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Clinical Cancer Research Vol. 12, 1728-1734, March 2006
© 2006 American Association for Cancer Research

Imaging, Diagnosis, Prognosis

Prognostic Relevance of AGR2 Expression in Breast Cancer

Florian Rudolf Fritzsche1, Edgar Dahl3, Stefan Pahl1, Mick Burkhardt1, Jun Luo4, Empar Mayordomo1, Tserenchunt Gansukh1, Anja Dankof1, Ruth Knuechel3, Carsten Denkert1, Klaus-Jürgen Winzer2, Manfred Dietel1 and Glen Kristiansen1,2

Authors' Affiliations: 1 Institute of Pathology and 2 Breast Centre, Charité, Universitätsmedizin Berlin, Berlin, Germany; 3 Institute of Pathology, University Hospital of the Rheinisch-Westfälische Technische Hochschule Aachen, Aachen, Germany; and 4 Department of Urology, Johns Hopkins Medical Institutions, Baltimore, Maryland

Requests for reprints: Glen Kristiansen, Institut für Pathologie, Charité, Universitätsmedizin Berlin, Schumannstrasse 20-21, 10117 Berlin, Germany. Phone: 49-30-450-536145; Fax: 49-30-450-563945; E-mail: glen.kristiansen{at}charite.de.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: We aimed to evaluate the expression of the human anterior gradient-2 (AGR2) in breast cancer on RNA and protein level and to correlate it with clinicopathologic data, including patient survival.

Experimental Design: AGR2 mRNA expression was assessed by reverse transcription-PCR in 25 breast cancer samples and normal tissues. A polyclonal rabbit AGR antiserum was used for immunohistochemistry on 155 clinicopathologically characterized cases. Statistical analyses were applied to test for prognostic and diagnostic associations.

Results: Immunohistochemical detection of AGR2 was statistically significantly associated with positive estrogen receptor status and lower tumor grade. AGR2-positive tumors showed significantly longer overall survival times in univariate analyses. For the subgroup of nodal-negative tumors, an independent prognostic value of AGR2 was found.

Conclusions: The expression of AGR2 in breast cancer is strongly associated with markers of tumor differentiation (estrogen receptor positivity, lower tumor grade). A prognostic effect of AGR2 for overall survival could be shown, which became independently significant for the group of nodal-negative tumors.

Despite considerable diagnostic and therapeutic advances in the treatment of breast cancer in recent years, there is still an urgent need for further molecular markers to provide the clinician with useful information concerning patient prognosis and possible therapeutic options. Kallikrein 5 (1), urokinase plasminogen activator, its inhibitor (2), tissue inhibitor of metalloproteinases 1 (3), Ep-CAM (4), osteopontin (5), CD24 (6), and SFRP1 (7) are just a few examples of a growing list of potentially useful prognostic markers in breast cancer. We and others recently described up-regulation of the androgen-inducible gene anterior gradient-2 (AGR2) in prostate cancer (6, 8). AGR2, which is also known as HAG-2 (9) or Gob-4 (10), is the human orthologue of the secreted Xenopus laevis AGR protein XAG-2. It has a putative role in ectodermal patterning of the frog embryo and is itself regulated by a number of fundamental embryonic molecules like noggin and chordin (11). XAG-2 expression induces cement gland differentiation and expression of neural marker genes in a fibroblast growth factor–dependent way (11). AGR2 has been shown previously to be coexpressed with estrogen receptor (ER) in breast cancer cell lines (9), suggesting involvement of AGR2 in the tumor biology of hormonally responsive breast cancers. This is in agreement with the finding that an antiestrogen-resistant derivative of the human T47D breast cancer cell line that has lost ER expression also has a strongly reduced AGR2 expression (12). The estrogen-dependent expression of AGR2 in breast cancer may be regulated by the four putative estrogen response elements present in the AGR2 promoter (12). Although the Xenopus XAG-2 protein was shown to be secreted (11), it is presently not clear whether the human AGR2 protein is secreted in normal and malignant breast tissue as well (13). Persson et al. (14) have shown by Basic Local Alignment Search Tool analysis (15) that AGR2 may represent a novel member of the protein disulfide isomerase family. Protein disulfide isomerases are oxidoreductases of the endoplasmic reticulum involved in protein maturation in the endoplasmic reticulum (16).

