BMC Cancer 2008, 8:36 http://www.biomedcentral.com/1471-2407/8/36 either because no DCIS cells were present in a section or complete absence of a core secondary to tissue exhaustion in the block. We attributed this to the nature of many of the samples that contained small and/or scattered areas of DCIS in the donor blocks. Because of this sample loss, there are different samples sizes for each molecular marker. Molecular markers that have been identified in invasive cancer as having predictive and prognostic value were chosen for our study. These included estrogen receptor alpha (ER-α), progesterone receptor (PR), HER-2/neu, COX-2, PPARγ, cyclin D1, p53, and p21. For all of the markers tested, we used standardized automated immu- nohistochemical techniques (DAKO Autostainer) along with heat induced antigen retrieval. The clones, dilutions and manufacturers of the primary antibodies used for the study are shown in Table 1. Immunohistochemical scoring of cases was performed by a breast pathologist (LKD) with additional review of equivocal cases by a second pathologist (EL), and final scoring by consensus. All scoring was blinded to patient information, including case-control status. Scoring of each marker was also blinded to the scoring for other markers. Specific scoring systems were used for the vari- ous sets of markers studied. Nuclear markers were consid- ered positive when the fraction of stained nuclei was ≥ 10% (ER-α, PR, p53, p21 and cyclin D1), regardless of the intensity of the immunostain. HER-2/neu cores were scored using a standard 0,1+, 2+ and 3+ membrane stain- ing intensity scale; 3+ staining of > 10% cells was required for positivity. COX-2 staining was scored using a system similar to that described for COX-2 evaluation of invasive breast cancer on TMA sections[25]. A three-tiered system was utilized for staining intensity: negative, weak and strong staining. Staining was considered positive if strong cytoplasmic granular staining in > 10% of tumor cells was present. Table 1: Antibodies and scoring system used for analysis PPARγ staining was evaluated using a descriptive scoring system taking into account the possibility of nuclear and cytoplasmic staining patterns as previously described for PPARγ staining in invasive breast carcinoma[26]. We clas- sified cases as: cytoplasmic staining only, combined nuclear and cytoplasmic staining and nuclear staining only. Staining was considered positive if > 10% of tumor cells displayed nuclear staining. Examples of DCIS show- ing positivity for COX-2 and PPARγ are shown in Figures 1 and 2. If two cores from the same patient were found to have dis- cordant IHC results, i.e. one core was read as positive and one as negative, or one core was scored as +3 and the sec- ond core as +2, the positive result or the higher IHC score was used for the analysis. Statistical analysis All markers were compared between cases and controls. Since there were only eight invasive recurrences, we did not attempt subset analyses of invasive versus non-inva- sive recurrences. The term "recurrence" is therefore used to encompass both invasive and non-invasive recurrence. Each marker was classified as positive or negative based on the criteria described above and in Table 1. After descriptive statistical analyses, to assess the distribution of each marker, and to check for outliers, a univariate analy- sis was performed for each marker. Continuous and ordi- nal measures were compared between cases and controls using the paired t-test or the Wilcoxon signed rank test. Dichotomous measures were compared using the chi- square test. After univariate analysis, multivariate analysis using conditional logistic regression (for matched sam- ples) was used to identify the markers that demonstrated odds ratios that were different from unity, using a thresh- old p value of 0.1. All p values are two-tailed. The associ- ations between positivity of various markers was explored using Spearman's rank correlation. Antibody* ER-α PR HER-2/neu COX-2 PPARγ P53 P21 Cyclin D1 Dilution 1:300 1:400 1:200 1:100 1:50 1:200 1:50 1:300 Clone 1D5 PgR636 ** CX229 E8 DO-7 SX118 DCS-6 Method of scoring > 10% positive cells – positive, < 10%- negative > 10% positive cells – positive, < 10%- negative Scored as +1, +2 and +3, +3- positive, +1/+2 -negative Strong positive, weak positive, negative Nuclear, cytoplasmic, nuclear+cytoplasmic, negative > 10% positive cells – positive, < 10% -negative > 10% positive cells – positive, < 10% -negative > 10% positive cells – positive, < 10% -negative *All antibodies were purchased from Dako Cytomation except COX-2 (Cayman Chemicals), PPARγ (Santa Cruz Biotechnologies), Cyclin E (Lab Vision Corporation) and CXCR4 (R&D Systems) ** DAKO polyclonal antibody A0485. Page 3 of 11 (page number not for citation purposes)