Zhou et al. BMC Cancer 2010, 10:653 http://www.biomedcentral.com/1471-2407/10/653 risk of disease recurrence, distant metastasis and shorter survival. The poorer outcome of patients with basal-like tumors has been shown in different patient populations [13-17]. However, the prognosis of basal-like DCIS still unknown. In this study, our aim was to investigate the prognosis of basal-like DCIS compared to other subtypes in a large population-based cohort. To classify the DCIS tumors, we used immunohistochemical (IHC) markers that have previously been used as a surrogate marker for the gene expression profiles [16-21]. Livasy et al., have earlier shown that IHC can be used for the identi- fication of basal like DCIS [18]. We identified the sub- types of DCIS in tumors from a cohort of 458 women with tumor material assembled in tissue micro arrays (TMA) [22]. To our knowledge, this study is the first to examine the prognosis of DCIS in relation to different molecular subtypes defined by IHC. Methods Patients We recruited all 458 women who were diagnosed with a primary DCIS between 1986 and 2004 in Uppland and Västmanland, Sweden. TMA construction H&E sections from all eligible cases were reviewed to select the area from which the cores for the TMAs would be taken. Prior to the TMA construction, all primary DCIS cases were histopathologically re-evaluated by one pathologist (KJ). Two cores of 1.0 mm in diameter were mounted into the recipient TMA blocks using a manual arraying device (MTA-1, Beecher Inc, WI, USA). The concordance of IHC staining between original whole sec- tion slides and TMA-slides and between biopsies from the same lesion in DCIS has earlier been evaluated in a subset of the cohort [23,24]. In total, ten TMA blocks were constructed from 458 cancer samples. IHC and silver-enhanced in situ hybridization (SISH) We performed IHC for estrogen receptor (ER), proges- terone receptor (PR), human epidermal growth factor receptor 2 (HER2), cytokeratin 5/6 (CK5/6) and epider- mal growth factor receptor (EGFR) on 4 μm paraffin sections cut from the TMAs. Immunostains for each marker were performed on a Dako Autostainer (Dako Corporation). IHC was conducted according to estab- lished protocols. Appropriate positive and negative con- trols were included in all staining runs. HER2 SISH was performed on an automated instru- ment, Ventana Benchmark (Ventana Medical Systems, Tucson, AZ), as per the manufacturer’s protocols for the INFORM HER2 DNA probe and chromosome 17 probes. Testing for the HER2 gene and chromosome Page 2 of 8 17 was performed on sequential sections. Both probes are labeled with dinitrophenol. Denaturation occurred on the instrument with enzyme digestion in protease 3 for 8 minutes. The detection system used a multimer labeled with goat antirabbit antibody horseradish peroxi- dase as the linking step. Visualization occurred with the sequential addition of silver acetate as the source of ionic silver, hydroquinone, and hydrogen peroxide to give a black metallic silver precipitate at the probe site. Counterstaining was performed with hematoxylin II on the instrument. The time taken for the complete run is 6.5 hours. Both HER2 and chromosome 17 detection were performed on the same slide run. Gene amplifica- tion was assessed using the American Society of Clinical Oncology/College of American Pathologists guideline and Australian HER2 Advisory Board criteria for single HER2 probe testing (diploid, 1 to 2.5 copies/nucleus; polysomy >2.5 to 4 copies/nucleus; equivocal, >4 to 6 copies/nucleus; low-level amplification, >6 to 10 copies/ nucleus; and high-level amplification >10 copies/ nucleus) and for dual HER2/CHR17 probe testing (non- amplified ratio <1.8; equivocal ratio, 1.8 to 2.2; gene amplification, >2.2). The status of HER2 protein expres- sion was assessed relying on SISH. For those cases on which SISH failed, the expression was based on IHC. Scoring and classification Stained TMA slides were scanned (ScanScope® XT, Aperio, USA) for evaluation of expression of ER, PR, HER2, CK5/6 and EGFR by ImageScope (Aperio, USA). Tumor cells that showed nuclear staining for ER or PR (> 10% of tumor cell nuclei staining) were considered ER or PR-positive respectively. Using the HerceptTest™- classification system, tumors were considered HER2 positive if the score was 3+. Cases were considered CK 5/6-positive or EGFR-positive if any cytoplasmic and/or membranous staining was detected in the tumor cells, even if focal. These latter IHC criteria are similar to those previously used for scoring these markers in inva- sive breast cancers [18,22,25,26]. If only one core included enough tumor tissue this was used for classifi- cation but at least 200 cells had to be present. Each tumor marker was scored by one single observer; ER, PR and HER2 by WZ, EGFR and CK5/6 by CJ and SISH by RMA. For this study, the IHC subtype definitions used were the refined definitions used in the analysis of invasive breast cancer: basal-like (ER-, HER2-, EGFR+ and/or cytokeratin 5/6+) [17]. Cases classified as negative for all the above markers were defined as unclassified is this study. Cases with missing data, due to lack of tumor tissue in the TMAs, were excluded from the further analyses. In the survival analyses we compared the basal-like group with all other DCIS together.