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1263
P-Cadherin Expression in Breast Carcinoma Indicates
Poor Survival
Alejandro Peralta Soler, M.D., Ph.D.1
Karen A. Knudsen, Ph.D.1
Hernando Salazar, M.D., M.P.H.2
Aaron C. Han, M.D., Ph.D.2
Albert A. Keshgegian, M.D., Ph.D.3
1
The Lankenau Medical Research Center, Wynnewood, Pennsylvania.
2
Department of Pathology, The Reading Hospital
and Medical Center, Reading, Pennsylvania.
3
Department of Pathology, The Lankenau Hospital, Wynnewood, Pennsylvania.
Presented in part as “Expression of E- and PCadherin in Breast Tumors” at the XXII Congress of
the International Academy of Pathology and the
13th World Congress of Academic and Environmental Pathology, Nice, France, October 18 –23,
1998, and as “P-Cadherin Expression in Breast
Cancer Indicates Poor Survival” at the 90th annual
meeting of the American Association for Cancer
Research, Philadelphia, PA, April 10 –14, 1999.
Supported by the John S. Sharpe Research Foundation of the Bryn Mawr Hospital.
The authors thank Gary D. Harner, Gwendolyn
Gilliard, and Elaine Johnston for technical assistance; Dr. Mike Free, Greg Maislin, and Jacqueline
Cater for statistical analysis; and the Editorial Office of the Lankenau Medical Research Center for
helping with the article preparation. They also
thank Drs. Margaret J. Wheelock and Keith R.
Johnson of the University of Toledo, Ohio, for
providing anti-P-cadherin monoclonal antibody
clone 6A9 and anti-g-catenin monoclonal antibody
clone 15F11.
Address for reprints: Alejandro Peralta Soler, M.D.,
Ph.D., The Lankenau Medical Research Center,
100 Lancaster Avenue, Wynnewood, PA 19096.
Received November 30, 1998; revision received
April 28, 1999; accepted April 28, 1999.
© 1999 American Cancer Society
BACKGROUND. The cadherin family of cell-cell adhesion molecules and their associated proteins, the catenins, are essential to embryonic development and the
maintenance of adult tissues. During development, the homotypic interaction of a
particular cadherin with an identical cadherin expressed on a neighboring cell
results in the sorting of cells to form distinctive tissues. Cadherins are believed to
be tumor suppressors, and their altered expression and function have been associated with tumor development.
METHODS. The authors examined the expression of P-cadherin, E-cadherin, and
N-cadherin, and a-catenin and b-catenin in 183 cases of invasive breast carcinoma
by immunohistochemistry on paraffin sections using specific antibodies and a
steam-based antigen retrieval method.
RESULTS. P-cadherin was positive in 95 cases and negative in 88 cases of breast
carcinoma. Positive P-cadherin expression in breast carcinoma showed a strong
correlation with poor patient prognosis. Five years after surgery, 90% of the
patients with P-cadherin negative tumors were alive in contrast to only 59% of
patients with P-cadherin positive tumors. The difference in survival reached statistical significance (P 5 0.0001) as early as 2 years after surgical treatment.
Expression of N-cadherin, a-catenin, and b-catenin did not correlate with patient
survival. Multivariable statistical analyses of the data showed that expression of
P-cadherin was independent of tumor size and lymph node metastases, but correlated inversely with estrogen/progesterone receptor status. In ductal carcinomas,
positive P-cadherin expression correlated with a higher histologic grade. In contrast, expression of E-cadherin was low in high grade ductal carcinomas but
negative tumors were uncommon. Negative or low E-cadherin expression did not
correlate with poor survival. In lobular carcinomas, E-cadherin expression frequently was negative or low, and P-cadherin always was negative.
CONCLUSIONS. Expression of P-cadherin in breast carcinoma is associated strongly
with poor survival and constitutes an independent prognostic predictor. P-cadherin expression is a better indicator of clinical outcome than alterations in the
expression of E-cadherin, N-cadherin, a-catenin, or b-catenin. Cancer 1999;86:
1263–72. © 1999 American Cancer Society.
