Clusterin Overexpression and Relapse-free Survival in Breast Cancer

Patients and Methods

Patients. Three hundred and fifty-two patients (median age, 46 years; age range, 25-79 years) with breast carcinomas were included in this study. They underwent breast surgery at the National Cancer Center (Gyeonggi, South Korea) and at Kangbuk Samsung Hospital (Seoul, South Korea) between 2000 and 2006. The mean follow-up was 53 months (range, 7 to 84 months). Relapse-free survival (RFS) was calculated as the period from surgery until the date of the first recurrence. The study was submitted to and met the guidelines of the local Institutional Review Committees.

Standard histopathological examination included the type of cancer, the pathological tumor stage assessed according to the criteria established by the 6th edition AJCC staging manual (6). Twenty-four (7%) out of 352 patients had in situ carcinoma. Histological types of invasive cancer were mucinous carcinoma in eight, medullary carcinoma in three, metaplastic carcinoma in one, tubulolobular carcinoma in one, lobular carcinoma in one and ductal not otherwise specified (NOS) in three hundred and four patients. The clinicopathological characteristics of the enrolled patients are shown in Table I.

Immunohistochemical staining. All of the tissues obtained from patients were routinely fixed in 10% buffered formalin and were embedded in paraffin blocks. Tissue array blocks containing breast cancer tissues of 6-mm diameter from enrolled cases were produced. The tissue microarray blocks were sectioned at a 4-μm thickness and were processed for immunohistochemical staining. Paraffin was removed from the tissue sections with xylene. The sections were rehydrated with graded ethanol and were immersed in Tris-buffered saline. The vendors that supplied the primary antibodies and the dilution factors used are listed in Table II. A biotinylated anti-mouse antibody was used as a secondary antibody and streptavidin horseradish peroxidase (Zymed Laboratories, San Francisco, CA, USA) was used following the instructions provided by the manufacturer. Finally, the sections were counterstained in Mayer's hematoxylin, dehydrated and cleared, and the sections were mounted for examination.

Clusterin expression was scored as follows: the staining intensity in the cytoplasm only was evaluated from 0 to 3 (representing none to strong staining, respectively) and the % cells in each intensity was obtained. The overall score was determined as follows: overall score = [(% cells with visual score 1) × 1] + [(% cells with visual score 2) × 2] + [(% cells with visual score 3) × 3]; expression was positive if the score was more than 70 (8). A cutoff value of 10% of the positively stained nuclei was used to define estrogen receptor (ER) and progesterone receptor (PR) positivity. Only cytoplasmic staining with any intensity in more than 30% of tumor cells was scored as positivity for bcl-2. Membranous staining for HER2 was scored as the following: 0, no staining or membranous staining in <10% of the cells; 1+, faint incomplete staining in 10% of the cells; 2+, weak to moderate complete staining in 10% of the cells; 3+, strong complete staining in 10% of the cells. HER2 overexpression was defined as score 3+. Cells stained for Ki-67 and p53 were counted and were expressed as a percentage. The Ki-67 labeling index was graded as low if the proportion of positive cells was <10% and high if ≥10%. p53 was scored as positive if more than 10% of cells were positive with strong intensity.

Statistical analysis. Statistical analysis was performed with the use of SPSS software, version 15.0 (SPSS, Chicago, IL, USA). Pearson's χ² tests were used to examine the correlation between the variables. The Cox proportional regression hazard model was used for survival analysis. Kaplan-Meier curves were plotted from data of RFS. A p-value <0.05 was considered statistically significant.