Illumina HumanHT-12 V4 BeadChips Research Articles

Response to intranasal Lactococcus lactis W136 probiotic supplementation in refractory CRS is associated with modulation of non-type 2 inflammation and epithelial regeneration.

We have previously documented that in individuals with chronic rhinosinusitis (CRS) refractory to surgery, intranasal application of live Lactococcus lactis W136, a probiotic bacterium, improves sinus-specific symptoms, SNOT-22, and mucosal aspect on endoscopy, accompanied by a reduction in sinus pathogens and an increase in protective bacteria. The present work explores the molecular mechanisms underpinning these observations using transcriptomics of the sinus mucosa. Epithelial brushings collected prospectively as a sub-study of the L. lactis W136 clinical trial were used to probe epithelial responses to microbiome supplementation using a hypothesis-free bioinformatic analysis of gene expression analysis. Samples from twenty-four patients with CRS refractory to medical and surgical management were prospectively collected during a clinical trial assessing the effect of 14 days of BID nasal irrigation with 1.2 billion CFU of live L. lactis W136 probiotic bacteria (CRSwNP = 17, CRSsNP = 7). Endoscopically guided sinus brushings were collected as part of the initial study, with brushings performed immediately before and after treatment. Following RNA extraction, samples were assessed using the Illumina HumanHT-12 V4 BeadChip. Differential gene expression was calculated, and pathway enrichment analysis was performed to identify potentially implicated processes. Differentially identified transcripts and pathways were assessed for the overall population and the clinical phenotypes of CRSwNP and CRSsNP. Patterns of response to treatment were similar across all groups, implicating pathways for the regulation of immunity and epithelial cell regulation. These resemble the patterns of improvement observed following successful treatment with endoscopic sinus surgery or azithromycin. Gene expression profiling following the application of live bacteria to the diseased sinus epithelium highlights the implication of multiple components of the inflammation-microbiome-epithelial barrier axis implicated in CRS. These effects appear to involve both epithelial restoration and modulation of innate and adaptive immunity, supporting the potential interest of targeting the sinus epithelium and the microbiome as potential CRS therapies.

Early blood transcriptomic signature predicts patients’ outcome after out-of-hospital cardiac arrest

BackgroundEarly prognostication is a major challenge after out-of-hospital cardiac arrest (OHCA). AimsWe hypothesized that a genome-wide analysis of blood gene expression could offer new prognostic tools and lines of research. MethodsSixty-nine patients were enrolled from an ancillary study of the clinical trial NCT00999583 that tested the effect of erythropoietin (EPO) after OHCA. Blood samples were collected in comatose survivors of OHCA at hospital admission and 1 and 3 days after resuscitation. Gene expression profiles were analyzed (Illumina HumanHT-12 V4 BeadChip; >34,000 genes). Patients were classified into two categories representing neurological favorable outcome (cerebral performance category [CPC] = 1-2) vs unfavorable outcome (CPC > 2) at Day 60 after OHCA. Differential and functional enrichment analyses were performed to compare transcriptomic profiles between these two categories. ResultsAmong the 69 enrolled patients, 33 and 36 patients were treated or not by EPO, respectively. Among them, 42% had a favorable neurological outcome in both groups. EPO did not affect the transcriptomic response at Day-0 and 1 after OHCA. In contrast, 76 transcripts differed at Day-0 between patients with unfavorable vs favorable neurological outcome. This signature persisted at Day-1 after OHCA. Functional enrichment analysis revealed a down-regulation of adaptive immunity with concomitant up-regulation of innate immunity and inflammation in patients with unfavorable vs favorable neurological outcome. The transcription of many genes of the HLA family was decreased in patients with unfavorable vs favorable neurological outcome. Concomitantly, neutrophil activation and inflammation were observed. Up-stream regulators analysis showed the implication of numerous factors involved in cell cycle and damages. A logistic regression including a set of genes allowed a reliable prediction of the clinical outcomes (specificity = 88%; Hit Rate = 83%). ConclusionsA transcriptomic signature involving a counterbalance between adaptive and innate immune responses is able to predict neurological outcome very early after hospital admission after OHCA. This deserves confirmation in a larger population.

