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We have previously identified a novel phenotypic dichotomy in breast cancer (BC) based on the response to a SRR2 (Sox2 regulatory region 2) reporter, with reporter responsive (RR) cells being more tumorigenic/stem-like than reporter unresponsive (RU) cells. Since the expression level of Sox2 is comparable between the two cell subsets, we hypothesized that post-translational modifications of Sox2 contribute to their differential reporter response and phenotypic differences. By liquid chromatography-mass spectrometry, we found Sox2 to be phosphorylated in RR but not RU cells. Threonine 116 is an important phosphorylation site, since transfection of the T116A mutant into RR cells significantly decreased the SRR2 reporter luciferase activity and the RR-associated phenotype. Oxidative stress-induced conversion of RU into RR cells was accompanied by Sox2 phosphorylation at T116 and increased Sox2-DNA binding. In a cohort of BC, we found significant correlations between the proportion of tumor cells immuno-reactive with anti-phosphorylated Sox2T116 and a high tumor grade (p = 0.006), vascular invasion (p = 0.001) and estrogen receptor expression (p = 0.032). In conclusion, our data suggests that phosphorylation of Sox2T116 contributes to the tumorigenic/stem-like features in RR cells. Detection of phospho-Sox2T116 may be useful in identifying a small subset of tumor cells carrying stem-like/tumorigenic features in BC. [ABSTRACT FROM AUTHOR]

Ki67 is a commonly used marker of cancer cell proliferation, and has significant prognostic value in breast cancer. In spite of its clinical importance, assessment of Ki67 remains a challenge, as current manual scoring methods have high inter- and intra-user variability. A major reason for this variability is selection bias, in that different observers will score different regions of the same tumor. Here, we developed an automated Ki67 scoring method that eliminates selection bias, by using whole-slide analysis to identify and score the tumor regions with the highest proliferative rates. The Ki67 indices calculated using this method were highly concordant with manual scoring by a pathologist (Pearson’s r = 0.909) and between users (Pearson’s r = 0.984). We assessed the clinical validity of this method by scoring Ki67 from 328 whole-slide sections of resected early-stage, hormone receptor-positive, human epidermal growth factor receptor 2-negative breast cancer. All patients had Oncotype DX testing performed (Genomic Health) and available Recurrence Scores. High Ki67 indices correlated significantly with several clinico-pathological correlates, including higher tumor grade (1 versus 3, P<0.001), higher mitotic score (1 versus 3, P<0.001), and lower Allred scores for estrogen and progesterone receptors (P = 0.002, 0.008). High Ki67 indices were also significantly correlated with higher Oncotype DX risk-of-recurrence group (low versus high, P<0.001). Ki67 index was the major contributor to a machine learning model which, when trained solely on clinico-pathological data and Ki67 scores, identified Oncotype DX high- and low-risk patients with 97% accuracy, 98% sensitivity and 80% specificity. Automated scoring of Ki67 can thus successfully address issues of consistency, reproducibility and accuracy, in a manner that integrates readily into the workflow of a pathology laboratory. Furthermore, automated Ki67 scores contribute significantly to models that predict risk of recurrence in breast cancer. [ABSTRACT FROM AUTHOR]

Background: Sox2, an embryonic stem cell marker, is aberrantly expressed in a subset of breast cancer (BC). While the aberrant expression of Sox2 has been shown to significantly correlate with a number of clinicopathologic parameters in BC, its biological significance in BC is incompletely understood.Methods: In-vitro invasion assay was used to evaluate whether the expression of Sox2 is linked to the invasiveness of MCF7 and ZR751 cells. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and/or Western blots were used to assess if Sox2 modulates the expression of factors known to regulate epithelial mesenchymal transition (EMT), such as Twist1. Chromatin immunoprecipitation (ChIP) was used to assess the binding of Sox2 to the promoter region of Twist1.Results: We found that siRNA knockdown of Sox2 expression significantly increased the invasiveness of MCF7 and ZR751 cells. However, when MCF7 cells were separated into two distinct subsets based on their differential responsiveness to the Sox2 reporter, the Sox2-mediated effects on invasiveness was observed only in 'reporter un-responsive' cells (RU cells) but not 'reporter responsive' cells (RR cells). Correlating with these findings, siRNA knockdown of Sox2 in RU cells, but not RR cells, dramatically increased the expression of Twist1. Accordingly, using ChIP, we found evidence that Sox2 binds to the promoter region of Twist1 in RU cells only. Lastly, siRNA knockdown of Twist1 largely abrogated the regulatory effect of Sox2 on the invasiveness in RU cells, suggesting that the observed Sox2-mediated effects are Twist1-dependent.Conclusion: Sox2 regulates the invasiveness of BC cells via a mechanism that is dependent on Twist1 and the transcriptional status of Sox2. Our results have further highlighted a new level of biological complexity and heterogeneity of BC cells that may carry significant clinical implications. [ABSTRACT FROM AUTHOR]