Clinicopathological significance of ataxia telangiectasia-mutated (ATM) kinase and ataxia telangiectasia-mutated and Rad3-related (ATR) kinase in MYC overexpressed breast cancers

Cell lines were purchased from American Type Culture Collection (ATCC, Manassas, USA). MDA-MB-231 and MDA-MB-468 cells were cultured in minimum essential amino acids medium supplemented with 1% L-glutamine and 1% non-essential amino acids. T47D cells were cultured in Dulbecco’s Modified Eagle’s medium. MCF-7 cells were grown in RPMI medium. All media were supplemented with 10% FBS and 1% penicillin streptomycin. Protein samples were prepared by lysing cells in RIPA buffer (Sigma–Aldrich) containing protease inhibitor (Sigma) and phosphatase inhibitor cocktail 1 and 2 (Sigma). Samples were run on SDS-PAGE gel (4–12%) bis-tris. Antibodies used were anti-MYC antibody (abcam, clone 9E10), ATM antibody (abcam clone Y170) and ATR antibody (cell signalling cat.no 2790S). Protein detection and quantification were determined by scanning the membranes on Licor-Odyssey’s Scanner (Licor, Biosciences) at the predefined intensity fluorescence.

MYC, ATM and ATR mRNA expressions were investigated in METABRIC (Molecular Taxonomy of Breast Cancer International Consortium) cohort. The METABRIC study protocol, detailing the molecular profiling methodology in a cohort of 1977 breast cancer samples is described by Curtis et al. [14]. Patient demographics are summarised in Supplementary Table S1 of supporting information. ER-positive and/or lymph node-negative patients did not receive adjuvant chemotherapy. ER-negative and/or lymph node-positive patients received adjuvant chemotherapy. For this cohort, the mRNA expression was hybridised to Illumina HT-12 v3 platform (Bead Arrays), and the data were pre-processed and normalised as described previously. Samples were classified into the intrinsic subtypes based on the PAM50 gene list. A description of the normalisation, segmentation and statistical analyses was previously described [14]. Real-time RT-qPCR was performed on the ABI Prism 7900HT sequence detection system (Applied Biosystems) using SYBR1 Green reporter. All the samples were analysed as triplicates. The Chi-square test was used for testing association between categorical variables, and a multivariate Cox model was fitted to the data using as endpoint breast cancer-specific death. X-tile (Version 3.6.1) was used to identify a cut-off in gene expression values such that the resulting subgroups had significantly different survival courses.

The study was performed in a consecutive series of 1650 patients with primary invasive breast carcinomas who were diagnosed between 1986 and 1999 and entered into the Nottingham Tenovus Primary Breast Carcinoma series. Patient demographics are summarised in Supplementary Table S2. This is a well-characterised series of patients with long-term follow-up that have been investigated in a wide range of biomarker studies [1, 2, 21]. All patients were treated in a uniform way in a single institution with standard surgery (mastectomy or wide local excision), followed by Radiotherapy. Prior to 1989, patients did not receive systemic adjuvant treatment (AT). After 1989, AT was scheduled based on prognostic and predictive factor status, including Nottingham Prognostic Index (NPI), oestrogen receptor-α (ER-α) status, and menopausal status. Patients with NPI scores of < 3.4 (low risk) did not receive AT. In pre-menopausal patients with NPI scores of ≥ 3.4 (high risk), classical Cyclophosphamide, Methotrexate, and 5-Fluorouracil (CMF), chemotherapy was given; patients with ER-α-positive tumours were also offered endocrine therapy. Postmenopausal patients with NPI scores of ≥ 3.4 and ER-α positivity were offered endocrine therapy, while ER-α-negative patients received classical CMF chemotherapy. Median follow-up was 111 months (range 1–233 months). Survival data, including breast cancer-specific survival (BCSS), disease-free survival (DFS), and development of loco-regional and distant metastases (DM), were maintained on a prospective basis. DFS was defined as the number of months from diagnosis to the occurrence of local recurrence, local lymph node (LN) relapse or DM relapse. Breast cancer-specific survival (BCSS) was defined as the number of months from diagnosis to the occurrence of BC-related death. Local recurrence-free survival (LRS) was defined as the number of months from diagnosis to the occurrence of local recurrence. DM-free survival was defined as the number of months from diagnosis to the occurrence of DM relapse. Survival was censored if the patient was still alive at the time of analysis, lost to follow-up, or died from other causes.

