We, and many others, have shown that image analysis of nuclear features (size, shape, chromatin texture, and ploidy) can be prognostic for survival of breast cancer patients (Chapman et al., 2007). without, radiation (Zellars et al., 2000). In breast carcinoma patients having lumpectomy and radiation therapy, overexpression of mutant p53 protein was reported to be significantly prognostic for ipsilateral breast tumor recurrence EP1013 (Turner et al., 2000). The accumulation of p53 of specimens on tissue microarrays was significantly associated with tumor specific survival of patients treated with hormone and/or cytotoxic adjuvant therapy (Torhorst et al., 2001). A positive correlation of stain intensity with survival was reported when low concentrations of antibody were used (McCabe et al., 2005; Henson, 2005). A non-linear relationship between stain intensity and patient survival has been reported; medium stain intensity was associated with better survival than high or low stain intensity (Camp et al., 2004). Only the latter two reports used image analysis to quantify p53 stain intensity, the others used visual judgment to score p53 staining. In contrast to reports of a positive prognostic value to immunohistologically detected p53, there have been reports of a lack of positive prognostic value of immunologically detected p53. P53 stain intensity, judged visually, was not correlated with overall survival, nor was there a concordance between p53 protein detected by immunochemistry and p53 gene mutations detected by DNA sequencing (Sj?gren et al., 1996). P53 was found not to be a significant risk factor for local recurrence after breast-conserving therapy and radiation therapy (Elkuizen et al., 1999). The percent of tumor cells stained with antibody to p53 was not significantly associated with recurrence free or overall survival TACSTD1 for patients who received mastectomy and chemotherapy but not radiotherapy (Reed et al., 2000). The proportion of tissues from invasive breast cancer that stained positively for p53 was only 18.7%, although the intensity of stain, judged visually, was associated with tumor histological grade (Bartley and Ross, 2002). The time to chest wall recurrence after mastectomy and radiotherapy was not significantly associated with p53 expression, although, overall and cause-specific survival after chest wall recurrence was significant (Haffty et al., 2004). In patients with early-onset breast cancer treated with surgery and radiotherapy, no significant correlation was found, between the expression of six immunochemical biomarkers, including p53, and local relapse (Choi et al, 2005). The difference conclusions EP1013 about the efficacy of using p53 immunohistochemistry for breast cancer prognosis have resulted in p53 not being recommended for use as a biomarker in breast cancer (Harris et al., 2007). These different conclusions may be related to differences between the studies in one or more factors. These factors include differences in clinical features of the patient cohorts (young or old age at diagnosis), treatment modalities (with or without adjuvant therapy), immunohistochemical methodology (different antibodies, high or low antibody concentration), methods of evaluating stain EP1013 intensity (visual judgment or computer-aided measurement), and statistical analysis of measurements (one or two cut points). In this study we have asked if p53 immunohistochemical stain intensity could be prognostic for a cohort of young breast cancer patients who EP1013 have been treated with radiation and adjuvant therapy. Stain intensity was decided quantitatively by image analysis of specimens on a tissue microarray (TMA). We found that p53 protein stain intensity measured by quantitative image analysis can distinguish patients with good and poor survival, and provide information beyond that provided by the patients estrogen receptor status and progesterone receptor status. The stratification of patients by p53 was confirmed by determining the Quantitative Nuclear Grade of their specimens, an independent technique that characterizes nuclear DNA rather than protein. 2. Materials and Methods 2.1 Tissue Microarray The tissue microarray (YTMA71) was produced at the.