistent with the association with p o o r e r length of survival, and could be argued to reflect many factors, including metastatic potential and chronology. Although several MUC1 antibodies have previously been used to compare MUC1 expression and survival in breast cancer patients, many of these studies are based on the analysis of relatively small numbers of patients with inadequate follow-up. The most comprehensive previous studies are those using antibodies N C R C l l I4'16 and BrE-3. 2° The association between a high proportion of tumor cells showing BC2 staining and p o o r e r disease-free and overall survival is in contradiction with studies using N C R C l l . Most studies using N C R C l l found better length of survival in patients with a high proportion of tumor cells expressing MUC1. 13 ' 16 ' 18 Similarly, survival was longer in two studies of patients with a high proportion of tumor cells staining with the DF3 antibody, a9'21 Paradoxically, tumors 1.0 W
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MONTHS FIGURE 12. Overall survival in breast cancer patients with greater than and less than 50% of tumor cells lining gland spaces showing apical membrane MUC1 mucin expression.
438
TABLE 6. Cox Proportional Hazards Regression Analysis of the Influence of Five Variables Significant in Univariate Analyses on Disease-Free and Overall Survival in Breast Cancer Patients (the Z Statistic and Probability for Each Variable Are Shown) Disease-Free Survival Variable Nodal metastases T u m o r grade T u m o r size Overall proportion positive for MUC1 Proportion showing cytoplasmic MUC1
Overall Survival
Z
P
Z
P
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<0.001 NS NS
5.72 2.24 0.07
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0.98
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1.06
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0.61
NS
lacking HMFG1 and HMFG2 have been shown to have both p o o r u and good 12 prognoses; yet in other studies results from these anttbodies were found to have no prognostic significance) 7'18 O t h e r MUC1 VNTR-binding antibodies, 115D8, 139H2, and F36/22, have been shown to have no prognostic utility. ~8'~9 However, a recent detailed study in a relatively large n u m b e r of patients using antibody BrE-3 demonstrated a p o o r e r length of survival in patients with a high cytoplasmic expression and better length of survival in patients with a high apical m e m b r a n e expression. 2° The current study also examined the prognostic importance of the proportion of tumor cells showing cytoplasmic and apical m e m b r a n e staining. The finding of p o o r prognosis in patients with high cytoplasmic mucin expression is consistent with the observations using antibody BrE-3. Although the extent of apical m e m b r a n e staining was of no prognostic significance in the current study, this was based on a careful measurement of the degree of staining of apical membranes, where present, not on the extent of glandular surfaces in the tumor. We believe the latter, which is a c o m p o n e n t in the assessment of tumor grade, may c o n f o u n d some other studies of MUC1 expression and prognosis. The somewhat arbitrary divisions into high and low percentages of cells showing MUC1 expression used for the statistical analyses in this study require prospective assessment on a further set of cases to verify the validity of these divisions. The apparent differences in reactivity between N C R C l l and antibodies BC2 and BrE-3 could result from a higher specificity of NCRC11 for the glycoform of MUC1 p r o d u c e d by welPdifferentiated tumor cells. However, BC2 reacts strongly with normal epithelial cells and also with the apical m e m b r a n e ofwell-different a t e d tumors, suggesting that a possible p o o r e r reactvity ofNCRC11 with cytoplasmic MUC1 may be the cause of the differential reactivity. This hypothesis can only be tested in a trial of these different antibodies on serial sections. Ceriani et al 2° hypothesize that BrE-3 binds less glycosylated (possibly less mature) forms of mucin present in the cytoplasm of some tumor cells and that failure of some tumors to glycosylate and transport the mucin to the cell m e m b r a n e (and thus increased cyto-
MUC1 MUCIN IN BREAST CANCER (McGuckin et al)
plasmic detection) is associated with poorer length of survival. Although a high expression of the MUC1 mucin, as detected by antibody BC2, in the current study was associated with a poor prognosis, generalizations regarding the implications of expression of this complex molecule by breast cancers cannot easily be made because of its structural complexity, highlighted by differences in reactivity of different antibodies.