Influence of Genetic Variations in Selenoprotein Genes on the Pattern of Gene Expression after Supplementation with Brazil Nuts.

Selenium (Se) is an essential micronutrient for human health. Its beneficial effects are exerted by selenoproteins, which can be quantified in blood and used as molecular biomarkers of Se status. We hypothesize that the presence of genetic polymorphisms in selenoprotein genes may: (1) influence the gene expression of specific selenoproteins and (2) influence the pattern of global gene expression after Brazil nut supplementation. The study was conducted with 130 healthy volunteers in Sao Paulo, Brazil, who consumed one Brazil nut (300 μg/Se) a day for eight weeks. Gene expression of GPX1 and SELENOP and genotyping were measured by real-time PCR using TaqMan Assays. Global gene expression was assessed by microarray using Illumina HumanHT-12 v4 BeadChips. Brazil nut supplementation significantly increased GPX1 mRNA expression only in subjects with CC genotype at rs1050450 (p < 0.05). SELENOP mRNA expression was significantly higher in A-carriers at rs7579 either before or after supplementation (p < 0.05). Genotype for rs713041 in GPX4 affected the pattern of blood cell global gene expression. Genetic variations in selenoprotein genes modulated both GPX1 and SELENOP selenoprotein gene expression and global gene expression in response to Brazil nut supplementation.

Persistence of Innate Immune Pathways in Late Stage Human Bacterial and Fungal Keratitis: Results from a Comparative Transcriptome Analysis.

Microbial keratitis (MK) is a major cause of blindness worldwide. Despite adequate antimicrobial treatment, tissue damage can ensue. We compared the human corneal transcriptional profile in late stage MK to normal corneal tissue to identify pathways involved in pathogenesis. Total RNA from MK tissue and normal cadaver corneas was used to determine transcriptome profiles with Illumina HumanHT-12 v4 beadchips. We performed differential expression and network analysis of genes in bacterial keratitis (BK) and fungal keratitis (FK) compared with control (C) samples. Results were validated by RTqPCR for 45 genes in an independent series of 183 MK patients. For the microarray transcriptome analysis, 27 samples were used: 12 controls, 7 BK culture positive for Streptococcus pneumoniae (n = 6), Pseudomonas aeruginosa (n = 1), and 8 FK, culture positive for Fusarium sp. (n = 5), Aspergillus sp. (n = 2), or Lasiodiplodia sp. (n = 1). There were 185 unique differentially expressed genes in BK, 50 in FK, and 339 common to both [i.e., genes with fold-change (FC) < −4 or ≥4 and false discovery rate (FDR) adjusted P < 0.05]. MMP9 had the highest FC in BK (91 FC, adj p = 3.64 E-12) and FK (FC 64, adj. p = 6.10 E-11), along with other MMPs (MMP1, MMP7, MMP10, MMP12), pro-inflammatory cytokines (IL1B, TNF), and PRRs (TLR2, TLR4). HIF1A and its induced genes were upregulated uniquely in BK. Immune/defense response and extracellular matrix terms were the most enriched Gene Ontology terms in both BK and FK. In the network analysis, chemokines were prominent for FK, and actin filament reorganization for BK. Microarray and RTqPCR results were highly correlated for the same samples tested with both assays, and with the larger RTqPCR series. In conclusion, we found a great deal of overlap in the gene expression profile of late stage BK and FK, however genes unique to fungal infection highlighted a corneal epithelial wound healing response and for bacterial infection the prominence of HIF1A-induced genes. These sets of genes may provide new targets for future research into therapeutic agents.

Apoptotic and genomic effects of corilagin on SKOV3 ovarian cancer cell line.