KEYWORDS: cadherins and catenins in breast carcinoma, P-cadherin, poor survival,
cap cell carcinoma of the breast.
he cadherins are calcium-dependent cell-cell adhesion proteins.1
The best characterized and most widely distributed members of
the family are the classical cadherins,2 a group that includes epithelial
(E)-, nerve (N)-, and placental (P)-cadherin. Intracellularly, the cadherins interact with several proteins termed catenins, including
a-catenin, b-catenin, g-catenin (plakoglobin), and p120ctn, a substrate
for the tyrosine kinase Src.3– 6 The catenins link the cadherins to the
cytoskeleton7 and mediate signal-transduction mechanisms that con-
T
1264
CANCER October 1, 1999 / Volume 86 / Number 7
trol cellular events, including cell polarity and differentiation, cell growth, and cell death. During development, the homotypic binding of a particular cadherin
to an identical cadherin expressed on an adjacent cell
produces the sorting of cells into distinctive tissues.1
In adult organisms, the cadherin-catenin adhesion
system maintains the differentiated state of the tissues
and suppresses tumor development.8 –12 Alterations in
the cadherin-catenin cell-cell adhesion system are
associated with loss of differentiation and tumor formation,13 increased invasiveness, metastasis,14 and
unfavorable prognosis for patients with breast carcinoma.15,16
In breast carcinoma, several lines of evidence indicate that reduced expression and function of cadherins are associated with tumor development and
invasion. E-cadherin is considered a tumor suppressor
in the breast, and breast carcinoma frequently exhibits
loss of heterozygosity on the long arm of chromosome
16 (16q), which contains the E-cadherin gene
(16q22.1). Low E-cadherin expression is found in half
of infiltrating ductal carcinomas17–19 as a result of
hypermethylation of the E-cadherin promoter region.20 Lobular carcinoma contains a high frequency
of E-cadherin mutations, resulting in decreased or
absent expression.21,22 Low expression of E-cadherin
in breast carcinoma has been associated with dedifferentiation,17 increased invasiveness,16 and high metastatic potential.23 Conversely, transfection of E-cadherin cDNA into invasive breast carcinoma cells
suppresses osteolytic bone metastases in mice,24 reduces invasiveness, and induces posttranscriptional
up-regulation of syndecan-1, a proteoglycan associated with cell differentiation and cell-matrix anchorage.25 Another member of the cadherin family, Hcadherin, a mesenchymal cadherin lacking the
cytoplasmic domain, also has a tumor-suppressing
role in breast carcinoma, and its expression is reduced
in human breast tumors.26 Loss or alterations in
the cadherin-associated proteins, a-catenin13 and
p120ctn,27 contribute to additional mechanisms of
poor cell adhesion and breast carcinogenesis. Deletions of a-catenin were found in some tumors.13,28,29
Alterations also have been found in b-catenin,30 a
mediator in the oncogenic Wnt-1 signaling pathway
that has been implicated in breast tumorigenesis in
mice.31,32
The expression and role of P-cadherin in breast
carcinoma is still understood poorly. In one study,
P-cadherin was not detected in a series of patients
with ductal carcinoma,19 but, in another study, strong
P-cadherin expression was found in some cases of
infiltrating ductal carcinoma, and it was associated
with reduced E-cadherin expression and advanced
histologic grade.33 In the current study, a comprehensive analysis was conducted of the expression of cadherins and catenins in breast carcinoma. P-cadherin
was expressed in more than half of the cases of invasive ductal carcinoma studied. More importantly, we
found that the strong expression of P-cadherin in
breast carcinoma correlates with poor patient outcome. Furthermore, our data indicate that the expression of P-cadherin represents a more accurate predictor of poor patient survival than the altered expression
of other cadherins and catenins.