REFERENCES 1. Zotter S, Hagelnan PC, Lossnitzer A, et ah Tissue and turnout distribution of human polymorphic epithelial mucin. Cancer Rev 1112:55-101, 1988 2. HilgersJ, Zotter S, Kenemans P: Polymorphic epithelial mucin and CA125-bearing glycoprotein in basic and applied carcinoma research. Cancer Rev 11-12:3-10, 1988 3. Gum JRJ: Mucin genes and the proteins they encode: Structure, diversity, and regulation. Am J Respir Cell Mol Biol 7:557-564, 1992 4. Toribara NW, Roberton AM, Ho SB, et ah Human gastric mucin: Identification of a unique species by expression cloning. J Biol Chem 268:5879-5885, 1993 5. Bobek LA, Tsai H, Biesbrock AR, et al: Molecular cloning, sequence, and specificity of expression of the gene encoding the low molecular weight human salivary mucin (MUC7). J Biol Chem 268:20563-20569, 1993 6. Walsh MD, McGuckin MA, Devine PL, et al: Expression of MUC2 epithelial mucin in breast carcinoma. J Clin Pathol 46:922925, 1993 7. Gendler SJ, Lancaster CA, Taylor-Papadimitriou J, et al: Molecular cloning and expression of human tumor-associated polymorphic epithelial mucin. J Biol Chem 265:15286-15293, 1990 8. McKenzie IFC, Xing P-X: Mucins and breast cancer: Recent immunological advances. Cancer Cells 2:75-78, 1990 9. Xing P-X, TjandraJJ, Stacker SA, et ah Monoclonal antibodies reactive with mucin expressed in breast cancer. Immunol Cell Biol 67:183-195, 1989 10. Xing P-X, Reynolds K, Tjandra JJ, et al: Synthetic peptides reactive with anti-human milk fat globule monoclonal antibodies. Cancer Res 50:89-96, 1990 11. Wilkinson MJS, Howell A, Harris M, et ah The prognostic significance of two epithelial membrane antigens expressed by human mammary carcinomas. I n t J Cancer 33:299-304, 1984 12. Berry N, Jones DB, SmallwoodJ, et al: The prognostic value of the monoclonal antibodies HMFG1 and HMFG2 in breast cancer. B r J Cancer 51:179-186, 1985 13. Ellis I t , Hinton CP, MacNayJ, et al: Immunocytochemical staining of breast carcinoma with the monoclonal antibody NCRCll, a new prognostic indicator. Br M e d J 290:881-883, 1985 14. Angus B, Napier J, Purvis J, et ah Survival in breast cancer related to tumour oestrogen receptor status and immunohistochemical staining for NCRCll. J Pathol 149:301-306, 1986 15. Wright C, Angus B, Napier J, et al: Prognostic factors in breast cancer: Immunohistochemical staining for SP1 and NCRCll related to survival, tumour epidermal growth factor receptor and oestrogen receptor status. J Pathol 153:325-331, 1987 16. Ellis I t , Bell J, ToddJM, et ah Evaluation of immunoreactivity with monoclonal antibody NCRC11 in breast carcinoma. BrJ Cancer 56:295-299, 1987
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17. Parham DM, Slidders W, Robertson AJ: Quantitation of human milk fat globule (HMFG1) expression in breast carcinoma and its association with survival. J Clin Pathol 41:875-879, 1988 18. Walker RA: Assessment of milk fat globule membrane antibodies and lectins as markers of short-term prognosis in breast cancer. Br J Cancer 62:462-466, 1990 19. Hayes DF, Mesa-Tejada R, Papsidero LD, et al: Prediction of prognosis in primary breast cancer by detection of a high molecular weight mucin-like antigen using monocloual antibodies DF3, F36/ 22, and CU18: A cancer and leukemia group B study. J Clin Oncol 9:1113-1123, 1991 20. Ceriani RL, Chan CM, Baratta FS, et ah Levels of expression of breast epithelial mucin detected by monoclonal antibody BrE-3 in breast cancer prognosis. I n t J Cancer 51:343-354, 1992 21. Byrne J, Horgan PG, England S, et ah An evaluation of the usefulness of primary tumour expression of MCA and CA15-3 as prognostic indicators in breast carcinoma. E u r J Surg Oncol 18:230-234, 1992 22. Baildam AD, Howell A, Barnes DM: The expression of milk fat globule antigens within human mammary tumours: Relationship to steroid hormone receptors and response to endocrine treatment. E u r J Cancer Clin Oncol 25:459-467, 1989 23. Hilkens-J, Bonfrer JMG, Kroezen V, et al: Comparison of circulating MAMa5 mad CEA levels and correlation with the estrogen receptor in patients with breast cancer. IntJ Cancer 39:431-435, 1987 24. LundyJ, Thor A, Meanza R, et al: Monoclonal antibody DF3 correlates with turnout differentiation and hormone receptor status in breast cancer patients. Breast Cancer Res Treat 5:266-276, 1985 25. Elston CW, Ellis I t : Pathology and breast screening. Histopathology 16:108-118, 1990 26. Kaplan EL, Meier P: Nonparametric estimation from incomplete observations. J Am Stat Assoc 53:457-481, 1958 27. Cox DR, t a k e s D. Analysis of Survival Data. London, Chapman and Hall, 1984 28. Jerome KR, Barnd DL, Bendt KM, et ah Cytotoxic T-lymphocytes derived from patients with breast adenocarcinoma recognize an epitope present on the protein core of a mucin molecule preferentially expressed by malignant cells. Cancer Res 51:2908-2916, 1991 29. Rughetti A, Turchi V, Ghetti CA, et al: Human B-cell immune response