BMC Cancer 2008, 8:36 http://www.biomedcentral.com/1471-2407/8/36 which is associated with an less than 2% ipsilateral recur- rence risk to breast conservation therapy (BCT) and endo- crine therapy where ipsilateral recurrence rates vary from 10%–40% [3,4]. Because of the increasing number of women being diagnosed with DCIS, the identification of those patients at high risk for a recurrence (invasive and non-invasive) is of increasing importance. Morphological factors such as grade and the presence of comedo-necrosis have been used thus far to predict recurrence, but lack sen- sitivity and are subject to variability of interpretation[5]. A number of molecular markers have been identified in invasive breast cancers that have predictive as well as prog- nostic value. Well known markers include the estrogen receptor α (ER) and progesterone receptor (PR) which are associated with improved outcomes and are predictive of response to tamoxifen therapy. HER-2/neu is a tyrosine kinase receptor related to the epidermal growth factor receptor family. Overexpression of HER-2/neu in invasive carcinoma is correlated with decreased relapse-free and overall survival, and resistance to hormonal and cytotoxic therapy[6]. In DCIS, HER-2/neu has been linked to adverse clinicopathologic characteristics including higher histological grade, comedo-necrosis and younger age. Additionally, HER-2/neu is found more frequently in DCIS associated with invasive cancer [7-10]. Cyclin D1, p53, and p21, are all proteins involved in cell cycle regu- lation and have prognostic value in invasive breast cancer as well [11-14]. All of these markers have been identified in DCIS recently, but their biologic significance is not fully understood, and long-term data relating these mark- ers to recurrence risk in DCIS are extremely scant[7,15- 17]. Two novel molecular markers are cycloxygenase-2 (COX-2) and peroxisome proliferator activated-receptor gamma (PPARγ). COX-2 overexpression has been shown to be up-regulated in many neoplastic and pre-neoplastic lesions including invasive breast cancer and DCIS [18-20]. COX-2 is the inducible form of cyclooxygenase, the rate- limiting enzyme for prostaglandin synthesis. PPARγs appear to play a role in the regulation of many physio- logic functions such as lipid metabolism, atherogenesis, inflammation and cellular differentiation[21,22]. PPARγ expression has been studied in invasive cancers but not in DCIS thus far. We have conducted an exploratory study to identify those molecular markers associated with increased or decreased ipsilateral in-breast recurrence after diagnosis of DCIS, in a population with long term follow-up; we opted for a case-control design because of the relative rarity of DCIS recurrence. Methods Following IRB approval of the study, women diagnosed with DCIS and treated with breast conservation, who sub- sequently developed ipsilateral recurrence of ductal carci- noma, either invasive or non-invasive, (cases) were identified from the Northwestern Memorial Hospital tumor registry and the Lynn Sage Breast Center database from 1992–2002. We have used the term "breast conserv- ing therapy" to include all women treated without mastec- tomy; of these 60% also received breast radiotherapy, which was not a standard component of BCT for DCIS during the earlier part of the study. Similarly, tamoxifen therapy was introduced into the treatment of DCIS over this time period, and just 32% of our study subjects were treated with tamoxifen. Only women who had their orig- inal surgery at Northwestern Memorial Hospital were included, because the tissue blocks at primary diagnosis were needed to create tissue microarrays. The recurrences were in the same region of the breast as the original DCIS, and were characterized as "local recurrences" by the treat- ing physicians. These cases were age-matched (within two years) to a set of control subjects diagnosed with DCIS and treated with breast conservation in the same calendar year, who did not recur over the same time period (con- trols). Matching for tumor size and grade was not possible because of the relative scarcity of younger women with DCIS and the limited number of women undergoing breast conservation early in study period. Clinical infor- mation recorded on chart review included age, race, age of menarche, first live birth, time to recurrence, type of sur- gery, the type of recurrence (DCIS or invasive cancer), use of adjuvant therapy in the form of radiation or endocrine therapy (Tamoxifen) and current follow-up status. Hema- toxylin and eosin stained paraffin sections of the case-con- trol groups were evaluated for size of DCIS and margin status. The grade of DCIS was classified according to the criteria of Page and Lagios [23]; since grading was not uni- formly practiced during the early part of the study period, and complete sets of slides were not available on every patient, we elected to review the grade on the TMA core samples for all study subjects. Free margins were defined as a tumor clearance of 2 mm or greater on review. Tissue microarray preparation and immunohistochemical analysis Areas of morphologically representative, non-necrotic sites in the tumor sections were chosen to prepare tissue microarrays (TMA). From each tumor block, two 1.5-mm cores were obtained using tissue arraying instrument (Beecher Instruments; Silver Spring, MD) TMA blocks were prepared using previously described protocol by Kononen et al[24]. Each TMA block consisted of 8–17 cores per block. The coordinates and clinicopathologic data were documented to identify the cores. Sections of 4- μm thickness were used for immunohistochemical analy- sis of markers with H&E-stained sections as morphologi- cal references for each core. Approximately 30% of the DCIS lesions were not represented on the TMA sections Page 2 of 11 (page number not for citation purposes)