Steven Pollock

The prognostic significance of tumour-stroma ratio in oestrogen receptor-positive breast cancer

Background:A high percentage of stroma predicts poor survival in triple-negative breast cancers but is diminished in studies of unselected cases. We determined the prognostic significance of tumour–stroma ratio (TSR) in oestrogen receptor (ER)-positive male and female breast carcinomas.Methods:TSR was measured in haematoxylin and eosin-stained tissue sections (118 female and 62 male). Relationship of TSR (cutoff 49%) to overall survival (OS) and relapse-free survival (RFS) was analysed.Results:Tumours with ⩾49% stroma were associated with better survival in female (OS P=0.008, HR=0.2–0.7; RFS P=0.006, HR=0.1–0.6) and male breast cancer (OS P=0.005, HR=0.05–0.6; RFS P=0.01, HR=0.87–5.6), confirmed in multivariate analysis.Conclusions:High stromal content was related to better survival in ER-positive breast cancers across both genders, contrasting data in triple-negative breast cancer and highlighting the importance of considering ER status when interpreting the prognostic value of TSR.

Phosphorylation of Estrogen Receptor β at Serine 105 Is Associated with Good Prognosis in Breast Cancer

Estrogen receptor (ER) action is modulated by posttranslational modifications. Although ERalpha phosphorylation correlates with patient outcome, ERbeta is similarly phosphorylated but its significance in breast cancer has not been addressed. We investigated whether ERbeta that is phosphorylated at serine 105 (S105-ERbeta) is expressed in breast cancer and assessed potential clinical implications of this phosphorylation. Following antibody validation, S105-ERbeta expression was studied in tissue microarrays comprising 108 tamoxifen-resistant and 351 tamoxifen-sensitive cases and analyzed against clinical data. S105-ERbeta regulation in vitro was assessed by Western blot, flow cytometry, and immunofluorescence. Nuclear S105-ERbeta was observed in breast carcinoma and was associated with better survival (Allred score > or =3), even in tamoxifen-resistant cases, and additionally correlated with ERbeta1 and ERbeta2 expression. Distinct S105-ERbeta nuclear speckles were seen in some higher grade tumors. S105-ERbeta levels increased in MCF-7 cells in response to 17beta-estradiol, the ERbeta-specific agonist diarylpropionitrile, and the partial ERbeta-agonist genistein. S105-ERbeta nuclear speckles were also seen in MCF-7 cells and markedly increased in size and number at 24 hours following 17beta-estradiol and, in particular diarylpropionitrile, treatment. These speckles were coexpressed with ERbeta1 and ERbeta2. Presence of S105-ERbeta in breast cancer and association with improved survival, even in endocrine resistant breast tumors suggest S105-ERbeta might be a useful additional prognostic marker in this disease.

The practicalities of using tissue slices as preclinical organotypic breast cancer models

Models considering breast cancer complexity cannot be easily or accurately replicated in routine cell line or animal models. We aimed to evaluate the practicality of organotypic tissue slice culture in breast cancer. Following ethical approval, 250 µm thick sections from surplus breast tumours (n=10) were prepared using a vibrating blade microtome. Triplicate tissue slices were placed in 6-well plates and cultured for up to 7 days±tamoxifen (1 nM) or doxorubicin (1 µM). Tissue slices were fixed and embedded before sectioning for morphological evaluation and immunohistochemistry. H&E showed good preservation of tissue morphology. Collagen production was evident. Biomarkers of proliferation and apoptosis could be evaluated using immunohistochemistry and used as surrogates to quantify drug effects. In summary, breast cancer tissue slices can be cultured in vitro as organotypic models. Nevertheless, although simple in concept, the delicacy of the model with regard to handling makes subsequent analytical processes challenging.

ERβ1 represses FOXM1 expression through targeting ERα to control cell proliferation in breast cancer.

In this study, we investigated the effects of ectopic estrogen receptor (ER)β1 expression in breast cancer cell lines and nude mice xenografts and observed that ERβ1 expression suppresses tumor growth and represses FOXM1 mRNA and protein expression in ERα-positive but not ERα-negative breast cancer cells. Furthermore, a significant inverse correlation exists between ERβ1 and FOXM1 expression at both protein and mRNA transcript levels in ERα-positive breast cancer patient samples. Ectopic ERβ1 expression resulted in decreased FOXM1 protein and mRNA expression only in ERα-positive but not ERα-negative breast carcinoma cell lines, suggesting that ERβ1 represses ERα-dependent FOXM1 transcription. Reporter gene assays showed that ERβ1 represses FOXM1 transcription through an estrogen-response element located within the proximal promoter region that is also targeted by ERα. The direct binding of ERβ1 to the FOXM1 promoter was confirmed by chromatin immunoprecipitation analysis, which also showed that ectopic expression of ERβ1 displaces ERα from the endogenous FOXM1 promoter. Forced expression of ERβ1 promoted growth suppression in MCF-7 cells, but the anti-proliferative effects of ERβ1 could be overridden by overexpression of FOXM1, indicating that FOXM1 is an important downstream target of ERβ1 signaling. Together, these findings define a key anti-proliferative role for ERβ1 in breast cancer development through negatively regulating FOXM1 expression.