to the polymorphic epithelial mucin. Cancer Res 53:245%2459, 1993 30. Slomiany BL, Slomiany A: Role of mucus in gastric mncosal protection. J Physiol Pharmacol 42:14%161, 1991 31. Hilkens J, Ligtenberg MJ, Vos HL, et al: Cell membraneassociated mucins and their adhesion-modulating property. Trends Biochem Sci 17:359-363, 1992 32. Ligtenberg MJ, Buijs F, Vos HL, et al: Suppression of cellular aggregation by high levels of episialin. Cancer Res 52:2318-2324, 1992 33. Hayes DF, Silberstein DS, Rodrique SW, et al: DF3 antigen, a human epithelial cell mucin, inhibits adhesion of eosinophils to antibody-coated targets. J Immunol 145:962-970, 1990 34. Taylor-Papadimitriou J: Report on the First International Workshop on Carcinoma Associated Mucins. IntJ Cancer 49:1-5,1991 35. Devine PL, McKenzie IFC: Mucins: Structure, function and association with malignancy. Bioessays 14:619-625, 1992 36. Peterson JA, Larocca D, Walkup G, et ah Molecular analysis of epitopic heterogeneity of the breast mucin, in Ceriani RL (ed): Breast Epithelial Antigens. New York, NY, Plenum, 1991, pp 55-68 37. Devine PL, Warren JA, Ward BG, et al: Glycosylation and the exposure of tumor-associated epitopes on mucins. J Tumor Marker Oncol 5:11-26, 1990 38. Devine PL, Warren JA, Clark BA, et al: The complexity of cancer-associated epitope expression on antigens produced by ovarian tumour cells. J Tumor Marker Oncol 5:321-339, 1990
nostically significant, but the proportion of cells that lined gland spaces showing apical membrane staining was of no prognostic significance. A high level of MUC1 expression was significantly associated with the presence of axillary node metastases and estrogen receptors but not with other tumor characteristics. HUM PATHOL 26:432439. Copyright © 1995 by W.B. Saunders Company Key words: breast cancer, mucin, prognosis, survival, MUC1. Abbreviations: VNTR, variable number of tandem repeats; HMFGM, human milk fat globule membrane; PBS, phosphate-buffered saline.
Mucins are high molecular weight glycoproteins f o u n d in the cell m e m b r a n e s of h u m a n epithelial cells f r o m the m a m m a r y gland, salivary gland, digestive tract, respiratory tract, kidney, bladder, prostate, uterus, and rete testis, a During malignant transformation m e m b r a n e expression of these mucins often changes f r o m apical to circumferential coincident with loss of polarity of the epithelial cell, and synthesis increases markedly, often resulting in large amounts of mucin being either shed or secreted by t u m o r cells. Glycosylation of the peptide backbone also is altered, resulting in novel carbohydrate epitopes or exposure of the peptide backbone. 2 Recently, the genes o f seven of these mucins have b e e n fully or partially cloned. ~5 In all of these mucins a substantial portion of the core protein consists of sequences that are repeated a large and variable n u m b e r of times, known as the variable n u m b e r of tand e m repeats, or VNTR, region. Many antimucin m o n o c l o n a l antibodies have b e e n described that react with breast carcinomas. Some of these have b e e n raised using breast t u m o r cells as immunogens, but m a n y have b e e n raised against o t h e r types of tumors or the h u m a n milk fat globule m e m brane (HMFGM). Although we have previously d e m o n strated the presence of the MUC2 mucin and its association with p o o r survival in breast cancer, 6 the mucin f o u n d most c o m m o n l y in breast carcinoma is the product of the MUC1 gene, t e r m e d polymorphic epithelial
mucin. 7 Almost all anti-MUC1 core protein m o n o c l o n a l antibodies described react with the VNTR region. 8 However, m o n o c l o n a l antibodies with similar m i n i m u m epitopes (as assessed by binding to synthetic peptides) can show differing reactivities with breast carcinoma. T h e IgG1 mouse monoclonal antibody, BC2, used in this study was raised using HMFGM as the i m m u n o g e n , and reacts with very high affinity to the peptide epitope, APDTR, on the VNTR of MUC1 mucins. 9,a° Several studies have e x a m i n e d the relationship between expression o f MUC1 mucins and survival in breast cancer patients. 11-21 However, the results f r o m these studies are often contradictory, and m a n y are based on the analysis of relatively small n u m b e r s of patients with inadequate follow-up. MUC1 expression by breast tumors and mucin levels in serum also have b e e n related to estrogen receptor s t a t u s , 11'12'14-16'22-24 also with contradictory findings. T h e current study assesses MUC1 mucin expression in a retrospective series of breast carcinomas and compares the prognostic significance of this expression with that of other prognostic factors, such as the presence ofaxillary n o d e metastases, histological type and grade, t u m o r size, presence of coexistent carcinoma in situ, estrogen receptor status, and m e n o p a u s a l status.
