Oxysterols synergize with statins by inhibiting SREBP-2 in ovarian cancer cells.

OBJECTIVE: Determine mechanisms responsible for enhanced statin efficacy in a novel statin combination we name STOX (STatin-OXysterol). METHODS: Ovarian cancer cell lines were treated with combinations of statins and oxysterols. Cell viability was determined by a modified MTT assay. Apoptosis was evaluated by immunoblotting of PARP and DAPI-mediated visualization of apoptotic nuclei. STOX effects on the expression of genes of the mevalonate pathway were assessed by real-time qPCR and immunoblotting. siRNA-mediated gene silencing was used to test the involvement of oxysterol-mediated repression of SREBP-2 in STOX synergy. The impact of statin-mediated inhibition of protein prenylation and on cholesterol homeostasis was evaluated. RESULTS: Oxysterols dramatically enhance cytotoxicity of statins in ovarian cancer cells through increased apoptosis. Decreased expression of SREBP-2 down-regulates the mevalonate pathway and prevents the active statin-induced sterol feedback, enhancing statin toxicity. Comparison of two ovarian cancer cell lines reveals two distinct mechanisms of statin induced toxicity, namely, dependence on protein geranylgeranylation and/or perturbation of cellular cholesterol levels. CONCLUSIONS: We provide evidence of statins' mechanisms of cytotoxicity in different ovarian cancer cells and discovered a new approach to significantly enhance the anti-tumor activity of statins. These observations provide a potential new path to improve statins as a treatment against ovarian cancer with obtainable dosages.

Patient Goal-directed Care in an Orthopaedic Spine Specialty Clinic.

INTRODUCTION: Using health-related goals to direct care could improve quality and reduce cost of medical care; however, the effect of these goals for patients with spinal pathologies is not well understood. The purpose of this study was to describe patient-reported goals by provider type and to evaluate the effect of patient-provider goal awareness on patient satisfaction and treatment pathway. METHODS: A pilot program was instituted in which all new or existing patients scheduled with either a single spine surgeon or a nonsurgical spine nurse practitioner were asked to complete a paper survey instrument regarding their goals of care before their visit. The patient goals were then discussed between the provider and the patient. Univariate and multivariate analyses were performed to evaluate relationships between patient goals, provider seen, diagnosis, and treatment recommendations. RESULTS: There were 703 respondents to the survey, of whom 416 were included for subgroup analysis. Patient-reported goals varied by provider type. When examining rates of recommended interventions by patient goals, notable differences were observed for 7 of the 13 goal categories. Significant differences in intervention recommendations by provider type existed for physical therapy, medications, MRI, and surgery (all P < 0.001). After controlling for other variables, seeing a surgeon, thoracolumbar pathology, and goals of "return to activity or social events I enjoy," and "learn about spine surgery" were significant independent predictors of recommendation for surgery (all odds ratio > 3 and P < 0.05). This model generated an area under the curve of 0.923 (95% confidence interval, 0.861 to 0.986), indicating outstanding discrimination in predicting recommendation for surgery. Patient satisfaction scores rose from 91.5% to 92.2%, but this difference was not statistically significant (P = 0.782). CONCLUSION: Specific patient-reported goals vary by provider type and are associated with specific diagnosis and treatment recommendations. Goal-directed care may improve the design of treatment pathways and the overall patient experience.

Order of concentric and eccentric muscle actions affects metabolic responses.

