Although an important biomarker in breast cancer, Ki67 lacks scoring standardization, which has limited its clinical use. Our previous study found variability when laboratories used their own scoring methods on centrally stained tissue microarray slides. In this current study, 16 laboratories from eight countries calibrated to a specific Ki67 scoring method and then scored 50 centrally MIB-1 stained tissue microarray cases. Simple instructions prescribed scoring pattern and staining thresholds for determination of the percentage of stained tumor cells. To calibrate, laboratories scored 18 'training' and 'test' web-based images. Software tracked object selection and scoring. Success for the calibration was prespecified as Root Mean Square Error of scores compared with reference o0.6 and Maximum Absolute Deviation from reference o 1.0 (log2-transformed data). Prespecified success criteria for tissue microarray scoring required intraclass correlation significantly 40.70 but aiming for observed intraclass correlation ≥ 0.90. Laboratory performance showed non-significant but promising trends of

The tumor suppressor gene maspin has been reported to inhibit the invasiveness and motility of breast cancer cells. It has been reported that maspin is expressed in normal human mammary epithelial cells but is downregulated during cancer progression, and that p53 could induce maspin expression by transcriptional activation. However, to date, the clinical significance of maspin expression and its correlation with p53 protein expression in human breast cancer patients have not been elucidated. One hundred and sixty-eight female patients diagnosed with invasive ductal carcinoma, who had undergone a mastectomy (154 patients) or breast-conserving surgery (14 patients), were followed up for 15-119 months (median: 87 months) postoperatively. Immunoreactivity for maspin and p53 antibodies with paraffin-embedded carcinoma tissue was investigated using labeled streptavidin-biotin methods. Tumors with more than 20% of positive cells were considered positive for the expression of maspin. The expression of maspin in carcinoma cells was found in 27.4% (46 of 168) and significantly correlated with larger tumor size (p = 0.008), higher histologic grade (p = 0.0001) and positive p53 status (p = 0.003). A significant inverse relationship was observed between the expression of maspin and estrogen receptor (p = 0.0004) or progesterone receptor status (p = 0.02). Univariate analysis by log-rank test revealed a significant association between the expression of maspin and shorter relapse-free survival (p < 0.0001) and overall survival (p < 0.0001). According to Cox's multivariate analysis, the expression of maspin had the most significant effect in relapse-free survival (p < 0.0001) and overall survival (p < 0.0001) followed by lymph node status. In turn, the expression of maspin in 58 cases of ductal carcinoma in situ were also investigated to explore whether the downregulation of maspin through cancer progression are true or not. However, there were no positive cases in our series. These results seem to be contrary to previous reports defining maspin as a tumor suppressor gene. Although more precise characterization of the maspin expression, especially gene analysis is essential, the present investigation suggests that the expression of maspin is not downregulated through malignant progression and that the immunohistochemic detection of maspin in carcinoma cells may be helpful for selecting the group of breast cancer patients with an aggressive phenotype.

CyclinD1 plays a critical role in regulating cell cycle progression. CyclinD1 mRNA and protein are overexpressed in approximately 50% of primary breast cancer cases. However, its clinical significance as a predictive factor remains unclear. One hundred and seventy-three female patients diagnosed with invasive ductal carcinoma who had undergone a mastectomy (161 patients) or breast-conserving surgery (12 patients) were followed up for 6 -119 months (median 86 months) postoperatively. Immunoreactivity for monoclonal anti-cyclinD1 antibody (clone DCS-6) with paraffin-embedded carcinoma tissues was investigated using a labeled streptavidin-biotin method. Overexpression of cyclinD1 was found in 42% (73 of 173), and strongly correlated with estrogen receptor (ER) expression (p < 0.000001). Univariate analysis revealed no association between overexpression of cyclinD1 and overall survival or relapse-free survival in all patient groups. However, in the ER-negative subgroup (n ‫؍‬ 75), overexpression of cyclinD1 was significantly correlated with shorter overall survival (p ‫؍‬ 0.018) and relapse-free survival (p ‫؍‬ 0.014) as well as the lymph node status and tumor size. In contrast, there were no significant associations between overexpression of cyclinD1 and clinical outcome in the ER-positive subgroup. According to Cox's multivariate analysis in the ER-negative subgroup, overexpression of cyclinD1 had the most significant effect on overall survival (p ‫؍‬ 0.02) and relapse-free survival (p ‫؍‬ 0.0058), followed by nodal status and histologic grade. These findings suggest that overexpression of cyclinD1 is an independent prognostic indicator in ER-negative breast cancer patients.

ALDH1 expression in primary cancer is an independent prognostic factor in node-positive breast cancer patients.

ALDH1 expression in carcinoma cells is an independent prognostic factor in TN breast cancer patients.