MATERIALS AND METHODS Patients
From the Department of Obstetrics and Gynecology and the Department of Surgery, University of Queensland, Royal Brisbane Hospital; the Department of Pathology, Royal Brisbane Hospital; and the Department of Gynecologic Oncology, Royal Women's Hospital, Herston, Australia. Accepted for publication August 8, 1994. Supported by a grant from the Queensland Cancer Fund and Australian National Health and Medical Research Council Project Grant No. 92-1140. Address correspondence and reprint requests to Michael A. McGuckin, BSc, PhD, Department of Obstetrics and Gynecology, University of Queensland, Clinical Sciences Building, Royal Brisbane Hospital, Herston, 4029, Australia. Copyright © 1995 by W.B. Saunders Company 0046-8177/95/2604-001355.00/0
432
One hundred seventy-one cases of invasive breast carcinoma diagnosed at the Department of Pathology of the Royal Brisbane Hospital between 1971 and 1986 were selected for this study. The cases were sequential and unselected other than being excluded if there was insufficient histological material (including axillary node biopsy) or inadequate followup information. Histological classification of these invasive cancers was performed by one investigator (RGW). The results of the clinical follow-up and immunohistochemistry study were independently collected. These cases included 140 ductal, 20 lobular, five medullary, one mucinous, two papillary, and one each of apocrine, tubulolobular, and squamous
MUC1 MUCIN IN BREASTCANCER (McGuckin et al) TABLE 1. Grouping of Tumor and Patient Characteristic Variables Used for Statistical Analyses Variable Number of axillary node metastases Tumor diameter Tumor grade Overall proportion of tumor cells stained by BC2 (%) Intensity of BC2 staining Proportion of tumor cells with cytoplasmic MUC1 (%) Proportion of tumor cells with apical membrane MUC1 (%) Menopausal status Histological type Estrogen receptor status Microvascular invasion Coexistent carcinoma in situ Age (yr)
Groupings 0, 1-3, >3 ~<10 mm, 11-20 mm, 21-50 nlm, >50 mlrl 1,2,3 <75, >75
of invasive tumor cells and in situ tumor cells showing MUC1 expression were classified separately into five groups: no staining, 0% to 25%, 26% to 50%, 51% to 75%, and 76% to 100% of cells positive. The proportion of cells showing cytoplasmic staining and the proportion of cells exposed to glandular spaces showing apical membrane staining were classified similarly. The intensity of staining for each of these classifications also was assessed using a five-point scale.
Statistical Analyses Chi-squared tests were used to investigate associations between MUC1 expression and other tumor characteristics. Univariate differences between recurrence-free and overall survival estimates for the variables listed in Table 1 were assessed using actuarial survival analyses and log-rank t e s t s . 26 Those parameters showing significant univariate differences in survival were included in multivariate Cox proportional hazard regression models. 27 Data were stored using the Paradox 3.0 relational database (Borland International, Scotts Valley, CA). All statistical analyses were performed using the SAS/STAT 6.04 statistical program (SAS Institute Inc, Cary, NC) on a microcomputer.
scores 0-2, scores 34 <50, >50 <50, >50 Premenopausal and perimenopausal, postmenopausal Ductal, lobular, other Positive, negative Present, absent Present, absent <50, />50
RESULTS cell carcinomas. Coexistent ductal carcinoma in situ was present in 54 of these cases and lobular carcinoma was present in six. Histological grading was performed using the Nottingham modification of the Bloom and Richardson system. 25 Peritumoral microvascular invasion was recorded where present. Data regarding tumor size, estrogen receptor status, menopausal status, and presence of axillary node metastases were obtained from clinical charts and pathology records. Estrogen receptor data, derived from a biochemical dextran-coated charcoal method, were available in only 100 of the cases.
D e t e c t i o n of MUC1 M u c i n by Immunohistochemistry All paraffin blocks containing tumor from each primary breast carcinoma were sectioned at 3 #m. Sections were stained with the BC2 monoclonal antibody using an avidinbiotin complex immunoperoxidase technique. Sections were dewaxed and rehydrated, treated for 10 minutes with 3% (v/ V) H202 in 18% (v/v) methanol in 0.1 m o l / L phosphate buffer containing 0.9% (w/v) NaC1, pH 7.4 (phosphate-buffered saline [PBS]), to limit endogenous peroxidase activity, washed three times in PBS, and then nonspecific binding blocked in 4% skim milk powder in PBS for 15 minutes. After brief washing sections were placed flat in a humidified box and incubated with BC2 (diluted 1:5,000 in PBS) monoclonal antibody mouse ascites for 45 minutes. The sections were washed thoroughly in PBS after each antibody incubation. Sections were subsequently incubated with 1:150 biotinylated sheep antimouse immunoglobulins (Amersham Australia Pty Ltd, Castle Hill, Australia) for 45 minutes and then with 1:150 streptavidin-horseradish peroxidase (Amersham Australia Pty Ltd) for 30 minutes. After washing peroxidase activity was detected using 3,3'-diaminobenzidine as a chromogen with H2O 2 as a substrate. The sections were counterstained with hematoxylin and mounted. As negative controls, serial sections were stained as above but with PBS substituted for the primary antibody. Immunoperoxidase-stained tumors were examined by two investigators (MAM and MDW). The overall proportions
433
Expression of MUC1 in Normal Breast Epithelium MUC1 was e x p r e s s e d by m o s t b r e a s t e p i t h e l i a l cells f o u n d a d j a c e n t to c a r c i n o m a s . E x p r e s s i o n was usually l i m i t e d to t h e apical m e m b r a n e o f t h e s e cells (Fig 1).
