ω-3PUFA supplementation ameliorates adipose tissue inflammation and insulin-stimulated glucose disposal in subjects with obesity: a potential role for apolipoprotein E.

BACKGROUND: Long chain omega-3 polyunsaturated fatty acids (ω-3PUFA) supplementation in animal models of diet-induced obesity has consistently shown to improve insulin sensitivity. The same is not always reported in human studies with insulin resistant (IR) subjects with obesity. OBJECTIVE: We studied whether high-dose ω-3PUFA supplementation for 3 months improves insulin sensitivity and adipose tissue (AT) inflammation in IR subjects with obesity. METHODS: Thirteen subjects (BMI = 39.3 ± 1.6 kg/m2) underwent 80 mU/m2·min euglycemic-hyperinsulinemic clamp with subcutaneous (Sc) AT biopsy before and after 3 months of ω-3PUFA (DHA and EPA, 4 g/daily) supplementation. Cytoadipokine plasma profiles were assessed before and after ω-3PUFA. AT-specific inflammatory gene expression was evaluated on Sc fat biopsies. Microarray analysis was performed on the fat biopsies collected during the program. RESULTS: Palmitic and stearic acid plasma levels were significantly reduced (P < 0.05) after ω-3PUFA. Gene expression of pro-inflammatory markers and adipokines were improved after ω-3PUFA (P < 0.05). Systemic inflammation was decreased after ω-3PUFA, as shown by cytokine assessment (P < 0.05). These changes were associated with a 25% increase in insulin-stimulated glucose disposal (4.7 ± 0.6 mg/kg ffm•min vs. 5.9 ± 0.9 mg/kg ffm•min) despite no change in body weight. Microarray analysis identified 53 probe sets significantly altered post- ω-3PUFA, with Apolipoprotein E (APOE) being one of the most upregulated genes. CONCLUSION: High dose of long chain ω-3PUFA supplementation modulates significant changes in plasma fatty acid profile, AT, and systemic inflammation. These findings are associated with significant improvement of insulin-stimulated glucose disposal. Unbiased microarray analysis of Sc fat biopsy identified APOE as among the most differentially regulated gene after ω-3PUFA supplementation. We speculate that ω-3PUFA increases macrophage-derived APOE mRNA levels with anti-inflammatory properties.

The Identification of Macrophage-enriched Glycoproteins Using Glycoproteomics.

Prostate cancer is a leading cause of cancer-related deaths of men in the United States. Whereas the localized disease is highly treatable by surgical resection and radiation, cancer that has metastasized remains incurable. Immune cells that primarily scavenge debris and promote prostate cancer angiogenesis and wound repair are M2 macrophages. They are phenotypically similar to M2 tumor-associated macrophages (M2-TAMs) and have been reported to associate with solid tumors and aide in proliferation, metastasis, and resistance to therapy. As an invasive species within the tumor microenvironment, this makes M2-TAMs an ideal therapeutic target in prostate cancer. To identify novel surface glycoproteins expressed on M2 macrophages, we developed a novel method of creating homogeneous populations of human macrophages from human CD14+ monocytes in vitro These homogeneous M1 macrophages secrete pro-inflammatory cytokines, and our M2 macrophages secrete anti-inflammatory cytokines as well as vascular endothelial growth factor (VEGF). To identify enriched surface glycoproteins, we then performed solid-phase extraction of N-linked glycopeptides followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) on our homogeneous macrophage populations. We discovered five novel peptides that are enriched exclusively on human M2 macrophages relative to human M1 macrophages and human CD14+ monocytes. Finally, we determined whether these surface glycoproteins, found enriched on M2 macrophages, were also expressed in human metastatic castrate-resistant prostate cancer (mCRPC) tissues. Using mCRPC tissues from rapid autopsies, we were able to determine M2 macrophage infiltration by using immunohistochemistry and flow cytometry. These findings highlight the presence of macrophage infiltration in human mCRPC but also surface glycoproteins that could be used for prognosis of localized disease and for targeting strategies.

Identifying global expression patterns and key regulators in epithelial to mesenchymal transition through multi-study integration.