In a recent study, Fletcher et al. showed immunohistochemical expression of AGR2 in 83% (n = 58) of breast cancer cases using a tissue microarray (17). They also found a significant correlation with ER expression and an inverse correlation with epidermal growth factor receptor expression. However, no significant association between AGR2 expression and tumor grade, patient age, and presence of axillary lymph node metastasis could be shown. The estrogen responsiveness of AGR2 was recently confirmed by Liu et al. (13) as the AGR2 mRNA expression in MCF-7 breast cancer cells increased >7-fold in the presence of estrogen. More importantly, their study showed that AGR2 can induce a metastatic phenotype in vivo. AGR2-transfected rat mammary cells (Rama 37) injected in the mammary fat pads of syngenic rats induced a high incidence of lung metastases. Because the incidence of primary tumors in this rat model was not increased, it can be concluded that AGR2 expression may be associated with metastasis but not with initiation of ER-positive tumors (13). The study of Liu et al. (13) also analyzed human breast tumors (n = 44) immunohistochemically and found a significant correlation between ER{alpha} positivity and AGR2 expression; however, no correlation to patient survival was analyzed.

In our study, we aimed to investigate the expression of AGR2 immunohistochemically in a large breast cancer collective (n = 155) and to evaluate prognostic properties by correlation with clinicopathologic variables. Furthermore, we have carefully analyzed the AGR2 mRNA expression by real-time PCR in ER{alpha}-positive and ER{alpha}-negative breast cancer. Our data indicate that AGR2 expression is associated with a positive ER status and also a favorable prognosis in patients with Unio Internationale Contra Cancrum stage (UICC) I disease.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Antibody specificity: Western blot of AGR2 in human prostate cancer cell lines. Cultured PC-3 cells were subjected to standard Western blot analysis as described (18). A polyclonal rabbit antibody (1:1,000 dilution) raised against an AGR2-specific peptide was used to detect AGR2 protein expression in human prostate cancer cell line PC-3 (positive control) and CWR22Rv1 (negative control). Protein levels of ß-actin were examined by reprobing the same blot and served as loading controls. Only one weak nonspecific band was detected (Fig. 1 ).


Figure 1
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  		Fig. 1. Western blot analysis of AGR2 expression in human prostate cancer cell lines. PC3 cells (A) strongly express AGR2 (positive control), whereas CWR22Rv1 cells (B) do not express detectable amounts of AGR2 (negative control). ß-Actin was used as loading control.

 
Expression analysis using a matched tumor/normal tissue array. The matched tumor/normal expression array from Clontech (Heidelberg, Germany) consists of 68 cDNAs, synthesized from human tumorigenic and corresponding normal tissue (http://www.clontech.com/techinfo/manuals/pdf/pt3424-1.pdf). Each pair was independently normalized based on the expression of three housekeeping genes and immobilized in separate dots (19). An AGR2-specific cDNA fragment (EcoRI/NotI fragment of IMAGE cDNA clone with accession no. AA625485) was radiolabeled using a Megaprime labeling kit (Amersham Biosciences, Braunschweig, Germany), hybridized overnight at 68°C using ExpressHyb Hybridization Solution (Clontech), washed, and exposed to Kodak XAR-5 X-ray film with an intensifying screen (Eastman Kodak Co., Rochester, NY).

RNA preparation from paraffin-embedded tissue specimens. Archival formalin-fixed, paraffin-embedded tissue from 25 well-characterized representative (44% pT1, 48% pN0, 40% G1-G2) breast cancer specimens and 16 normal breast tissues, both from the archives of the Institute of Pathology of the Rheinisch-Westfälische Technische Hochschule Aachen, were used. For each formalin-fixed, paraffin-embedded tissue specimen, six 4-µm-thick tissue sections were cut with a microtome (Leica Microsystems, Bensheim, Germany) and transferred to a water bath filled with diethylpyrocarbonate-treated water. Sections were mounted on standard glass slides and dried for 1 hour at 60°C. Sections were deparaffinized and rehydrated as follows: 2 x 15 minutes in xylole; 2 x 15 minutes in 100% ethanol; and short rinses in 96%, 70%, and 50% ethanol followed by immersion in distilled water. Tissue material was transferred to a microcentrifuge tube and RNA was extracted according to the Trizol protocol supplied by the manufacturer (Life Technologies, Mannheim, Germany). cDNA was synthesized according to the protocol supplied with the Clontech RT-for-PCR-kit.

