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.

We co-assessed PD-L1 expression and CD8+ tumor-infiltrating lymphocytes in gastric cancer (GC), and categorized into 4 microenvironment immune types. Immunohistochemistry (PD-L1, CD8, Foxp3, E-cadherin, and p53), PD-L1 mRNA in situ hybridization (ISH), microsatellite instability (MSI), and EBV ISH were performed in 392 stage II/III GCs treated with curative surgery and fluoropyrimidine-based adjuvant chemotherapy, and two public genome databases were analyzed for validation. PD-L1+ was found in 98/392 GCs (25.0%). The proportions of immune types are as follows: PD-L1+/CD8High, 22.7%; PD-L1−/CD8Low, 22.7%; PD-L1+/CD8Low, 2.3%; PD-L1−/CD8High, 52.3%. PD-L1+/CD8High type accounted for majority of EBV+ and MSI-high (MSI-H) GCs (92.0% and 66.7%, respectively), and genome analysis from public datasets demonstrated similar pattern. PD-L1−/CD8High showed the best overall survival (OS) and PD-L1−/CD8Low the worst (P < 0.001). PD-L1 expression alone was not associated with OS, however, PD-L1−/CD8High type compared to PD-L1+/CD8High was independent favorable prognostic factor of OS by multivariate analysis (P = 0.042). Adaptation of recent molecular classification based on EBV, MSI, E-cadherin, and p53 showed no significant survival differences. These findings support the close relationship between PD-L1/CD8 status based immune types and EBV+, MSI-H GCs, and their prognostic significance in stage II/III GCs.

BackgroundAnti-EGFR antibody–based treatment is an important therapeutic strategy for advanced colorectal cancer (CRC); despite this, several mutations—including KRAS, BRAF, and PIK3CA mutations, and HER2 amplification—are associated with the mechanisms underlying the development of resistance to anti-EGFR therapy. The aim of our study was to investigate the frequencies and clinical implications of these genetic alterations in advanced CRC.MethodsKRAS, BRAF, and PIK3CA mutations were determined by Cobas real-time polymerase chain reaction (PCR) in 191 advanced CRC patients with distant metastasis. Microsatellite instability (MSI) status was determined by a fragmentation assay and HER2 amplification was assessed by silver in situ hybridization. In addition, KRAS mutations were investigated by the Sanger sequencing method in 97 of 191 CRC cases.ResultsMutations in KRAS, BRAF, and PIK3CA were found in 104 (54.5%), 6 (3.1%), and 25 (13.1%) cases of advanced CRC, respectively. MSI-high status and HER2 amplification were observed in 3 (1.6%) and 16 (8.4%) cases, respectively. PIK3CA mutations were more frequently found in KRAS mutant type (18.3%) than KRAS wild type (6.9%) (P = 0.020). In contrast, HER2 amplifications and BRAF mutations were associated with KRAS wild type with borderline significance (P = 0.052 and 0.094, respectively). In combined analyses with KRAS, BRAF and HER2 status, BRAF mutations or HER2 amplifications were associated with the worst prognosis in the wild type KRAS group (P = 0.004). When comparing the efficacy of detection methods, the results of real time PCR analysis revealed 56 of 97 (57.7%) CRC cases with KRAS mutations, whereas Sanger sequencing revealed 49 cases (50.5%).ConclusionsKRAS mutations were found in 54.5% of advanced CRC patients. Our results support that subgrouping using PIK3CA and BRAF mutation or HER2 amplification status, in addition to KRAS mutation status, is helpful for managing advanced CRC patients.

The aim of this study was to determine the clinicopathological significance of programmed cell death ligand 1 (PD-L1) expression in glioblastoma (GBM). In a retrospective cohort of 115 consecutive patients with GBM, PD-L1 expression was determined using immunohistochemistry (IHC). Membranous and fibrillary PD-L1 staining of any intensity in > 5% neoplastic cells and tumour infiltrating immune cells (TIIs) was considered positive staining. In addition, isocitrate dehydrogenase-1 (IDH-1) (R132H) expression and cluster of differentiation 3 (CD3)-positive T-cell infiltration were investigated using IHC. O(6)-methylguanine-DNA methyltransferase (MGMT) promoter methylation assay and fluorescence in situ hybridization (FISH) for the assessment of 1p/19q deletion were performed. Expression of PD-L1 in tumour cells and TIIs was found in 37 (32.2%) and 6 (5.2%) patients, respectively. Kaplan-Meier analysis indicated that PD-L1 expression in tumour cells was significantly associated with poor overall survival (OS) (P = 0.017), though multivariate Cox analysis did not confirm this association (hazard ratio 1.204; P = 0.615). PD-L1 expression in TIIs did not correlate with the patient prognosis (P = 0.545). In addition, MGMT methylation and IDH-1 (R132H) expression were associated with a better prognosis (P < 0.001 and P = 0.024, respectively). The expression of PD-L1 was associated with CD3-positive T-cell infiltration (P < 0.001), and IDH-1 wild type status (P = 0.008). A deeper insight into PD-L1 expression could help to ensure the success of future immunotherapy in GBM. Our study suggested that PD-L1 target therapy might be beneficial for PD-L1-expressing GBM patients with a poor prognosis.