BACKGROUND: Epithelial to mesenchymal transition (EMT) is the process by which stationary epithelial cells transdifferentiate to mesenchymal cells with increased motility. EMT is integral in early stages of development and wound healing. Studies have shown that EMT could be a critical early event in tumor metastasis that is involved in acquisition of migratory and invasive properties in multiple carcinomas. METHODS: In this study, we used 15 published gene expression microarray datasets from Gene Expression Omnibus (GEO) that represent 12 cell lines from 6 cancer types across 95 observations (45 unique samples and 50 replicates) with different modes of induction of EMT or the reverse transition, mesenchymal to epithelial transition (MET). We integrated multiple gene expression datasets while considering study differences, batch effects, and noise in gene expression measurements. A universal differential EMT gene list was obtained by normalizing and correcting the data using four approaches, computing differential expression from each, and identifying a consensus ranking. We confirmed our discovery of novel EMT genes at mRNA and protein levels in an in vitro EMT model of prostate cancer - PC3 epi, EMT and Taxol resistant cell lines. We validate our discovery of C1orf116 as a novel EMT regulator by siRNA knockdown of C1orf116 in PC3 epithelial cells. RESULTS: Among differentially expressed genes, we found known epithelial and mesenchymal marker genes such as CDH1 and ZEB1. Additionally, we discovered genes known in a subset of carcinomas that were unknown in prostate cancer. This included epithelial specific LSR and S100A14 and mesenchymal specific DPYSL3. Furthermore, we also discovered novel EMT genes including a poorly-characterized gene C1orf116. We show that decreased expression of C1orf116 is associated with poor prognosis in lung and prostate cancer patients. We demonstrate that knockdown of C1orf116 expression induced expression of mesenchymal genes in epithelial prostate cancer cell line PC3-epi cells, suggesting it as a candidate driver of the epithelial phenotype. CONCLUSIONS: This comprehensive approach of statistical analysis and functional validation identified global expression patterns in EMT and candidate regulatory genes, thereby both extending current knowledge and identifying novel drivers of EMT.

The presence of androgen receptor elements regulates ZEB1 expression in the absence of androgen receptor.

Zinc finger E-box binding homeobox 1 (ZEB1) is a transcription factor that plays a central role in the epithelial to mesenchymal transition (EMT) of cancer cell lines. Studies on its regulation have mostly focused on the negative 3'UTR binding of miR200c. Interestingly, it has been previously reported that androgen receptor (AR) regulates ZEB1 expression in breast and prostate cancers. In order to validate this, various ZEB1 promoter deletions were cloned into a luciferase reporter system to elucidate the contribution of two putative androgen response elements (AREs). The in vivo contribution of AR was also assessed in cell lines after R1881 treatment using qPCR with prostate specific antigen (PSA) as the positive control. We discovered that AR upregulates the levels of expression of ZEB1 10-fold on a luciferase promoter that only contains the distal ARE. However, when the proximal ARE is included, no additional activation is apparent with AR or its hormone independent variant, AR-V7. Furthermore, we demonstrate here that a promoter construct containing both AREs activates transcription of ZEB1 even in the AR-null cell lines DU145 and PC3. Incubation of the AR-positive cell line, LNCaP with R1881, failed to substantially increase the expression levels of ZEB1. Despite the presence of AREs in the promoter region, it appears that ZEB1 expression can be induced even without AR. In addition, the region around the distal ARE is a potent repressor in AR-null cell lines.

Linking adipose tissue eosinophils, IL-4, and leptin in human obesity and insulin resistance.

BACKGROUNDObesity is a multifactorial disease with adverse health implications including insulin resistance (IR). In patients with obesity, the presence of high circulating levels of leptin, deemed hyperleptinemia, is associated with IR. Recent data in mice with diet-induced obesity (DIO) show that a partial reduction in leptin levels improves IR. Additional animal studies demonstrate that IL-4 decreases leptin levels. In rodents, resident adipose tissue eosinophils (AT-EOS) are the main source of IL-4 and are instrumental in maintaining metabolic homeostasis. A marked reduction in AT-EOS content is observed in animal models of DIO. These observations have not been explored in humans.METHODSWe analyzed AT from individuals with obesity and age-matched lean counterparts for AT-EOS content, IL-4, circulating leptin levels, and measures of IR.RESULTSOur results show that individuals with obesity (n = 15) had a significant reduction in AT-EOS content (P < 0.01), decreased AT-IL-4 gene expression (P = 0.02), and decreased IL-4 plasma levels (P < 0.05) in addition to expected IR (P < 0.001) and hyperleptinemia (P < 0.01) compared with lean subjects (n = 15). AT-EOS content inversely correlated with BMI (P = 0.002) and IR (P = 0.005). Ex vivo AT explants and in vitro cell culture of primary human mature adipocytes exposed to either IL-4 or EOS conditioned media produced less leptin (P < 0.05).CONCLUSIONOur results suggest that IL-4 acts as a link between EOS, AT, and leptin production. Future studies exploring this interaction may identify an avenue for the treatment of obesity and its complications through amelioration of hyperleptinemia.TRIAL REGISTRATIONClinicaltrials.gov NCT02378077 & NCT04234295.

Comprehensive NGS profiling to enable detection of ALK gene rearrangements and MET amplifications in non-small cell lung cancer.

