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Clinical Cancer Research Vol. 11, 4083-4089, June 1, 2005
© 2005 American Association for Cancer Research

Imaging, Diagnosis, Prognosis

Automated Quantitative Analysis of E-Cadherin Expression in Lymph Node Metastases Is Predictive of Survival in Invasive Ductal Breast Cancer

Malini Harigopal1, Aaron J. Berger1, Robert L. Camp1, David L. Rimm1 and Harriet M. Kluger2

Authors' Affiliations: Departments of 1 Pathology and 2 Medicine, Yale University School of Medicine, New Haven, Connecticut

Requests for reprints: Harriet M. Kluger, Section of Medical Oncology, Yale Cancer Center, Yale University School of Medicine, 310 Cedar Street, New Haven, CT 06510. Phone: 203-785-6221; Fax: 203-785-7531; E-mail: Harriet.Kluger{at}yale.edu.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: The tumor suppressor adhesion molecule E-cadherin is believed to have an anti-invasive role in breast cancer. Lymph node involvement is the best prognostic marker known, yet there is variability in outcome among node-positive patients. We investigated the relationship between E-cadherin expression in primary invasive ductal tumors and corresponding nodal metastases, and determined the prognostic value of E-cadherin expression in node-positive breast cancer.

Experimental Design: Membrane E-cadherin expression was studied by immunohistochemical staining of tissue microarrays with fluorescent-labeled antibodies. An objective method of automated quantitative analysis (AQUA) was used. AQUA uses cytokeratin to define pixels as breast cancer (tumor mask) within the array spot, and measures E-cadherin expression using a Cy5-conjugated antibody within the mask.

Results: We employed a tissue microarray containing 207 primary and matched nodal metastases suitable for AQUA analysis. There was no significant difference in mean staining intensity between the primary and nodal specimens (P = 0.8). A scattergram was generated which identified a subset of patients (25%) with high E-cadherin expression in nodal metastases, and this top quartile had improved survival (P = 0.028). On univariate analysis, increased E-cadherin expression in nodal metastases was strongly associated with improved survival (P = 0.007), whereas expression in primary tumors was not (P = 0.13). On multivariate analysis, nodal E-cadherin expression retained its independent association with survival, as did tumor size and HER2/neu status.

Conclusions: Strong E-cadherin expression in lymph node metastases was highly predictive of improved survival. This suggests that expression of adhesion molecules at metastatic sites portends less aggressive tumor behavior.

Key Words: E-cadherin • breast cancer • prognosis • immunohistochemistry • Cell adhesion • Breast cancer • Preclinical studies: biomarkers and prevention • Methodology for microarrays

The primary cause of morbidity and mortality in breast cancer patients is tumor invasion and subsequent distant metastasis. The risk of metastatic disease is estimated by factors such as number of positive lymph nodes, tumor size, tumor grade, hormone receptor status, and ploidy. Lymph node involvement is the most important prognostic marker, yet there is heterogeneity in prognosis among women within the node-positive group, with some patients surviving with local therapy alone, and others developing metastatic disease despite aggressive systemic therapy. We currently have no means by which to reliably select adjuvant chemotherapy regimens for node-positive patients. A recent clinical trial showed that aggressive, dose-dense chemotherapy yields a small (<10%), but significant improvement in the disease-free and overall survival in node-positive patients, and dose-dense chemotherapy is gaining wide acceptance in the U.S. (1). Earlier studies revealed a subset of node-positive patients who fare well without chemotherapy, particularly those with estrogen receptor–positive breast cancer, who might survive with adjuvant tamoxifen alone (2). Therefore, there is an urgent need to identify additional prognostic markers in order to improve patient selection for more toxic chemotherapy regimens.

