RhoC protein, a known marker of metastases in aggressive breast cancers and melanoma, has also been found to be overexpressed in certain head and neck cancers, thus we investigated the correlation between RhoC expression and the metastatic behavior of head and neck squamous cell carcinoma. Selective inhibition of RhoC expression was achieved using lentiviral small hairpin RNA (shRNA) transduced and tracked with green fluorescent protein to achieve 70% to 80% RhoC inhibition. Fluorescence microscopy of the RhoC knockdown stable clones showed strong green fluorescence in the majority of cells, signifying a high efficiency of transduction. Importantly, quantitative real-time PCR showed no significant decrease in the mRNA expression levels of other members of the Ras superfamily. Cell motility and invasion were markedly diminished in RhoC-depleted cell lines as compared with control transduced lines. H&E staining of lung tissue obtained from severe combined immunodeficiency mice, which had been implanted with RhoC knockdown cells, showed a marked decrease in lung metastasis and inflammation of the blood vessels. The cultured lung tissue showed a significant decrease in cell growth in mice implanted with RhoC-depleted cell lines as compared with shRNA-scrambled sequence control lines. Microscopic studies of CD31 expression revealed substantial quantitative and qualitative differences in the primary tumor microvessel density as compared with parental and shRNA-scrambled controls. This study is the first of its kind to establish the involvement of RhoC specifically in head and neck metastasis. These findings suggest that RhoC warrants further investigation to delineate its robustness as a novel potentially therapeutic target. (Mol Cancer Res 2009;7(11):1771-80)

Programmed cell death 1 (PD-1) and its ligand (PD-L1) are key physiologic suppressors of the cytotoxic immune reaction. However, to date, the combination of PD1/PD-L1 expression and tumor-infiltrating lymphocytes (TILs) and antigen-presenting cells has been only minimally reported in breast carcinoma, in particular in relation to HER2-positive cases. The goal of this study was to evaluate both cellular tumoral immune reaction and PD-L1/PD1 distribution in HER2-positive cases, as well as any associations with clinical outcome using conventional chemotherapy combined with HER2 blocking. Multicolor immunohistochemical multiplex assays simultaneously demonstrating PD1, PD-L1, and CD8 or PD-L1, CD3, and CD163 were performed on tissue microarrays (TMA) representing 216 pretreatment cases of HER2-positive invasive breast carcinoma. PD-L1 expression was identified in 38 cases (18%), including 12 cases (6%) with PD-L1 labeling of tumor cells and 26 cases (12%) with PD-L1 labeling of immune cells only. Ten of 12 cases with PD-L1 staining of tumor cells showed staining of associated immune cells as well. With this assay method, PD1 was detectable in many fewer cases (6 cases or 3%). PD-L1 expression was positively associated with high Nottingham grade, negative ER and PR, the absence of lymph node metastasis, and high levels of CD8 cells. The overall survival by univariate analysis was positively associated with lower tumor stage, the absence of lymph node metastasis, PD-L1 expression, and high levels of CD8 cells. Therefore, our data suggest cytotoxic immune reaction mediated by CD8-positive T cells and PD-L1 expression may predict a better outcome in patients with HER2-positive breast carcinoma managed with conventional chemotherapy and HER2-blocking therapy. These findings recommend clinical trials utilizing checkpoint blocking immunotherapy in some form for HER2-positive breast cancer.

HER2 ITH analyses conducted with GPA method revealed that HER2 ITH is an independent factor predicting incomplete response to anti-HER2 neoadjuvant chemotherapy.

Intermediate neural progenitors (INPs) need to avoid both dedifferentiation and differentiation during neurogenesis, but the underlying mechanisms are not well understood. In Drosophila, the Ets protein Pointed P1 (PntP1) is required to generate INPs from type II neuroblasts. Here, we investigated how PntP1 promotes INP generation. By generating pntP1-specific mutants and using RNAi knockdown, we show that the loss of PntP1 leads to both an increase in type II neuroblast number and the elimination of INPs. The elimination of INPs results from the premature differentiation of INPs due to ectopic Prospero expression in newly generated immature INPs (imINPs), whereas the increase in type II neuroblasts results from the dedifferentiation of imINPs due to loss of Earmuff at later stages of imINP development. Furthermore, reducing Buttonhead enhances the loss of INPs in pntP1 mutants, suggesting that PntP1 and Buttonhead act cooperatively to prevent premature INP differentiation. Our results demonstrate that PntP1 prevents both the premature differentiation and the dedifferentiation of INPs by regulating the expression of distinct target genes at different stages of imINP development.

Objective: The Afirma gene expression classifier (GEC) is a molecular test to further classify indeterminate fine-needle aspiration (FNA) as benign or suspicious for malignancy. Study Design: A total of 158 FNAs with Bethesda category III/IV cytology were sent for an Afirma GEC test. We correlated the Afirma GEC results with surgical outcome and also compared the data after Afirma's implementation with the data before. Results: Among the 158 FNAs, the Afirma result was benign in 63 (40%), suspicious in 85 (54%) and unsatisfactory in 10 (6%). In total, 73 (86%) suspicious Afirma cases had surgery and 28 (38%) showed carcinoma. In contrast, only 8 (13%) benign Afirma cases had surgery and all of them were benign. The sensitivity, specificity, negative predictive value and positive predictive value (PPV) of Afirma were 100, 15, 100 and 38%, respectively. The PPV was 20% in cases with follicular lesion of undetermined significance, but was 50% in cases suspicious for follicular neoplasm (SFN). The surgical excisional rate was significantly decreased in SFN cases after the Afirma test. Conclusions: The Afirma GEC is useful for further risk stratifying SFN cases.

Our data have shown that examination of intratumoral CD8 cells together with PD-L1 expression proves useful in predicting response to anti-HER2 targeted therapy in patients with HER2 breast cancer.

FNAs of cervical lymph nodes yield adequate materials for reliable HR-HPV testing in metastatic HNSCCs and should be incorporated into routine pathologic evaluation for these patients. The high concordance between p16 IHC and HR-HPV ISH suggests p16 IHC may serve as a surrogate for HR-HPV ISH in metastatic HNSCC cases with oropharyngeal origin and is helpful to pinpoint primary tumor site. Diagn. Cytopathol. 2016;44:792-798. © 2016 Wiley Periodicals, Inc.

Recent studies indicate that activation of hypothalamic Agouti-related protein (Agrp) neurons can increase forage-related/repetitive behavior and decrease anxiety levels. However, the impact of physiological hunger states and food deprivation on anxiety-related behaviors have not been clarified. In the present study, we evaluated changes in anxiety levels induced by physiological hunger states and food deprivation, and identified the neuron population involved. Ad libitum fed and fasted mice were tested in the open field and elevated plus-maze behavioral tests. The DREADD approach was applied to selectively inhibit and stimulate neurons expressing Agrp in hypothalamic arcuate nucleus in Agrp-Cre transgenic mice. We found that anxiety levels were significantly reduced in the late light period when mice have increased need for food and increased Agrp neurons firing, in contrast to the levels in the early light period. Consistently, we also found that anxiety was potently reduced in 24-h fasted mice, relative to 12-h fasted mice or fed ad libitum. Mechanistically, we found that chemogenetic activation of Agrp neurons reduced anxiety in fed mice, and inactivation of Agrp neurons reduced fasting-induced anxiolytic effects. Our results suggest that anxiety levels may vary physiologically with the increasing need for food, and are influenced by acute fasting in a time-dependent manner. Agrp neurons contribute to fasting-induced anxiolytic effects, supporting the notion that Agrp neuron may serve as an entry point for the treatment of energy states-related anxiety disorders.