Abeer M. Shaaban

Nuclear and Cytoplasmic Expression of ERβ1, ERβ2, and ERβ5 Identifies Distinct Prognostic Outcome for Breast Cancer Patients

Purpose: Previous conflicting results about the prognostic significance of estrogen receptor (ER)-β in breast cancer may be explained by contribution of isoforms, of which five exist. Our aim was to elucidate the prognostic significance of ERβ1, ERβ2, and ERβ5 by immunohistochemistry in a large cohort of breast carcinomas with long-term follow-up. Experimental Design: Tissue microarrays were stained with ERβ1, ERβ2, and ERβ5 antibodies and scored as percentage of positive tumor cells and using the Allred system. Nuclear and cytoplasmic staining was evaluated and correlated with histopathologic characteristics, overall survival (OS), and disease-free survival (DFS). Results: Nuclear ERβ2 and ERβ5, but not ERβ1, significantly correlated with OS (P = 0.006, P = 0.039, and P = 0.099, respectively), and ERβ2 additionally with DFS (P = 0.013). ERβ2 also predicted response to endocrine therapy (P = 0.036); correlated positively with ERα, progesterone receptor, androgen receptor, and BRCA1; and correlated inversely with metastasis and vascular invasion. Tumors coexpressing ERβ2 and ERα had better OS and DFS. Cytoplasmic ERβ2 expression, alone or combined with nuclear staining, predicted significantly worse OS. Notably, patients with only cytoplasmic ERβ2 expression had significantly worse outcome (P = 0.0014). Conclusions: This is the first study elucidating the prognostic role of ERβ1, ERβ2, and ERβ5 in a large breast cancer series. ERβ2 is a powerful prognostic indicator in breast cancer, but nuclear and cytoplasmic expression differentially affect outcome. Measuring these in clinical breast cancer could provide a more comprehensive picture of patient outcome, complementing ERα.

'Declining estrogen receptor-beta expression defines malignant progression of human breast neoplasia'

It has been shown that the risk of breast cancer developing in certain morphologically identifiable benign breast lesions correlates with expression of estrogen receptor alpha (ER-&agr;). Although ER-&agr; and ER-&bgr; genes share a large degree of homology, it is generally thought that their distribution and functions are substantially different in many tissues. Recent development of reliable antibodies to ER-&bgr; has provided this first opportunity to test the hypothesis that the likelihood of malignant transformation in morphologically benign breast lesions can be accurately defined by the distribution and level of ER-&bgr; expression relative to that of ER-&agr;. Using a monoclonal antibody, ER-&bgr; protein expression has been analyzed in 53 normal breasts and compared with a cohort of histologically distinct breast lesions of different prognostic risk (54 hyperplasia of usual type, 35 ductal carcinoma in situ, and 141 invasive cancers). All of these tissues were also assessed for ER-&agr;. Expression of ER-&bgr; protein was also analyzed in an additional spectrum of benign breast lesions with low or negligible risk of progression to malignancy. The median proportion of cells expressing ER-&bgr; was highest in normal breast lobules (median 94.33%, interquartile range 78.25–99.00) but declined significantly through usual ductal hyperplasia (median 76.67, interquartile range 49.17–95.00, P = 0.002) and ductal carcinoma in situ (median 70.00, interquartile range 59.00–85.00, P = 0.009) to invasive cancer (median 60.00, interquartile range 50.00–80.00, P < 0.001). An appreciable proportion (33.81%) of ER-&agr;-negative invasive cancers expressed ER-&bgr;. A high but variable level of ER-&bgr; expression occurred in the benign lesions. The data from the intact histologic tissues were evaluated with respect to the relative expression of ER-&agr; and ER-&bgr; in five mammary cell lines of different behavioral phenotype (MCF7, ZR-75, T47D, MDAMB231, HUMA121). The highly significant differences in expression and distinct tissue distributions of ER-&agr; and ER-&bgr; within the histologic lesions of defined risk, together with the data from the cell lines, support the original hypothesis that the tissue concentration, relative occurrence, and/or interaction of these two types of estrogen receptor may play an important role in modulating mammary tumorigenesis.

