p53 accumulation is a strong predictor of recurrence in estrogen receptor-positive breast cancer patients treated with aromatase inhibitors.
Bottom Line: High p53 expression was positively correlated with tumor grade, HER2 score and Ki-67 expression.Significant association was observed between disease-free survival and high p53 expression in multivariate analysis (P < 0.0001).Compared with women without recurrence, women with early recurrence had significantly higher p53 expression (P < 0.0001), as did women with late recurrence (P = 0.037).
Affiliation: Breast and Endocrine Surgery, Hokkaido University Hospital, Sapporo, Japan.Show MeSH
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Mentions: One 4-μm section of each submitted paraffin block was stained first with hematoxylin–eosin to verify that an adequate number of carcinoma cells were present and that the fixation quality was adequate for IHC analysis. Serial sections (4 μm) were prepared from selected blocks and float-mounted on adhesive-coated glass slides for IHC13. The IHC status of ER, PR and HER2 was determined using the PATHWAY rabbit monoclonal antibodies (clone SP1, 1E2 and 4B5, respectively) and iView DAB Detection Kit (Ventana Medical Systems, Inc., Tucson, AZ, USA). Expression of ER and PR were scored by assigning proportion and intensity scores, according to Allred's procedure.14 In brief, a proportion score represented the estimated proportion of tumor cells staining positive as follows: 0, none; 1, <1/100; 2, 1/100–1/10; 3, 1/10–1/3; 4, 1/3–2/3; and 5, >2/3. Any brown nuclear staining in breast epithelium counted towards the proportion score. An intensity score represented the average intensity of the positive cells as follows: 0, none; 1, weak; 2, intermediate; and 3, strong. The proportion and intensity scores were then added to obtain a total score that could range from 0 to 8. Tumors with ≥1% positive cells (proportion score ≥2) were evaluated as positive. To determine the level of HER2 expression, the membrane staining pattern was estimated and scored on a scale of 0 to 3+. For Ki-67 and p53 staining, antigens were retrieved in Dako EnVision FLEX Target Retrieval Solution, high pH (pH 9.0), using Dako PT Link for 20 min at 97°C according to the manufacturer's instructions (Dako, Glostrup, Denmark). The IHC for Ki-67 was performed using a mouse monoclonal anti-human Ki-67 antibody (MIB-1, Dako) at 1:200 dilution for 30 min at room temperature and the Dako Envision FLEX system was used for visualization. The labeling index (LI) was assessed as the percentage of tumor cells showing definite nuclear staining among >1000 invasive tumor cells15 analyzed using NanoZoomer 2.0-HT (Hamamatsu photonics, Hamamatsu, Japan) for slide scanning and Tissue Studio (Definiens, Munich, Germany) for automated scoring. If there were clear hot spots, data from these areas were assessed. The IHC for p53 was performed using a mouse monoclonal anti-human p53 antibody (DO-7, Dako) at 1:200 dilution for 30 min at room temperature with the Dako Envision FLEX system for visualization. p53 protein expression was measured as the percentage of cells showing definite nuclear staining. p53 was considered positive if there was ≥10% positive nuclear staining regardless of the intensity.12,16–18 Representative images of staining for Ki-67 and p53 are shown in Fig.1.
Affiliation: Breast and Endocrine Surgery, Hokkaido University Hospital, Sapporo, Japan.