High nuclear MSK1 is associated with longer survival in breast cancer patients

This immunohistochemical-based study was performed using a cohort of early stage breast cancer patients (n = 1902) treated at Nottingham University Hospitals, with long-term follow-up, between 1986 and 1998. Information on clinical history, outcome and tumour characteristics was collected and assessed in a standardised manner, including age at diagnosis, tumour size, histologic stage and grade, Nottingham prognostic index (NPI), lymphovascular invasion (LVI), oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) status. ER, PR and HER2 status were available for this cohort and have been described previously (Abdel-Fatah et al. 2010). HER2 positivity expression was determined by immunohistochemistry (IHC) with fluorescence in situ hybridisation (FISH), and FISH was performed for the cases with IHC scored as 2+. Basal-like phenotype was defined as the expression of cytokeratin (CK)-5/6 and/or CK-14 expression in 10% or more of invasive tumour cells, irrespective of ER, PR or HER2 status (Rakha et al. 2006). The median age of the patients was 55 years (ranging from 18 to 72), and 63.2% (1203 of 1902) of patients had stage I disease. Breast cancer-specific survival was defined as the time interval (in months) from the date of primary surgery to death resultant from breast cancer. The median follow-up time was 177 months (ranging from 1 to 308 months). Patients were managed under a uniform protocol, where all underwent wide local excision (n = 819, 43.1%) or mastectomy (n = 1067, 56.1%), and approximately half of the patients received radiotherapy (n = 1025, 53.9%). Systemic adjuvant treatment was given based on ER, menopausal status and NPI values. Patients with ER-positive disease were chosen for hormone therapy (n = 674, 35.4%). Patients with ER negative or premenopausal status were chosen for CMF chemotherapy (cyclophosphamide, methotrexate and 5-fluorouracil, n = 320, 16.8%), if they had an NPI value of 3.4 or above, whereas patients who had an NPI value less than 3.4 did not receive adjuvant chemotherapy. This study is reported in accordance with REMARK criteria (McShane et al. 2005). Nottingham Research Ethics Committee 2 approved the project under “Development of a molecular genetic classification of breast cancer R&D “(No. 03HI01 REC Ref.C202313)”. The clinicopathologic variables of the patient cohort are shown in Table 1.

MSK1 protein expression was investigated using freshly cut 4 µm sections from tissue microarrays and assessed by immunohistochemistry. A single 0.6 mm tissue core was used for each patient with the core being taken from a representative tumour area as assessed by a specialist breast cancer histopathologist, as described previously (Abd El-Rehim et al. 2005). A range of antibodies were assessed for suitability with primary antibody specificity being confirmed by Western blotting, using a range of breast cancer cell line lysates, prior to immunohistochemistry (data not shown). TMA sections were dewaxed in xylene and rehydrated in ethanol, followed by antigen retrieval, in 0.01 mol/L sodium citrate buffer (pH6.0), using a microwave, 750W for 10 min then 450W for 10 min. Staining was achieved using a Novolink Polymer Detection System (Leica, Denmark) according to the manufacturers’ instructions. Briefly, endogenous peroxidase activity was neutralised with Peroxidase Block reagent for 5 min at room temperature, followed by Protein Block reagent for 5 min at room temperature, to minimise non-specific interactions of the subsequent detection reagents. Primary antibody (anti-MSK1, 1: 200, Bethyl Laboratories, USA, 9252) was diluted in Bond Primary Antibody Diluent (Leica, Denmark) and applied to the tissue for 1 h at room temperature. Immunohistochemical reactions were visualised with 3, 3′-diaminobenzidine and counterstained with haematoxylin, dehydrated and fixed in xylene followed by mounting with DPX. Positive controls constituted early stage breast tumour composite sections of varying grades, used in initial optimisations, and negative control omitted primary antibody.

Staining was assessed at 200× magnification following high-resolution scanning (Nanozoomer Digital Pathology Scanner, Hamamatsu Photonics). As no cytoplasmic staining was observed, only nuclear staining intensity was assessed. The staining intensity was categorised as: none (0), weak (1), medium (2) and strong (3). The immunohistochemistry H-scores were calculated by multiplying the percentage of positive tumour cell nuclei by the staining intensity, giving rise to a score ranging between 0 and 300. The immunohistochemistry methodology was described previously (Woolston et al. 2011a, b). 30% of cases were scored by a second independent assessor, blind to survival endpoints, clinicopathological variables, and other assessors H-scores. Good concordance was demonstrated between both scorers (single measure intraclass correlation coefficient was 0.881). X-tile software was used to generate a non-biased cut point that was used to dichotomise immunohistochemical scores (Camp et al. 2004).

Pearson’s chi-squared test of association (χ²) was used to examine the relationship between categorised protein expression and clinicopathologic factors, or Fisher’s exact test if a cell count in a 2 × 2 table was less than 5. Kaplan–Meier survival curves were plotted and significance was determined using the Log-rank test. The Cox proportional hazards regression model was applied in multivariate survival analysis. All differences were considered statistically significant at the level of P < 0.05. Statistical analysis was performed using SPSS 22.0 software.