MATERIALS AND METHODS
Clinical Material
Five-micron-thick sections were obtained from formalin fixed, paraffin embedded, archival tissues from
invasive breast carcinomas obtained from the Department of Pathology of The Bryn Mawr Hospital, Bryn
Mawr, Pennsylvania. The tumor tissues were obtained
from a series used in a previous study.34 The samples
used in that study included biopsy, lumpectomy, and
mastectomy specimens that were large enough to provide material for flow cytometry and for biochemical
and immunocytochemical analysis of hormone receptors. Out of the original 300 cases stained by immunohistochemistry, 183 cases were selected for this
study based on a minimum of a 5-year patient follow-up after surgery for living patients and breast
carcinoma as the cause of death for those patients that
died. The surgical procedures were performed between 1989 and 1992. Patient status was classified as
died of disease, alive with disease, or alive with no
evidence of disease. The original pathology data included tumor size, histologic grade, estrogen and progesterone receptor status, and number of lymph node
metastases. Histologic grading was according to the
Bloom and Richardson method,35 as modified by Elston and Ellis. Tumors were classified as Grade 1 (well
differentiated), Grade 2 (moderately differentiated),
and Grade 3 (poorly differentiated). Histopathologic
diagnoses included 160 invasive ductal carcinomas, 18
invasive lobular carcinomas, 4 medullary carcinomas,
and 1 metaplastic carcinoma. Three cases of normal
breast tissues from reduction mammoplasty also were
included in the study.
Antibodies
A mouse monoclonal antibody (MoAb) against P-cadherin was purchased from Transduction Laboratories
(Lexington, KY) and was used at a 2 mg/mL. Anti-Pcadherin MoAb clone 6A9 was a gift of Dr. M.J. Wheelock (University of Toledo, OH) and was used as conditioned supernatant fluid. Anti-E-cadherin MoAb
clone HECD-1 was purchased from Zymed Laboratory
P-Cadherin in Breast Carcinoma/Peralta Soler et al.
(San Francisco, CA) and was used at 5 mg/mL. AntiN-cadherin MoAbs 13A9 and 3B9 (Zymed Laboratory)
were developed in our laboratories and were used as
conditioned supernatant fluids.5 They recognize intracellular domains of human N-cadherin in paraffin sections without cross-reactivity to other cadherins.36 Anti-a-catenin MoAb was purchased from Zymed
Laboratory and was used at 1 mg/mL. Anti-b-catenin
MoAb 5H10 (Zymed Laboratory) was developed in
collaboration with Drs. Margaret J. Wheelock and
Keith R. Johnson (University of Toledo) and was used
as conditioned supernatant fluid.37 This antibody recognizes b-catenin in routinely processed paraffin sections of tumors from archival tissues. For g-catenin
(plakoglobin), we used MoAb 15F11,38 (a gift from Drs.
Margaret J. Wheelock and Keith R. Johnson, University
of Toledo). The absence of cross-reactivity of the antibodies has been tested previously by Western immunoblotting.4,5,36 –38 All of the antibodies have been
used previously in immunohistochemistry on formalin fixed, paraffin embedded human tissues.5,39
1265
TABLE 1
Semiquantitative Evaluation of P-Cadherin Expression and
Patient Statusa
P-cadherin
Total
ANED (%)
AWD (%)
DOD (%)
Negative
Total positive
Level 1
Level 2
Level 3
Level 4
88
95
16
38
21
20
76 (86.5)
47 (49.5)
11 (69)
26 (68)
7 (33)
3 (15)
4 (4.5)
9 (9.5)
2 (12)
0 (0)
3 (14.5)
4 (20)
8 (9)
39 (41)
3 (19)
12 (32)
11 (52.5)
13 (65)
ANED: alive with no evidence of disease; AWD: alive with disease; DOD: died of disease.
a
P-cadherin positive tumors were separated into 4 levels based on the percentage of positive cells;
Level 4, 75–100% positive cells; Level 3, 50–75% positive cells; Level 2, 20–50% positive cells; Level 1,
10–20% positive cells. Patient status was classified as (died of disease), alive with disease, and alive with
no evidence of disease. Note a 86.5% disease free survival rate in patients with P-cadherin negative
tumors 5 years after surgery compared with a 15% disease free survival rate in patients with high levels
of P-cadherin expression (Level 4).
(plasma membrane, cytoplasm, nucleus) and staining
intensity were recorded for tumor and nontumor tissues.