Quantitative reverse transcription-PCR. AGR2 mRNA expression was analyzed using intron-spanning primers on the LightCycler system (Roche Diagnostics, Mannheim, Germany). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used as reference to obtain relative expression values. Primers used in this study are presented in Table 1 . Real-time reverse transcription-PCR (RT-PCR) was carried out with Fast Start DNA master hybridization probes (Roche Diagnostics). The conditions were as follows: initial denaturation in one cycle of 15 minutes at 95°C, followed by 40 cycles at 95°C for 20 seconds, 60°C for 20 seconds, and 72°C for 30 seconds. Gene expression was quantified by the comparative CT method (20).


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  		Table 1. AGR2 and GAPDH primers used for real-time RT-PCR

 
Patients. For immunohistochemistry, our study included 155 patients with breast cancer diagnosed at the Institute of Pathology, University Hospital Charité, Berlin, Germany, between 1991 and 1997. Patient age at the time of diagnosis ranged from 30 to 81 years with a mean of 59 years. Clinical follow-up data, including overall survival and disease-free survival, were available for all cases. The median observation time for overall survival was 75 months for patients still alive at the time of analysis, and ranged from 1 to 162 months. Thirty patients (19.4%) died during follow-up and 59 patients (38.1%) experienced disease progression, defined by either metastatic disease or local recurrence. For statistical analysis of the effect of adjuvant therapy, we arranged the patients into two groups: the first group (n = 69) had received either no adjuvant therapy, radiotherapy, or systemic therapy, excluding tamoxifen. The second group had received tamoxifen with or without an additional systemic or local therapy (n = 80). For six patients, no data on adjuvant therapy were available.

The selection of cases for this study was based on availability of tissue and these were not stratified for any known preoperative or pathologic prognostic factors. Patients with systemic disease (pM1) at the time of diagnosis were excluded. Histologic typing of tumors was carried out according to the criteria of WHO (21). Tumor stage was determined according to the guidelines of the UICC (22). Tumors were graded according to Bloom and Richardson in the modification of Elston and Ellis (23). Data regarding the ER status, the expression of Her-2/neu (c-erbB2), and the proliferative fraction (Mib-1) were gathered from the archival pathology reports. The clinicopathologic data of the cases are described in Table 2 .


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  		Table 2. Clinicopathologic variables and association with AGR2 expression of the tumor set

 
Immunohistochemistry. Immunohistochemical analysis was carried out on formalin-fixed, paraffin-embedded archival tissue blocks. These were cut, mounted on slides, deparaffinized with xylene, and gradually rehydrated. Antigen retrieval was achieved by pressure cooking in 0.01 mol/L citrate buffer for 5 minutes. For AGR2/Gob-4, we generated polyclonal rabbit antisera against peptides derived from the AGR2 protein sequence (dilution 1:250; ref. 24). The primary antibody was incubated at room temperature for 1 hour. As a negative control, four slides were processed without primary antibody. Detection took place by the conventional labeled streptavidin-biotin method (DAKO, Hamburg, Germany) with alkaline phosphatase as the reporting enzyme according to the instructions of the manufacturer. Fast-Red (Sigma-Aldrich, Munich, Germany) served as chromogen. Afterward, the slides were briefly counterstained with hematoxylin and aqueously mounted.