Introduction: Next-generation sequencing (NGS) is currently widely used for biomarker studies and molecular profiling to identify concurrent alterations that can lead to the better characterization of a tumor's molecular landscape. However, further evaluation of technical aspects related to the detection of gene rearrangements and copy number alterations is warranted. Methods: There were 12 ALK rearrangement-positive tumor specimens from patients with non-small cell lung cancer (NSCLC) previously detected via fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), and an RNA-based NGS assay, and 26 MET high gene copy number (GCN) cases detected by FISH, selected for this retrospective study. All 38 pre-characterized cases were reassessed utilizing the PGDx™ elio™ tissue complete assay, a 505 gene targeted NGS panel, to evaluate concordance with these conventional diagnostic techniques. Results: The detection of ALK rearrangements using the DNA-based NGS assay demonstrated excellent sensitivity with the added benefit of characterizing gene fusion partners and genomic breakpoints. MET copy number alterations were also detected; however, some discordances were observed likely attributed to differences in algorithm, reporting thresholds and gene copy number state. TMB was also assessed by the assay and correlated to the presence of NSCLC driver alterations and was found to be significantly lower in cases with NGS-confirmed canonical driver mutations compared with those without (p=0.0019). Discussion: Overall, this study validates NGS as an accurate approach for detecting structural variants while also highlighting the need for further optimization to enable harmonization across methodologies for amplifications.

IL-4 induces proliferation in prostate cancer PC3 cells under nutrient-depletion stress through the activation of the JNK-pathway and survivin up-regulation.

Interleukin (IL)-4 plays a critical role in the regulation of immune responses and has been detected at high levels in the tumor microenvironment of cancer patients where it correlates with the grade of malignancy. The direct effect of IL-4 on cancer cells has been associated with increased cell survival; however, its role in cancer cell proliferation and related mechanisms is still unclear. Here it was shown that in a nutrient-depleted environment, IL-4 induces proliferation in prostate cancer PC3 cells. In these cells, under nutrient-depletion stress, IL-4 activates mitogen-activated protein kinases (MAPKs), including Erk, p38, and JNK. Using MAP-signaling-specific inhibitors, it was shown that IL-4-induced proliferation is mediated by JNK activation. In fact, JNK-inhibitor-V (JNKi-V) stunted IL-4-mediated cell proliferation. Furthermore, it was found that IL-4 induces survivin up-regulation in nutrient-depleted cancer cells. Using survivin-short-hairpin-RNAs (shRNAs), it was demonstrated that in this milieu survivin expression above a threshold limit is critical to the mechanism of IL-4-mediated proliferation. In addition, the significance of survivin up-regulation in a stressed environment was assessed in prostate cancer mouse xenografts. It was found that survivin knockdown decreases tumor progression in correlation with cancer cell proliferation. Furthermore, under nutrient depletion stress, IL -4 could induce proliferation in cancer cells from multiple origins: MDA-MB-231 (breast), A253 (head and neck), and SKOV-3 (ovarian). Overall, these findings suggest that in a tumor microenvironment under stress conditions, IL-4 triggers a simultaneous activation of the JNK-pathway and the up-regulation of survivin turning on a cancer proliferation mechanism.

Automated next-generation profiling of genomic alterations in human cancers.

The lack of validated, distributed comprehensive genomic profiling assays for patients with cancer inhibits access to precision oncology treatment. To address this, we describe elio tissue complete, which has been FDA-cleared for examination of 505 cancer-related genes. Independent analyses of clinically and biologically relevant sequence changes across 170 clinical tumor samples using MSK-IMPACT, FoundationOne, and PCR-based methods reveals a positive percent agreement of >97%. We observe high concordance with whole-exome sequencing for evaluation of tumor mutational burden for 307 solid tumors (Pearson r = 0.95) and comparison of the elio tissue complete microsatellite instability detection approach with an independent PCR assay for 223 samples displays a positive percent agreement of 99%. Finally, evaluation of amplifications and translocations against DNA- and RNA-based approaches exhibits >98% negative percent agreement and positive percent agreement of 86% and 82%, respectively. These methods provide an approach for pan-solid tumor comprehensive genomic profiling with high analytical performance.

Are you responsible for medical director duties in anatomic pathology--and why should you care?

For many pathologists, the roles of the medical director are not clearly distinguished from other medical leadership and management duties, such as those of the chair of pathology or a section head of anatomic pathology. We describe the evolution in the understanding of the roles of the medical director as defined by the Clinical Laboratory Improvement Amendments of 1988 and separate from the roles of the chair of pathology, department chair, division chair, and a section head in anatomic pathology in our institution. Delineation of the roles of medical director, department chair, chair of anatomic pathology division, and a section head of a subdivision in anatomic pathology has helped us to understand the roles and ensure that all duties are fulfilled for patient care and for compliance and regulation.

Am I demoralizing my staff?--10 questions to ask.

Examine 10 questions that all physician leaders should ask themselves periodically to ensure that they are leading, and not bringing down, their staff.

