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Clinical Research |
Department of Pathology, Yale University School of Medicine, New Haven, Connecticut
Requests for reprints: David L. Rimm, Department of Pathology, Yale University School of Medicine, 310 Cedar Street, PO Box 208023, New Haven, CT 06520-8023. Phone: 203-737-4204; Fax: 203-737-5089; E-mail: david.rimm{at}yale.edu.
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Introduction |
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Previous immunohistochemical analyses of ß-catenin in breast cancer tumors have shown mixed results regarding localization and relationship with patient outcome. Dillon et al. (6) showed no relationship between ß-catenin expression and outcome in a cohort of 91 patients. Nakopoulou et al. (7) examined 51 invasive ductal carcinomas by semiquantitative immunohistochemical analysis and found that membranous expression was significantly associated with low histologic grade tumors (P = 0.04) and positive estrogen receptor (ER) expression (P = 0.045). Abnormal (reduced) ß-catenin expression was significantly associated with a high rate of infiltrating lymph nodes (P = 0.016), advanced stage of disease (P = 0.02), and presence of lymph node metastases (P = 0.016). An examination of 121 breast cancer tumors by Karayiannakis et al. (8) showed retention of normal membranous ß-catenin staining in 32% of invasive ductal carcinomas but did not report correlation with outcome. In an examination of 90 primary breast cancers by Bukholm et al. (9), 55.6% of tumors had reduced or absent membranous staining but there was no relationship with expression and metastasis. Semiquantitative immunohistochemical analysis of 55 invasive ductal breast tumors by Bankfalvi et al. (10) showed that 36% had reduced membranous ß-catenin expression, all of which were in less differentiated tumors (grades 2 and 3). The reduced ß-catenin expression correlated with positive lymph node status (P = 0.03) but there was no significant relationship between ß-catenin expression and patient survival. Thus, the overall conclusion of this series of studies was that decreased ß-catenin staining was associated with poor prognostic factors.
Then, in complete contradiction to previous studies, an analysis of 123 primary breast cancer tumors by Lin et al. (11) of Mien-Chie Hung's group showed that high ß-catenin expression in nuclei was associated with poorer prognosis in patients compared with tumors with membranous ß-catenin expression (P < 0.0001) and that it was an independent predictive marker in multivariate analysis (P = 0.001). Although nuclear staining has been reported in many tumor types, especially colon (12, 13), this group was the first and only group to find it in breast cancer with prognostic value, although another paper assessing 40 breast cancer samples designated 62.5% as cytoplasmic/nuclear ß-catenin positive (14). Several additional articles report a lack of nuclear ß-catenin staining. Pedersen et al. (15) reported no relationship to patient survival in 61 breast cancer tumors by the same method as the Hung group. A similar immunohistochemical breast cancer tumor study looking at protein expression correlations between wnt signaling pathway components by Wong et al. (16) also did not see any ß-catenin nuclear staining. The largest cohort to date to assess ß-catenin expression in breast cancer was that of Gillet et al. (17). They found no significant difference in patient outcome between low (22%) and high (88%) membranous staining, with negligible nuclear ß-catenin staining in 470 patients. Thus, there is one report of nuclear expression of ß-catenin with a compelling association with outcome. These conflicting observations led us to reexamine ß-catenin expression in a large cohort of breast cancer tumors using more quantitative methods.
Tissue microarrays (18–20) are an efficient way to evaluate hundreds of tumors without extensive damage to the original tumor sample. Additional benefits to this method are that all samples are treated under identical antigen retrieval and staining conditions. Use of a grid format allows for each tumor to be linked to its respective clinical and pathologic information in a database. Several studies have shown that a single breast cancer tissue microarray core is sufficient to represent the expression seen on whole sections on a population level with clinical end points (21–23).
A set of algorithms called automated quantitative analysis (AQUA) has been developed in our laboratory that allows for rapid analysis of immunofluorescence on tissue microarrays (12). Automated analysis reduces the amount of human variability in scoring immunohistochemical staining by eye and results in a continuous range of protein expression AQUA scores of rather than ordinal (0, +1, +2, and +3) scores. AQUA allows for quantification of the marker (protein of interest) within user-defined subcellular compartments within the tumor region of each tissue microarray spot. Figure 1 shows an illustration of how AQUA works with an example of ER staining on a breast cancer tissue microarray core.