BackgroundGlioblastomas may develop de novo (primary glioblastomas, P-GBLs) or through progression from lower-grade astrocytomas (secondary glioblastomas, S-GBLs). The aim of this study was to compare the immunohistochemical classification of glioblastomas with clinically determined P-GBLs and S-GBLs to identify the best combination of antibodies for immunohistochemical classification.MethodsWe evaluated the immunohistochemical expression of epidermal growth factor receptor (EGFR), p53, and isocitrate dehydrogenase 1 (IDH-1) in 150 glioblastoma cases.ResultsAccording to clinical history, the glioblastomas analyzed in this study consisted of 146 P-GBLs and 4 S-GBLs. Immunohistochemical expression of EGFR, p53, and IDH-1 was observed in 62.6%, 49.3%, and 11.1%, respectively. Immunohistochemical profiles of EGFR(+)/p53(-), IDH-1(-)/EGFR(+)/p53(-), and EGFR(-)/p53(+) were noted in 41.3%, 40.2%, and 28.7%, respectively. Expression of IDH-1 and EGFR(-)/p53(+) was positively correlated with young age. The typical immunohistochemical features of S-GBLs comprised IDH-1(+)/EGFR(-)/p53(+), and were noted in 3.6% of clinically P-GBLs. The combination of IDH-1(-) or EGFR(+) was the best set of immunohistochemical stains for identifying P-GBLs, whereas the combination of IDH-1(+) and EGFR(-) was best for identifying S-GBLs.ConclusionsWe recommend a combination of IDH-1 and EGFR for immunohistochemical classification of glioblastomas. We expect our results to be useful for determining treatment strategies for glioblastoma patients.

Hepatic steatosis significantly developed 3 months after cholecystectomy. Therefore, cholecystectomy might be considered a risk factor for hepatic steatosis, but the relationship should be confirmed with long-term follow-up from a large group of patients.

Neuregulin 1 (NRG1), a ligand for human epidermal growth factor (HER) 3 and HER4, can activates cell signaling pathways to promote carcinogenesis and metastasis. To investigate the clinicopathologic significance of NRG1 and its receptors, immunohistochemistry was performed for NRG1, HER3, and HER4 in 502 consecutive gastric cancers (GCs). Furthermore, HER2, microsatellite instability (MSI), and Epstein-Barr virus (EBV) status were investigated. NRG1 gene copy number (GCN) was determined by dual-color fluorescence in situ hybridization (FISH) in 388 available GCs. NRG1 overexpression was observed in 141 (28.1%) GCs and significantly associated with aggressive features, including infiltrative tumor growth, lymphovascular, and neural invasion, high pathologic stage, and poor prognosis (all P < 0.05), but not associated with EBV, MSI, or HER2 status. HER3 cytoplasmic and membranous expression were observed in 157 (31.3%) and 13 (2.6%), respectively. HER4 cytoplasmic expression was observed in 277 (55.2%), including 115 (22.9%) cases with nuclear expression. In contrast to NRG1, cytoplasmic expression of HER3 and HER4 proteins were not associated with survival, but GC patients with HER3 membranous expression showed significantly worse survival. In addition, HER4 nuclear expression was inveresely correlated with patients outcome in GC. NRG1 overexpression was also closely correlated with HER3 (P = 0.034) and HER4 (P < 0.001) cytoplasmic expression. NRG1 GCN gain (GCN ≥ 2.5) was detected in 14.7% patients, including two cases of amplification, and was moderately correlated with NRG1 overexpression (κ, 0.459; P < 0.001). Multivariate analysis identified NRG1 overexpression as an independent prognostic factor for survival (P = 0.040), unlike HER3 and HER4 expression. In 14 HER2positive GC with trastzumab combined chemotherapy, coexpression of NRG1 and HER3 was detected in 2 (14.3%) cases, and these GC patients group with coexpression of NRG1 and HER3 also showed a shorter PFS (P = 0.005).Although our results indicate a lack of prognostic significance of HER3 and HER4 overexpression in GC, overexpression of their ligand, NRG1, was associated with aggressive clinical features and represented an independent unfavorable prognostic factor. Therefore, NRG1 is a potential prognostic and therapeutic biomarker in GC patients.--

The purpose of this study was to interpret students' participation in terms of social and argumentation norms to improve understanding of social interaction in scientific argumentation. Therefore, the study sought to identify social and argumentation norms formed in group argumentation and to explore changes in students' participation as lessons progressed. Twelve lessons that included argumentation were delivered to 44 eighth graders in Korea. In each lesson, small group argumentation tasks were introduced after the teacher had explained the main concepts or after student-centred hands-on activities. We analysed argumentation in one focus group based on various data, including audiotaped and videotaped conversations, field notes and student interviews. In early lessons, discussions were always teacher-initiated and led by a high-achieving student, while other students rarely presented ideas. Moreover, students struggled to seek answers in a textbook and often used analogies and common sense to explain phenomena. They tended to accept others' opinions unquestioningly or ignore other low achievers' ideas in small group argumentation. In later lessons, we observed studentinitiated and more equally distributed discussions, in which students were likely to make claims or statements actively based on experimental results and scientific knowledge. Along with these changes in discussion style, some students were seen to support the building of social norms and argumentation norms in a group. Also, performing tasks and receiving guidance from the teacher helped to build students' epistemological norms about scientific argumentation.