Differential regulation of oestrogen receptor β isoforms by 5′ untranslated regions in cancer

Oestrogen receptors (ERs) are critical regulators of the behaviour of many cancers. Despite this, the roles and regulation of one of the two known ERs – ERβ– are poorly understood. This is partly because analyses have been confused by discrepancies between ERβ expression at mRNA and proteins levels, and because ERβ is expressed as several functionally distinct isoforms. We investigated human ERβ 5′ untranslated regions (UTRs) and their influences on ERβ expression and function. We demonstrate that two alternative ERβ 5′UTRs have potent and differential influences on expression acting at the level of translation. We show that their influences are modulated by cellular context and in carcinogenesis, and demonstrate the contributions of both upstream open reading frames and RNA secondary structure. These regulatory mechanisms offer explanations for the non‐concordance of ERβ mRNA and protein. Importantly, we also demonstrate that 5′UTRs allow the first reported mechanisms for differential regulation of the expression of the ERβ isoforms 1, 2 and 5, and thereby have critical influences on ERβ function.

Epithelial–mesenchymal interactions in breast cancer: evidence for a role of nuclear localized β-catenin in carcinoma-associated fibroblasts

Verghese E T, Shenoy H, Cookson V J, Green C A, Howarth J, Partanen R H, Pollock S, Waterworth A, Speirs V, Hughes T A & Hanby A M
(2011) Histopathology59, 609–618

Problems (and solutions) in the study of male breast cancer.

Owing to its rarity, large-scale retrospective studies in male breast cancer have suffered from the small numbers of cases available for study from any one center. Here we describe our experience in establishing a large collection of male breast cancers in tissue microarray format suitable for biomarker analysis by immunohistochemistry.

The value of archival tissue blocks in understanding breast cancer biology

Pathological reporting of breast cancer has evolved alongside scientific advances. Such advances have led to recognition of different molecular classes of breast cancer resulting in improved disease management. The aim of this study was to establish whether these advances could be applied to archival breast cancer cases dating from the 1940s to assess historical trends. Important observations included the marked differences in pathological reporting, size of tumour and in ERα expression throughout the decades.

Adding value to rare tissue samples donated to biobanks: characterisation of breast tissue and primary cell cultures obtained from a female-to-male transgender patient

Abstract Biobanks provide a window of opportunity to store and add value to material from rare cases allowing their future use in biomedical research. One such example is the opportunityto obtain good quality tissue from patients undergoing gender re-assignment. Following patient agreement to donate tissue samples to our biobank we catalogued the histological appearance, defined the expression of the hormone receptors ERα, PR, AR and the proliferation marker Ki67, and generated and characterised primary cell cultures in a female to male (FTM) transgender patient referred to our unit for surgery. Immunohistochemistry was performed for ERα, PR and AR and the proliferation marker Ki67. Hormone receptor expression was confined to epithelial cells lining the breast ducts. Ki67 immunoreactivity was sparse indicating little proliferation of luminal epithelium, consistent with normal mammary gland. Cultures of epithelial cells and fibroblasts were derived from surplus tissue. The latter lacked expression of epithelial markers and hormone receptors but exhibited expression of vimentin. Culture of the former on Matrigel saw an outgrowth of more rounded “epithelial-like” cells. Immunofluoresence characterisation showed a mixed phenotype with expression of vimentin and both myoepithelial and luminal epithelial markers. Sporadic weak ERα expression and moderate PR expression was seen. In summary, as well as routinely collecting tissue and blood samples, we have characterised and stored tissue and cells from a FTM transgender patient, adding value to this resource which,available from the Breast Cancer Campaign Tissue Bank for those interested in further studying the biology of FTM transgender tissue.

Comparative Biomarker Analysis in 523 Matched Male and Female Breast Cancers.

Incidence rates of male breast cancer (MBC) are rising. MBC etiology is poorly understood with most of our current knowledge regarding its biology, natural history and treatment extrapolated from our knowledge of female breast cancer (FBC). Retrospective studies on MBC have suffered from small numbers of cases available from any one centre thus a significant problem in studying this disease is accruing sufficiently large numbers to allow comparative analysis of possible prognostic markers. Using a co-ordinated multi-centre approach, the aim of this study was to conduct the first large scale study to address the relevance of the expression of recognised biomarkers in FBC in the same disease in males. Five hundred and twenty three cases were obtained retrospectively and assimilated into TMAs, including 260 MBCs and 263 cases of stage-matched FBCs. MBC comprised 21 grade 1, 121 grade 2, 68 grade 3, 50 unknown, mean age 67 (range 39-90) with 167 ductal, 4 lobular, 10 papillary, 10, mucinous, 4 DCIS, 1 mixed and 64 unknown. FBC comprised 29 grade 1, 140 grade 2, 94 grade, mean age 58 (range 27-92) with 220 ductal, 23 lobular, 14 mixed and 6 unknown. Four µm TMA sections were analysed using the following biomarkers: hormone receptors (ERα, ERβ1, ERβ2, ERβ5, total PR, PRA, PRB, AR), apoptosis markers (p53, bcl2), basal (CK5/6, CK14) and luminal epithelial markers (CK18, CK19), E-cadherin and HER2. Biomarkers were scored according to published criteria; for ERβ isoforms both nuclear and cytoplasmic immunoreactivity was determined Statistical analysis was conducted using SPSS. Luminal A (ERα+, and/or PR+, HER2-) was seen in 93% of MBC vs. 84% of FBC, Luminal B (ERα+, and/or PR+, HER2+) or HER2 subgroup (ERα-, PR-, HER2+) was not seen in MBC but found in 6% and 2% of FBC, respectively. Basal-like tumours (ERα-, PR-, HER2-, CK5/6+) were infrequent (MBC 2%, FBC 1%) and in MBC these tumours also expressed ERβ isoforms. No differences were observed in grade, stage or LN status between genders. Univariate analysis showed ERα, ERβ1, ERβ5, PRA, AR, p53 were significantly associated with FBC while cytoplasmic ERβ2, bcl2 and e-cadherin were associated with MBC (all P Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2109.