Several biomarkers have been studied to identify poor prognostic subgroups. One such prognostic marker, which is mechanistically important in breast cancer invasion, is E-cadherin, a calcium-regulated cell surface glycoprotein. E-cadherin is a cell adhesion molecule that is expressed in normal breast epithelium, benign breast lesions and breast carcinoma. E-cadherin is essential in maintaining cell-cell contact in epithelial cells via the formation of complexes with cytoplasmic catenins and actin cytoskeleton. It is a tumor suppressor gene, and is believed to have a crucial anti-invasive role, as evidenced from studies on highly invasive breast cancer cell lines (3–6). Loss of E-cadherin function, by either mutation or down-regulation is therefore regarded as one of the mechanisms by which tumor cells invade and metastasize (3, 7–10). Numerous studies have addressed the prognostic implications of loss of E-cadherin in carcinomas of the breast, stomach, lung, bladder, kidney, and prostate (11–13). Researchers have shown that tumors with decreased E-cadherin expression tend to be more infiltrative and more likely to metastasize to lymph nodes (14). Several studies have linked reduced E-cadherin expression with increased tumor grade and poor outcome, whereas others have shown variable results with no prognostic value for E-cadherin (15, 16). The majority of these studies have been done on relatively small cohorts of patients with short follow-up. Moreover, slide-to-slide variability in staining and subjectivity of interpretation of staining intensity limit the value of these studies.

Although in vitro studies in cell lines have provided evidence of an association between reduced E-cadherin expression and invasion, this association has not consistently been shown in vivo (17–21). Few studies have specifically looked at the expression pattern of E-cadherin in breast primaries and patient matched nodal metastasis and/or distant metastasis (21, 22). Despite a large body of literature on the prognostic value of E-cadherin in breast cancer, the role of E-cadherin as a prognostic marker is at best ambiguous and needs clarification (5, 10, 11, 23).

In the present study, we used tissue microarrays containing primary breast tumors and matched nodal metastases to overcome the difficulties of slide-to-slide staining variability. We used our automated quantitative analysis (AQUA) method of tissue microarray analysis (24), which enables us to identify subtle differences in staining intensity not discernable by the naked eye in an objective and reproducible fashion. We evaluated E-cadherin as a prognostic marker in node-positive breast cancer, and we further studied the biological importance of E-cadherin by comparing levels of E-cadherin expression in primary tumors and lymph node metastases.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Tissue microarray design and construction
Case selection. Paraffin-embedded, formalin-fixed specimens from 341 sequential node-positive breast cancer patients (280 invasive ductal and 61 invasive lobular cases), were identified from the archives of the Yale University Department of Pathology from 1962 to 1979, with a mean follow-up time of 20 years. Yale University Institutional Review Board permission was obtained for collection of the specimens and the clinical data. Complete treatment information was unavailable, but most patients received treatment with local radiation, and 15% were given chemotherapy. Patients diagnosed after 1978 also received tamoxifen. Following a pathologist's review, regions of invasive carcinoma in primary tumors and matched lymph nodes were selected. A tissue microarray was constructed placing a core side by side from each primary and matched lymph node. Details of tissue microarray construction have been described extensively in previous publications (25–28). Samples from normal breast tissue, included in the array, served as positive controls for E-cadherin. The 280 invasive ductal carcinomas included pure ductal (238), ductal with lobular features (32), colloid (9), and anaplastic carcinoma (1).

Fluorescent immunohistochemistry and AQUA. Immunohistochemical staining was done as previously described (29). Briefly, slides were deparaffinized in two changes of xylene and rehydrated through changes of ethanol with decreasing concentrations. Slides were pressure-cooked for antigen retrieval in citrate buffer (pH 6.0). Endogenous peroxidase activity was quenched in methanol and 3% hydrogen peroxide for 30 minutes. Slides were incubated at 4°C overnight in a humidity tray with the primary antibody [mouse monoclonal antihuman E-cadherin antibody, 1:400 (Transduction Labs, Inc, San Diego, CA), and a rabbit polyclonal primary anticytokeratin antibody, AE1/AE3, 1:200 (Dako Corporation, Carpinteria, CA)] was used to identify tumor cells. Corresponding secondary antibodies were applied for 1 hour at room temperature in bovine serum albumin/TBS:Alexa 488-conjugated goat anti-rabbit (1:100, Molecular Probes, Eugene, OR) and Envision anti-mouse (Dako). 4,6-Diamidino-2-phenylindole (DAPI) was included with the secondary antibodies to visualize nuclei. Fluorescent chromogen Cy-5-tyramide (NEN Life Science Products, Boston, MA) was used for target identification.