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Materials and Methods |
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ß-Catenin detection by ELISA. ß-Catenin protein was detected in the cell lines by ELISA using the DuoSet IC Human Total ß-catenin ELISA (R&D Systems, Minneapolis, MN). Ten-centimeter plates of the cell lines were washed in cold 1x PBS and then incubated for 30 minutes at 4°C in radioimmunoprecipitation assay buffer supplemented with a protease inhibitor cocktail, which included Pefabloc and CLAP (chymostatin, leupeptin, antipain, and pepstatin A). Total protein was measured using the BCA protein detection method (Pierce). Cell lysates were aliquoted and stored at –80°C. Cell lysates were diluted to 0.1 mg/mL in lysis buffer for use in the ß-catenin ELISA. The ELISA was done as per the instructions of the manufacturer; all standards and samples were done in duplicate. ß-Catenin concentrations in the cell lysates were determined from the resultant standard curve and are expressed as ng/mg protein lysate.
Tissue microarray construction and cohort details. Tissue microarrays were constructed from archival formalin-fixed, paraffin-embedded breast cancer specimens as previously described and reviewed (25, 26). Briefly, representative regions were selected for coring by pathologists based on the corresponding H&E-stained full sections. The tissue microarray was constructed with single 0.6-mm-diameter cores of each case spaced 0.8 mm apart in a grid format using a Tissue Microarrayer (Beecher Instruments, Sun Prairie, WI). The tissue microarray block was then cut into 5 µm sections with a microtome, adhered to the slide by an adhesive tape-transfer method (Instrumedics, Inc., Hackensack, NJ) and UV crosslinked. The breast cancer cohort consists of 688 samples of invasive ductal carcinoma selected from the Yale University Department of Pathology archives as available from 1961 to 1983. The mean follow-up time of this cohort is 12.8 years and the mean age of diagnosis is 58.1 years. The median follow-up time is 8.9 years and the median age of diagnosis is 58.0 years. Of the 656 patients with follow-up time, 328 were censored at 20 years and 276 were uncensored at 20 years. Of the 328 censored patients, their median follow-up was 21.4 years, with the minimum at 4.2 months. This cohort contains approximately half node-positive specimens and half node-negative specimens, which have been used previously in other studies from our laboratory (27–32). Complete clinical treatment information was not available for this cohort; however, some limited information was available for the node-positive patients. Most node-negative patients were treated with local radiation, and none received Herceptin. About 15% of the node-positive patients were treated with chemotherapy (primarily Adriamycin, cytoxan, and 5-fluorouracil), and subsequently 
27% received tamoxifen (post-1978). The node-negative patients were largely treated only by surgical resection.
Cell line microarray construction. The cell lines were cultured on 75 cm2 tissue culture flasks as described above. Once the cells reached 90% confluence, the cells were washed twice in 1x PBS and 5 mL neutral buffered formalin were added to the cells for 5 minutes at room temperature. After 5 minutes, the cells were removed from the flask with a cell scraper. The cells were stored at 4°C in the neutral buffered formalin. To make a cell line microarray, a donor block of each of the cell lines was first made; this was done as follows: the cells collected in neutral buffered formalin were centrifuged at room temperature at 1,500 rpm for 10 minutes. The supernatant was removed and the cells were resuspended in 500 µL of 80% ethanol. The cells were then centrifuged at 12,000 rpm for 5 minutes after which the supernatant was removed. The resultant pellet was mixed with 50 to 150 µL warm (55°C) 1% low melt agarose (made in 1x PBS) and the cell slurry was transferred to the cap of a 1.5 mL Eppendorf tube and allowed to solidify at room temperature; this step is done as quickly as possible to avoid the agarose solidifying before being added to the Eppendorf cap. Once the cell block had solidified, it was removed from the Eppendorf cap and placed in a tissue cassette, processed, and embedded in paraffin. A cell line microarray of 3-fold redundancy of the cell lines was constructed in the same method as the tissue microarray described above.