BACKGROUND: Resistance exercise provides an effective stimulus for improving the metabolic plasticity of skeletal muscle, and the type of acute muscle contraction plays an important role in determining specific responses and adaptations. The purpose of the current investigation was to examine the effect of contraction order on metabolic responses by comparing monophasic concentric and eccentric squats versus a protocol incorporating alternated concentric and eccentric repetitions. METHODS: Twelve recreationally active men (21.1±1.1yr) performed three nearly identical squat protocols on separate days. Protocols varied only with contraction-type, including 4 sets × 10 reps concentric-only (CON), eccentric-only (ECC), and BOTH which alternated 5 concentric followed by 5 eccentric reps (CON-ECC; sets 1 and 3) and vice versa (ECC-CON; sets 2 and 4). The experimental trials were performed once weekly in a randomized, counter-balanced order, and expired gases were collected using a two-way non-rebreathing mask and oxygen consumption quantified with indirect calorimetry. Subjects raised (CON) and lowered (ECC) the load in 2s, and all sets (2 min) and repetitions (8 s) were separated by standardized rest intervals. RESULTS: From the BOTH protocol, the increase in metabolic rate was significantly greater (P≤0.05) during squats performed with CON-ECC order (0.60±0.11 L·min-1) compared to ECC-CON (0.44±0.07 L·min-1), but excess postexercise oxygen consumption (EPOC) was opposite, with significantly greater metabolic rate during the 2-minute rest intervals after ECC-CON squats (0.46±0.09 L·min-1) compared to CON-ECC (0.25±0.05 L·min-1). Metabolic rates during and after squats were significantly greater (P≤0.05) with CON (0.63±0.09; 0.49±0.10 L·min-1) compared to ECC (0.34±0.04; 0.20±0.04 L·min-1), respectively. CONCLUSIONS: These data present an interesting paradigm regarding the contraction-dependent metabolic responses to monophasic resistance exercise and suggest a greater EPOC following concentric versus eccentric muscle actions.

Leveraging Aging in Place Through Sensor-Enhanced In-Home Monitoring.

Seniors expect to age in place, which means living in their own homes as long as possible with familiar facilities and environments. Due to the capability of continuous and unobtrusive monitoring, the sensor-enhanced in-ho monitoring is regarded as a promising solution to support aging in place. In this paper, by reviewing three influential projects in this field of in-home monitoring for aging in place, we present our opinions and suggestions on the development of informatics-supported aging in place for its practical application in healthcare such as diagnosis and nursing in the era of data science. To promote the practical usage of in-home monitoring in aging, we highlight the gap between demands and available approaches. We conclude that in the next stage we should design demand-oriented system, conduct evidence-based research and accelerate interdisciplinary collaboration.

Clinical Acquired Resistance to RAF Inhibitor Combinations in BRAF-Mutant Colorectal Cancer through MAPK Pathway Alterations.

UNLABELLED: BRAF mutations occur in approximately 10% of colorectal cancers. Although RAF inhibitor monotherapy is highly effective in BRAF-mutant melanoma, response rates in BRAF-mutant colorectal cancer are poor. Recent clinical trials of combined RAF/EGFR or RAF/MEK inhibition have produced improved efficacy, but patients ultimately develop resistance. To identify molecular alterations driving clinical acquired resistance, we performed whole-exome sequencing on paired pretreatment and postprogression tumor biopsies from patients with BRAF-mutant colorectal cancer treated with RAF inhibitor combinations. We identified alterations in MAPK pathway genes in resistant tumors not present in matched pretreatment tumors, including KRAS amplification, BRAF amplification, and a MEK1 mutation. These alterations conferred resistance to RAF/EGFR or RAF/MEK combinations through sustained MAPK pathway activity, but an ERK inhibitor could suppress MAPK activity and overcome resistance. Identification of MAPK pathway reactivating alterations upon clinical acquired resistance underscores the MAPK pathway as a critical target in BRAF-mutant colorectal cancer and suggests therapeutic options to overcome resistance. SIGNIFICANCE: RAF inhibitor combinations represent promising approaches in clinical development for BRAF-mutant colorectal cancer. Initial characterization of clinical acquired resistance mechanisms to these regimens identified several MAPK pathway alterations driving resistance by reactivating MAPK signaling, highlighting the critical dependence of BRAF-mutant colorectal cancers on MAPK signaling and offering potential strategies to overcome resistance.

Hobnail variant of papillary thyroid carcinoma: an institutional case series and molecular profile.