Histopathologic types of benign breast lesions and the risk of breast cancer - Case-control study

The hypothetical multistep model of carcinogenesis indicates that breast cancer develops via a series of intermediate hyperplastic lesions through in situ to invasive carcinoma. To identify the risk inherent within the different morphologic lesions, we have analyzed the data from 674 benign biopsy specimens comprising 120 cases who subsequently developed breast cancer and 382 controls (matched for age and date of biopsy) spanning a period up to 20 years of follow-up (mean 66.95 months). In this series we have confirmed an increased risk associated with certain types of benign breast lesions. Atypical lobular hyperplasia was the most significant risk factor for breast cancer with more unfavorable outcome in patients <50 years of age (p = 0.003) and a relative risk (RR) of 4.55 (confidence interval [CI] 1.77–11.69). Hyperplasia of usual type showed an RR of 1.53 (CL 1.10–2.13) with a statistically worse probability of survival (cancer-free time) for patients >50 years. For atypical ductal hyperplasia the RR was 2.03 (CI 0.80–1.39). Blunt duct adenosis was significantly more common in cases progressing to breast cancer compared with controls, showing an RR of 2.08 (CI 1.12–2.85). We describe in detail the criteria of morphologic changes observed in blunt duct adenosis and define, for the first time, the level of risk associated with each of its six subtypes. Improved knowledge of breast carcinogenesis will provide insight for defining high-risk groups thus resulting in improved screening and management regimens.

Breast Cancer Risk in Usual Ductal Hyperplasia Is Defined by Estrogen Receptor-α and Ki-67 Expression

The hypothetical multistep model for breast carcinogenesis indicates that invasive carcinoma arises via a series of intermediate hyperplastic lesions through various grades of atypia to in situ and invasive carcinoma. Non-atypical hyperplasia [hyperplasia of usual type (HUT)] is a nonobligate precursor of breast cancer. Although its further morphological subclassification is unlikely, refining is more likely to depend on defining biological markers of risk. Having assembled a cohort of benign proliferative breast lesions of known outcome, we studied the expression of estrogen receptor-alpha (ER-alpha) and Ki-67 using morphometric image analysis as well as dual-labeled immunofluorescence in HUT foci and in surrounding normal lobules of 25 patients that progressed to breast cancer and 19 controls. Those patients that progressed to breast cancer (cases) showed significantly higher ER-alpha [median, 57.00% of cells within individual HUT foci; interquartile range (IQ), 33.48 to 67.78] and Ki-67 (median, 3.82%; IQ, 0.85 to 11.28) expression in their HUT foci compared with controls (ER-alpha median, 30.27%; IQ, 19.75 to 52.50 and Ki-67 median, 0.77%; IQ, 0.0458 to 1.72, P = 0.008 and <0.001). No significant difference in expression of dual-stained cells was found between cases and controls. Although normal lobules from cases showed higher ER-alpha expression compared with controls, this was not statistically significant. Our data point to a previously undescribed hormone-dependent pathway in this particular group of breast neoplasms and suggest the possibility of selective hormonal therapy to suppress the proliferative potential of these benign but high-risk breast lesions. The findings of this study might have important implications for improving breast cancer screening and management strategies.

Wild-type oestrogen receptor beta (ERβ1) mRNA and protein expression in Tamoxifen-treated post-menopausal breast cancers

This study has tested the hypothesis that comparison of protein and mRNA expression for ERα and ERβ1 by human breast cancers provides novel information relating to the clinical and pathological characteristics of human breast cancers. Expression of ERα and ERβ1 was identified in 167 invasive cancers from postmenopausal women treated only with endocrine therapy. The cohort included 143 cases receiving only adjuvant Tamoxifen following surgery. ERα and ERβ1 expression was analysed by immunohistochemistry and reverse transcription RT–PCR and compared with clinical progression of individual cancers. ERα protein was closely associated with the corresponding RNA detected by RT–PCR (Chi-square, P<0.001). In contrast, ERβ1 protein and mRNA were inconsistent. Although an association was identified between ERα and ERβ mRNAs (Chi-square, P<0.001) and between ERα protein and ERβ1 mRNA (Chi-square, P<0.027), no association was identified for the ERα and ERβ1 proteins detected by immunohistochemistry. ERβ1 was not associated with outcome. However, in the absence of ERα, ERβ1 protein expression was associated with elevated cell proliferation. There was a trend for the ERβ1 protein-positive cases to have a worse outcome, both within the group as a whole as well as within the ERα-positive Tamoxifen-treated cases. This study has confirmed the hypothesis that expression of ERα is an important determinant of breast cancer progression, and has further demonstrated that ERβ1 may play a role in the response of breast cancers to endocrine therapy.