Immunofluorescence staining. The tissue microarray and cell line microarray slides were deparaffinized by two xylene rinses followed by two rinses with 100% ethanol. Antigen retrieval was done by boiling the slides in a pressure cooker in a sodium citrate buffer (pH 6.0). After rinsing briefly in 1x TBS, a 30-minute incubation with 2.5% hydrogen peroxide/methanol was used to block endogenous peroxidases. To reduce nonspecific background staining, slides were incubated with 0.3% bovine serum albumin/1x TBS for 1 hour at room temperature, followed by a series of 2-minute rinses in 1x TBS, 1x TBS/0.01% Triton, and 1x TBS (TBS washes). Slides were incubated overnight at 4°C with a monoclonal mouse anticytokeratin antibody (clone AE1/AE3, DAKO, Carpinteria, CA, 1:100) for the HER2 slides and a rabbit anticytokeratin antibody (1:100, DAKO) for the ß-catenin, ER, PR, and Ki-67 slides. Slides were hybridized with either HER2 (c-erbB-2 oncoprotein, 1:8,000 dilution for 1 hour, DAKO), ß-catenin (clone 14, 1:500 overnight incubation, BD Biosciences, San Jose, CA), ER (clone ID5, 1:50 dilution for 1 hour, DAKO), PR (clone 636, 1:50 dilution for 1 hour, DAKO), or Ki-67 (clone B56, 1:200 overnight incubation, BD Biosciences). Slides were washed in 1x TBS rinses and incubated with secondary antibodies for 1 hour at room temperature as follows: Alexa 488 goat anti-mouse or Alexa 488 goat anti-rabbit (1:100, Molecular Probes, Eugene, OR) for detecting cytokeratin and species-specific horseradish peroxidase (HRP) with a dextran-polymer backbone (Envision, DAKO) along with 4',6-diamidino-2-phenylindole (DAPI, 1:100, DAKO) for visualization of nuclei. For Ki-67 detection, double amplification was needed. This was accomplished by following the Envision reagent step with incubation with biotin tyramide (1:50 dilution for 10 minutes, Perkin-Elmer, Wellesley, MA) and then HRP-streptavidin (1:200 dilution for 30 minutes, Perkin-Elmer). All slides were washed with TBS rinses followed by a 10-minute incubation with Cy-5 tyramide (1:50 dilution in Amplification Diluent, Perkin-Elmer). The slides were mounted in 0.6% n-propyl gallate (an antifade mounting medium) and coverslipped.
AQUA analysis of tissue and cell line microarrays. The AQUA software linked to the fluorescence microscopy system allowed for quantification of the protein of interest within the tumor region of each tissue microarray core (Fig. 1; ref. 12). Briefly, pan-cytokeratin is used to separate epithelial tumor from surrounding stroma, and different fluorescent tags (i.e., DAPI) are used to demarcate subcellular compartments (Fig. 1A and D). Due to the thickness of the tissue sections and the resulting overlap of subcellular compartments, an exponential subtraction algorithm (RESA) is used to subtract an out-of-focus image from an in-focus image, thereby providing improved pixel assignment to subcellular compartments. At the Cy-5 wavelength, which is outside the range of tissue autofluorescence, the target of interest is tagged and measured within the subcellular compartments by the PLACE algorithm (see Fig. 1). As illustrated by the images of ER staining in the diagram of the AQUA method in Fig. 1, the first step of quantification was the capture of the cytokeratin staining for each breast cancer core (Fig. 1A). Nuclear regions were delineated by taking an image of DAPI staining (Fig. 1B), which shows stromal nuclei as well as epithelial nuclei and defines a nuclear compartment. Coalescence of cytokeratin staining at the membrane was used to define the membrane compartment for analysis of HER2 and ß-catenin staining. The image of the marker of interest [ß-catenin, ER, HER2, Ki-67, or progesterone receptor (PR)] were captured at the Cy-5 wavelength (Fig. 1C). In-focus and out-of-focus images were captured at each wavelength. The compartments used for analyses were nuclear and membranous ß-catenin staining, nuclear ER staining, nuclear progesterone staining, nuclear Ki-67 staining, and membranous HER2 staining. The resulting AQUA score is the measurement of the biomarker pixel intensity within a compartment divided by the total area of the compartment (to normalize for differences in tumor area in each spot). Tissue microarray cores without sufficient breast tumor epithelium (<5% of total area is under the keratin mask) were excluded from the analysis, which reduced the total number of specimens evaluated for each of the biomarkers to between 452 and 576 samples included in later analyses.
