Tumor-associated macrophages (TAMs) are involved in tumor progression by promoting epithelial-mesenchymal transition (EMT), tumor cell invasion, migration and angiogenesis. However, in breast cancer, the clinical relevance of the TAM infiltration according to distinct histologic locations (intratumoral vs. stromal) and hormone receptor status is unclear. We investigated the significance of the levels of TAM infiltration in distinct histologic locations in invasive breast cancer. We also examined the relationship of the TAM levels with the clinicopathologic features of tumors, expression of EMT markers, and clinical outcomes. Finally, we analyzed the prognostic value of TAM levels according to hormone receptor status. High levels of infiltration of intratumoral, stromal and total TAMs were associated with high histologic grade, p53 overexpression, high Ki-67 proliferation index and negative hormone receptor status. Infiltration of TAMs was also correlated with overexpression of vimentin, smooth muscle actin and alteration of β-catenin. Overall, a high level of infiltration of intratumoral TAMs was associated with poor disease-free survival, and was found to be an independent prognostic factor. In subgroup analyses by hormone receptor status, a high level of infiltration of intratumoral TAM was an independent prognostic factor in the hormone receptor-positive subgroup, but not in the hormone-receptor negative subgroup. Our findings suggest that intratumoral TAMs play an important role in tumor progression in breast cancer, especially in the hormone receptor-positive group, and the level of TAM infiltration may be used as a prognostic factor and even a therapeutic target in breast cancer.

MicroRNAs (miRNAs) are involved in the progression of breast cancer. Some miRNAs, especially the miR-200 family, miR-9, and miR-155 have been reported to be associated with epithelial-mesenchymal transition (EMT) and breast cancer stem cell (BCSC) phenotypes. This study was designed to evaluate the expression levels of these miRNAs in human breast cancer samples and analyzed their relationship with clinicopathologic features of the tumor including breast cancer subtype, EMT, BCSC phenotype, and prognosis. Expression levels of the miR-200 family, miR-9, and miR-155 were quantified using qRT-PCR. Breast cancer subtype, BCSC phenotype (CD44+/CD24- and ALDH1+), and expression of EMT markers (vimentin expression and E-cadherin loss) were evaluated by immunohistochemistry. miR-9 was more highly expressed in HER2+ and triple-negative subtypes than in luminal subtypes. Its expression level was significantly higher in tumors with high T stage, high histologic grade, p53 overexpression, and high proliferation index. Expression of miR-9 was also higher in tumors showing the CD44+/CD24- phenotype, vimentin expression, and E-cadherin loss. Furthermore, high level of miR-9 expression was found to be an independent prognostic factor for poor disease-free survival of the patients. Expression of miR-200a and miR-141 was highest in luminal A subtype, and miR-155 expression was highest in triple-negative subtype. Although the expression levels of some miR-200 family members and miR-155 showed difference with regard to EMT or BCSC phenotype, they were not associated with patients' prognosis. In conclusion, overexpression of miR-9 is found in tumors with aggressive phenotypes and is associated with poor prognosis in breast cancer, suggesting that it may serve as a potential biomarker for breast cancer progression and a target for treatment.

The transcription factors of embryonic stem cells, such as Oct4, Sox2, Nanog, Bmi1, and Klf4, are known to be associated with stemness, epithelial–mesenchymal transition and aggressive tumor behavior. This study was designed to evaluate the clinicopathological significance of their expression in breast cancer. Immunohistochemistry for Oct4, Sox2, Nanog, Bmi1, and Klf4 was performed in 319 cases of invasive breast cancer. The relationship between the expression of these markers and clinicopathologic features of the tumors, including breast cancer stem cell phenotype and epithelial–mesenchymal transition marker expression, and their prognostic value in breast cancer, were analyzed. Expression of Oct4 and Sox2 was commonly associated with high histologic grade and high Ki-67 index in the whole group and in the hormone receptor-positive subgroup. On the other hand, expression of Nanog, Bmi1, and Klf4 was inversely correlated with aggressive features of the breast cancer. Oct4 expression was associated with ALDH1 expression but not with epithelial–mesenchymal transition marker expression. In survival analysis, Oct4 expression was independently associated with poor prognosis in the whole group and in the hormone receptor-positive subgroup, but not in hormone receptor-negative subgroup. Particularly, Oct4 expression was associated with poor clinical outcome in patients with hormone receptor-positive breast cancer treated with tamoxifen. Our results indicate that Oct4 expression is associated with aggressive features, ALDH1 expression, tamoxifen resistance and poor clinical outcomes in hormone receptor-positive breast cancer, and thus may be useful as a predictive and prognostic marker in this subgroup of breast cancer.