The rising incidence of male breast cancer

Although breast cancer rates are declining in Western societies, quoted figures are restricted to female breast cancer. Recently in this journal, Stang and Thomssen reversed this trend by analysing male breast cancer data from the SEER Program of the National Cancer Institute, which showed an increased incidence in male breast cancer of 1.0 per 100,000 in the late 1970s to around 1.2 per 100,000 at the start of this decade [1]. In the UK around 350 cases of male breast cancer are diagnosed annually [2]. To determine if this figure is changing, we reviewed data obtained from members of the United Kingdom Association of Cancer Registries (UKACR). In most cases data was provided directly from UKACR members both as absolute numbers (1981–2004) and European Age Standardised Rates (EASR; 1991– 2004). As shown in Fig. 1, male breast cancer incidence is increasing in the UK, paralleling US data which shows a concordant increase over a similar time period [1, 3]. This begs the question of why male rates are rising. Age is the single biggest risk factor for male breast cancer. We are an ageing population and increased male breast cancer may well parallel increased longevity. However this increase is also reflected in ASR rates, which accounts for this. Nowadays, men are more health conscious and with increased public awareness of breast cancer in general e.g. through pink ribbon campaigns, they may be much more likely to seek medical attention for breast symptoms than ever before, which could contribute to increased diagnosis. Men who have been repeatedly exposed to radiation from a young age and over a long time period are at greater risk of developing breast cancer [4]. One might expect that males living within the fallout zone from the 1986 Chernobyl nuclear disaster (which included the UK) may be at greater risk. Although reports are sketchy, an article published in Croatian suggests that post-Chernobyl, the ratio of male breast cancer increased from 1:139 to 1:79 [5]. Rising levels of obesity resulting from physical inactivity and poor diet is a serious public health issue in both the US and UK and may be contributory to increasing male breast cancer. High estrogen levels are linked to breast cancer and local estrogen biosynthesis by breast tissue adipocytes may be an important factor in obese individuals. Increased use of pesticides, many of which are weakly estrogenic, by the agricultural industry and their subsequent introduction into the food chain could also add to rising trends. Increased alcohol intake has been associated with male breast cancer [6] and this is probably indirectly linked to alcohol-induced liver damage and subsequent hyperestrogenism. Liver cirrhosis is associated with increased estrogen levels which could increase breast cancer risk. There are suggestions that this risk is higher when cirrhotic patients live longer [7]. The so-called ‘binge drinking’ culture that has developed in recent years, particularly in the UK could also be a casual factor. Although male breast cancer is rare, for the patient a diagnosis is often unexpected but it should be regarded as no less important than other ‘male’ illnesses. Its rarity precludes randomised clinical trials but its rising trend, which seems likely to continue, suggests coordinated multi-centre approaches are necessary to accumulate V. Speirs (&) Leeds Institute of Molecular Medicine, Wellcome Trust Brenner Building, St James’s University Hospital, Leeds LS9 7TF, UK e-mail: v.speirs@leeds.ac.uk

Combined analysis of eIF4E and 4E-binding protein expression predicts breast cancer survival and estimates eIF4E activity

Increased eukaryotic translation initiation factor 4E (eIF4E) expression occurs in many cancers, and makes fundamental contributions to carcinogenesis by stimulating the expression of cancer-related genes at post-transcriptional levels. This key role is highlighted by the facts that eIF4E levels can predict prognosis, and that eIF4E is an established therapeutic target. However, eIF4E activity is a complex function of expression levels and phosphorylation statuses of eIF4E and eIF4E-binding proteins (4E-BPs). Our hypothesis was that the combined analyses of these pathway components would allow insights into eIF4E activity and its influence on cancer. We have determined expression levels of eIF4E, 4E-BP1, 4E-BP2 and phosphorylated 4E-BP1 within 424 breast tumours, and have carried out analyses to combine these and relate the product to patient survival, in order to estimate eIF4E activity. We show that this analysis gives greater prognostic insights than that of eIF4E alone. We show that eIF4E and 4E-BP expression are positively associated, and that 4E-BP2 has a stronger influence on cancer behaviour than 4E-BP1. Finally, we examine eIF4E, estimated eIF4E activity, and phosphorylated 4E-BP1 as potential predictive biomarkers for eIF4E-targeted therapies, and show that each determines selection of different patient groups. We conclude that eIF4Es influence on cancer survival is modulated substantially by 4E-BPs, and that combined pathway analyses can estimate functional eIF4E.