Evaluation of the immunohistochemical stainings. The immunostains were examined by three pathologists who were blinded to patient outcome. We aimed to keep our scoring system of the AGR2 stainings simple to minimize interobserver variability and to enhance the reproducibility of our findings in future studies. An immunoreactive score was established, as described by Remmele and Stegner (25). The staining intensity was classified into four categories: negative (0), weakly positive (1), moderately positive (2), and strongly positive (3). The percentage of tumor cells staining positively was grouped accordingly: 0% (0), 1% to 10% (1), 10% to 50% (2), 51% to 80% (3), and 81% to 100% (4). The immunoreactive score was computed as the product of the categorized staining intensity and percentage of positive cells.

Statistical analysis. Statistical analysis was done using SPSS, version 13.0. Fisher's exact and {chi}2 tests were applied to assess the statistical significance of the associations between expression of AGR2 and various clinicopathologic variables. Wilcoxon test and Mann-Whitney U test were used to compare expression levels. Correlations were calculated according to Spearman. Univariate survival analysis was carried out according to Kaplan-Meier, whereas differences in survival curves were assessed with the log-rank test. Cox regression analysis was used for multivariate survival analyses. P values <0.05 were considered significant.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
AGR2 expression analysis on the RNA level. To analyze the expression of AGR2 in various solid tumors, including those of the breast, an AGR2-specific probe was hybridized onto an array containing 68 cDNA pair samples derived from multiple human tumors and corresponding normal tissues from individual patients (matched tumor/normal array; Clontech). As shown in Fig. 2 , AGR2 is clearly up-regulated in 6 of 9 tumors derived from the breast but also in 3 of 7 endometrial tumors and 2 of 14 tumors derived from the kidney when compared with the corresponding normal cDNA. Furthermore, AGR2 was found to be expressed in A594 cells (see Fig. 2, cell line 6), a human alveolar type II epithelium-like lung carcinoma cell line.


Figure 2
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  		Fig. 2. Expression analysis of AGR2 using the matched tumor/normal array. A, organization of the cDNA samples derived from tumor and normal tissue of individual patients on the array. N, normal; T, tumor. 1 to 9, cDNAs derived from the following cell lines: 1, HeLa; 2, Daudi; 3, K562; 4, HL60; 5, G361; 6, A594; 7, Molt4; 8, SW480; 9, Raji. B, hybridization results obtained with the AGR2 probe. The filter used contains nine breast cancer samples.

 
AGR2 expression was further analyzed by real-time RT-PCR in a set of 25 primary breast cancer specimens and 16 normal breast tissues. The result of the LightCycler expression analysis is diagrammatically presented in Fig. 3 . Consistent with the data obtained by the matched tumor/normal array, AGR2 expression in breast tumors was considerably up-regulated compared with its expression in normal breast tissue (median expression level, 10.9 versus 1.0) demonstrating an ~10-fold up-regulation in the tumor. The statistical significance of this up-regulation was P = 0.006 according to the Mann-Whitney U test for independent variables. Defining a real-time PCR {delta}CT value of ≤8 (i.e., at least the 1/256 expression of the reference gene GAPDH) as the cutoff level for undoubtful AGR2 expression in a tissue, we could show AGR2 mRNA in 6 of 16 normal breast tissues (38%) and 22 of 25 breast tumor samples analyzed (88%).


Figure 3
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  		Fig. 3. Abundant up-regulation of AGR2 expression in breast cancer as shown by quantitative RT-PCR. Median AGR2 expression was >10-fold up-regulated in breast tumors (samples 1-25) compared with normal breast tissues (samples A-P). Calculation of error bars according to Applied Biosystems user manual.