Immunohistochemistry
Immunohistochemistry was performed in most cases
in an automated immunostainer (Biotek Techmate
500; Ventana Medical Systems, Tuczon, AZ) using an
avidin-biotin system, following the manufacturer’s instructions (Vector Laboratories, Burlingame, CA). A
heat-induced antigen retrieval method40 was applied.
Deparaffinized 5-mm-thick sections were placed in
prewarmed 0.1 M citrate buffer, pH 6.0 (Dako Corp.,
Carpinteria, CA) for 20 minutes in a steamer (Black
and Decker, Shelton, CT), as described previously.39,41
Primary antibodies were incubated for 1 hour at room
temperature. When immunohistochemistry was performed manually, the primary antibodies were incubated overnight at 4°C in a humid chamber. Sections
were counterstained with hematoxylin.
Data Recording
The identities of patients were kept confidential, and
the samples were coded in accordance with the guidelines of the Institutional Review Board of the Bryn
Mawr Hospital for the use of human tissues for research. The follow-up data were provided by the Tumor Registry of the Bryn Mawr Hospital. A semiquantitative analysis of the immunohistochemistry was
performed to determine the approximate percentage
of cells expressing each cadherin and catenin. Cadherin and catenin positive tumors were classified into
4 levels based on the percentage of positive cells: Level
1, 10 –20% positive cells; Level 2, 20 –50% positive cells;
Level 3, 50 –75% positive cells; and Level 4, 75–100%
positive cells. Also, the patterns of cellular distribution
Statistical Methods
Relative mortality rates comparing patients with and
without P-cadherin expression were estimated by incidence density ratios and 95% confidence intervals.
Potential confounding or effect-modifying variables
(including tumor grade, tumor size, lymph node involvement, and estrogen/progesterone receptor status) were investigated by using Cox proportional hazards regression models.42 Two patients (1.1%) were
missing estrogen/progesterone receptor status and
were excluded from multivariable analyses. P-cadherin positive and P-cadherin negative groups were
compared with regard to each factor by using chisquare statistics and generalized Fisher exact tests or
two-group t-tests, as appropriate.43
Multivariable analyses were performed by constructing adjusted incidence density ratios to compare
patients with positive P cadherin expression with patients with negative P-cadherin expression while controlling for each covariate factor one at a time. Multivariable models were estimated to assess the impact of
P-cadherin expression on survival after adjusting for
multiple covariates. After assessing associations
among covariate factors, a final multivariable model
was constructed that included P-cadherin expression,
tumor grade category, and lymph node status categories.
RESULTS
Both anti-P-cadherin MoAbs used in this study (the
MoAb from Transduction Laboratories and MoAb
1266
CANCER October 1, 1999 / Volume 86 / Number 7
FIGURE 1. Kaplan–Meier survival
curve of patients with placental (P)-cadherin positive (solid squares) and P-cadherin negative (open squares) tumors.
The difference in survival reached statistical significance 2 years after surgical
treatment (P 5 0.0001).
6A9) produced similar staining patterns. Western blot
analysis showed that the anti-P- and anti-E-cadherin
MoAbs used in this study did not have cross-reactivity.
Both anti-P-cadherin MoAbs recognize a 118-kilodalton (kDa) protein, and the anti-E-cadherin MoAb
(Zymed Laboratory) recognizes a 120-kDa protein in
cells induced to express either P-cadherin or E-cadherin (data not shown). Anti-N-cadherin MoAbs, 13A9
and 3B9, have been shown previously to recognize a
135-kDa protein corresponding to N-cadherin.38 Both
anti-N-cadherin MoAbs used here produced similar
staining patterns. Anti-g-catenin (plakoglobin) MoAb
was used only in a limited number of cases, and the
data were not included in the correlative analysis with
patient outcome.
P-cadherin positive cells were found in 52% of the
cases, and a semiquantitative analysis of the expression was recorded as the percentage of positive tumor
cells (see Data Recording, above). When semiquantitative evaluation of P-cadherin expression was correlated with the clinical status of the patients (Table 1),
the results showed that, 5 years after surgery, 41% of
the patients with P-cadherin positive tumors died of
the disease, 9.5% were alive with disease, and 49.5%
were alive with no evidence of disease. In contrast,
only 9% of the patients with P-cadherin negative tumors died of the disease, 4.5% were alive with disease,
and 86.5% were alive with no evidence of disease.