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The changes in DNA methylation status in cancer cells are characterized by hypermethylation of promoter CpG islands and diffuse genomic hypomethylation. Alu and long interspersed nucleotide element-1 (LINE-1) are non-coding genomic repetitive sequences and methylation of these elements can be used as a surrogate marker for genome-wide methylation status. This study was designed to evaluate the changes of Alu and LINE-1 hypomethylation during breast cancer progression from normal to pre-invasive lesions and invasive breast cancer (IBC), and their relationship with characteristics of IBC. We analyzed the methylation status of Alu and LINE-1 in 145 cases of breast samples including normal breast tissue, atypical ductal hyperplasia/flat epithelial atypia (ADH/FEA), ductal carcinoma in situ (DCIS) and IBC, and another set of 129 cases of IBC by pyrosequencing. Alu methylation showed no significant changes during multistep progression of breast cancer, although it tended to decrease during the transition from DCIS to IBC. In contrast, LINE-1 methylation significantly decreased from normal to ADH/FEA, while it was similar in ADH/FEA, DCIS and IBC. In IBC, Alu hypomethylation correlated with negative estrogen receptor (ER) status, and LINE-1 hypomethylation was associated with negative ER status, ERBB2 (HER2) amplification and p53 overexpression. Alu and LINE-1 methylation status was significantly different between breast cancer subtypes, and the HER2 enriched subtype had lowest methylation levels. In survival analyses, low Alu methylation status tended to be associated with poor disease-free survival of the patients. Our findings suggest that LINE-1 hypomethylation is an early event and Alu hypomethylation is probably a late event during breast cancer progression, and prominent hypomethylation of Alu and LINE-1 in HER2 enriched subtype may be related to chromosomal instability of this specific subtype.

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PurposeThe microRNA-221/222 (miR-221/222) gene cluster has been reported to be associated with the promotion of epithelial-mesenchymal transition (EMT), downregulation of estrogen receptor-α, and tamoxifen resistance in breast cancer. We studied the expression of miR-222 in human breast cancer samples to analyze its relationship with clinicopathologic features of the tumor, including estrogen receptor status, expression of EMT markers, and clinical outcomes.MethodsQuantitative real-time polymerase chain reaction was performed to detect the expression of miR-222 in 197 invasive breast cancers. Expression of EMT markers (vimentin, smooth muscle actin, osteonectin, N-cadherin, and E-cadherin) was evaluated using immunohistochemistry.ResultsHigh miR-222 levels were associated with high T stage, high histologic grade, high Ki-67 proliferation index, and HER2 gene amplification. Its expression was significantly higher in the luminal B and human epidermal growth factor receptor 2-positive (HER2+) subtypes than in the luminal A and triple-negative subtypes. In the hormone receptor-positive subgroup, there was a significant negative correlation between miR-222 and estrogen receptor expression, and miR-222 expression was associated with EMT marker expression. In the group as a whole, high miR-222 expression was not associated with clinical outcome. However, subgroup analyses by hormone receptor status revealed that high miR-222 expression was a poor prognostic factor in the hormone receptor-positive subgroup, but not in the hormone receptor-negative subgroup.ConclusionThis study showed that miR-222 is associated with down-regulation of the estrogen receptor, EMT, and tumor progression in hormone receptor-positive breast cancer, indicating that miR-222 might be associated with endocrine therapy resistance and poor clinical outcome in hormone receptor-positive breast cancer.

Purpose: Gastric cancer remains the leading cause of cancer-related deaths in Northeast Asia. Population-based endoscopic screenings in the region have yielded successful results in early detection of gastric tumors. Endoscopic screening rates are continuously increasing, and there is a need for an automatic computerized diagnostic system to reduce the diagnostic burden. In this study, we developed an algorithm to classify gastric epithelial tumors automatically and assessed its performance in a large series of gastric biopsies and its benefits as an assistance tool. Experimental Design: Using 2,434 whole-slide images, we developed an algorithm based on convolutional neural networks to classify a gastric biopsy image into one of three categories: negative for dysplasia (NFD), tubular adenoma, or carcinoma. The performance of the algorithm was evaluated by using 7,440 biopsy specimens collected prospectively. The impact of algorithm-assisted diagnosis was assessed by six pathologists using 150 gastric biopsy cases. Results: Diagnostic performance evaluated by the AUROC curve in the prospective study was 0.9790 for two-tier classification: negative (NFD) versus positive (all cases except NFD). When limited to epithelial tumors, the sensitivity and specificity were 1.000 and 0.9749. Algorithm-assisted digital image viewer (DV) resulted in 47% reduction in review time per image compared with DV only and 58% decrease to microscopy. Conclusions: Our algorithm has demonstrated high accuracy in classifying epithelial tumors and its benefits as an assistance tool, which can serve as a potential screening aid system in diagnosing gastric biopsy specimens.