BMC Cancer 2008, 8:36 http://www.biomedcentral.com/1471-2407/8/36 significant (p = 0.024). Interestingly, expression was lim- ited to grade 2 and 3 DCIS with a highly significant inter- action (p < 0.00001) between PPARγ and grade. The odds ratio for recurrence was 0.11 (95% CI 0.012–1.00, p = 0.050) indicating a protective effect of nuclear PPARγ against recurrence. There was no association between age or HER-2/neu status and PPARγ expression. The protective effect of PPARγ positivity was not changed in multivariate modeling with tamoxifen, radiation therapy, tumor size, estrogen receptor status and margin status. Among the group where PPARγ data was missing as a result of processing-related core loss, 12 samples were from con- trols and 6 were from cases. Similar to the core loss pattern seen with COX-2 staining, the tumor size in case lesions with core loss was similar to that of control lesions with core loss during PPARγ staining (6.8 mm, range 4–20 mm in cases and 8.7 mm, range 2–20 mm in controls). Results of combined analysis of Grade, COX-2 and PPARγ expression We performed a multivariate analysis including the four parameters which were significant at a p value of 0.1 or lower (size, grade COX-2, PPARγ). COX-2 expression con- tinued to be significantly associated with increased risk of any ipsilateral in-breast recurrence, with an OR of 7.89 (95%CI 1.7–36.2). Tumor size remained positively, but not significantly, associated with recurrence risk, with a 5% increase in risk for each mm increase in size (OR 1.05, 95% CI 0.97–1.1). The association of grade and ipsilateral breast recurrence also remains statistically non-signifi- cant, although the point estimate remained elevated, with an OR of 1.58 (95% CI 0.5–4.9). In this multivariate model, PPARγ expression remained negatively associated with recurrence, with an OR of 0.17 but this was no longer statistically significant (95% CI 0.02–1.8), (see Table 4). Associations between markers Relationships between the markers tested were explored to look for consistency with known correlations, and to Table 4: DCIS recurrence risk related to histologic grade, COX- 2 positivity, and PPARγ positivity. Univariate Analysis Size Grade COX-2 PPARγ Multivariate Analysis Size Grade COX-2 PPARγ OR 1.05 1.92 5.11 0.11 1.05 1.58 7.90 0.17 95% CI 0.99–1.10 0.95–3.87 1.7–15.5 0.01–1.0 0.97–1.15 0.50–4.93 1.72–36.23 0.06–1.84 p value 0.081 0.069 0.004 0.05 0.237 0.434 0.008 0.144 identify additional associations which may be of biologi- cal interest. These are shown in Table 5. COX-2 expression and PPARγ expression in DCIS had a significant inverse relationship, consistent with their opposite effects on recurrence risk As expected, estrogen and progesterone receptor positivity was highly and significantly correlated (p < .0001), and progesterone receptor expression dis- played a significant inverse relation with HER-2/neu pos- itivity. The expected association between ER positivity and Cyclin D1 was also observed [27,28], as was the positive association between ER and p21 positivity. [16]. There were weaker, although significant associations of p53 pos- itivity with ER and cyclin D1 positivity, which are of uncertain significance. Also noted was a strong and signif- icant association between p21 and cyclin D1 positivity, consistent with the suggestion that cyclin D1 expression may be indirectly induced as a result of the accumulation of wild-type p53 through p21 [16]. Discussion Our results show that strong cytoplasmic COX-2 expres- sion by immunohistochemistry is an independent predic- tor of increased risk of ipsilateral in-breast recurrence. Furthermore, even though the number of cases was small, COX-2 expression appears to be associated with the devel- opment invasive recurrence. The risk of recurrence associ- ated with strong COX-2 positivity was independent of tumor size, margin status, estrogen receptor positivity, use of radiotherapy, and tamoxifen use. This report is corrob- orated by a recent study, where the COX-2 positivity rate in recurrent DCIS (62%) was very similar to ours (67%) although COX-2 positivity by itself was not a statistically significant predictor of recurrence in that study (p = 0.08)[29]. Other studies have shown an association between COX-2 overexpression and an aggressive pheno- type in invasive breast cancer[30,31] and several studies suggest that COX-2 expression is associated with a worse prognosis [32-34]. Together, these findings are exciting for several reasons. COX-2 could potentially be incorpo- rated into the clinic as a prognostic marker for ipsilateral in-breast recurrence, especially invasive recurrence. Excel- lent antibodies exist for COX-2 staining, it is easily repro- ducible and scoring is straightforward. Prostaglandin E , 2 the primary product of COX-2, induces inflammation and can act as a mediator in signal transduction pathways that modulate cellular adhesion and cell growth[35,36]. COX- 2 inhibitors were being actively investigated in clinical tri- als for treatment of breast cancer and as chemopreventive agents in high risk women[37], until toxicity concerns interrupted many of these trials. Our results provide added incentive to develop and test new agents to target this pathway, known to be of importance in several epi- thelial malignancies. We found that roughly half of the ER positive DCIS lesions in our study overexpressed COX-2 protein, the rate limiting enzyme in prostaglandin synthe- Page 6 of 11 (page number not for citation purposes)