Möllerström et al. BMC Cancer 2010, 10:296 http://www.biomedcentral.com/1471-2407/10/296 Table 2: Technical data of the laboratory procedure for each specific antibody Antibody AdipoR1 Adora1 BTG2 CD46 Antibody dilution 1:300 1:500 1:1000 1:40 Manufacturer Phoenix Pharmaceuticals Catalogue number H-001-44 Genway Biotech 18-461-10001 Genway Biotech 18-003-42396 BD Biosciences 555948 Type of antibody Rabbit polyclonal Rabbit polyclonal Rabbit polyclonal Mouse monoclonal Time of Pre-treatment incubation (RT) buffer visualization system 30 min 30 min 30 min 30 min Borgs decloaker Borgs decloaker S2367 S1699 LSAB LSAB Envision Envision Page 4 of 11 dance index (C-index) [14]. The time-dependent AUC characterises the temporal changes in predictive accu- racy. Concordance index offers an easy way to interpret global accuracy measure that varies between 0.5 and 1. A concordance index of 1 means that with 100% precision we can rank the patient's survival time given the recorded marker information. If the concordance index converges to 0.5 the ranking of survival times becomes more and more driven by chance, and becomes completely random at 0.5. To quantify the impact of a single marker on the predictive accuracy we removed one marker at the time from the final model and refitted a Proportional Hazards Model and re-estimated the C-index. An estimation of the correlation between expression of the different pro- teins were performed using Pearson correlation. Results Of the 144 specimens present in the tissue microarray, 136-141 were interpretable for protein expression (Table 3). Excluded samples had few tumour cells, large tissue loss or affluence of necrotic tissue. BTG2 was expressed in both the cytoplasm and the cell membrane (Figure 1). In one sample, BTG2 showed expression in the cytoplasm and the membrane in one area and was expressed in the membrane exclusively in another area (Figure 2). The proportion of BTG2 protein expression was higher in tumours from 5-year overall survivors than among the tumours from deceased patients. The overall expression differed significantly between alive and dead patients (P = 0.026), although there was a stronger association with membrane specific expression (P = 0.013) (Table 3). Kaplan-Meier curves visualize the difference in overall survival between patients with tumours positive versus negative for overall and cell membrane specific BTG2 expression in Figure 3. Moreover, the difference between the curves was significant using the Breslow-Wilcoxon test [12] for both overall BTG2 (P = 0.011) and cell mem- brane expression (P = 0.015). Cytoplasm or cell mem- brane expression of BTG2 was observed in 78% of the samples and cell membrane specific expression in 39% of the samples. None of the remaining three analysed pro- teins (ADORA1, ADORA1 and CD46) showed a statisti- cally significant difference in expression between alive and dead patients in this study (Table 3). The ADIPOR1 protein was expressed in the cytoplasm in 18% of the samples. The majority of the positive samples showed primarily granular staining (Figure 4a). Approximately 24% of the samples were positive for ADORA1 staining, also displaying primarily granular staining in the cyto- plasm (Figure 4b). Fourteen percent of the samples expressed CD46 in the cell membrane (Figure 4c). The multivariate analysis showed that the model con- taining BTG2 expression had better predictive power than the model built on current classical pathological markers (Table 4). The BTG2 model revealed a C-value of 0.781 compared to the slightly lower C-value of 0.772 for the model of currently used markers. If only the markers displaying statistical significance are used from the model of current prognostic markers (HER2 expression and patient age), a C-value of 0.739 was achieved. Figure 5 shows the classification accuracy of a Cox-regression model based on the current markers compared to the classification accuracy of a new model based on the new markers considered in the present study. Over the whole time-span considered the new markers offer superior classification accuracy. For both the new markers and the old ones the classification accuracy shows a slight decreasing trend with time. The strongest correlation of protein expression were between ADIPOR1 and ADORA1 (k = 0.749) and between BTG2 overall expres- sion and BTG2 cytoplasm expression (k = 0.723) (Figure 6). Discussion In the present investigation, the expression of four pro- teins (ADIPOR1, ADORA1, BTG2 and CD46) was stud- ied by IHC using tissue microarrays. The aim was to evaluate the association between protein expression of these four genes and 5-year overall survival in breast can- cer patients. Protein expression of BTG2 was found to be