Albergaria et al. BMC Cancer 2011, 11:299 http://www.biomedcentral.com/1471-2407/11/299 Patients’ ages ranged from 28 to 92 years old, mostly submitted to therapeutic surgery and/or surgery plus chemotherapy in the case of lymph node-positive patients. The formalin-fixed paraffin-embedded histolo- gical sections were reviewed and the diagnoses con- firmed by two trained pathologists (FS and FM). The tumours were characterized for clinical and pathological parameters - namely age, tumour size, lymph-node sta- tus, and histological grade (Table 1). Whenever was pos- sible, NPI was calculated for each of the patients by using the following equation: NPI = 0.2 × tumour size (cm) + grade (1-3) + lymph node status (1-3) [47]. Patient follow-up information was available for 455 cases, ranging from a 1 to 120 months after the diagno- sis. Overall and disease-free survival time was defined as the time from the date of surgery to the date of death or to the date of breast cancer derived relapse/metasta- sis, respectively. An additional cohort of 168 TNBC patients was included in this study and analysed separately. This cohort resulted from the combination of the 89 triple negative tumours from the former series, plus 25 cases from the Divino Espirito Santo Hospital (Ponta Del- gada-Portugal), 29 cases from the Federal University of Santa Catarina (Florianopolis-Brazil) and 25 cases from the São João Hospital (Porto-Portugal). All these TNBC specimens were evaluated and classified accordingly with the same criteria and by the same panel of patholo- gists. Patients followed the same therapeutic regimen design as the general series. This study was conducted under the national regula- tive law for the handling of biological specimens from tumour banks, being the samples exclusively available for research purposes in retrospective studies. Tissue microarray construction and immunohistochemistry Representative tumour areas were selected on haema- toxylin-eosin-stained sections. At least two tissue cores (0.6 mm in diameter) were obtained from each selected specimen and deposited into a recipient paraffin block, using a tissue microarray (TMA) workstation (Manual Tissue Arrayer, Beecher Instruments, Inc.). The TMA blocks were designed and built as previously described [20] and non-neoplastic tissue cores were included as controls. In order to classify all breast cancer tumours according with the molecular subtype, immunohistochemistry was performed and the expression of breast cancer biomar- kers [49], namely the hormonal receptors ER and PR, the tyrosine kinase receptors HER2 and EGFR, the basal cytokeratins CK5 and CK14, and also P-cadherin and vimentin were evaluated. Immunohistochemical expres- sion was detected using HRP polymer (Cytomation Page 3 of 10 Table 1 Patients characteristics and tumour parameters Variable (N = 467) Age at Diagnosis, years Mean and standard deviation Range Tumour Size (cm) Mean and standard deviation Range T1: < 2 cm T2: 2-5 cm T3: > 5 cm Not assessed Lymph Node Invasion Present Absent Not assessed Histological Grade Grade I Grade II Grade III Not assessed Oestrogen receptor Positive Negative Not assessed Progesterone Receptor Positive Negative Not assessed HER2 Positive Negative Not assessed Nottingham Prognostic Index NPI < 3.4 3.4 ≤ NPI ≤ 5.4 NPI > 5.4 Not assessed Molecular Subtype Luminal HER2 Over-expressing Triple Negative Data 59 ± 13 64 (Min 28; Max 92) 3.110 cm ± 2.00 cm 15.6 (Min 0.4; max 16) 101 (24.7%) 245 (59.9%) 63 (15.4%) 58 207 (56.6%) 159 (43.4%) 101 81 (18.3%) 135 (30.5%) 227 (51.2%) 24 309 (66.5%) 156 (33.5%) 2 228 (48.9%) 238 (51.1%) 1 68 (14.7%) 395 (85.3%) 4 99 (24.4%) 188 (46.4%) 118 (29.2%) 62 343 (73.6%) 33 (7.1%) 90 (19.3%) Envision System HRP, DAKO, Carpinteria, CA), accord- ing with the manufacturer’s instructions. Both methods used diaminobenzidine as chromogen. Immunohistochemical evaluation The expression of ER, PR, HER2, EGFR, CK5, CK14, P- cadherin and vimentin was evaluated according with the grading systems previously described [49]. These immu- nohistochemical results were used to classify the