Statistical analysis. Statistical analyses using Cox proportional hazards and Kaplan-Meier survival curves (with their significance analyzed by the Mantel-Cox log-rank test) were completed using Statview 5.0.1 (SAS Institute, Inc., Cary, NC). The cohort was split into a matched training and validation set using a function of the X-tile software (24) as a method for selection of optimal cutpoints.1 This is achieved by the calculation of a log-rank 
2 statistic for every possible division of the cohort expression data. It is then divided into either two or three optimal groups that are ordinal indicators of risk based on the continuous input variable. The results are plotted on a two-dimensional graph in which each pixel is colored proportionally to its 2 value. The maximum 2 value is automatically determined by X-tile and is designated as the cutpoint for optimal parsing of the protein expression data. X-tile also allows for training set/validation set analysis to validate the cutpoint selection. The training set and validation set were randomly assigned to generate two groupings with equivalent baseline survival curves for all patients with values for all three markers and both censor and follow-up information.
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The optimal AQUA low cutpoint as determined from the ß-catenin training set was 19.17 ng/mg and the high cutpoint was 109.53 ng/mg (n = 222, Fig. 4A and B
). The green color of the pixels in the X-tile plot shows that patients with high levels of membranous ß-catenin have a better prognosis than those with lower levels in a continuous manner at many different possible cutpoints. The low population consists of 58 patients (36.21% censored), the middle population has 124 patients (54.84% censored), and the high population has 40 patients (70.00% censored). Because 2 values were used to determine the optimal cutpoint, it is not statistically valid to test this same data for significance by the log-rank test. Instead, the cutpoints must be tested on a unique, nonoverlapping validation set (n = 223; Fig. 4C and D). Using the cutpoints from the training set, we find 58 patients classified as low (36.21% censored), 125 classified as middle (55.20% censored), and 40 patients classified as high (67.50% censored). This resulted in a Kaplan-Meier log-rank test 2 value of 13.764 and a P value of 0.0010, suggesting that a highly statistically significant worse outcome is associated with decreased expression of ß-catenin in the membranous compartment. Note that very few cases showed nuclear expression and no associations with outcome were found for this subset.
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Discussion |
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The predominant staining pattern for ß-catenin this cohort was membranous. This is consistent with most of the other studies in the literature, as well as a previous study of breast cancer tumors from our laboratory by Chung et al. (28). However, the paper from the Hung group is in direct conflict with this result (11). Furthermore, there is abundant evidence from more basic experiments suggesting an important role for wnt signaling in breast cancer (reviewed in refs. 4, 5). For example, transgenic wnt expression leads to mammary lobular hyperplasia and ultimately carcinoma as reviewed by Smalley and Dale (34, 35).
Furthermore, transgenic mutant ß-catenin expressed in mouse mammary glands leads to carcinoma (36). If wnt signaling is important, then ß-catenin should be present in the nucleus to activate the lymphoid enhancer factor/T-cell factor transcriptional complex. There are a number of possible explanations for this apparent discrepancy. One possibility is that the level of ß-catenin in the nucleus is too low to be detected by our assay. However, to our knowledge, no one has measured the level of ß-catenin in the nucleus of cells in the wnt model systems. Furthermore, in breast cancers from mice expressing mutant ß-catenin, immunofluorescent assays did not reveal either mutant or wild-type ß-catenin in the nucleus (36). A second possibility is that it is not present in the nucleus, but rather wnt signals via some alternative mechanism (noncanonical wnt signaling; ref. 37). A third possible explanation is the antibody selection. Because different antibodies have different affinity, it is possible that different studies would show disparate results as a function of antibody selection. We feel this is less likely because this antibody had previously shown nuclear and membranous staining in both colon carcinoma (12) and melanoma samples (38). Furthermore, the antibody used by the Hung group is not published in Lin et al, so it is difficult to compare to other studies. In summary, we believe that the minimal amount of ß-catenin found in the nucleus in breast cancer samples is reflective of the biology rather than an antibody detection artifact.