Expression of MUC1 in C a r c i n o m a In Situ BC2 s t a i n i n g was d e t e c t e d in 40 o f 60 cases (67%) o f c o e x i s t e n t c a r c i n o m a in situ o f e i t h e r d u c t a l o r l o b u lar type. I n c r i b r i f o r m a n d m i c r o p a p i l l a r y cases a p i c a l m e m b r a n e s t a i n i n g was c h a r a c t e r i s t i c (Fig 2), w h e r e a s in c o m e d o a n d solid h i s t o t y p e s i n t e n s e diffuse cytop l a s m i c s t a i n i n g also was o f t e n s e e n (Fig 3). I n s o m e cases MUC1 was d e t e c t e d in a l a r g e p e r c e n t a g e o f cells in in situ lesions w h e n t h e invasive t u m o r cells s h o w e d o n l y a low level o f e x p r e s s i o n .
FIGURE 1. Apical staining of normal breast duct epithelium. (Immunoperoxidase, BC2 antibody; original magnification × 100.)
HUMAN PATHOLOGY
Volume 26, No. 4 (April 1995)
TABLE 2. Percentage of Invasive Breast Cancers Showing MUC1 Expression, Cytoplasmic Expression, and Apical and Circumferential M e m b r a n e Expression Percentage of Cells Positive (%) Classification
0
1-25
26-50
51-75
76-100
Overall Cytoplasmic Apical membrane
1 8 13
12 14 22
8 20 21
19 29 13
60 29 30
A s s o c i a t i o n s B e t w e e n MUC1 Expression a n d O t h e r Tumor C h a r a c t e r i s t i c s
MUC1, as detected by antibody BC2, was expressed in all but two of the 171 invasive carcinomas. However, the pattern of expression and the p r o p o r t i o n of positive t u m o r cells varied greatly between tumors. Table 2 shows the p r o p o r t i o n of t u m o r cells showing MUC1 expression and the p r o p o r t i o n showing expression in the cytoplasm and in the apical m e m b r a n e . In some tumors expression was restricted to the apical m e m b r a n e of t u m o r cells lining gland spaces (Fig 4). In other tumors strong diffuse cytoplasmic staining was p r o m i n e n t in the presence (Fig 5) or absence of m e m b r a n e staining. Twenty-five tumors showed apical staining in the absence of cytoplasmic staining, whereas only 11 tumors showed cytoplasmic staining in the absence of m e m b r a n e staining. Cytoplasmic MUC1 expression was intense in most lobular carcinomas, and in these tumors intracytoplasmic vacuoles also were often present (Fig 6).
Data for the overall p r o p o r t i o n o f cells stained with BC2 were divided into two classes (tumors showing staining in less than 75% and in greater than 75% of cells) on the basis of survival analysis results (see Results). Associations between overall MUC1 expression and other t u m o r characteristics are shown in Table 3. Weak significant associations were f o u n d between a high level of MUC] expression and both estrogen receptor and axillary n o d e positivity. MUC] expression was high in 76% of estrogen receptor-positive tumors c o m p a r e d with 54% of estrogen receptor-negative tumors. MUC1 expression was high in 76% of tumors f r o m patients with m o r e than three nodes containing metastases c o m p a r e d with 64% of patients with one to three nodes involved and 51% of patients with no nodal metastases. Although there were variations in MUC1 expression in different t u m o r size groups, these did not follow a consistent pattern. No significant associations were f o u n d between overall MUC1 expression and histological type, t u m o r grade, m e n o p a u s a l status, the presence of microvascular invasion, or coexistent carcin o m a in situ. T h e p r o p o r t i o n of cells showing cytoplasmic MUC1 expression was not significantly associated with any other t u m o r characteristics. T h e p r o p o r t i o n of cells showing staining of apical m e m branes also was not associated with any characteristics
FIGURE 3. Comedo ductal carcinoma in situ of the breast showing cytoplasmic and luminal membrane expression of MUC1 mucin. (Immunoperoxidase, BC2 antibody; original magnification ×200.)
FIGURE 4. Infiltrating ductal carcinoma of the breast with MUC 1 mucin expression limited to apical staining of tumor cells lining gland spaces. (Immunoperoxidase, BC2 antibody; original magnification x 160.)
FIGURE 2. Micropapillary ductal carcinoma in situ of the breast showing apical membrane expression of MUC1 mucin. (Immunoperoxidase, BC2 antibody; original magnification x160.)
Expression o f MUC1 in I n v a s i v e C a n c e r
434
MUC1 MUCIN IN BREASTCANCER (McGuckin et al)
TABLE 3.
Associations Between the Proportion of Breast Tumor Cells Showing MUC1 Expression as Defined by Monoclonal Antibody BC2 and Other Tumor Characteristics (the Number of Tumors in Each Classification Is Shown) Parameter Estrogen receptors No. of axillary n o d e metastases T u m o r size (mm)
FIGURE 5. Infiltrating ductal carcinoma of the breast showing diffuse cytoplasmic and circumferential membrane expression of MUC1 mucin. (Immunoperoxidase, BC2 antibody; original
magnification × 160.)
