Jung et al. BMC Cancer 2012, 12:382 http://www.biomedcentral.com/1471-2407/12/382 Page 6 of 11 Figure 2 Example of a recurrently altered region on 17q12. The bottom plot represents the copy number profile of 48 breast cancers around 7q12 as a heat map. Intensity ratios are plotted in different color scales, reflecting the extent of genomic gains (red) and losses (green), as indicated on the reference color bar. The upper plot illustrates examples of intensity ratios of individual breast cancers. The red zone represents RARs defined using our criteria (frequency > 30% and P < 0.05; NEXUS software v3.1). X-axis, genomic position (Mb); Y-axis, Log2Ratio. RARs associated with prognosis in EBC Univariate survival analysis was performed to screen RARs that have potential implications on patient sur- vival. Univariate analysis was also performed to identify clinicopathologic features (age at diagnosis, stage, ER, PR, and HER2) for inclusion as covariates for Cox regres- sion. The event was defined as a death within ten years of the diagnosis. Among the genetic and clinical factors, RAR-G12 (16p11.2) and RAR-G13 (17q12) were signifi- cantly associated with poor survival in the discovery set of 48 EBCs (P = 0.0186 and P = 0.0186, respectively) (Figures 4A and B). The role of combinations of the 23 RARs and their association with poor survival was also explored, as described in the Methods. As a result, eight com- bination rules were found to be potentially associa- ted with death events (Laplace accuracy score >0.75) (Additional file 1: Table S6). All eight poor survival- associated rules contained ‘RAR-G12 and RAR-G13 positives,’ suggesting that the co-occurrence of these two alterations may affect EBC prognosis. Indeed, on univariate survival analysis, RAR-G12 and −13 double- positives had a significantly worse prognosis compared with the prognosis of others (P = 0.0001) (Figure 4C). Among the clinicopathologic features, none of them was associated with survival. Replication of the poor prognosis-associated RARs In order to validate the prognosis-associated RARs iden- tified in the discovery set, a genomic qPCR system was designed to target the NUPR1 gene located in RAR-G12 and the ERBB2 gene located in RAR-G13 (Figure 5A). For independent validation, the DNA extracted from FFPE samples of 97 EBCs (replication set) was used. On univariate survival analysis of the replication set, patients positive for RAR-G13 (ERBB2) were found to have a sig- nificantly poorer survival than patients without the ERBB2 gain (P = 0.0038) (Figure 5B). Patients positive for RAR-G12 (NUPR1) also showed poorer survival than negative patients, but only with borderline significance (P = 0.0839) (Figure 5C). As expected, the patients posi- tive for both NUPR1 and ERBB2 gains had a significantly poorer prognosis than patients in the replication set that did not have this combination (P = 0.0014) (Figure 5D). When survival curves were compared based on a more detailed RAR status (positive for both RAR-G12 and −13, positive for either RAR-G12 or −13, and negative for both), the survival probabilities among the three