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EZH2 and ALDH-1 mark breast epithelium at risk for breast cancer development

Lakshmi P Kunju, M.D.,1,* Cynthia Cookingham, M.D.,1,* Kathy A. Toy, M.S.,4 Wei Chen, Ph.D.,2 Michael S. Sabel,3,4 and Celina G. Kleer, M.D.1,4

Abstract

It is well established that benign proliferative lesions and atypical hyperplasia increase the risk of breast cancer, which can develop in either breast. At present there is no radiologic, pathologic, or molecular marker capable of distinguishing which proliferative or atypical lesions will progress to carcinoma. EZH2, a protein involved in stem cell renewal and carcinogenesis is upregulated in morphologically normal breast epithelium from BRCA1 mutation carriers. Here, we tested the hypothesis that EZH2 expression alone or in combination with the breast stem cell marker aldehyde dehydrogenase-1 (ALDH-1) may identify benign breast biopsies that progress to breast cancer in the future. Benign breast biopsies from 59 women who subsequently developed (study group, n=29) or did not develop (control group, n=30) breast cancer in the same time period were subjected to immunohistochemical analyses of EZH2 and ALDH-1 proteins. When present, EZH2 was expressed in the nuclei of benign epithelial cells while ALDH-1 was expressed in the cytoplasm of epithelial cells and/or in the stroma. EZH2, epithelial ALDH-1 and expanded stromal ALDH-1 positive cells were present in 95%, 43%, and 69% of the study group biopsies, compared to 16%, 13% and 37% of the control biopsies, respectively (p<0.05 for all). The mean percentage of EZH2 positive cells was higher in the study group than in the control group (34% and 6%, respectively). EZH2 expression was associated with breast cancer development (p= 8.2 × 10−6) and with younger age at cancer diagnosis (p = 0.0086). Both stromal and epithelial ALDH-1 were associated with development of breast cancer (p= 0.001 and p = 0.049, respectively). Our study provides first evidence that EZH2 and epithelial and stromal ALDH-1 detection in benign breast biopsies may predict increased risk for breast cancer, with implications for breast cancer prevention.

Introduction

Benign breast disease is an important risk factor for a later breast cancer, which can develop in either breast (1, 2). Compelling data indicates that atypical ductal hyperplasia, atypical lobular hyperplasia as well as proliferative lesions (prominent ductal hyperplasia, papilloma, radial scar, and sclerosing adenosis) increase the risk of breast cancer (2, 3). The identification of benign breast disease has become more common as the use of mammography has increased. Having accurate risk estimates for women who receive this diagnosis is necessary but not available at present.

The Polycomb group (PcG) proteins form chromatin-modifying complexes essential for embryonic development and stem cell renewal and are deregulated in cancer (4). There is increasing evidence that the oncogenic role of the PcG protein EZH2 may be ascribed to its role in stem cell maintenance (5, 6). Our group and other investigators have found that EZH2 has potent oncogenic properties in the breast (5, 712). We have reported that EZH2 protein expression is elevated in benign appearing breast epithelium from prophylactic mastectomies from BRCA1 mutation carriers (13). Holst et al showed that a small number of cells from the normal breast epithelium can bypass senescence in vitro, and are associated with pre-neoplastic phenotypes (14). These cells exist in vivo as well, as rare foci of morphologically normal cells in disease-free breast. The same group recently showed that loss of p16INK4A results in overexpression of EZH2, recruitment of DNA-methyltransferases and DNA hypermethylation, and proposed that these epigenetic events are early events in breast cancer initiation (15).

Activation of the enzymatic activity of aldehyde dehydrogenase 1 (ALDH-1) in breast epithelium has been shown to be a marker of breast cancer stem cells and to play a functional role in stem cell renewal and differentiation (1618). Expression of ALDH-1 protein in breast carcinomas detected by immunohistochemistry was associated with poor clinical outcome (6, 16, 17). We have noted that ALDH-1 is expressed by intralobular stromal cells, and that its expression is expanded beyond the intralobular stroma in tissue samples. In this study, we tested the hypothesis that expression of EZH2 alone or in combination with epithelial and/or stromal ALDH-1 protein may identify benign proliferative and atypical lesions at increased risk for breast cancer.

Materials and Methods

Case Selection

The Surgical Pathology files at the University of Michigan were searched following Institutional Board Review Approval for breast tissue samples from women who had undergone a breast biopsy for a benign breast lesion between 1970 and 2003 and who subsequently developed breast cancer (study group). As a control group, we included benign breast samples from women who had undergone a breast biopsy during the same time frame and did not subsequently develop breast cancer.