T u m o r grade
Histological type
Menopausal status
with the exception of tumor grade. Grade 1 tumors showed a higher proportion of cells with apical membrane staining than grade 2 and 3 tumors. MUC1 Expression R e l a t e d to Survival
The results of univariate analyses of recurrencefree and overall survival are presented in Tables 4 and 5, respectively. The number of lymph nodes containing metastases was the strongest predictor of both recurrence-free and overall survival. Other significant variables were tumor size, grade, and the overall proportion of cells showing MUC1 expression for overall survival, and both the overall proportion of cells showing MUC1 expression and the proportion showing cytoplasmic MUC1 for recurrence-free survival. Overall MUC1 expression was classified into tumors showing less than and greater than 75% of cells positive after consideration of actuarial survival curves for the four quartiles. The poorer overall and recurrence-free survivals (P <
FIGURE6. Lobular carcinoma of the breast showing both diffuse and vacuolar cytoplasmic expressionof MUC1 mucin. (Im-
munoperoxidase, BC2 antibody; original magnification × 160.)
435
Carcinoma in situ Microvascular invasion
Classification Negative Positive 0 1-3 >3 1-10 11-20 21-50 >50 1 2 3 Ductal Lobular Other Pre/periPostPresent Absent Present Absent
BC2 <75%
BC2 >75%
25 11 41 17 10 1 37 17 13 17 29 22 57 4 7 17 51 25 43 21 47
30 34 42 30 31 9 39 41 14 31 52 20 83 16 4 16 87 43 60 19 84
X2, P 4.74, .029 7.52, .023
9.66, .022
3.70, NS
5.93, NS
2.36, NS 0.43, NS 3.54, NS
NOTE. Chi-squared tests were used to test for significant differences in the proportions of MUCl-positive t u m o r s within the classifications of o t h e r t u m o r characteristics.
.05) for patients with greater than 75% of tumor cells showing MUC1 expression are demonstrated by the survival curves in Figs 7 and 8. Cytoplasmic and apical MUC1 expression was classified into tumors showing less than and greater than 50% of cells positive after consideration of actuarial survival curves for the four quartiles. The poorer recurrence-free survival (P < .05) and the trend for poorer overall survival (not significant, P = .076) for patients showing high levels of cytoplasmic MUC1 expression are demonstrated in Figs 9 and 10. The proportion of cells showing apical membrane staining showed no prognostic significance (Figs 11 and 12). Intensity of BC2 staining, menopausal status, histological type, estrogen receptor status, and the presence of microvascular invasion and carcinoma in situ had no significant effect on recurrence-free or overall survival in univariate analyses. The number of lymph nodes with metastatic carcinoma, tumor size and grade, the overall proportion of cells showing MUC1 expression, and the proportion showing cytoplasmic MUC1 expression were combined in proportional hazards multivariate regression analyses for both overall and recurrence-free survivals (Table 6). In these analyses the only variable to show prognostic significance independent from the highly significant nodal status was histological grade in the overall survival model only. Tumor size and the proportion of cells showing overall or cytoplasmic MUC1 expression were not significant prognostic indicators independent of nodal status.
HUMAN PATHOLOGY
Volume 26, No. 4 (April 1995)
TABLE 4. Univariate Analyses of the Effect of 13 Variables on Recurrence-Free Survival (5-Year and 10-Year Actuarial Survival Estimates [Mean + SEM] Are Shown)
Variable N u m b e r o f axillary node metastases P r o p o r t i o n of cells with MUC1 e x p r e s s i o n (%) P r o p o r t i o n o f cells with c y t o p l a s m i c MUC1 e x p r e s s i o n (%) T u m o r size Tumor grade I n t e n s i t y o f BC2 stain Microvascular invasion
Age P r o p o r t i o n o f cells showing apical membrane MUC1 expression Presence of c a r c i n o m a in situ Estrogen receptors M e n o p a u s a l status H i s t o l o g i c a l type
5-Year Survival
10-Year Survival
(%)
(%)
X 2, P
TABLE S. Univariate Analyses of the Effect of 13 Variables on Overall Survival (5-Year and 10-Year Actuarial Survival Estimates [Mean _+ SEM] Are Shown) 5-Year Survival (%)
10-Year Survival (%)
0 1-3 >3 1 2 3 <75 >75
94 ± 5 82 -+ 12 51 -+ 15 92 ± 8 8 O _+ 9 69 ± 15 86 _+ 8 77 _+ 8
78 _+ 13 54 + 22 1 7 ± 13 73 _+ 18 49+14 60_+ 18 68 + 15 50 _+ 15
59.85, <.0001
~10 10-20 21-50 >50
90 ± 19 82± 8 85 -+ 10 62 + 18
72 + 35 59_+ 15 59 _+ 19 49 _+ 20
3.85, .0499
Variable
0 1-3 >3 <75 >75
85 + 63 _+ 34_+ 76 _+ 61 -+
7 14 15 10 10
72 -+ 12 44 _+ 21 13_+ 12 65 + 14 41 _+ 14
54.78, < . 0 0 0 1
<50 >50
74 -+ 10 62 _+ 10
59 -4- 16 46 _+ 12
4.82, .028
Number of axillary node metastases Tumor grade
6.91, .009 Proportion of cells with MUC1 expression (%) Tumor size (ram)
Proportion of cells with cytoplasmic MUC1 expression Menopausal status
2.00, NS 1.79, NS 1.39, NS 1.28, NS
0.43, NS 0.38, NS 0.002, NS
4.31, .038
1.83, NS 1.75, NS 0.65, NS
Coexistent carcinoma in situ Estrogen receptors Histological type Microvascular invasion Intensity of BC2 stain Proportion of cells showing apical membrane MUC1 expression
0.65, NS
4.50, .034
3.16, NS
Age
0.96, NS 0.95, NS
X2, P
0.62, NS 0.39, NS 0.26, NS 0.02, NS 0.004, NS
N O T E . L o g - r a n k tests w e r e u s e d to test for d i f f e r e n c e s in survival; X 2 a n d p r o b a b i l i t y are shown.