High P-cadherin expression correlated with poor survival: 65% of the patients with tumors expressing a
high level of P-cadherin (Level 4) died of the disease
within 5 years after surgery compared with 19%
among patients with low-expressing (Level 1) tumors
TABLE 2
Correlation between P-Cadherin Expression and
Estrogen/Progesterone Receptor Status
Status
ER/PR negativea
ER/PR positiveb
P-cadherin positivec
P-cadherin negative
50
17
43
71
ER: estrogen receptor; PR: progesterone receptor.
a
These include tumors negative for both receptors and tumors negative for either estrogen receptor or
progesterone receptor.
b
These include tumors positive for both receptors.
c
Two of the 95 P-cadherin positive patients did not have hormone receptor data.
TABLE 3
Correlation between P-Cadherin Expression and Histologic Grade in
Invasive Breast Carcinomas
Histologic tumor grade
P-cadherin
Total
Grade 1
(%)
Grade 2
(%)
Grade 3
(%)
Negative
Positive
88
95
20 (23)
8 (8)
55 (63)
57 (60)
13 (14)
30 (32)
(Table 1). The results of a Kaplan–Meier survival curve
(Fig. 1) showed that the number of patients with Pcadherin positive tumors that died of the disease was
significantly higher than that for patients with P-cadherin negative tumors as early as 2 years after surgical
treatment (P 5 0.0001). The comparison between patients with positive and negative P-cadherin-express-
P-Cadherin in Breast Carcinoma/Peralta Soler et al.
1267
TABLE 4
Two-Variable Regression Analysis of Mortality Rates Modeling P-Cadherin Expression plus One Covariate
Covariate (adjusted one at a time with
P-cadherin expression)
Covariate
Unadjusted mortality ratio
Grade
3 vs. 1
2 vs. 1
Tumor size
Positive lymph nodes
.5 positive vs. negative
1–5 positive vs. negative
Negative ER/PR receptor status
P value
P-cadherin expression
IDR (95% CI)a
,0.029
,0.158
,0.002
9.5 (1.3–71.9)
4.2 (0.6–31.6)
1.5 (1.2–1.9)b,c
,0.0001
,0.002
,0.01
6.9 (2.7–17.9)
4.2 (1.7–10.4)
2.2 (1.2–4.2)
P value
IDR (95% CI)a
,0.0001
5.5 (2.6–11.7)
,0.0001
5.5 (2.5–12.3)
,0.0001
6.1 (2.8–13.2)
,0.0001
5.0 (2.3–10.8)
,0.0002
4.4 (2.0–9.7)
IDR: incidence density ratio; 95% CI: 95% confidence interval.
a
Incidence density ratio computed by exponentiating estimated slope coefficient from Cox regression.
b
Incidence density ratio is per 1 standard deviation (16.8 mm) increase in size.
c
Includes 15 imputed size values.
ing tumors was performed for each covariate factor.
Table 2 shows the correlation between P-cadherin
expression and estrogen/progesterone receptor status. There was a greater proportion of patients in the
positive P-cadherin group with tumors with negative
estrogen/progesterone receptor status compared with
the negative P-cadherin group. Estrogen or progesterone receptor negative status was found in 53% of the
P-cadherin positive tumors. In contrast, only 19% of
the P-cadherin negative tumors were estrogen/progesterone receptor negative. A Fisher exact test
showed that this difference was statistically significant
(P , 0.0001).