Morphologic Review

Histopathologic evaluation was performed independently and blindly by three practicing pathologists (LPK, CC, and CGK) with expertise in breast disease. Differences in opinion in rare instances were resolved by consensus evaluation of the case with the senior pathologist (CGK). Morphologic parameters evaluated in this study included: a) presence of non-proliferative fibrocystic changes including simple cysts, apocrine metaplasia and stromal fibrosis; b) proliferative lesions including adenosis, papillomas and intraductal hyperplasia; c) presence of epithelial atypia including flat epithelial atypia and atypical ductal hyperplasia. In the current study atypical ductal hyperplasia and flat epithelial atypia were diagnosed following the criteria established by the World Health Organization (19) and according to the classification system outlined by Schnitt et al (20) respectively.

At present there is no unequivocal agreement on whether quantitative criteria should be applied to separate atypical ductal hyperplasia from low-grade ductal carcinoma in situ. Thus, atypical ductal hyperplasia was diagnosed when there was a proliferation of evenly distributed monomorphic cells growing in different patterns (micropapillary, cribriform) but that coexisted with usual ductal hyperplasia, and/or there was partial ductal involvement. Flat epithelial atypia lesions were characterized by variably distended terminal duct lobular units in which the native epithelial cells are replaced by one to several layers of a monotonous, atypical cuboidal to columnar cell population with apical snouts, secretory or floccular material in the lumen, often with microcalcifications. Flat epithelial atypia by definition, unlike atypical ductal hyperplasia, have a flat growth pattern with no complex architecture.

Clinical Information

Relevant clinical information including age of patients at the time of breast biopsy with benign breast disease as well as age at time of diagnosis of cancer as well as details of breast cancer such as type, size, Nottingham grade, TNM stage, hormone receptors, (estrogen receptor (ER) and progesterone receptor (PR)) status, HER-2/neu and axillary lymph node involvement were recorded for all cases.

Immunohistochemistry

Immunohistochemistry was performed on tissue sections using standard biotin-avidin complex technique utilizing a monoclonal antibody against EZH2 (1:300, BD Biosciences, San Diego, CA) and ALDH-1 (1: 5000, BD Biosciences, San Diego, CA). EZH2 expression was recorded as percentage of EZH2 expressing epithelial cells with nuclear expression as previously performed (13). Epithelial expression of ALDH-1 was scored as positive or negative. ALDH-1 staining when present in epithelial cells was clearly cytoplasmic and noted in at least 5% of epithelial cells in both groups (16). ALDH-1 expression was considered expanded when it extended beyond the intralobular stroma into the interlobular stroma. EZH2 and ALDH-1 expression was evaluated in epithelial cells including proliferative lesions and atypical hyperplasia. Foci with maximum intensity staining were scored. Slides were reviewed in a blinded manner.

Statistical Analysis

ALDH-1 scores of the study and control groups were tabulated and Fisher's exact test was performed to access the association between ALDH-1 and cancer development. In addition to this, treating ALDH-1 expression as ordinary, logistic regression analysis was also performed. Mean EZH2 expression was compared between the study and control groups using unpaired two sample t test. The possible association between EZH2 expression and group status was analyzed using logistic regression. Based on this logistic prediction model, receiver operating characteristic (ROC) curve was plotted to evaluate the performance of the model. Leave-one-out cross validation procedure was applied to avoid over-fitting. Additional clinical variables were compared between the two groups. Similarly, Fisher's exact test and unpaired two sample t –test were applied to categorical and continuous variables respectively.

Results

Histopathological and Clinical Characteristics of the Study Cohorts

To test the hypothesis that EZH2 expression alone or in combination with the breast cancer stem cell marker ALDH-1 may identify benign breast epithelium, proliferative and atypical lesions that will progress to breast cancer in the future, we studied benign breast biopsies from 59 women who subsequently developed (study group, n=29) or did not develop (control group, n=30) breast cancer in the same time period. These women did not have personal history of breast cancer. Detailed histopathologic and immunohistochemical analyses for EZH2 and ALDH-1 were performed. The histologic classifications included no significant abnormality, non-proliferative fibrocystic changes, proliferative fibrocystic changes and epithelial atypia (atypical ductal hyperplasia and/or flat epithelial atypia).

Benign biopsies from women in the study group had increased incidence of proliferative and atypical lesions compared to the controls and exceeded those reported in the general population (19). The study group showed proliferative lesions and epithelial atypia in 9/29 (31%) and 7/29 (24%) cases respectively compared to 6/30 (24%) and 3/30 (10%) in the control group. These differences were not statistically significant (Chi Square test p=0.31, Fisher's exact test p=0.24) (Table 1).