 
AGR2 immunostaining in normal breast tissue, and intraductal and invasive carcinoma. The immunohistochemical staining for AGR2 was highly restricted to the secretory epithelial cells of normal breast glands, and cells of intraductal carcinoma and invasive carcinoma, respectively. Blood vessels, connective tissue cells, and inflammatory cells were negative. The immunohistochemical staining showed a slightly granular cytoplasmic appearance. In normal breast tissue adjacent to the tumor, weak AGR2 expression was seen in 121 (89.0%) of 136 conclusive cases (Fig. 4A ). Intraductal carcinoma revealed a mostly moderate to strong cytoplasmic expression in 87 (92.6%, n = 94; Fig. 4B) and invasive carcinoma in 135 (87.1%, n = 155) cases (Fig. 4C). The immunoreactive score for AGR2 was significantly higher in intraductal carcinoma (median 8) and invasive carcinoma (median 6) compared with adjacent normal tissue (median 4, Wilcoxon test: P = 0.016). Expression of AGR2 was statistically significantly correlated with higher pT status, nodal status, lower proliferative fraction (Mib-1), and positive ER status. There was no significant correlation with tumor grade or c-erbB2 status (Table 3 ). For further statistical analyses, we delineated groups of tumors with no (immunoreactive score = 0) versus positive (immunoreactive score = 1-12) AGR2 expression. In crosstables, we found AGR2 expression significantly associated with positive ER status and lower histologic tumor grade (Table 2).


Figure 4
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  		Fig. 4. AGR2 immunohistochemistry. A, whereas secretory epithelia (bold arrows) show a homogenous cytoplasmic staining for AGR2, myoepithelial cells (small arrows) are AGR2 negative. B, strong cytoplasmic staining of intraductal carcinoma with intraluminal cellular debris. C, strong cytoplasmic staining of invasive ductal carcinoma (small arrows) surrounding AGR2-negative normal parenchymal tissue (white arrows). D, strong cytoplasmic staining of invasive lobular carcinoma (short arrows) with adjacent lobular carcinoma in situ (long arrows). Original magnification, x200 (A and D), x400 (B and C).

 

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  		Table 3. Correlation of AGR2 expression in breast cancer with conventional clinical or tumor variables

 
AGR2 expression and survival times. In univariate survival analysis, cumulative survival curves were calculated according to the Kaplan-Meier method. Differences in survival were assessed with the log-rank test. We analyzed the effect of AGR2, patient age, histologic tumor type, pT status, pN status, tumor grade, ER status, cerbB2 status, and type of therapy on overall survival time and disease-free survival time. The conventional prognostic markers pT status, nodal status, histologic grade, and ER status reached significance for overall and disease-free survival. Patients treated with tamoxifen had significantly longer overall survival times. For patients with AGR2-expressing breast carcinomas, significantly longer overall survival times (5-year survival rate 88% versus 73%, P = 0.035) became apparent, whereas disease-free survival times (P = 0.528) remained insignificant (Fig. 5A and B ). In Cox multivariate analysis, AGR2 failed significance (data not shown).


Figure 5
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  		Fig. 5. Kaplan-Meier curves with univariate analyses (log rank) for patients without AGR2 expression (dotted line) versus AGR2-expressing tumors (bold line). A and B, overall survival time (A) and disease-free survival time (B) analyses showing a significantly longer overall survival times of tumors with AGR2 expression. C and D, overall survival time analyses in the subgroups of small (pT1) tumors (C) and nodal-negative patients (D) showing significantly longer survival times for AGR2-expressing tumors.

 
Survival analysis in patient subgroups. To evaluate the prognostic value of AGR2 expression in selective patient groups, we repeated the univariate and multivariate survival analysis in subgroups stratified according to pT status, pN status, tumor grade, ER status, and cerbB2 status, respectively. A trend toward longer disease-free survival times was revealed in various subgroups that failed significance (data not shown). In the subgroups of small tumors (pT1, P = 0.001), nodal-negative tumors (pN0, P = 0.001) and tumors of UICC stage I (P = 0.002) significance was reached for overall survival time (Fig. 5). For the subgroup of nodal-negative tumors, the significant prognostic effect of AGR2 (P = 0.044) was confirmed by Cox multivariate analysis (Table 4 ), whereas it failed significance in the other subgroups (data not shown).