A FACS-based approach to obtain viable eosinophils from human adipose tissue.

Eosinophils have been widely investigated in asthma and allergic diseases. More recently, new insights into the biology of these cells has illustrated eosinophils contribute to homeostatic functions in health such as regulation of adipose tissue glucose metabolism. Human translational studies are limited by the difficulty of obtaining cells taken directly from their tissue environment, relying instead on eosinophils isolated from peripheral blood. Isolation techniques for tissue-derived eosinophils can result in unwanted cell or ribonuclease activation, leading to poor cell viability or RNA quality, which may impair analysis of effector activities of these cells. Here we demonstrate a technique to obtain eosinophils from human adipose tissue samples for the purpose of downstream molecular analysis. From as little as 2 g of intact human adipose tissue, greater than 104 eosinophils were purified by fluorescence-activated cell sorting (FACS) protocol resulting in ≥ 99% purity and ≥ 95% viable eosinophils. We demonstrated that the isolated eosinophils could undergo epigenetic analysis to determine differences in DNA methylation in various settings. Here we focused on comparing eosinophils isolated from human peripheral blood vs human adipose tissue. Our results open the door to future mechanistic investigations to better understand the role of tissue resident eosinophils in different context.

"Rainbow Draws" in the Emergency Department: Clinical Utility and Staff Perceptions.

BACKGROUND: Collecting a predefined set of blood tubes (the "rainbow draw") is a common but controversial practice in many emergency departments (EDs), with limited data to support it. We determined the actual utilization of rainbow draw tubes at a single facility and evaluated the perceptions of ED staff regarding the utility of rainbow draws. METHODS: We analyzed 2 weeks of ED visits (1326 visits by 1240 unique patients) to determine blood tube utilization for initial and add-on testing, as well as the incidence of additional venipunctures. We also surveyed ED staff regarding aspects of ED phlebotomy and test ordering. Utilization data analysis was structured to satisfy specific concerns addressed in the ED staff survey. RESULTS: Observed tube utilization data showed that fluoride/oxalate, citrate, and serum separator tubes were frequently discarded unused, and that the actual utility of the rainbow draw for add-on testing and avoiding additional venipunctures was low. ED staff perceived that the rainbow draw was highly valuable, both to expedite add-on testing and to avoid additional venipunctures. Contrasting the objective (utilization data) and subjective (survey results) to drive changes in the standard ED blood collection reduced the estimated waste blood by 175 L/year. CONCLUSIONS: Comparison of perceptions and objective utilization data drove process changes that were mutually agreeable to ED and laboratory staff. Although specifics of ED and laboratory work flows vary between institutions, the principles and strategy of this study are widely applicable.

Vertical versus lateral thinking.

Traditional medical training may actually impede a physician's ability to solve complex problems because it is based on vertical, linear and traditional thinking.

Burnout in pathology: suggestions for individual and systemwide solutions.

Burnout, a syndrome characterized by exhaustion, cynicism, and reduced effectiveness, does not spare pathologists, including cytopathologists, residents, and fellows. Burnout extracts a huge physician toll, resulting in decreased quality of care, poorer patient safety, increased physician turnover, and diminished patient satisfaction. In this review, we describe the drivers of burnout and suggest both individual and systemwide solutions that some centers have implemented to reverse the trend of increasing physician burnout.

A Flow Cytometry-Based Assay for Procoagulant Platelet Polyphosphate.

BACKGROUND: Platelet polyphosphate is an inorganic procoagulant polymer of orthophosphate units that is stored in dense granules and is released upon platelet activation. Here, we describe an assay to measure polyphosphate on the surface of procoagulant human platelets. METHODS AND RESULTS: Recombinant Escherichia coli-expressed exopolyphosphatase deletion mutant PPX_Δ12 labeled with fluorescent Alexa488 dye was used as a polyphosphate probe in flow cytometry. PPX_Δ12-Alexa488-signal dose-dependently increased with long-chain polyphosphate binding to platelets. In contrast, short-chain polyphosphate that is found in the supernatant of activated platelets, did not bind to the platelet surface. Both exopolyphosphatase treatment and polyphosphate pre-incubation abolished PPX_Δ12-Alexa488 binding to polyphosphate on platelets. Stimulation of platelets with thrombin receptor agonist Trap6, and P2Y12 receptor activator ADP increased polyphosphate accumulation on platelet surfaces and PPX_Δ12-Alexa488 signal in a dose-dependent manner. CONCLUSION: This study indicates that long-chain polyphosphate binds to platelet plasma membranes and presents a promising diagnostic assay to measure this interaction on human platelets in platelet-rich plasma. Future investigations will aim to determine if polyphosphate can be used as a novel biomarker of thrombosis. © 2016 International Clinical Cytometry Society.