AQUA. AQUA has been described in detail in prior publications (24–27, 29). In brief, AQUA consists of two steps, image acquisition and image analysis. In the first step, high-resolution (1,024 x 1,024 pixel; 0.5 µm resolution) monochromatic images of each histospot (tissue core) were acquired. In-focus and out-of-focus images were acquired for each spot. Areas of tumor are distinguished from stromal elements and lymphocytes by creating a mask from the cell surface cytokeratin signal. DAPI was used to identify nuclei. There was no nuclear E-cadherin staining.

Algorithmic analysis of images. A percentage of each out-of-focus image is subtracted from the corresponding in-focus image, based on a pixel-by-pixel analysis of the two images (using the rapid exponential subtraction algorithm). The ratio of the highest to lowest intensity pixels in the in-focus image represents the signal-to-noise ratio of the image. All out-of-focus information is eliminated by the rapid exponential subtraction algorithm. The pixel locale assignment for compartmentalization of expression algorithm assigns each pixel in the image to a specific subcellular compartment. Pixels that cannot be assigned to a compartment with 95% confidence intervals are discarded. The E-cadherin signal from the membrane area of tumor cells was scored on a unit scale of 0 to 255, and expressed as signal intensity divided by the membrane area.

Statistical analysis. Overall survival analysis was assessed by Kaplan-Meier analysis with Mantel-Cox log rank score for statistical significance. Relative risk was assessed by univariate and multivariate analyses. All statistical analyses were done by JMP v.5 and StatView software programs (SAS Institute, Inc., Cary, NC). A Wilcoxon paired t test was used to compare the mean expression level of E-cadherin in primary tumors and matched nodal metastases. Patients were deemed "uncensored" if they had died of breast cancer within 20 years of their initial diagnosis. All other patients were "censored."


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Sixty-one cases of lobular carcinomas had very low E-cadherin expression, and were removed from the analysis. To validate our breast cancer cohort, we assessed the association of several traditional histopathologic prognostic variables with outcome. Markers associated with poor outcome include large tumor size (P = 0.02), high nuclear grade (P = 0.0005), number of involved lymph nodes (P = 0.004), race (P = 0.0017), PR status (P = 0.0116), and HER2/neu expression (P = 0.0009). These results are shown in Table 1.


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  		Table 1. Univariate survival analysis of commonly used clinical and pathologic variables with 20 year follow-up

 
To assess the reproducibility of AQUA, E-cadherin expression was measured on two separate slides containing cores from the same patients. The matching scores correlated well (R = 0.82). To confirm the validity of AQUA, we compared the quartiles of our AQUA scores to semiquantitative, pathologist-based by-eye scores, and found very strong correlations (P < 0.0001 by t tests and P < 0.0001 by ANOVA).

Of the 280 invasive ductal carcinoma specimens, 207 (73.9%) primary tumors and 222 (79.2%) nodal metastases were suitable for AQUA analysis. Excluded spots had insufficient tumor (<10% of the histospot), absent cores or necrotic tumor. Normal ducts and lobules within the tissue microarrays showed strong E-cadherin expression, and served as positive internal controls. There was no E-cadherin expression in stromal cells. The E-cadherin membrane expression in tumor mask by AQUA varied from 4.03 to 144.50 for primary tumors and 4.10 to 166.54 for nodal metastases. Examples of high and low E-cadherin expression in lymph node metastases are shown in Fig. 1A-D. Histograms showing the frequency distribution of scores for E-cadherin expression in primary tumors and nodal metastases are shown in Fig. 2A and B. A Wilcoxon paired t test was done on the 202 cases that had scores for both primary tumors and matched nodal metastases. There was no significant difference between the staining intensity of primary and metastatic specimens (P = 0.8). A mean AQUA score of 26.7 was obtained for primaries and 26.3 for nodal metastases, as shown in Fig. 2C.



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  		Fig. 1. Membrane E-cadherin expression in lymph node metastases using cytokeratin to the define tumor mask, DAPI to define the nuclear compartment and Cy5 for target (E-cadherin) identification. High (A and C) and low (B and D) E-cadherin expression in lymph nodal metastases at 10x and 60x magnification, respectively.

 


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  		Fig. 2. A and B, histogram showing frequency distribution of continuous scores for E-cadherin expression in primary tumors (A) and nodal metastases (B). C, box-and-whisker plot comparing the mean intensity of staining between primary tumors and nodal metastases.