Tumour Marker Prognostic Studies (REMARK) criteria, recommended by McShane et al. [28], were followed throughout this study. Ethical approval was obtained from the Nottingham Research Ethics Committee (C202313).

Tumours were arrayed in tissue microarrays (TMAs) constructed with 0.6 mm cores sampled from the periphery of the tumours. The TMAs were immunohistochemically profiled for MYC, ATM and ATR and other biological antibodies as previously described [1, 2, 21]. Immunohistochemical staining was performed using the Thermo Scientific Shandon Sequenza chamber system (REF: 72110017), in combination with the Novolink Max Polymer Detection System (RE7280-K: 1250 tests), and the Leica Bond Primary Antibody Diluent (AR9352), each used according to the manufacturer’s instructions (Leica Microsystems). Leica Autostainer XL machine was used to dewax and rehydrate the slides. Pre-treatment antigen retrieval was performed on the TMA sections using sodium citrate buffer (pH 6.0) and heated for 20 min at 95 °C in a microwave (Whirpool JT359 Jet Chef 1000W). A set of slides were incubated for 18 h at 4 °C with the primary mouse monoclonal anti-ATM antibody, clone Y170 (Ab32420, Abcam, Cambridge, UK), at a dilution of 1:100. A set of slides were incubated for 18 h at 4 °C with the primary mouse monoclonal anti-ATR antibody, clone 1E9 (H00000545-M03, Novus Biologicals, Cambridge, UK), at a dilution of 1:20. A set of slides were incubated for 45 min at 4 °C with mouse monoclonal primary antibody for c-MYC (Clone 9E100; Abcam Ltd) at a dilution of 1: 100.

Whole field inspection of the core was scored and intensities of nuclear staining were grouped as follows: 0 = no staining, 1 = weak staining, 2 = moderate staining, 3 = strong staining. The percentage of each category was estimated (0–100%). H-score (range 0–300) was calculated by multiplying intensity of staining and percentage staining. X-tile (version 3.6.1, Yale University, USA) was used to identify a cut-off for ATM protein expression. The percentage of positive cells was used, with a cut-off of < 25% cells being classed as low, and ≥ 25% as high for ATM protein level. X-tile (version 3.6.1, Yale University, USA) was used to identify a cut-off for protein expression. H-score of ≥ 60 was taken as the cut-off for high ATR expression. Assessment of MYC staining was estimated subjectively on intensity corresponding to negative, weak, moderate and strong nuclear and/or cytoplasmic staining. Dichotomisation of c-MYC protein expression was based on the mean resulting in negative/weak (MYC negative) and moderate/strong (MYC positive) groups, which were selected prior to analysis.

Data analysis was performed using SPSS (SPSS, version 17 Chicago, IL). Where appropriate, Pearson’s Chi-square, Fisher’s exact, Student’s t and ANOVA one-way tests were used. Cumulative survival probabilities were estimated using the Kaplan–Meier method, and differences between survival rates were tested for significance using the log-rank test. Multivariate analysis for survival was performed using the Cox proportional hazard model. The proportional hazards assumption was tested using standard log–log plots. Hazard ratios (HR) and 95% confidence intervals (95% CI) were estimated for each variable. All tests were two-sided with a 95% CI and a p value < 0.05 considered significant. For multiple comparisons, p values were adjusted according to Benjamini–Hochberg method [23].