El on SNAI1 because of the statistical significant association with decreased CDH1 expression. Using a previously published [18] and commercially available Snail1-antibody, we could validate theCDH1, CDH2, SNAI1, TWIST1 in Colorectal AdenomasFigure 7. Correlation of qRT-PCR and immunohistochemistry. Y-axis: relative amount of 
target mRNA in the qRT-PCR on a log.scale; X-axis: positive or negative immunostaining for target protein. Bars indicate median value. A: Adenomas positive for E-Cadherin protein in the immunohistochemistry had a significantly lower expression of CDH1 mRNA in the qRT-PCR, 
compared to immunohistochemically negative ones (p = 0.003). B: Adenomas with positive nuclear Snail1 staining in the immunohistochemistry had significantly higher amounts of SNAI1 mRNA in the qRT-PCR, compared to adenomas negative for Snail1 protein in the immunohistochemistry (p = 0.001). C: Adenomas positive for nuclear Snail1 staining in the immunohistochemistry showed significantly lower amounts of CDH1 mRNA in the qRT-PCR, compared to those negative for Snail1 protein in the immunohistochemistry (p = 0.02). doi:10.1371/journal.pone.0046665.gmRNA data of SNAI1. We also observed a trend between nuclear Snail1 protein expression and decreased CDH1 protein (Ecadherin) expression. It is important to stress that the Snail1 immunohistochemistry displayed a nuclear localization reflecting its function as transcription factor. According to our data, SNAI1, but not TWIST1, seems to contribute significantly to the down-regulation of E-cadherin in benign colorectal adenomas, in which decreased E-cadherin levels have already been described [24,25,26,27]. Does that mean that EMT processes driving metastasis are already active in a benign premalignant condition? In Epigenetics transgenic mouse Autophagy models of cancer it could indeed be demonstrated that tumor cells disseminate in morphologically benign appearing hyperplastic lesions [28,29]. Subsequent molecular analyses of the hyperplastic tissue in the breast cancer mouse model revealed a strong up-regulation ofTWIST1 as well as of proteolytic enzymes suggesting EMT as an underlying mechanism for early tumor cell spread [29]. In human cancer however, only very few data are available indicating that tumor cell dissemination could take place in morphologically noninvasive tumors. There are convincing data demonstrating that patients with DCIS already harbour cytokeratin-positive DTC in their bone marrow [29,30]. In the case of colorectal cancer progression, Steinert et al. [31] reported about cytokeratin-positive as well as EpCAM-positive bone marrow DTC in a small series of patients with colorectal adenomas. These data by Steinert et al were controversially discussed and technical concerns were raised [32]. Therefore, a note of caution is required; especially since no independent and confirmatory data for the prevalence of bone marrow DTCs in adenoma patients are available. However, Pantel et al. just recently demonstrated using the FDA clearedCDH1, CDH2, SNAI1, TWIST1 in Colorectal AdenomasFigure 8. Correlation of Snail1 and E-cadherin protein expression in the immunochemistry. Y-axis: number of adenomas; white column = Snail1 negative; black column = Snail1 positive (p = 0.095). doi:10.1371/journal.pone.0046665.gCellSearch system that EpCAM positive circulating tumor cells (CTCs) can be already detected in the peripheral blood of benign colon diseases including adenomas [33]. But regarding the low annual transition rate o.El on SNAI1 because of the statistical significant association with decreased CDH1 expression. Using a previously published [18] and commercially available Snail1-antibody, we could validate theCDH1, CDH2, SNAI1, TWIST1 in Colorectal AdenomasFigure 7. Correlation of qRT-PCR and immunohistochemistry. Y-axis: relative amount of target mRNA in the qRT-PCR on a log.scale; X-axis: positive or negative immunostaining for target protein. Bars indicate median value. A: Adenomas positive for E-Cadherin protein in the immunohistochemistry had a significantly lower expression of CDH1 mRNA in the qRT-PCR, compared to immunohistochemically negative ones (p = 0.003). B: Adenomas with positive nuclear Snail1 staining in the immunohistochemistry had significantly higher amounts of SNAI1 mRNA in the qRT-PCR, compared to adenomas negative for Snail1 protein in the immunohistochemistry (p = 0.001). C: Adenomas positive for nuclear Snail1 staining in the immunohistochemistry showed significantly lower amounts of CDH1 mRNA in the qRT-PCR, compared to those negative for Snail1 protein in the immunohistochemistry (p = 0.02). doi:10.1371/journal.pone.0046665.gmRNA data of SNAI1. We also observed a trend between nuclear Snail1 protein expression and decreased CDH1 protein (Ecadherin) expression. It is important to stress that the Snail1 immunohistochemistry displayed a nuclear localization reflecting its function as transcription factor. According to our data, SNAI1, but not TWIST1, seems to contribute significantly to the down-regulation of E-cadherin in benign colorectal adenomas, in which decreased E-cadherin levels have already been described [24,25,26,27]. Does that mean that EMT processes driving metastasis are already active in a benign premalignant condition? In transgenic mouse models of cancer it could indeed be demonstrated that tumor cells disseminate in morphologically benign appearing hyperplastic lesions [28,29]. Subsequent molecular analyses of the hyperplastic tissue in the breast cancer mouse model revealed a strong up-regulation ofTWIST1 as well as of proteolytic enzymes suggesting EMT as an underlying mechanism for early tumor cell spread [29]. In human cancer however, only very few data are available indicating that tumor cell dissemination could take place in morphologically noninvasive tumors. There are convincing data demonstrating that patients with DCIS already harbour cytokeratin-positive DTC in their bone marrow [29,30]. In the case of colorectal cancer progression, Steinert et al. [31] reported about cytokeratin-positive as well as EpCAM-positive bone marrow DTC in a small series of patients with colorectal adenomas. These data by Steinert et al were controversially discussed and technical concerns were raised [32]. Therefore, a note of caution is required; especially since no independent and confirmatory data for the prevalence of bone marrow DTCs in adenoma patients are available. However, Pantel et al. just recently demonstrated using the FDA clearedCDH1, CDH2, SNAI1, TWIST1 in Colorectal AdenomasFigure 8. Correlation of Snail1 and E-cadherin protein expression in the immunochemistry. Y-axis: number of adenomas; white column = Snail1 negative; black column = Snail1 positive (p = 0.095). doi:10.1371/journal.pone.0046665.gCellSearch system that EpCAM positive circulating tumor cells (CTCs) can be already detected in the peripheral blood of benign colon diseases including adenomas [33]. But regarding the low annual transition rate o.