Table 3 shows the frequency of P-cadherin expression in invasive ductal carcinoma divided according to
the histologic tumor grade. Grade 3 (poorly differentiated) tumors were more frequently P-cadherin positive than negative. In contrast, Grade 1 (well differentiated) tumors were more frequently P-cadherin
negative. A Wilcoxon signed rank test for ranked qualified data with ties and a Fisher exact test showed a
significant difference (P 5 0.0006 and P 5 0.002, respectively) between the distribution of P-cadherin
positive and P-cadherin negative tumors when
grouped according to histologic grade. However, there
was no difference in the distribution of P-cadherin
positive and P-cadherin negative tumors in Grade 2
(moderately differentiated) tumors. This finding is
particularly significant, because 55% of the patients
who died of the disease had Grade 2 tumors, and 90%
of the tumors from those patients were P-cadherin
positive. Furthermore, positive lymph node status was
not significantly different between P-cadherin positive
(47%) and P cadherin negative (41%) patients with
TABLE 5
Multivariable Cox Regression Analysis of Mortality Rates
Variable
P value
IDR (95% CI)a
P-cadherin expression
Grade
3 vs. 1
2 vs. 1
Positive lymph nodes
.5 positive vs. negative
1–5 positive vs. negative
,0.0001
5.4 (2.4–12.2)
,0.036
,0.161
8.8 (1.2–67.3)
4.2 (0.6–31.7)
,0.0006
,0.003
5.4 (2.1–14.0)
4.0 (1.6–10.0)
IDR: incidence density ratio; 95% CI: 95% confidence interval.
a
Incidence density ratio was computed by exponentiating estimated slope coefficient from Cox
regression.
Grade 2 tumors. Also, there was no significant difference in tumor size between patients with P-cadherin
positive (25.04 mm 6 14.41 mm) and P-cadherin negative (24.33 mm 6 14.11 mm) Grade 2 tumors. When
all of the tumor samples were studied together, regardless of their histologic grade, a t-test analysis of
tumor size and a Fisher exact test for the presence of
lymph node metastases did not correlate with P-cadherin expression (P 5 0.516 and P 5 0.103, respectively), although the value of this analysis is relative,
because the criteria originally used for the selection of
the tumors was based on their large size.34
Multivariable analyses of assessment of mortality
was performed in patients with P-cadherin positive
and P-cadherin negative tumors (Table 4). The unadjusted mortality ratio comparing patients with P-cadherin positive tumors with patients with P-cadherin
negative tumors was 5.5 (95% confidence interval,
2.6 –11.7). These results were statistically significant
1268
CANCER October 1, 1999 / Volume 86 / Number 7
FIGURE 2. Immunohistochemical patterns of P-cadherin expression in breast carcinoma detected with anti-P-cadherin monoclonal antibody (MoAb; Transduction
Laboratories, Lexington, KY). (A) P-cadherin in the plasma membrane in invasive cells of a ductal carcinoma. (B) Cytoplasmic P-cadherin expression in invasive ductal
carcinoma. (C,D) P-cadherin expression mostly in the peripheral cells of invading ductal carcinoma nests. (E) P-cadherin negative invasive ductal carcinoma (small
arrow). Note the positive myoepithelial cells (large arrow) in nonneoplastic breast ducts. (F) Lobular carcinoma negative for P-cadherin in both the in situ and invasive
cells. Note the P-cadherin positive myoepithelial cells (arrow).
(P , 0.0001), indicating that the probability of dying of
breast carcinoma is 5.5 times greater in patients with
P-cadherin positive tumors compared with patients
with P-cadherin negative tumors. The mortality ratio
values determined by P-cadherin expression ranged
from 4.4 to 6.1 after adjusting for 1 covariate factor at
a time. All of the covariates analyzed, including tumor
grade, tumor size, positive lymph node status, and
negative estrogen/progesterone receptor status, also
were statistically significant predictors of patient out-
come when controlling for P-cadherin expression.
More importantly, there were no statistically significant interactions between any of the covariates and
P-cadherin expression. A multiple variable model that
included all factors simultaneously resulted in an adjusted mortality ratio for P-cadherin expression of 4.8
(range, 2.0 –11.3) with P 5 0.0003. A model including
P-cadherin expression, tumor grade, and lymph node
status (Table 5) showed that all three factors that were
included did not modify the predictive value of P-
P-Cadherin in Breast Carcinoma/Peralta Soler et al.
cadherin. These results indicate that expression of
P-cadherin in breast carcinoma constitutes an independent predictor of poor patient outcome.