Table 1
Clinical Characteristics and histological findings at time of initial biopsy in Study and Control Groups

The mean age of women at the time of initial benign biopsy was higher in the study group compared to the control group (52.3 ± 11.9 years and 46.9 ± 10.6 years, respectively). The mean age at the time of cancer diagnosis of women in the study group was 59 ±11.1 years, with a mean time frame of 6.7 ± 4.5 years to develop cancer from the initial benign biopsy. The mean follow-up time for control group is 8.3 ± 5.3 years. Clinical and histological features at the time of initial breast biopsy are shown in Table 1.

Most breast carcinomas that developed in the study group were invasive ductal (86%), predominantly moderate to high- grade (Nottingham grade 2 or 3, 66%), and ER positive (69%). Table 2 summarizes the clinical and pathological characteristics of the invasive carcinomas.

Table 2
Pathological Features of Breast Carcinomas that Developed in the 29 Women in Study Group

The percentage of EZH2 positive cells marks increased breast cancer risk and younger age at breast cancer diagnosis

EZH2 expression was observed mainly in the nucleus of epithelial cells as described previously (8, 9). EZH2 positive cells were present in 95% of biopsies in study group compared to 16% of the control biopsies. The mean percentage of EZH2 positive cells in the study group was 34% (± 27%) compared to 6% ( ±10%) in the control group (p = 8.2 × 10−6 and 7.3 × 10−4, two sample t test and logistic regression, respectively). EZH2 expression was associated with development of carcinoma in the absence of atypical ductal hyperplasia in the biopsy (logistic regression, p=0.004). Figure 1A shows representative pictures of benign breast biopsies expressing EZH2. A logistic prediction model based on EZH2 expression further confirmed our observation that EZH2 expression may predict future breast cancer diagnosis; when plotted on a ROC (receiver operator characteristics) curve the area under the curve (AUC) for EZH2 expression is 0.88 (Figure 1B).

Figure 1
EZH2 expression in benign breast and breast cancer development

We next evaluated the relationship between EZH2 expression and patient age at the time of benign breast biopsy and time of cancer diagnosis, and to the time interval required to develop breast cancer after a benign biopsy. EZH2 upregulation was significantly associated with younger age at time of benign breast biopsy (p=0.002) as well as younger age at cancer diagnosis (p = 0.0086) in the study group. The mean interval to develop breast cancer after a benign biopsy was 6.7 years. EZH2 expression was not associated with patient age at the time of initial benign breast biopsy in the control group.

ALDH-1 expression is associated with increased breast cancer risk

ALDH-1 showed cytoplasmic expression in epithelial cells as described previously (6, 16, 17). We noted that stromal cells also express ALDH-1. Figure 2 shows examples of ALDH-1 expression in benign biopsies. We evaluated both epithelial and stromal ALDH-1 expression in benign biopsies from the study and control groups. Epithelial ALDH-1 was present in 43% and 13% of biopsies in study and control group, respectively. There was a modest association between ALDH-1 and cancer development (Fisher's exact test, p-value = 0.049; logistic regression, p = 0.02). While stromal ALDH-1 staining was seen in the intralobular stromal cells in most cases, expansion of this population was significantly associated with increased risk for breast cancer (Fischer's exact test, p=0.0099) (Figure 2 a, b, c, d). Of note, both epithelial and stromal ALDH-1 were associated with development of carcinoma in the absence of atypical ductal hyperplasia in the biopsy (logistic regression, p=0.02 and p=0.007, respectively). When analyzed together, epithelial and/or stromal ALDH-1 did not add significant value to the already significant effect of EZH2 in predicting breast cancer development. These findings are summarized in Table 3.

Figure 2
ALDH-1 expression in epithelial and stromal cells of benign breast biopsies
Table 3
Expression of EZH2, epithelial and stromal ALDH-1 in benign biopsies

Discussion

In the current era of mammographic screening increasing number of women are being diagnosed with benign breast disease. While the presence of atypical hyperplasia and proliferative disease without atypia, age at the time of biopsy and/or family history are determinants of breast cancer risk in women with benign breast biopsies, there are no means to identify which patients will progress to carcinoma (13). Identification of clinically useful molecular markers of increased breast cancer risk, especially those that can be applied in precancerous states would help guide clinical decisions.

The Polycomb group protein (PcG) EZH2 functions in multimeric complexes that regulate gene expression to maintain cell identity and differentiation. EZH2 has been implicated in stem cell regulation and malignancy including breast cancer (7, 911, 2124). A recent report provided evidence that EZH2 is important for tissue derived stem cell maintenance (10).Our group has discovered that EZH2 is upregulated in morphologically normal breast epithelium at increased risk for breast cancer (13, 25). Specifically, we have shown that EZH2 is overexpressed in prophylactic mastectomies from BRCA1 carriers compared with normal breasts from women who underwent a breast reduction and had no personal history of breast cancer (13, 25). These findings suggest that EZH2 up regulation may occur in histologically normal epithelium at higher risk of breast cancer and prompted us to undertake the present study.

