Jung et al. BMC Cancer 2012, 12:382 http://www.biomedcentral.com/1471-2407/12/382 HER2 amplification/overexpression is known to occur at an early developmental stage of ductal carcinoma in situ (DCIS). Loss of 16q, where potential tumor suppressor genes such as E-cadherin (CDH1) and CDH13 are located, is also known to be a major event in low-grade invasive ductal carcinoma [12,13]. Especially, recent larger-scale studies have elucidated the molecular com- plexity of breast cancer and suggested novel genetic sub- groups [14-18]. However, since most of them studied Caucasians or Hispanics, the profiles of chromosomal alterations and their biological implications in Asians are relatively less well studied. In this study, we aimed to describe commonly occur- ring chromosomal alterations in EBC (stage I and II) and to explore the implications of recurrently altered regions (RAR) on patient prognosis. For this purpose, we analyzed DNA copy number alterations (CNAs) across the whole genome using oligoarray-comparative genomic hybridization (CGH) in a discovery set of EBC patients. RARs in the discovery set that were found to be significantly associated with prognosis were validated in an independent replication set. Our results will con- tribute to a better understanding of early tumorigenesis in breast cancer and will help to predict the prognosis of EBC patients. Methods Patients and tumor specimens As a discovery set for the whole genome array-CGH ana- lysis, frozen tumor tissues were obtained from 48 EBC patients who underwent surgical resection at Dankook University Hospital in Cheonan, Korea (from 1998 to 2002). As an independent replication set, 97 formalin- fixed, paraffin-embedded (FFPE) EBC tissue samples (from 1996 to 2002) were obtained from Seoul St. Mary’s Hospital, Korea. Patient survival status was obtained in 2010 from the Korean Central Cancer Registry, Ministry of Health and Welfare, Korea. All breast cancers were stage I, IIA, or IIB. This study was performed under ap- proval from the Institutional Review Board of the Catholic University Medical College of Korea (CUMC06U015). Tumor stage was determined according to the standard AJCC guidelines for tumor-node-metastasis classification (sixth edition). Clinicopathologic characteristics of the study subjects are summarized separately for the discovery and replication sets in Table 1. Hormone receptor status for ER, PR and HER2 was obtained through a medical rec- ord review and for the cases without the hormone recep- tor status, immunohistochemical (IHC) staining for ER, PR and HER2 was performed. Based on the IHC measure- ment, breast cancer cases were categorized into four dif- ferent molecular subtypes as described elsewhere: luminal type A (ER + and/or PR +, HER2 -: Luminal A), luminal type B (ER + and/or PR +, HER2 +: Luminal B), Her2 Table 1 General characteristics of the study subjects Characteristics Discovery set (n = 48) Replication set (n = 97) Age group < 50 years ≥ 50 years Stage Stage I Stage II Stage IIA Stage IIB Positive Negative ER status PR status Positive Negative HER2 status Positive Negative Subtype Luminal A Luminal B HER2 TNBC 26(54.2%) 48(49.5%) 11(22.9%) 37(77.1%) 26 11 25(52.1%) 23(47.9%) 35(72.9%) 13(27.1%) 11(22.9%) 37(77.1%) 29(60.4%) 10(20.8%) 1(2.1%) 8(16.7%) Page 2 of 11 22(45.8%) 49(50.5%) 25(25.8%) 72(74.2%) 50 22 54(55.7%) 43(44.3%) 59(60.8%) 38(39.2%) 23(23.7%) 74(76.3%) 53(54.6%) 16(16.5%) 7(7.2%) 21(21.6%) overexpressed (ER - and PR -, HER2 +: HER2), and triple negative (ER -/PR -/HER2 -: TNBC) [19]. For array-CGH analysis, 10-μm-thick frozen sections of tumor cell-rich areas (>60%) were microdissected. Gen- omic DNA was extracted from these sections using a DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany). For genomic real-time quantitative PCR (qPCR) analysis, 10-μm-thick paraffin sections of tumor cell-rich areas (>60%) in the replication set were microdissected. After paraffin removal, genomic DNA was extracted using a DNeasy Blood & Tissue Kit (Qiagen). Genomic DNA from a healthy female individual was used as the normal reference for all array-CGH experiments. Genomic DNA extracted from the blood of a Korean female individual without breast cancer was used as universal normal reference for all the array-CGH experiments. Array-CGH and data processing For array-CGH analysis, 30K whole-genome human oli- goarrays (Human OneArray™; Phalanx Biotech, Palo Alto, CA) were used. Oligoarray-CGH was performed as described elsewhere [20]. In brief, 1 μg of genomic DNA from tumor tissue was labeled with Cy3-dCTP. The reference DNA was labeled with Cy5-dCTP (GeneChem,