BMC Cancer 2008, 8:36 http://www.biomedcentral.com/1471-2407/8/36 It is not surprising to find an inverse relationship between expression of COX-2 and PPARγ, given their respective biologic functions. In invasive breast cancer, PPARγ mRNA is significantly decreased compared to normal tis- sues[52,53] and COX-2 levels have been shown to be upregulated in breast cancer in multiple studies[54,55]. This inverse relationship has been demonstrated previ- ously in invasive carcinoma[52]. To our knowledge this is the first time this relationship has been shown in DCIS. Evidence for a coordinated relationship between COX-2 and PPARγ comes from studies that showing activation of PPARγ suppresses transcriptional activation of COX-2 in cell culture models[41]. Another theory for this inverse relationship has to do with one of the products of consti- tutive Cyclooxygenase-1, PGJ2, a PPARγ ligand, which is downregulated in a paracrine fashion by PGE2, the major product of inducible COX-2[56]. We devised a scoring system, combining COX-2 and PPARγ status with size and grade, two important prognostic factors in DCIS. The limitations of our study include the relatively small size, which was related to the need for tissue from the time of the initial diagnosis, limiting eligibility to patients hav- ing their initial surgery at Northwestern Memorial Hospi- tal. Tumor size and grade, both of which have been found to predict recurrence risk in other studies, demonstrated a similar trend in this study, but were of borderline signifi- cance, most likely related to the small size of the study. We found that 25% of our patients had grade 3 DCIS, whereas the proportion of grade 3 DCIS in published series ranges from 28% to 50% [57-59] or higher [29]. Since grading of DCIS was not uniformly practiced at NMH over the course of the study, we reviewed grade on all study subjects, based on the TMA core samples. It is possible that the evaluation of DCIS grade on the TMA cores accounted for the high number of grade 2 cases, since heterogeneous distribution of necrosis in the DCIS lesion may not have been captured in the TMA cores, leading to the misclassi- fication of some grade 3 lesions. This may also have reduced our power to detect the effect of grade on recur- rence risk, and the high proportion of HER-2/nue positiv- ity among grade 2 lesions may be explained on the same basis. However, we do see the expected inverse relation- ship between hormone receptor positivity and HER-2/neu positivity [60], which is highly significant for progester- one receptor, which tends to validate our HER-2/neu scor- ing. Adjuvant therapy parameters such as tamoxifen and radi- otherapy, were also not associated with recurrence, and interactions of these important variables with expression of candidate biomarkers such as COX-2 and PPARγ can- not be meaningfully explored. However, it is not surpris- ing that neither the radiation, nor the tamoxifen benefit seen in large randomized trials is recapitulated in this small case-control study, where the use of radiotherapy (65 vs. 61%) and tamoxifen (32 vs.32%) was equally fre- quent among cases and controls. Since radiotherapy and tamoxifen exposure were not parameters under study in this case-control design, this balance between cases and controls is an advantage in terms of examining the effects of other parameters. Our rate of invasive recurrence was somewhat lower than reported in large trials [48], not unexpected with a single institution sample where the majority of patients received radiotherapy, and consistent with the proportion of invasive recurrences seen in the radiotherapy arm of NSABP B-18 [61]. Another challenge was the missing data on approximately 30% of tissue cores from the tissue microarrays. We chose to construct tissue microarrays rather than using whole sections because of the greater efficiency of this approach, in order to evaluate multiple biomarkers in this explora- tory study. Our success in retaining target tissue through multiple sections of the TMA blocks was approximately 70%, which compares favorably with other reports in the literature regarding target retention in TMAs of pre-inva- sive lesions [62-64]. The retention rate for pre-invasive lesions of interest in these studies ranged from 52%[64] to 79% [63]; Yang et al noted that target retention for non- invasive targets (DCIS and terminal duct lobular units) remained at about 79% for the first 30 sections, and then declined to 64% at section 40 and 52% at section 100. We noted the same trend, with attrition of target retention as the sectioning progressed through a TMA block. We used a core size of 1.5 mm, and achieved a target retention rate close to that of Yang et. al., who advocates a core size of 2 mm. Target retention was not related to lesion size, in agreement with Yang et. al. [63] We did not observe an effect on recurrence risk of the additional markers that we tested (ER, PR, HER-2/neu, cyclin D, p53, p21). This is consistent with findings from two other studies of multiple biomarkers and recurrence risk, which included several of the markers that we have examined [58,60]. These authors found no significant impact of ER, PR, or HER-2/neu expression on recurrence risk in multivariate analyses, although Ringberg et. al. did observe a significant relation between a biologic index consisting of seven different markers and risk of DCIS recurrence on women who did not receive breast irradia- tion. In another study, absence of HER-4 expression was found to be a predictor of recurrence risk [65]. However, our biomarker findings are validated by the observation of many of the expected inter-relations between various biomarkers, as shown in Table 5. Proteins along the ER axis were significantly correlated: ER with PR, cyclin D1 and p21; PR with p21, and inversely with HER2/neu; cyc- lin D1 with p21. The lack of association of PPARγ with ER or PR is likely related to the small number of PPARγ posi- Page 8 of 11 (page number not for citation purposes)