ESM_1. Detailed description of: a) clinical specimens’ resource and characteristics; b) immunohistochemistry staining protocol and, c) statistical analysis.

Title: Biological and clinical implications of nicastrin expression in invasive breast cancer

Journal: Breast Cancer Research and Treatment

Aleksandra Filipović1, Julian Hendrik Gronau1, Andrew R Green4, Jayson Wang2, Sabari Vallath3, Sabeena Rasul1, Ian O Ellis4, Ernesto Yagüe1, Justin Sturge1, R. Charles Coombes1

Authors affiliations: 1 Section of Oncology, Division of Cancer, Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, London, UK. 2Department of Pathology, Hammersmith Hospital, London, UK 3Centre for Tumour Biology, Institute of Cancer & CR-UK Clinical Centre, Bart’s & The London, Queen Mary School of Medicine & Dentistry, John Vane Science Centre, Charterhouse Square, London, UK. 4Department of Cellular Pathology, Queen’s Medical Centre, Nottingham University Hospital NHS Trust, Nottingham, UK

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a)Clinical specimens and tissue microarrays. Tissue microarrays (TMAs) containing 1050 primary operable BC cases from the Nottingham Tenovus Primary Breast Carcinoma Series (26) were employed. The cohort comprised women aged up to 70 years, who presented between 1986 and 1999. This well- characterized resource contains information on patients’ clinical and pathological data including histological tumor type, primary tumor size, lymph node status, histological grade and data on other BC relevant biomarkers. Patients within the good prognosis group (Nottingham Prognostic Index (NPI) ≤ 3.4) did not receive adjuvant therapy (AT). Hormonal therapy (HT) was prescribed to patients with ERα +ve tumors and NPI scores > 3.4 (moderate and poor prognostic groups). Pre-menopausal patients within the moderate and poor prognosis groups were candidates for cyclophosphamide, metotrexate and 5-fluorouracil (CMF) chemotherapy. Conversely, postmenopausal patients with moderate or poor NPI and ERα +ve were offered HT, while ERα -ve patients received CMF chemotherapy. Data collected included overall survival, breast cancer specific survival (BCSS), disease-free interval (DFI) and time to development of loco-regional and distant metastasis (DM). Clinical datawere maintained on a prospective basis. Median follow-up was 124 months (range 1 to 233). Breast cancer specific survival (BCSS) was defined as the time (in months) from date of the primary surgical treatment to the time of death from BC. DFI was defined as the interval (in months) from the date of the primary surgery to the first loco-regional or distant metastasis. Human tissue was used in accordance with the regulations of the local Research Ethics Committee of Imperial College NHS Healthcare Trust without patient identifiers being provided. Archival formalin-fixed, paraffin-embedded blocks from reduction mammoplasty (n = 40) were obtained from the Pathology Department, Charing CrossHospital, London. TMAs containing normal (BN481) and malignant breast (MC5001) tissue cores were purchased from US Biomax Inc., (USA).

b)Immunohistochemistry. Anti-nicastrin pAb N-1660 (Sigma, UK) was optimised to a working concentration of 1:200 on full-face excisional BC tissue sections. Subsequently, a 13-slide series BC TMA (n = 1050 cases) comprising 4 m thick formalin fixed paraffin embedded tissue cores were immunostained using this anti-nicastrin pAb for 3h at room temperature without antigen retrieval. Secondary labelling using indirect streptavidin-avidin-biotin was performed followed by detection with diaminobenzidine chromogen. Negative controls were performed by omission of the primary antibody. Immunostained sections were counterstained with hematoxilin. Nicastrin immunoreactivity was detected in the cytoplasm and cell membrane in breast epithelial cells, and was scored based on staining intensity ranging from 0 to +3; 0 = null, +1 = low, +2 = intermediate and +3 = high level of staining intensity. The percentage of nicastrin positive tumor cells scored as high (+2 or +3 in at least 66% of cells) or low/none was calculated for each tissue core on the TMAs by two independent investigators (AF and JW). High resolution digital imaging (NanoZomer, Hamamatsu Photonics, Welwyn Garden City, UK) at 20x magnification with a web-based interface (Distiller, SlidePath Ltd., Dublin, Ireland) was used. All cases were scored without prior knowledge of the clinicopathological or outcome data.

c)Statistical Analysis. Statistical analysis was performed using SPSS 16.0 statistical software (SPSS Inc., Chicago, IL, USA). Cut-off values for different biomarkers included in the study were chosen before statistical analysis. Analysis of categorical variables was performed with the appropriate statistical test. Survival curves were analysed using the Kaplan-Meier method with significance determined by the Log Rank test. Multivariate analysis was performed by Cox hazard analysis. A P value ≤ 0.05 (two-sided) was considered significant.