Tumor-infiltrating lymphocytes (TILs) have potential value for stratifying the treatment of breast cancer (BC), though their precise use remains unclear. We aimed to investigate the utility of TILs using an alternative approach in different settings. We reviewed patients with triple-negative (TN) or human epithelial growth factor receptor 2 (HER2)-positive invasive ductal carcinomas from a single institutional cohort and classified archived hematoxylin–eosin-stained samples in terms of TIL score as low (<10 %), intermediate, and high (>50 %). The prognostic and predictive values of TILs were analyzed retrospectively in both adjuvant and neo-adjuvant settings. In the adjuvant setting, the presence of TILs at primary surgery was a significant favorable prognostic factor among 154 TNBCs [relapse-free survival (RFS): p = 0.015], but not among 183 HER2+ BCs (RFS: p = 0.097). The TNBC low-TIL group tended to relapse earlier. In the neo-adjuvant setting, detection of TILs on biopsy before primary systemic therapy was associated with the ratio of patients achieving pathological complete response among 48 TNBCs (p = 0.024), but not among 58 HER2+ BCs (p = 0.30). The presence of TILs in surgical specimens after systemic therapy had prognostic value in HER2+ BC (RFS: p = 0.007). The impact of TILs differs between patients with TN and HER2+ BC treated with standard therapies. Our three-grade scale for TILs may contribute to our understanding of the importance of the tumor microenvironment in routine practice. TILs after primary systemic therapy may be useful for the further stratification of treatment of HER2+ BC.Electronic supplementary materialThe online version of this article (doi:10.1007/s10549-016-3848-2) contains supplementary material, which is available to authorized users.

Epidermal growth factor receptor (EGF‐R) and its ligand, transforming growth factor‐α (TGF‐α), play an important role through the autocrine growth‐regulation system in several human cancers, including breast cancer. However, the clinical significance of co‐expression of EGF‐R and TGF‐α has not been elucidated. One hundred seventy‐three female patients diagnosed as invasive ductal carcinoma who had undergone a mastectomy (159 patients) or breast‐conserving surgery (14 patients) were followed up for 81 to 119 months (median 94 months) post‐operatively. Immunoreactivity for EGF‐R, TGF‐α, p53 and c‐erbB‐2 with paraffin‐embedded carcinoma tissue was investigated using labeled streptavidin‐biotin methods. Positive rates of carcinoma cells were 27%, 33%, 32% and 26% for EGF‐R, TGF‐α, p53 and c‐erbB‐2, respectively. Expression of EGF‐R only was observed in 16% (28/173), of TGF‐α only in 22% (38/173), of both EGF‐R and TGF‐α in 11% (19/173) and of neither in 51% (88/173). By univariate analysis, significant differences in overall survival and disease‐free survival were noted according to the co‐expression of EGF‐R and TGF‐α (p < 0.0001, p < 0.0001), co‐expression of EGF‐R and c‐erbB‐2 (p = 0.0029, p = 0.0028), nodal status (p = 0.0028, p = 0.0001), tumor size (p = 0.0001, p < 0.0001) and c‐erbB‐2 expression (p = 0.0034, p = 0.018), respectively. The status of p53 expression (p = 0.01), estrogen receptor (p = 0.042) and progesterone receptor (p = 0.046) showed significant differences in overall survival. According to Cox's multivariate analysis, co‐expression of EGF‐R and TGF‐α had the most significant effect on disease‐free survival (p < 0.0001) and overall survival (p < 0.0001), followed by nodal status. Co‐expression of EGF‐R and TGF‐α by immunohistochemical detection is an independent prognostic indicator, and it may be helpful for determining the group of breast‐cancer patients with an aggressive phenotype. Int. J. Cancer 89:484–487, 2000. © 2000 Wiley‐Liss, Inc.

Expression of estrogen receptor beta (ERbeta) protein in human breast cancer and correlation with clinicopathological factors have been reported by many investigators, but many of them used ERbeta antibodies that react with both wild-type ERbeta (ERbetawt) and splicing variant isoform. Therefore, the frequency and correlation with clinicopathological factors of ERbetawt expression remain to be established. In the present study a monoclonal antibody EMR02, specific for ERbetawt, was used in formalin-fixed paraffin-embedded sections from 225 female primary breast cancer patients diagnosed as having invasive ductal carcinoma. Expression of ERalpha, progesterone receptor (PgR) and HER2/neu were also investigated by immunohistochemistry. For ERbetawt, ERalpha and PgR, positivity was defined as nuclear staining in >10% of the cancer cells. HER2/neu overexpression was defined as a Hercep test score 3+. Positivity for ERbetawt, ERalpha, PgR and HER2/neu overexpression was 55%, 74%, 61% and 25%, respectively. The expression of ERbetawt had a positive correlation with ERalpha (P=0.018) and PgR (P=0.02). There was significant positive correlation between ERbetawt expression and HER2/neu overexpression (P<0.0001). According to multivariate logistic regression analysis the most significant association was between ERbetawt expression and HER2/neu overexpression (P<0.0001). These results suggest that clinical significances of ERbetawt expression in human breast cancer patients may be more complex.