The Chung et al. (28) breast cancer study from our laboratory used only the node-negative cases and showed no relationship between expression and outcome. That study investigated ß-catenin expression in conjunction with other components of the wnt signaling pathway. There was no marked nuclear staining in the tumors and there was no prognostic value of membranous staining of ß-catenin in this cohort. We feel that this is an example of how quantitative immunofluorescence is a more sensitive and objective measure of protein expression than semiquantitiative subjective brown stain by-eye scoring of samples. We have shown previously that AQUA analysis is able to find associations between protein expression and patient outcome that were not seen when the same samples were evaluated by semiquantitative by-eye analysis (12, 27). When the continuous ß-catenin expression data in this study was analyzed on the entire cohort separating patients by nodal status, membranous ß-catenin was a stronger predictor of survival in univariate analysis for the 230 node-positive cases (P = 0.0077, data not shown) than in the 221 node-negative cases (P = 0.0158, data not shown). Therefore, it is not surprising that this relationship was missed by semiquantitative immunohistochemical analysis of ß-catenin only in node-negative cases.
This study is the largest quantitative cohort examination of ß-catenin protein levels and patient outcome to date. The use of AQUA has allowed us to generate an objective, continuous data set of protein expression levels for biomarkers of interest rather than ordinal semiquantitative scores. Use of ß-catenin ELISAs to determine protein concentration in cell lines, followed by AQUA analysis of those same cell lines as cores in tissue microarrays, allowed us to generate a standard curve of ß-catenin protein expression. This allowed us to quantitatively determine the ß-catenin protein concentration in the tumor tissue microarray cores. Our results show that high levels of membranous ß-catenin protein expression predict better prognosis both through univariate analysis and training set/validation set analysis by X-tile. ß-Catenin is an independent predictor of patient outcome as shown by multivariate analysis that included standard clinicopathologic variables and standard breast cancer biomarker assessments on a continuous level.
Although there is currently no Food and Drug Administration–approved therapeutic targeting ß-catenin (perhaps by stabilizing it on the membrane), studies are under way that are targeting the wnt pathway. Furthermore, even in the absence of predictive value, this study suggests prognostic value of assessment of membranous ß-catenin expression. We believe that it could be valuable as an independent marker or as a component of a series of markers designed to predict recurrence or outcome in breast cancer.
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Acknowledgments |
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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.
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Footnotes |
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1 X-tile software can be downloaded at http://www.tissuearray.org/rimmlab/. 
Received 1/10/06. Revised 2/14/06. Accepted 3/ 9/06.
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N89 ß-catenin induces precocious development, differentiation, and neoplasia in mammary gland. J Cell Biol 2001;153:555–68.This article has been cited by other articles:
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  E. Lopez-Knowles, S. J. Zardawi, C. M. McNeil, E. K.A. Millar, P. Crea, E. A. Musgrove, R. L. Sutherland, and S. A. O'Toole Cytoplasmic Localization of {beta}-Catenin is a Marker of Poor Outcome in Breast Cancer Patients Cancer Epidemiol. Biomarkers Prev., January 1, 2010; 19(1): 301 - 309. [Abstract] [Full Text] [PDF]
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 Y. Nadler, A. M. Gonzalez, R. L. Camp, D. L. Rimm, H. M. Kluger, and Y. Kluger Growth factor receptor-bound protein-7 (Grb7) as a prognostic marker and therapeutic target in breast cancer Ann. Onc., August 28, 2009; (2009) mdp346v1. [Abstract] [Full Text] [PDF]