NOTE. Log-rank tests were used to test for differences in survival; X2 and probability are shown.
DISCUSSION
T h e staining patterns observed using the m o n o clonal antibody BC2 in this study a p p e a r similar to those obtained in previous studies using different m o n o clonal antibodies reactive with the MUC1 core protein.
T h e MUC1 epithelial mucin, detected by the antibody BC2, was expressed in n o r m a l breast, carcinoma in situ, and almost all invasive breast cancers. A high level of expression by invasive cancers was associated with p o o r e r overall and disease-free survivals. Much is yet to be understood regarding the roles of this cornplex molecule in both normal and malignant epithelial cells, and arguments can be p r o p o s e d classifying the presence of mucins as b o t h g o o d and bad prognostic indicators. Association with good prognosis is consistent with observations that expression of this mucin may be a m a r k e r of differentiation in breast cancer a6 as well as with the demonstration of b o t h T- and B-cell i m m u n e responses to MUC1 peptide epitopes in breast and ovarian cancer. 28'29 Conversely, these molecules may act to protect t u m o r cells f r o m adverse physicochemical conditions (such as low p H ) 3° may be involved in cellular adhesion i m p o r t a n t in invasion and metastasis, 31'3z and also could act as i m m u n e blocking factors. 31'33 In addition to identification of the mucin core proteins, the detection of specific carbohydrate structures, which themselves may have biological importance, expressed on MUC1 and other mucins using carbohydrate-specific antibodies and lectins adds complexity to these considerations. 436
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-
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MONTHS F I G U R E 7. Disease-free survival in breast cancer patients with greater than a n d less than 75% of tumor cells showing MUC1 expression.
MUC1 MUCIN IN BREAST CANCER (McGuckin et al)
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h
0.6
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L > 75%
nO
0.4
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20
I 40
I 60
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80
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X 2= 3.16,
1.0
I
I
0.8
N.S.
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-'4
0.6
50%
< !
%
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-
0.2
-
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120
I
I
I
I
I
'1
20
40
60
80
100
120
MONTHS
MONTHS FIGURE 8, Overall survival in breast cancer patients with greater than and less than 75% of tumor cells showing MUC1 mucin expression.
FIGURE 10. Overall survival in breast cancer patients with
MUC1 localization varying from a restricted presence on apical cell membranes to strong diffuse cytoplasmic staining, in addition to heterogeneity between tumors in the proportion of tumor cells showing MUC1 expression, also was reported in studies using antibodies HMFG1 and HMFG2, u'lz'lyAs NCRC11,1~-163s DF3, t9'21 F36/22,19 115D8 and 139H2, as and BrE-3. 21 These antibodies have all been shown to react with synthetic peptides based on the VNTR sequence of MUC1.34-~6 However, in immunohistochemical studies combining more than one antibody, significant differences in reactivity between antibodies within individual breast cancer sections have been described. 16'a7 Differences in antibody reactivity also have been demonstrated using enzymelinked i m m u n o s o r b e n t assay and Western blotting techniques on tumor-derived MUC1 mucins, sT'3s Despite
reacting with similar m i n i m u m epitopes in peptide studies, these antibodies show differences in reactivity with native mucin, probably caused by the conformational presentation of the peptide epitopes or the influence of adjacent oligosaccharides. Only studies using antibody NCRC11 have consistently shown any association between MUC1 expression and tumor grade. A high proportion of tumor cells staining with this antibody was associated with low grade in several studies, la16,ls In one of these studies NCRC11 staining was highly significantly associated with grade, but staining with HMFG1, HMFG2, 115D8, and 139H2 were not, further highlighting differences in reactivity with these antibodies. 18 In the current study the overall proportion of cells staining with BC2 was not associated with tumor grade, but the proportion of cells showing
t,l
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-
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2= 4.82,
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~
,'-'
< 50%
greater than and less than 50% of tumor cells showing cytoplasmic MUC1 mucin expression.
0.6
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°
°
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0.4
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0.2 0.0 0
I
I
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1
I
I
20
40
60
80
100
120
MONTHS
MONTHS
FIGURE 11. Disease-free survival in breast cancer patients with greater than and less than 50% of tumor cells lining gland spaces showing apical membrane MUC1 mucin expression.
FIGURE 9. Disease-free survival in breast cancer patients with greater than and less than 50% of tumor cells showing cytoplasmic MUC1 mucin expression.
