0.41 1.01 0.61 0.32 0.19 0.73 1.80 0.41 1.06 0.61 0.32 0.19 2.65 2.48 12.39 4.34 2.13 1.17 8.90 15.98 2.44 13.04 4.24 2.15 1.14 20.15 0.986 0.048 0.325 0.690 0.105 0.141 0.003 0.993 0.040 0.343 0.709 0.094 0.0001 Jung et al. BMC Cancer 2012, 12:382 http://www.biomedcentral.com/1471-2407/12/382 Table 4 Results of multivariate aalysis Hazard ratio 95% confidence interval P value* Lower Upper RAR-G12*RAR-G13 RAR-G12*RAR-G13: NUPR1 (RAR-G12) and ERBB2 (RAR-G13) copy number gain double-positives. *Significant result (P < 0.05). that these copy number alterations were acquired at an earlier stage of EBC. In particular, 6 of the 15 earlier event RARs, RAR-G2 (1q21.2-q21.3), RAR-G7 (8q24.13), RAR-G8 (8q24.13-21), RAR-G9 (8q24.3), RAR-G10 (8q24.3), and RAR-L1 (8p23.1-p22), appeared in over 50% of cases. Some genes located in these six RARs have been suggested to be involved in early breast tumorigen- esis. For instance, the PTK2 gene located on 8q24.3 (RAR-G9) is a member of the focal adhesion kinase (FAK) subfamily of protein tyrosine kinases. Overexpres- sion of FAK was suggested to be an early event in DCIS tumorigenesis [26]. Although the protein levels of poten- tial cancer-related genes in these six highly common loci were not examined in this study, our data suggest that the six alterations may be commonly occurring genetic events in the initial stage of breast cancer development. Based on our findings, two RARs on 17q25 can be con- sidered relatively late events in breast tumorigenesis, since the RARs on 17q25 (RAR-G14 and -G15) were scarcely observed in stage I (<10%), but were quite fre- quent (>45%) in stage II. Interestingly, a copy number gain on 17q25.3 was reported to be one of the recurrence-associated chromosomal alterations in one previous report on Korean women with breast cancer [27]. When we assessed the prognostic implications of RARs, RAR-G12 (16p11.2) and RAR-G13 (17q12) were significantly associated with poorer prognosis in the dis- covery set. A number of cancer-related genes are located in these two RARs: NUPR1, MVP, MAPK3, FUS, and Variable Using each RAR Age Stage ER PR HER2 RAR-G12 RAR-G13 Age Stage ER PR HER2 Using RAR double-positives 1.01 3.54 1.63 0.82 0.48 2.55 5.36 1.00 3.73 1.60 0.84 0.46 7.31 Page 9 of 11 PYCARD are located in RAR-G12 while ERBB2, GRB7, and PPP1R1B are located in RAR-G13. Among these po- tential cancer-related genes, Nupr1 is known to interact with various molecules involved in cell cycle regulation, programmed cell death and transcription activity. For these reasons, Nupr1 is a potential molecular target in the development of anticancer drugs [28]. Although the NUPR1 gene has been suggested to be responsible for the growth and progression of many cancers including breast cancer [29,30], the prognostic implications of the NUPR1 gene in EBC have not been reported. Amplifica- tion and overexpression of the ERBB2 oncogene in RAR-G13 (17q12) is known to be associated with high recurrence rates and reduced breast cancer survival [31-33]. The frequent copy number gains (38%) and amplification (29%) of ERBB2 in this study are consist- ent with previous studies on breast cancer [11,34]. In a replication analysis by genomic qPCR, the prog- nostic implication of ERBB2 gain (RAR-G13) was suc- cessfully replicated in the larger replication set, but that of the NUPR1 gain (RAR-G12) was not. We hypothe- sized that the NUPR1 gain itself might not be an influen- tial alteration, but that EBC prognosis is more strongly affected by the co-occurrence of NUPR1 with a strong driver mutation (ERBB2). Association-rule mining results also supported the predictive power of their co- occurrence for poor prognosis. As expected, when these two RARs were combined and used as an independent factor, the hazard ratio increased in an additive manner. A stronger significance level was also achieved on Cox regression analysis compared with when only ERBB2 was used, which may reflect the multigenic nature of cancer. In this study, 191 high-level CNAs (158 amplifications and 33 HDs) were detected by array-CGH, and 5 of them were detected in more than 10% of the samples. A substantial number of the high-level CNAs overlap data- base of genomic variants (DGV, http://projects.tcag.ca/ variation/) entries and the copy number variants (CNVs) identified from Koreans [35]. Although the limitations of DGV are well known in terms of accuracy and overesti- mation, we cannot rule out the possibility that some high-level CNAs identified in this study are copy CNVs because we used DNA from a single individual as a uni- versal reference. All five of the common amplifications (observed in >10% of the samples) also overlap the CNV loci in DGV. However, four of them, except for one very small (0.02 Mb) amplification on 16p11.2, were reported to be amplifications or copy number gains in breast cancer by a recent high-resolution array-CGH analysis [15-17,36], suggesting that these four common amplifi- cations are likely CNAs. The amplification frequency of ERBB2 in this study was largely similar to the previous studies including Koreans [37-39].