Albergaria et al. BMC Cancer 2011, 11:299 http://www.biomedcentral.com/1471-2407/11/299 tumours into the different breast cancer subtypes, namely Luminal, HER2-OE and Triple-negative (TNBC). Statistical analysis Statistical analysis was performed using SPSS statistics 17.0 software (SPSS Inc., Chicago, IL, USA). Continuous variables were presented as the mean standard deviation, and categorical variables were presented as number (percentage). Mean differences for continuous variables such as tumour size was performed using unpaired T- test with a 95% confidence interval. Association between different expression subtypes and the clinicopathological features were assessed by Pearson correlation and chi- squared tests. Survival curves were estimated by the Kaplan-Meier method using the long-rank test to assess significant dif- ferences in survival. Cox regression models were fitted to estimate hazard ratios [(HR, 95% confidence interval (CI))] for the classical prognostic factors that constitute the Nottingham Prognostic Index, namely, tumour size, tumour grade and lymph-node status (LNS). Similarly, Cox regression was used to calculate HR for the NPI scoring in different expression subtypes. For all analysis, a significant level of 5% was considered. Results NPI as a predictor of survival in breast cancer subtypes Using the follow-up survival data from 455 breast can- cer patients, we stratified the breast-cancer-specific out- come, according with the NPI status. The NPI ranged from 2-8.4 (mean 4.8). In the whole series, and in line with what has been shown, NPI was able to stratify breast cancer patients into good, moderate and worse prognosis in terms of overall and disease-free survival (Figure 1A and 1B, respectively). The outcome stratifica- tion ability of NPI was also tested in specific breast can- cer expression profiles. The same strength of statistical significance was also observed in the subset of luminal tumours (Figure 1C and 1D). An interesting finding was the one observed in the HER2-OE subgroup, where none of the cases was scored as NPI < 3.4, reinforcing the poor prognosis of this breast cancer subtype. Here, although the Kaplan-Meier curves showed a clear differ- ence between the survival of NPI > 5.4 and 3.4 < NPI < 5.4 patients, especially concerning overall-survival, the statistical value was not significant (Figure 1E and 1F). In the cohort of TNBC, unequivocal patient outcome stratification was draw by the NPI status. Notably, only one case was scored with NPI < 3.4, confirming the aggressive behaviour pattern of these tumours. In fact, in both overall and disease-free survival Kaplan-Meier curves (Figure 1G and 1H), TNBC patients with NPI > 5.4 are clearly separated from the good/moderate out- come curve, reinforcing not only the worse survival of Page 4 of 10 the high-scored-NPI TNBC patients, but also suggesting the value of NPI as a predictor of survival in TNBC patients. Tumour size, histological grade and LNS in Luminal, HER2-OE and TN breast cancer Each clinical-pathological feature that constitutes the NPI equation was explored in the context of luminal, HER2-OE and TNBC groups. The mean tumour size was calculated for the three breast cancer subgroups. As shown in Table 2 the mean tumour size of TNBC was visibly higher compared to the average size of HER2-OE and luminal tumours, which displayed the lowest mean size. Using unpaired comparison analysis (T-test, 95% CI), to evaluate the significance of the difference found in tumour size between TNBC and each of the other subtypes, we showed that tumour size of TNBC was sta- tistically different from the luminal subtype (p < 0.001) and from the HER2-OE subtype (p = 0.05). Concerning histological grade, Chi-square test showed that there was a significant association between grade and the dif- ferent molecular subtypes (p < 0.0001). The Table 2 shows that 78% and 71% of the HER2-OE and TNBC patients, respectively, were high-grade tumours, while the frequency distribution of tumour grade among lumi- nal subtype tumours was rather homogeneous. Interest- ingly, a non-statistically significant association between LNS and the different expression subtypes was observed, showing that the occurrence of lymph-node metastiza- tion is a similarly frequent event across all the molecular subtypes, but most importantly, that the extension of these lymph-node involvement is as relevant in TNBC tumours (26.4%) as it is for luminal (29.4%) or HER2- OE breast cancers (33.3%) (Table 2). High-scored-NPI lesions and its relation with tumour size, grade and LNS in a subset of TNBC Using only the cohort of 164 TNBC patients, we evalu- ated the association of each of the Nottingham Prognos- tic components to the NPI augmentation. A boxplot graphic (Additional file 1) was draw to show the signifi- cant association of tumour size, histological grade and lymph node status to high scores of NPI in TNBC. Using Chi-square test we observed a strong association between larger tumours (p < 0.0001), displaying high histological grade (p < 0.0001) and with extensive lymph node invasion (p < 0.0001), with the worst outcome group, represented by NPI > 5.4 (Additional file 1). Besides the evidence that nearly 72% of TNBC are grade III tumours, therefore clearly contributing for a high NPI, it is however important to stress that the contribu- tion of LNS also clearly associates with high NPI. More- over, similarly with what was shown for tumour larger than 5 cm, all the TNBC with more than 3 metastatic