BACKGROUND: Papillary thyroid carcinoma (PTC) is increasing in incidence while mortality is unchanged. Identifying patients with higher risk of recurrence and death is essential. Case series identify the hobnail variant of PTC (HVPTC), which is characterized by micropapillary architecture, apocrine features, and loss of cellular polarity. Herein, we describe the clinical course, pathologic features, and mutational profile of patients at our institution with HVPTC. METHODS: A query into the surgical pathologic database (2009-2012) was performed, and clinicopathologic data were collected on all patients carrying the diagnosis of HVPTC. BRAF(V600E) testing was performed on paraffin-embedded blocks using SNaPshot mutational analysis. RESULTS: Twelve patients with HVPTC were identified, with an average age of 54.1±18.8 years. Seven patients (63.6%) were AJCC Stage III or IV at presentation. Tumors were large (3.7±2.0 cm), some were multifocal (33.3%), and frequently with extrathyroidal extension (58.3%), lymphovascular invasion (41.7%), and lymph node metastasis (75%). Forty percent of the patients had concomitant tall cell features (TCF), and two had small foci of undifferentiated (anaplastic) thyroid carcinoma (ATC). Eighty percent of tumors undergoing mutational analysis had the BRAF(V600E) mutation, and the remaining 20% harbored a RET/PTC1 gene rearrangement. No other known thyroid cancer mutations were identified on SNaPshot analysis. At median follow-up of 26 months, four patients had recurrent or persistent disease, one of whom died from the disease one year after surgery. CONCLUSIONS: The hobnail variant of PTC has an aggressive behavior, with a high incidence of infiltrative tumors and metastatic disease. Strikingly, all tumors in our series harbored a PTC-associated genetic abnormality, either a BRAF(V600E) mutation (80%) or a RET/PTC1 rearrangement (20%). This histologic variant warrants further study, and patients with this diagnosis should be observed closely for recurrence.

VE1 antibody immunoreactivity in normal anterior pituitary and adrenal cortex without detectable BRAF V600E mutations.

OBJECTIVES: The VE1 monoclonal antibody was developed to recognize the V600E mutation in BRAF, which is found in various tumors. METHODS: We report that the VE1 antibody stains normal anterior pituitary gland and adrenal cortex, which lack detectable BRAF V600E mutations. RESULTS: Staining with the VE1 antibody was seen in the adenohypophysis and correlated well with adrenocorticotropic hormone (ACTH)-positive cells. ACTH-positive cells were typically most concentrated in the central mucoid wedge and pars intermedia, and VE1 staining was strong in these regions. Moreover, VE1 staining was seen in ACTH-expressing pituitary adenomas without detectable BRAF mutations. VE1 staining of the adrenal cortex was also significant, with the strongest staining seen in the inner segment of the zona fasciculata. Parathyroid glands, pancreatic islets, or parafollicular C cells in the thyroid showed no VE1 staining. CONCLUSIONS: Overall, VE1 staining of endocrine tissues strongly suggests limitations on the use of this antibody for the detection of BRAF mutations.

Identification of oncogenic mutations and gene fusions in the follicular variant of papillary thyroid carcinoma.

BACKGROUND: The diagnosis of the follicular variant of papillary thyroid carcinoma (FVPTC) is increasingly common. Recent studies have suggested that FVPTC is heterogeneous and comprises multiple tumor types with distinct biological behaviors and underlying genetics. OBJECTIVES: The purpose of this work was to identify the prevalence of mutations and gene fusions in known oncogenes in a panel representative of the common spectrum of FVPTC diagnosed at an academic medical center and correlate the clinical and pathological features obtained at the initial diagnosis with the tumor genotype. MATERIALS AND METHODS: We performed SNaPshot genotyping on a panel of 129 FVPTCs of ≥1 cm for 90 point mutations or small deletions in known oncogenes and tumor suppressors and identified gene fusions using an anchored multiplex PCR assay targeting a panel of rearranged oncogenes. RESULTS: We identified a mutation or gene fusion in 70% (89 of 127) of cases. Mutations targeting the RAS family of oncogenes were the most frequently observed class of alterations, present in 36% (46 of 127) of cases, followed by BRAF mutation, present in 30% (38 of 127). We also detected oncogenic rearrangements not previously associated with FVPTC, including TFG-ALK and CREB3L2-PPARγ. BRAF mutation was significantly associated with unencapsulated tumor status. CONCLUSIONS: These data support the hypothesis that FVPTC is composed of distinct biological entities, with one class being identified by BRAF mutation and support the use of clinical genotyping assays that detect a diverse array of rearrangements involving ALK and PPARγ. Additional studies are necessary to identify genetic drivers in the 30% of FVPTCs with no known oncogenic alteration and to better predict behavior in tumors with known genotypes.