Distribution of P-cadherin in tumor cells showed
several distinctive tissue patterns (Fig. 2). In invasive,
P-cadherin positive ductal carcinomas, P-cadherin
was either predominantly at the plasma membrane or
in the cytoplasm. P-cadherin-expressing carcinoma
cells often were located at the periphery of invasive
cell clusters. P-cadherin frequently was expressed by
cells penetrating the stroma in early invasive ductal
carcinomas (not shown), suggesting an association
between P-cadherin expression and invasion. Myoepithelial cells always were positive for P-cadherin, both
in nontumoral ducts and in ducts and lobules containing in situ carcinoma. In P-cadherin negative invasive ductal carcinomas, P-cadherin positive mesenchymal spindle cells, resembling myoepithelial cells,
were observed occasionally around the tumor cells.
Lobular carcinomas were negative for P-cadherin, and
negative in situ lobular carcinomas often were surrounded by P-cadherin positive myoepithelial cells.
Expression of E-cadherin was positive in 98% of
ductal carcinomas, and E-cadherin expression was
correlated inversely with tumor grade. High levels of
E-cadherin expression (Levels 3 and 4) were observed
in 83% of patients with Grade 1 (well differentiated),
77% of patients with Grade 2 (moderately differentiated), and 51% of patients with Grade 3 (poorly differentiated) tumors. Low E-cadherin expression (Levels 1
and 2) was found in 44% of patients with Grade 3
tumors. However, only 5% of Grade 3 tumors were
E-cadherin negative. In contrast to ductal carcinomas,
44% of lobular carcinomas were E-cadherin negative,
and 39% had very low expression (Level 1). When the
semiquantitative analysis of E-cadherin expression
was correlated with patient survival, unexpectedly,
there was not a significant association between low
E-cadherin expression and poor survival. Five years
after surgery, 60% of the patients with E-cadherin
negative tumors and 68% of the patients with tumors
expressing low E-cadherin (Level 1) were alive. Within
5 years after surgery, 20% of the patients with Ecadherin negative tumors and 26% and of the patients
with E-cadherin positive tumors died of the disease
(Table 6).
N-cadherin was found in 48% of the tumors, but
its expression was restricted to a small population of
cells and was mostly cytoplasmic (Fig. 3). There was
no correlation between the expression of N-cadherin
and patient survival (not shown). The expression of
a-catenin and b-catenin (Fig. 3) was variable. The
staining of b-catenin was particularly intense in some
ductal carcinoma samples. Low or negative b-catenin
1269
TABLE 6
Semiquantitative Evaluation of E-Cadherin Expression and
Patient Status
E-cadherina
Total
ANED
(%)
AWD
(%)
DOD
(%)
Negative
Total positive
Level 1
Level 2
Level 3
Level 4
10
173
19
30
64
60
6 (60)
117 (68)
13 (68)
16 (53)
38 (59.5)
50 (83.5)
2 (20)
11 (6)
3 (16)
0 (0)
6 (9.5)
2 (3.5)
2 (20)
45 (26)
3 (16)
14 (47)
20 (31)
8 (13)
ANED: alive with no evidence of disease; AWD: alive with disease; DOD: died of disease.
a
See Table 1 for definition of levels of cadherin expression and patient status.
expression in ductal carcinoma samples was very uncommon. In contrast, low expression was frequent in
lobular carcinoma samples (not shown). Semiquantitative evaluation of a-catenin and b-catenin expression was difficult, and we did not find a correlation
between catenin staining and patient survival (not
shown).