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L. Jilaveanu, C. Zito, S. J. Lee, K. L. Nathanson, R. L. Camp, D. L. Rimm, K. T. Flaherty, and H. M. Kluger Expression of Sorafenib Targets in Melanoma Patients Treated with Carboplatin, Paclitaxel and Sorafenib Clin. Cancer Res., February 1, 2009; 15(3): 1076 - 1085. [Abstract] [Full Text] [PDF]
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A. Safholm, J. Tuomela, J. Rosenkvist, J. Dejmek, P. Harkonen, and T. Andersson The Wnt-5a-Derived Hexapeptide Foxy-5 Inhibits Breast Cancer Metastasis In vivo by Targeting Cell Motility Clin. Cancer Res., October 15, 2008; 14(20): 6556 - 6563. [Abstract] [Full Text] [PDF]
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Y. Nadler, R. L. Camp, C. Schwartz, D. L. Rimm, H. M. Kluger, and Y. Kluger Expression of Aurora A (but Not Aurora B) Is Predictive of Survival in Breast Cancer Clin. Cancer Res., July 15, 2008; 14(14): 4455 - 4462. [Abstract] [Full Text] [PDF]
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G. M. Kreizenbeck, A. J. Berger, A. Subtil, D. L. Rimm, and B. E. Gould Rothberg Prognostic Significance of Cadherin-Based Adhesion Molecules in Cutaneous Malignant Melanoma Cancer Epidemiol. Biomarkers Prev., April 1, 2008; 17(4): 949 - 958. [Abstract] [Full Text] [PDF]
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 A. Plotnikov, Y. Li, T. H. Tran, W. Tang, J. P. Palazzo, H. Rui, and S. Y. Fuchs Oncogene-Mediated Inhibition of Glycogen Synthase Kinase 3{beta} Impairs Degradation of Prolactin Receptor Cancer Res., March 1, 2008; 68(5): 1354 - 1361. [Abstract] [Full Text] [PDF]
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C. B. Moeder, J. M. Giltnane, M. Harigopal, A. Molinaro, A. Robinson, K. Gelmon, D. Huntsman, R. L. Camp, and D. L. Rimm Quantitative Justification of the Change From 10% to 30% for Human Epidermal Growth Factor Receptor 2 Scoring in the American Society of Clinical Oncology/College of American Pathologists Guidelines: Tumor Heterogeneity in Breast Cancer and Its Implications for Tissue Microarray Based Assessment of Outcome J. Clin. Oncol., December 1, 2007; 25(34): 5418 - 5425. [Abstract] [Full Text] [PDF]
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H. Wang, D. Teske, A. Tess, R. Kohlhepp, Y. Choi, C. Kendziorski, and A. R. Moser Identification of Novel Modifier Loci of ApcMin Affecting Mammary Tumor Development Cancer Res., December 1, 2007; 67(23): 11226 - 11233. [Abstract] [Full Text] [PDF]
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D. B. Seligson, F. Hongo, S. Huerta-Yepez, Y. Mizutani, T. Miki, H. Yu, S. Horvath, D. Chia, L. Goodglick, and B. Bonavida Expression of X-Linked Inhibitor of Apoptosis Protein Is a Strong Predictor of Human Prostate Cancer Recurrence Clin. Cancer Res., October 15, 2007; 13(20): 6056 - 6063. [Abstract] [Full Text] [PDF]
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J. M. Giltnane, L. Ryden, M. Cregger, P.-O. Bendahl, K. Jirstrom, and D. L. Rimm Quantitative Measurement of Epidermal Growth Factor Receptor Is a Negative Predictive Factor for Tamoxifen Response in Hormone Receptor Positive Premenopausal Breast Cancer J. Clin. Oncol., July 20, 2007; 25(21): 3007 - 3014. [Abstract] [Full Text] [PDF]
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D. Huang, G. P. Casale, J. Tian, N. K. Wehbi, N. A. Abrahams, Z. Kaleem, L. M. Smith, S. L. Johansson, J. E. Elkahwaji, and G. P. Hemstreet III Quantitative Fluorescence Imaging Analysis for Cancer Biomarker Discovery: Application to {beta}-Catenin in Archived Prostate Specimens Cancer Epidemiol. Biomarkers Prev., July 1, 2007; 16(7): 1371 - 1381. [Abstract] [Full Text] [PDF]
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D. L. Rimm, J. M. Giltnane, C. Moeder, M. Harigopal, G. G. Chung, R. L. Camp, and B. Burtness Bimodal Population or Pathologist Artifact? J. Clin. Oncol., June 10, 2007; 25(17): 2487 - 2488. [Full Text] [PDF]
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E. Pick, Y. Kluger, J. M. Giltnane, C. Moeder, R. L. Camp, D. L. Rimm, and H. M. Kluger High HSP90 Expression Is Associated with Decreased Survival in Breast Cancer Cancer Res., April 1, 2007; 67(7): 2932 - 2937. [Abstract] [Full Text] [PDF]
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