Elsevier

Human Pathology

Volume 39, Issue 10, October 2008, Pages 1431-1437
Human Pathology

Original contribution
The combination of high cyclin E and Skp2 expression in breast cancer is associated with a poor prognosis and the basal phenotype,☆☆

https://doi.org/10.1016/j.humpath.2008.03.004Get rights and content

Summary

Gene expression studies have identified a basal phenotype of breast cancer; these are hormone receptor and HER2-negative cancers with poor prognosis. High levels of cyclin E and Skp2, and low levels of p27 have previously been individually associated with both basal-like breast cancer and a poor outcome after diagnosis. The goal of this study was to first confirm the prognostic value of these biomolecular markers using a breast cancer tissue microarray. Second, we also test the hypothesis that the combined phenotype of high cyclin E, low p27, and high Skp2 would be a strong predictor of outcome and would be closely associated with the basal phenotype of breast cancer. Our cohort consisted of 438 cases of breast cancer and the median follow-up was 15.4 years. The tissue microarray was constructed from archival tumor blocks and we used commercially available antibodies for biomarker immunostaining. Cyclin E was positive in 46% of cases, p27 was negative in 62%, and Skp2 was positive in 35%. We found cyclin E and Skp2 to be prognostic for breast cancer–specific survival in univariate analyses, but p27 was not prognostic. The strongest predictor of outcome was the combination of cyclin E positive and Skp2 positive (difference in survival of 19% at 10 years, P = .0009). This combination was present in 78 (27%) of 288 cases for which data on both biomarkers were available. This combination was also highly associated with young age at diagnosis, grade 3 tumors, ER-negative status, HER2-negative status, and the basal biomarkers epidermal growth factor receptor and cytokeratin 5/6. However, in a multivariate model including standard clinicopathologic variables, this combination was not found to have independent prognostic significance. In conclusion, the combination of high cyclin E and Skp2 expression predicts for poor prognosis in breast cancer in univariate analysis only, it is associated with high risk features, and it is associated with the basal phenotype.

Introduction

Gene expression analyses of multiple breast cancer cohorts have consistently demonstrated a separation of estrogen receptor (ER)–negative breast cancers into HER2 positive and basal subtypes [1], [2], [3]. The basal breast cancers are associated with a particularly poor prognosis, and, as they are both ER negative and HER2 negative, these patients do not benefit from adjuvant hormonal therapy or trastuzumab. Currently, there is uncertainty regarding the identification of the basal breast cancers—the “gold standard” definition is by gene expression profile, but proposed immunohistochemical surrogate panels include the triple-negative definition (ER negative, PR negative, and HER2 negative) and more specific panels incorporating positive markers of the basal phenotype (ER negative, HER2 negative, and either epidermal growth factor receptor [EGFR] or CK5/6 positive) [4], [5]. Further study into the molecular characteristics of basal breast cancers may lead to the discovery of potential therapeutic targets. Recent studies characterizing this subtype have found that, when studied individually, the expression of cyclin E and Skp2, and the absence of p27 (the product of the KIP1 gene) are each associated with basal breast cancers [6], [7]. In addition, these 3 proteins are linked through a common molecular pathway that regulates the cell cycle at the G1/S checkpoint. Loss of this checkpoint results in genomic instability and the development of neoplasia [8].

Cyclin E and its associated cyclin-dependent kinase, CDK2, promote cell-cycle progression. Overexpression of cyclin E was shown to have independent prognostic significance in a cohort of patients with breast cancer [9], [10]. p27 is an inhibitor of the cyclin E/CDK2 complex and as such functions as a tumor suppressor [11], [12]. In addition, some studies suggest that p27 can predict response to chemotherapy [13] and hormone therapy [14], [15]. Skp2 is a ubiquitin ligase that targets p27 for degradation [16] and also has an oncogenic role in breast cancer [7], [17]. Although cyclin E, p27, and Skp2 have not yet seen widespread use as clinical biomarkers, they have the potential to improve prognostic models and possibly predict response to adjuvant treatment. However, some follow-up studies have failed to confirm these previous findings. In this study, we use tissue microarrays (TMAs) to analyze the expression of these 3 biomarkers on a cohort of 438 breast cancer samples linked to long-term clinical outcome data. By combining these biologically linked biomarkers, we aimed to create a more specific test to isolate a clinically important breast cancer subgroup. We hypothesized that the novel combination of cyclin E positive, p27 negative, and Skp2 positive, which we will refer to as the 3-marker phenotype, would identify a particularly high-risk subgroup of breast cancer and be associated with the basal breast cancer phenotype.

Section snippets

Materials and methods

Our study population is a single institution cohort comprising 438 patients who had surgery for breast neoplasia at the Vancouver General Hospital between 1974 and 1995. Detailed clinicopathologic and outcome data were available for all patients. Because our patients were accrued from an extended period, and the treatment of breast cancer has changed significantly, we did not include treatment data in our analysis. Patients presenting with metastatic disease, patients with in situ disease only,

Results

The mean age of our patient cohort was 60 years and the median follow-up 15 years. Fifty-two percent had tumors greater than 2 cm, and 54% had grade 3 tumors. Thirty-five percent were node negative, 80% were ER positive, and 14% were HER2 positive.

For the biomarkers specific to this study, cyclin E was positive in 46% of cases, Skp2 was positive in 35%, and p27 was negative in 62% (Table 1). Representative images are presented in Fig. 1 and all primary image data are available at //www.gpecimage.ubc.ca/tma/web/viewer.php

Discussion

There have been several published studies that reported on the prognostic value of cyclin E, p27, and Skp2 as individual biomarkers in breast cancer. All 3 proteins are linked through a common biological pathway, playing a central role in cell-cycle regulation. This is the first study that has combined these markers as a 3-marker phenotype.

Cyclin E forms a complex with CDK2 and promotes G1/S cell-cycle progression. The CCNE gene is amplified in some breast cancer cell lines [27], and it is

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    ☆☆

    This work was supported by a grant from the Canadian Breast Cancer Research Alliance to WDF and TON. The Genetic Pathology Evaluation Centre is supported by an unrestricted educational grant from Sanofi-Aventis.

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