Several insights emerge from our study. Benign breast biopsies from women who subsequently develop breast cancer exhibited proliferative breast disease, atypical ductal hyperplasia and/or flat epithelial atypia more frequently than biopsies from women who did not develop breast cancer. This finding fully supports previous studies which have shown that atypical hyperplasia confers the highest relative risk of breast cancer in women with benign breast disease (2, 3).

An important novel observation is that EZH2 is overexpressed in benign breast biopsies of women who subsequently develop breast cancer compared to the control group (95% vs. 16%, p-value = 8.2 × 10−6). Although the ability of EZH2 expression to predict future breast cancer development may be linked to cell proliferation, its role as a predictor of breast cancer may depend on its effect on stem cell survival and alteration of DNA repair pathways (10, 11, 22, 26, 27).

A young age at breast cancer diagnosis has been associated with worse outcome in breast cancer. Younger women exhibit worse survival compared with older women (28, 29). This association is likely to be related to tumor biology as breast carcinomas among young women are more likely lymph node positive, hormone receptor negative, and possess complex molecular alterations (30, 31). We have previously reported that EZH2 overexpression is associated with worse prognosis in patients with breast cancer (8). Here, we found that EZH2 protein is significantly overexpressed in younger women both at the time of benign (p=0.0023) and breast cancer diagnoses (p=0.0086). Taken together, our data allow us to postulate that EZH2 may play distinct roles at different stages of breast cancer progression, in benign epithelium and in breast cancer cells. We are currently investigating this hypothesis.

In view of the potential role of EZH2 in cancer stem cells (10) we evaluated the utility of the breast cancer stem cell marker candidate ALDH-1 in identifying benign breast tissue at increased risk for carcinoma. Measurement of aldehyde dehydrogenase isoform 1 (ALDH-1) enzymatic activity has been shown to distinguish benign and breast cancer stem cells and other neoplasms including human hematopoietic malignancies (32, 33). ALDH-1 plays a functional role in stem cell differentiation. It has been demonstrated that expression of ALDH-1 in the cytoplasm of cancer cells is associated with poor clinical outcome in breast cancer patients and that high ALDH-1 activity selects for both normal and tumorigenic human mammary epithelial cells with stem/progenitor properties(16). Recently, Resetkova et al showed that tumoral stromal expression of ALDH-1 by immunohistochemistry was associated with survival of triple negative invasive breast carcinomas (6). Here, we report that ALDH-1 is expressed in epithelial and stromal cells in benign breast tissues, and discovered that ALDH-1 positivity in breast epithelial cells is associated with increased risk of breast cancer. Of note, expansion of the stromal cell population expressing ALDH-1 beyond the intralobular stroma is also associated with cancer development. Taken together, these data support a role for the microenvironment in promoting tumor development before morphological abnormalities of carcinoma become evident. These data also support the utility of immunohistochemistry in the evaluation of ALDH-1 as a putative marker of breast cancer stem cells.

The limited number of cases in our study precludes adequate multivariable statistical analyses to investigate whether EZH2 and/or ALDH-1 are associated with breast cancer independently of epithelial atypias and other clinical features. However, when biopsies containing atypical ductal hyperplasia were excluded from the analysis, EZH2, epithelial and stromal ALDH-1 remained significantly associated with breast cancer diagnosis.

In summary, benign breast biopsies in women who subsequently develop breast cancer show increased incidence of proliferative and atypical lesions compared to the controls. Epithelial atypia including atypical ductal hyperplasia and/or flat epithelial atypia are more common in these biopsies and exceed those reported in the general population. We have characterized for the first time the expression of EZH2 and candidate breast stem cell marker ALDH-1 in benign breast disease. Our data show that EZH2 and ALDH-1 up regulation is frequently noted in high risk proliferative lesions in benign breast lobules and is significantly associated with subsequent development of breast cancer at a younger age. Our data pave the way for larger prospective validation studies as these findings may have clinical implications in the evaluation and management of women with benign breast biopsies.

Acknowledgments

Financial Support: This work was supported by NIH grants 2R01CA107469, R01CA125577, and U01CA154224 (to CGK), and by the National Institutes of Health through the University of Michigan's Cancer Center Support Grant (5 P30 CA46592).

Footnotes

Presented in part at 98th United States and Canadian Academy of Pathology (USCAP) Meeting, Boston, MA, Feb 2009

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