 
Using the Cox univariate survival analysis of continuous, raw AQUA scores, E-cadherin expression in primary tumors was not significantly associated with survival (P = 0.13), whereas E-cadherin expression in nodal metastases was a very strong predictor of survival (P = 0.007), as shown in Table 2. Using the {chi}2 test, we found no association between E-cadherin expression and other patient characteristics such as number of involved nodes, nuclear grade, histologic grade, tumor size, race, HER2/neu, estrogen receptor, and laterality.


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  		Table 2. Cox univariate survival analysis

 
Using the Cox proportional hazards model, we did multivariate analyses to assess the independent predictive value of E-cadherin expression in nodal metastases. E-cadherin expression in nodal metastases retained its independent predictive value, as did tumor size and HER2/neu status, as shown in Table 3.


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  		Table 3. Multivariate analysis of 20-year survival as determined by Cox regression analysis

 
A scattergram of mean intensity of E-cadherin expression in primary tumors and nodal metastases was generated, as shown in Fig. 3, with the asterisks representing the top quartile of nodal expressers. Figure 4 shows an example of a primary tumor with low E-cadherin expression and a corresponding lymph node with high expression.



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  		Fig. 3. Scattergram comparing the intensity of E-cadherin expression between primary tumors and nodal metastases. *, the top quartile of E-cadherin expressers in nodal metastases.

 


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  		Fig. 4. A, low/absent E-cadherin expression in a primary specimen (inset, cytokeratin expression from the histospot). B, re-expression in a lymph node from the same patient.

 
We divided the patients by quartiles of AQUA nodal E-cadherin scores, and did Kaplan-Meier survival analyses. As shown in the inset of Fig. 5, the lower three quartiles clearly overlap in terms of survival. We therefore separated the top quartile of nodal expressers from the rest of the cohort. Kaplan-Meier survival analysis as seen in Fig. 5 showed that patients in the top quartile (high expressers) had superior survival compared with the other patients (log rank P = 0.028). The relative risk for death among low expressers was 1.72 (95% confidence intervals, 1.1-2.68), as shown in Table 2. On multivariate analysis, high E-cadherin expression retained its independent predictive value when analyzed as a continuous variable, as well as when dividing the data in a binary fashion between "high" and "low" expressers.



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  		Fig. 5. Kaplan-Meier survival curves of E-cadherin levels in the top 25% of nodal expressers and the remainder of the cohort show that higher E-cadherin expression in nodal metastasis was associated with improved survival (P = 0.028). Inset, Kaplan-Meier survival curves for all four quartiles.

 
The decision to divide the continuous E-cadherin scores by quartiles is somewhat arbitrary. We therefore wanted to evaluate other cut-points. Using the newly developed software program X-tile (30), we found that the optimal cut-point is at the 13th percentile. With this cut-point, we found that the lower 87% of nodal E-cadherin expressers had worse survival (relative risk, 2.985; 95% confidence intervals, 1.46-6.1; P = 0.0027).


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
This is the first study using the AQUA method on a large cohort breast cancer tissue microarray to study the relationship between E-cadherin expression in node-positive patients, with matched primary tumors and nodal metastases, under uniform testing conditions. AQUA enables us to measure E-cadherin expression as a continuous variable rather than arbitrarily dividing the expression into nominal categories. Several previous studies have evaluated the prognostic value of E-cadherin by using an immunohistochemical staining index, which combines staining intensities and the fraction of positive cancer cells (5, 22). Although this system gives some measure of quantification, it is still dependent on the observer interpretation, and not completely objective and quantitative. Previously, Chairpin et al. (11) employed quantitative image analysis on whole sections from frozen tissue, which suffers from lack of uniform conditions.

As expected, we found low E-cadherin expression in the lobular primary tumors and matched lymph nodes. These cases were removed from the analysis. We compared E-cadherin expression in primary invasive ductal tumors and matched lymph node metastases, and found no significant difference in mean expression. We further studied the prognostic importance of E-cadherin expression, and found that whereas increased expression in the primary tumors was not associated with outcome, increased expression in lymph nodes was a strong predictor of improved outcome.

When using continuous output scores, one has to determine cut-points. We found that with use of our newly developed X-tile method (30), the validated optimal cut-point yielded superior results in terms of prediction of outcome when compared with the arbitrary use of quartiles as cut-points.