A multi-centre investigation towards reaching a consensus on the immunohistochemical detection of ERbeta in archival formalin-fixed paraffin embedded human breast tissue

SummaryEstrogen receptor (ER) α is a well-established independent prognostic factor in breast cancer whose presence determines the clinical implications of adjuvant endocrine therapy. A second receptor, ERb has been described, and a number of studies have examined its expression in breast tissue. However elucidation of the role played by ERb has been hampered by published immunohistochemical studies employing a variety of protocols and scoring systems such that inter-laboratory comparisons are difficult. Here we present a multi-centre study designed to critically evaluate inter-laboratory differences in methodology. Six UK and Irish centres participated in this study. A small series of breast cancers were stained using centre-specific laboratory protocols and scored using both centrespecific and standard scoring protocols. There was generally poor agreement as to what constituted a positive or negative case when centre-specific scoring systems were used with less than half of all cases in agreement. Concordance was improved when a standard scoring system was used but varied according to threshold for positivity employed and primary antibody. Our results emphasise the need for further studies addressing the role of ERb to be based on a wider consensus on criteria for positivity. Ideally this should be based on calibration against clinical outcome.

Carcinoembryonic antigen cell adhesion molecule 6 predicts breast cancer recurrence following adjuvant tamoxifen.

Purpose: Tamoxifen remains therapy of choice for premenopausal estrogen receptor α–positive breast cancer. However, resistance and recurrence are serious problems. Our previous work indicated that carcinoembryonic antigen cell adhesion molecule 6 (CEACAM6) was significantly up-regulated in tamoxifen-resistant (TAMr) MCF-7 derivatives. The aim of this study was to determine the functional role of CEACAM6 in endocrine-resistant breast cancer and to retrospectively test whether it was predictive of resistance in a large cohort of breast cancers with long-term follow-up. Experimental Design: siRNA silencing of CEACAM6 was done in TAMr cells and effects on clonogenicity and endocrine sensitivity were determined. CEACAM6 immunohistochemistry was done on a tissue microarray comprising 108 relapsed primary human breast cancers and 243 tamoxifen-sensitive controls. Results: siRNA-mediated silencing of CEACAM6 reduced both clonogenicity and anchorage-dependent and anchorage-independent growth of TAMr cells. Importantly, CEACAM6 silencing restored sensitivity of TAMr cells to 4-hydroxytamoxifen and proliferative response to 17β-estradiol. Immunohistochemistry showed significantly more CEACAM expression in the relapsed group compared with nonrelapsed controls [35 of 108 (33.3%) and 32 of 243 (13.2%), respectively; odds ratio, 3.16 (95% confidence interval, 1.83-5.47); P < 0.0001]. Additionally, we derived an outcome predictor model based on CEACAM expression that restratified patients in the Nottingham prognostic index intermediate-risk group into either higher-risk or lower-risk group. Conclusions: Our data support an important role for CEACAM6 in endocrine resistance, which can serve as a powerful predictor of future recurrence.

Increased risk of malignant progression in benign proliferating breast lesions defined by expression of heat shock protein 27.

Heat shock protein 27 (hsp-27) is a regulator of oestrogen receptor (ER) expression and a modulator of intracellular homeostasis. In this laboratory, Shaaban et al demonstrated the importance of ER-α, together with Ki67, in enhancing the progression of benign breast lesions of defined morphological types. To better understand the mechanisms by which ER-α promotes breast neoplasia, this study was performed to test the hypothesis that the roles of ER-α and hsp-27 may be defined by their quantitative expression in proliferative breast lesions of varying histological risk. The expression of hsp-27 was identified using a specific monoclonal antibody and analysed to assess the proportion of positive epithelial cells using digitised morphometric image analysis. The expression of ER-α was analysed by immunohistochemistry and Western blotting in a variety of benign (HUMA121) and malignant mammary cell lines, including ER-α(+) (MCF7, ZR-75, T47D) and ER-α(−) (MDA-MB 231) breast cancer cell lines. The data confirm that, during progression from normal through proliferative breast lesions to in situ cancer, there was a significant increase in both the proportion and the optical density of the epithelial cells expressing hsp-27. The mean levels of expression ranged from 7.4% of the total number of epithelial cells in normal lobules to 25.17% of epithelial cells in hyperplasias of usual type (HUT) to 61.1% of epithelial cells in ductal carcinoma in situ (P<0.001). The study has confirmed the expression of hsp-27 to be closely associated with ER-α(+) expression, and that its regulated expression occurs early along the mammary oncogenic pathway, supporting the initial hypothesis. It is our proposal that the differential expression of hsp-27 modulates the phenotypic behaviour of morphologically benign epithelial cells and hence may be an important determinant in initiating, or promoting, a population of human mammary cancers.