Anchored multiplex PCR for targeted next-generation sequencing.

We describe a rapid target enrichment method for next-generation sequencing, termed anchored multiplex PCR (AMP), that is compatible with low nucleic acid input from formalin-fixed paraffin-embedded (FFPE) specimens. AMP is effective in detecting gene rearrangements (without prior knowledge of the fusion partners), single nucleotide variants, insertions, deletions and copy number changes. Validation of a gene rearrangement panel using 319 FFPE samples showed 100% sensitivity (95% confidence limit: 96.5-100%) and 100% specificity (95% confidence limit: 99.3-100%) compared with reference assays. On the basis of our experience with performing AMP on 986 clinical FFPE samples, we show its potential as both a robust clinical assay and a powerful discovery tool, which we used to identify new therapeutically important gene fusions: ARHGEF2-NTRK1 and CHTOP-NTRK1 in glioblastoma, MSN-ROS1, TRIM4-BRAF, VAMP2-NRG1, TPM3-NTRK1 and RUFY2-RET in lung cancer, FGFR2-CREB5 in cholangiocarcinoma and PPL-NTRK1 in thyroid carcinoma. AMP is a scalable and efficient next-generation sequencing target enrichment method for research and clinical applications.

Comparison of 2 monoclonal antibodies for immunohistochemical detection of BRAF V600E mutation in malignant melanoma, pulmonary carcinoma, gastrointestinal carcinoma, thyroid carcinoma, and gliomas.

BRAF mutation is seen in a variety of human neoplasms including cutaneous malignant melanoma, papillary thyroid carcinoma, colorectal carcinoma, non-small cell lung carcinoma, pleomorphic xanthoastrocytoma, and others. Currently, there are 2 commercially available monoclonal antibodies for the detection of BRAF V600E mutation; however, a full and practical comparison of their performance in various tumor types on an automated staining platform has not been done. We investigated their sensitivity and specificity in detecting the BRAF V600E mutation in a series of 152 tumors including 31 malignant melanomas, 25 lung carcinomas, 32 gastrointestinal carcinomas, 23 thyroid carcinomas, 35 gliomas, and 6 other malignancies. In this series, the concordance rate between immunohistochemistry (IHC) and mutational analyses was 97% (148/152) for VE1 and 88% (131/149) for anti-B-Raf. The sensitivity and specificity were 98% (60/61) and 97% (88/91) for monoclonal VE1 and 95% (58/61) and 83% (73/88) for anti-B-Raf, respectively. There were 4 cases with discordant IHC and mutational results for monoclonal VE1 in contrast to 18 cases for anti-B-Raf. Our studies showed that IHC with monoclonal VE1 has a better performance compared with anti-B-Raf in an automated staining platform and confirmed that clone VE1 provides excellent sensitivity and specificity for detecting the BRAF V600E mutation in a variety of tumor types in a clinical setting.

Effect of delayed auditory feedback on normal speakers at two speech rates.

This study investigated the effect of short and long auditory feedback delays at two speech rates with normal speakers. Seventeen participants spoke under delayed auditory feedback (DAF) at 0, 25, 50, and 200 ms at normal and fast rates of speech. Significantly two to three times more dysfluencies were displayed at 200 ms (p<0.05) relative to no delay or the shorter delays. There were significantly more dysfluencies observed at the fast rate of speech (p = 0.028). These findings implicate the peripheral feedback system(s) of fluent speakers for the disruptive effects of DAF on normal speech production at long auditory feedback delays. Considering the contrast in fluency/dysfluency exhibited between normal speakers and those who stutter at short and long delays, it appears that speech disruption of normal speakers under DAF is a poor analog of stuttering.