437
HUMAN PATHOLOGY
Volume26, No. 4 (April 1995)
apical m e m b r a n e staining was higher in grade 1 than in higher grade tumors. This observation in well-differentiated tumors is consistent with the pattern of MUC1 expression in normal breast epithelial cells. In the current study estrogen receptor positivity was associated with a higher proportion of tumor cells showing BC2 staining. Several studies have addressed expression of MUC1 in relation to expression of estrogen receptors with conflicting results. Large studies using antibody N C R C l l found a positive association between a high level of NCRC11 positivity and estrogen receptor positivity, 14'16 although other studies with smaller numbers of patients failed to demonstrate this association) s'~5 The associations, if any, between tumor differentiation, estrogen receptor expression, and MUC1 production remain indefinite. In the current study a high proportion of cells positive using BC2 also was associated with the presence of nodal metastases, whereas studies using NCRC11 and other MUC1 antibodies showed no association with nodal metastases. ~6'18 The association with nodal metastases is consistent with the association with p o o r e r length of survival, and could be argued to reflect many factors, including metastatic potential and chronology. Although several MUC1 antibodies have previously been used to compare MUC1 expression and survival in breast cancer patients, many of these studies are based on the analysis of relatively small numbers of patients with inadequate follow-up. The most comprehensive previous studies are those using antibodies N C R C l l I4'16 and BrE-3. 2° The association between a high proportion of tumor cells showing BC2 staining and p o o r e r disease-free and overall survival is in contradiction with studies using N C R C l l . Most studies using N C R C l l found better length of survival in patients with a high proportion of tumor cells expressing MUC1. 13 ' 16 ' 18 Similarly, survival was longer in two studies of patients with a high proportion of tumor cells staining with the DF3 antibody, a9'21 Paradoxically, tumors 1.0 W
>
0.8
•Q•c2
X 2= 0 . 0 0 4 ,
N.S.
-
d
< 50~
,<
z 0
0.6 > 50~
i--
r~ O n O
n,-
n
0.4
0,2
--
0.0 0
I
I
I
I
I
I
20
40
60
80
100
120
MONTHS FIGURE 12. Overall survival in breast cancer patients with greater than and less than 50% of tumor cells lining gland spaces showing apical membrane MUC1 mucin expression.
438
TABLE 6. Cox Proportional Hazards Regression Analysis of the Influence of Five Variables Significant in Univariate Analyses on Disease-Free and Overall Survival in Breast Cancer Patients (the Z Statistic and Probability for Each Variable Are Shown) Disease-Free Survival Variable Nodal metastases T u m o r grade T u m o r size Overall proportion positive for MUC1 Proportion showing cytoplasmic MUC1
Overall Survival
Z
P
Z
P
5.58 0.97 0.39
<0.001 NS NS
5.72 2.24 0.07
<.001 <.05 NS
0.98
NS
0.14
NS
1.06
NS
0.61
NS
lacking HMFG1 and HMFG2 have been shown to have both p o o r u and good 12 prognoses; yet in other studies results from these anttbodies were found to have no prognostic significance) 7'18 O t h e r MUC1 VNTR-binding antibodies, 115D8, 139H2, and F36/22, have been shown to have no prognostic utility. ~8'~9 However, a recent detailed study in a relatively large n u m b e r of patients using antibody BrE-3 demonstrated a p o o r e r length of survival in patients with a high cytoplasmic expression and better length of survival in patients with a high apical m e m b r a n e expression. 2° The current study also examined the prognostic importance of the proportion of tumor cells showing cytoplasmic and apical m e m b r a n e staining. The finding of p o o r prognosis in patients with high cytoplasmic mucin expression is consistent with the observations using antibody BrE-3. Although the extent of apical m e m b r a n e staining was of no prognostic significance in the current study, this was based on a careful measurement of the degree of staining of apical membranes, where present, not on the extent of glandular surfaces in the tumor. We believe the latter, which is a c o m p o n e n t in the assessment of tumor grade, may c o n f o u n d some other studies of MUC1 expression and prognosis. The somewhat arbitrary divisions into high and low percentages of cells showing MUC1 expression used for the statistical analyses in this study require prospective assessment on a further set of cases to verify the validity of these divisions. The apparent differences in reactivity between N C R C l l and antibodies BC2 and BrE-3 could result from a higher specificity of NCRC11 for the glycoform of MUC1 p r o d u c e d by welPdifferentiated tumor cells. However, BC2 reacts strongly with normal epithelial cells and also with the apical m e m b r a n e ofwell-different a t e d tumors, suggesting that a possible p o o r e r reactvity ofNCRC11 with cytoplasmic MUC1 may be the cause of the differential reactivity. This hypothesis can only be tested in a trial of these different antibodies on serial sections. Ceriani et al 2° hypothesize that BrE-3 binds less glycosylated (possibly less mature) forms of mucin present in the cytoplasm of some tumor cells and that failure of some tumors to glycosylate and transport the mucin to the cell m e m b r a n e (and thus increased cyto-
MUC1 MUCIN IN BREAST CANCER (McGuckin et al)
plasmic detection) is associated with poorer length of survival. Although a high expression of the MUC1 mucin, as detected by antibody BC2, in the current study was associated with a poor prognosis, generalizations regarding the implications of expression of this complex molecule by breast cancers cannot easily be made because of its structural complexity, highlighted by differences in reactivity of different antibodies.
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