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  		Table 4. Cox multivariate analysis for overall survival in the subgroup of nodal-negative tumors

 

   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
AGR2, the human orthologue of XAG2 protein, is a small, possibly secreted molecule (13) of yet weakly defined functions that is widely expressed in human tissues. Whether it acts as a protein disulfide isomerase, as has been suggested by Persson et al. (14), has to await functional studies. Analyzing a dot blot array with cDNAs from 11 different human tumor entities and corresponding normal tissues we detected a very strong AGR2 mRNA expression in normal human colon, stomach, rectum, prostate, and breast. These organs secrete a variety of small molecules necessary to maintain tissue homeostasis and integrity. Our data are in agreement with previous findings that AGR2 is predominately expressed in tissue that contain mucus-secreting cells (10) and/or function as endocrine organs (26, 27). Currently, nothing is known about the function of XAG-2 in adult organs of Xenopus. However, in the Xenopus embryo, XAG-2 is responsible for patterning the cement gland, which is a mucus-secreting tissue as well. The cement gland is involved in the attachment of the Xenopus embryo to a solid support before swimming and feeding. In contrast to the situation in frogs, the biological function of the AGR2 protein in human cancer is still largely unknown.

In this study, we carefully and quantitatively analyzed the expression of AGR2 in human breast cancer both on the RNA and protein level and correlated these expression data to clinicohistopathologic data. The analysis of the matched tumor/normal array showed that AGR2 up-regulation in breast cancer is very significant compared with that in other tumor entities because six of nine matching tumor/normal pairs exhibited a strong up-regulation in the tumor. Additionally, a significant up-regulation of AGR2 was found in endometrial and renal tumors, which awaits further study. The up-regulation of AGR2 in breast cancer could be further confirmed by quantitative real-time RT-PCR in a set of 25 primary invasive ductal carcinomas that were compared with normal breast tissue. Liu et al. (13) recently published a similar quantification of AGR2 mRNA in breast cancer using semiquantitative RT-PCR with only 25 cycles. They found AGR2 expression in three of nine (33%) normal breast tissues compared with 6 of 16 (38%) normal breast tissues in this study. In the tumor tissue, Liu et al. (13) detected AGR2 expression in 44 of 56 cases (79%) compared with 22 of 25 (88%) tumor samples in our study. When Liu et al. quantified AGR2 expression according to the ER status, they detected a considerable lower expression of AGR2 mRNA in ER-negative tumors (13 of 22, 59%) compared with ER-positive tumors (31 of 34, 91%). In analogy, we found AGR2 expression on protein level significantly correlated to a positive ER status. Still, in the group of ER-negative tumors, 31 of 45 (68.9%) showed AGR2 expression, which suggests that AGR2 expression in human tumors is only partially dependent on the presence of a functional ER.

Our immunohistochemical study on AGR2 protein expression in breast cancer specimens (n = 155) is the largest analysis described thus far. We could confirm the findings of Fletcher et al. (9), who found a clear correlation between AGR2 expression and ER status. However, in our larger data set, we found additionally significant associations with the tumor grade and the proliferation rate. The coexpression of AGR2 and ER, the higher expression rates seen in tumors with a lower proliferative fraction, and the association with lower tumor grades indicate that AGR2 is a marker of differentiation, although further functional studies are clearly needed to underscore these findings. We also found the somehow contradictory significant correlations of higher AGR2 expression with pT status and positive nodal status that elude a convincing explanation thus far.

Importantly, we found that AGR2 might be a new prognostic marker in breast cancer for the subgroups of nodal negative, pT1, and Unio Internationale Contra Cancrum stage I tumors. For the group of nodal-negative breast tumors, we could show an independent prognostic value for AGR2 expression being associated with favorable overall survival (P = 0.044). Further studies are clearly needed to verify these findings to establish AGR2 as a prognostic marker in breast cancer and to clarify its role in carcinogenesis by functional analysis.


   Acknowledgments
 
We thank Britta Beyer (Charité Berlin) and Inge Losen (Rheinisch-Westfälische Technische Hochschule Aachen) for excellent technical assistance and Ilka Olson for discussions.


   Footnotes
 
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: F. R. Fritzsche, E. Dahl, and S. Pahl contributed equally to this work.

Received 9/20/05; revised 1/ 6/06; accepted 1/11/06.


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 Materials and Methods
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The Adenocarcinoma-Associated Antigen, AGR2, Promotes Tumor Growth, Cell Migration, and Cellular Transformation
Cancer Res., January 15, 2008; 68(2): 492 - 497.
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