Although tissue microarrays enable us to simultaneously evaluate expression of a marker on a large cohort of cases under uniform conditions, they also carry the disadvantage of potential tumor heterogeneity. However, the large cohort size should correct for problems associated with intralesional heterogeneity (31).

As can be seen in Fig. 3, there is a small subset of patients with differences in expression between primary and nodal metastases. These differences might be due to changes at the transcriptional level. These subsets were too small to evaluate as separate prognostic groups.

Previous studies in breast cancer have shown that loss of E-cadherin in primary tumors may be a transient phenomenon, enabling cells to break away and to be subsequently reexpressed in metastatic sites (21), possibly by facilitating lymphatic tumor emboli (32). Kowalski et al. and Bukholm et al. specifically evaluated paired primary breast tumors and matched distant metastases, and found that in a subset of patients the metastases had stronger E-cadherin expression than the primary specimens, consistent with our findings (20, 21). Mareel et al. showed high E-cadherin expression in ex vivo culture from invasive tumors or metastases, which were noninvasive in vitro (33). The authors explain this intriguing finding by transient down-regulation of E-cadherin, without irreversible mutations of the gene. Decreased expression could be due to gene hypermethylation or transcriptional suppression. Several E-cadherin transcription repressors (SNAIL, SLUG, E12/E47, ZEB-1, and SIP1) have been shown to act by binding to the proximal E-boxes (E-pal; ref. 34). SLUG-induced suppression of E-cadherin in Madin-Darby canine kidney cells has been shown at the transcriptional level, and is associated with tumor aggression. Re-expression in MDCK cells has been shown in metastases in mouse models (35).

SNAIL and SIP-1 have been shown to directly suppress E-cadherin gene transcription and increase cellular invasion via up-regulation of matrix metalloproteinase activity (36). Expression of SLUG has been shown to correlate strongly with loss of E-cadherin expression, and has been implicated as an in vivo repressor of E-cadherin expression in breast carcinoma (34). We speculate that similar suppression of E-cadherin by transcription factors may occur when breast cancers develop invasive and metastatic capabilities, and might be the basis for the transient down-regulation and up-regulation of E-cadherin in primary and metastatic breast carcinoma.

Chromosome banding and CGH analysis of primary tumors and corresponding nodal metastases have shown more genetic heterogeneity among the neoplastic cells in the primary specimens than in metastases (20, 37–40). We hypothesize, based on prior experimental data, that the few cases where E-cadherin was absent in the primary but strongly expressed in nodal metastases, could also be explained by genetic heterogeneity in the primary specimen and clonal selection following metastases, as well as by transcriptional regulation. Therefore, measurement of E-cadherin expression at metastatic sites may be more predictive of outcome. In conclusion, it seems that retention or reexpression of E-cadherin in nodal metastases (including those tumors that may have transient decreased expression and subsequent increased levels in nodal tumor) portends a less aggressive tumor behavior.

Finally, measurement of E-cadherin expression in lymph node metastases might have important clinical implications. With the indiscriminant use of aggressive adjuvant chemotherapy regimens for node-positive breast cancer patients, we are overtreating some patients who would have equal benefit from less aggressive regimens (1). Additional studies are needed to prospectively evaluate nodal E-cadherin levels in order to identify patients who are likely to have an indolent natural history of disease, with the ultimate goal of reserving aggressive systemic therapy for those with the poorest prognosis.


   Acknowledgments
 
The authors acknowledge with appreciation the assistance of Kyle A. DiVito and Maciej Zerkowski with presentation of the figures, as well as the statistical advice rendered by Adam Mendizabal, EMMES Corporation.


   Footnotes
 
Grant support: NIH/National Institute of General Medical Sciences Medical Scientist Training Program grant GM07205 (to A.J. Berger). NIH grant K0-8 ES11571 and the Breast Cancer Alliance (to R.L. Camp). The Patrick and Catherine Weldon Donaghue Foundation for Medical Research and grants from the DOD and NIH including NIH R21 CA100825-01 (to D.L. Rimm). The Susan G. Komen Foundation, the C.J. Swebilius Foundation for Translational Research, and the Breast Cancer Alliance (to H.M. Kluger).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: M. Harigopal and A.J. Berger contributed equally to this work.

Received 10/27/04; revised 2/11/05; accepted 3/ 2/05.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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