DISCUSSION
The results of this study show that the expression of
P-cadherin in breast carcinoma is a valuable indicator
of poor patient survival. P-cadherin expression is a
better indicator of poor survival than the altered expression of other cadherins and catenins, including a
decrease in E-cadherin expression. In previous studies, assessment of the down-regulation or altered subcellular distribution of E-cadherin, a-catenin, and
b-catenin as prognostic markers of breast carcinoma
has proven problematic. Although E-cadherin frequently is mutated in lobular carcinomas of the
breast, lack of expression is rare. The value of assessing reduced E-cadherin expression as a predictor of
shorter survival for patients with breast carcinoma
remains controversial. Although decreased E-cadherin
in breast carcinoma cells has been associated with
dedifferentiation,17 invasiveness,16 and metastases,23
low E-cadherin expression did not correlate with patient prognosis.27 In another study, the down-regulation of E-cadherin alone failed to correlate with the
metastatic capacity of breast carcinomas, and the concomitant evaluation of catenins was necessary for predicting the development of metastases.29 In contrast, a
recent study correlated reduced E-cadherin expression with shorter overall patient survival, although the
study was limited to a population of lymph node negative patients.44 These opposite findings may reflect
the difficulty in evaluating decreased protein expression in tumor cells, which requires optimal tissue pro-
1270
CANCER October 1, 1999 / Volume 86 / Number 7
FIGURE 3. Immunohistochemical expression of epithelial (E)-cadherin
(Zymed Laboratory, Inc., San Francisco,
CA MoAb), b-catenin (5H10 MoAb), and
nerve (N)-cadherin (13A9 MoAb) in patients with breast carcinoma. (A) Cell
membrane and cytoplasmic distribution
of E-cadherin in ductal carcinoma. (B)
shows strong b-catenin expression in
an invasive ductal carcinoma. (C) Low
E-cadherin expression in lobular carcinoma cells (small arrow) growing under
the epithelial layer of a duct. Note the
high level of E-cadherin in the ductal
nonneoplastic epithelial cells (large arrow). (D) Focal cytoplasmic expression
of N-cadherin in an invasive ductal carcinoma.
cessing and computer-assisted analysis of the immunohistochemical staining. Alternatively, they may
indicate the current poor understanding of the dynamics in the regulation of E-cadherin expression
during the different phases of tumor invasion and
metastasis. The expression of N-cadherin in breast
carcinoma has not been evaluated previously. In this
study, N-cadherin expression failed to correlate with
poor patient survival.
The role of P-cadherin in breast carcinoma is not
known. However, the phenotypical characteristics of
cells expressing P-cadherin during breast develop-
ment provide clues that might help to explain the
aggressive behavior of P-cadherin expressing tumors.
During the development of the mouse mammary
gland, P-cadherin and E-cadherin are expressed differentially in the breast epithelium. P-cadherin is expressed by the cap cells, a layer of growing cells located at the tip of the end buds, and E-cadherin is
expressed by the differentiated epithelial cells facing
the lumen of ducts.45 The spatially selective expression of E-cadherin and P-cadherin is required for both
mammary tissue integrity and normal DNA synthesis.45 The P-cadherin expressing cap cells are consid-
P-Cadherin in Breast Carcinoma/Peralta Soler et al.
ered to be a stem cell population in the breast.46,47
They have a higher proliferation rate and lower frequency of apoptosis than the luminal epithelial cells48
as a result of paracrine regulation by growth factors,
including transforming growth factor-a and epidermal
growth factor. These growth factors stimulate the advance of ductal structures into the surrounding stroma.49 Cap cells differentiate into myoepithelial cells,
contributing to the morphogenesis of the branching
mammary gland.46 Evidence that P-cadherin plays a
critical role in maintaining the growth of the ductal
structures of the breast is supported further by the
mammary phenotype of the P-cadherin gene knockout mouse model. P-cadherin-deficient mice show abnormal breast development. The mammary glands of
virgin females do not have a ductal morphology, and,
instead, they resemble those of pregnant mice.50
The data from the current study that show expression of P-cadherin in a subset of patients with aggressive breast carcinomas suggest a histogenetic origin in
cap cells or acquisition of a tumor phenotype with
characteristics similar to cap cells. Additional data
support this postulate. Cap cells do not express estrogen receptors,51 a common characteristic of tumors
expressing P-cadherin. P-cadherin expressing tumors
are highly aggressive, and the paracrine growth factors
that stimulate the growth of cap cells also are associated with the up-regulation of matrix metalloproteinases52 and increased invasiveness in breast carcinomas.53 In addition, the presence of prostate specific
antigen (PSA), which has been found by polymerase
chain reaction in '30% of breast carcinomas,54 indicates favorable patient prognosis55 and is expressed in
a mutually exclusive manner with P-cadherin in prostate and prostate carcinomas.56 We propose that it is
appropriate to reevaluate the current classification of
breast tumors in light of more recent information on
tumor progeny and prognostic significance of the different phenotypes.
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