Corilagin is a member of the tannin family and has been isolated from traditional Chinese medicinal plants, such as Phyllanthus spp. Corilagin has anti-inflammatory, antioxidative, antiatherogenic, and antihypertensive effects in various experimental models. In this research, we aimed to investigate for the first time whether corilagin had apoptotic and genomic effects in ovarian cancer treatment in the same study. The potential apoptotic of corilagin was investigated using a WST1 cell proliferation test, caspase 3, and mitochondrial membrane potential JC1 assays in a time- and dose-dependent manner. Genomic changes in expression levels against corilagin treatment were measured using an Illumina human HT-12V4 BeadChip microarray. Bioinformatic data analyses were performed using GenomeStudio and Ingenuity Pathway Analysis software. The data of our study demonstrated that there were statistically significant time- and dose-dependent increases in caspase 3 enzymatic activity and loss of mitochondrial membrane potential in line with decreases in cancer cell proliferation. According to gene-ontology analysis, we found that adherens junctions, antigen processing and presentation, and the phosphatidylinositol signaling system were the most statistically significant networks in response to corilagin treatment on SKOV3 cells, in a time- and dose-dependent manner. The apoptotic and genome-wide effects of corilagin on ovarian cancer cells were examined in detail for the first time in the literature. The results of our study suggest that corilagin might have the potential to be used as a new treatment option for epithelial ovarian cancer.

P3.01-019 Desmoplasia is Associated with Poor Prognosis and Carcinoma-Associated Fibroblast Heterogeneity in Non-Small Cell Lung Cancer: Topic: Morphology

Cancer-associated fibroblasts (CAFs) are known to influence tumor development, progression and metastasis. Their characteristics and prognostic role in non-small cell lung cancer (NSCLC) patients have been recognized. However, the functional heterogeneity of CAFs between patients and its genetic basis is less understood. Two pathologists scored for desmoplasia on Hematoxylin-Eosin stained sections of resected lung tumors from two patient cohorts: one consisting of 171 NSCLC patients (128 adenocarcinoma, 43 squamous carcinoma) and the second of 24 primary cultures of CAFs. Percent area of desmoplasia among total tumor stroma was used to define high desmoplasia (HD) versus low desmoplasia (LD). The desmoplasia and survival analysis were assessed for 171 NSCLC patients. Gene expression data on RNA extracted from CAFs in contracted gels following 24 hours incubation was obtained using Illumina Human HT-12v4 Bead Chips array and was preprocessed and normalized using RMA and values were log2 transformed. Significant genes whose expression is strongly correlated (Spearman correlation coefficient and p value) with percent of desmoplasia were identified in both cohorts. The gene set enrichment analysis (GSEA) was applied to test for the enrichment of CAF cohort significant genes in 171 NSCLC cohort. Additionally, CAF significant genes were subjected to pathway enrichment analysis using Pathway Data Integration Portal ver. 2.5 (http://ophid.utoronto.ca/pahtDIP). The prognosis of adenocarcinoma patients with HD had poorer outcome in comparison to the patients with LD (disease free survival at 3 years 34% vs. 75% p=0.00045 and relapse rate 41% vs. 14%, p=0.0051). In the CAF cohort, the number of genes that are significantly associated with desmoplasia for enrichment are 356. Using GSEA, these genes were enriched in 171 NSCLC cohort (with a p value of 0.045). Protein-protein interaction (PPI) partners of these 356 genes were acquired using Integrated Interaction Database – IID (version 2016-03, http://dcv.uhnres.utoronto.ca/iid/). Obtained genes were then ranked according to their degree, i.e., number of PPIs. Top 44 (top 1%) of the genes were then selected to pathway enrichment analysis using pathDIP version 2.5. 245 pathways that significantly enriched by these 44 genes (FDR < 0.01) were obtained. Many of these pathways are known to be involved in lung cancer. We demonstrated that the prognosis of lung adenocarcinoma patients with HD had poorer outcome in comparison to the patients with LD. Furthermore, PPI analysis of CAF genes associated with HD reveals enrichment of many cancer-related pathways, suggesting their high relevance to lung cancer.

Whole Blood Gene Expression Profiles of Patients with a Past Aneurysmal Subarachnoid Hemorrhage.

BackgroundThe pathogenesis of development and rupture of intracranial aneurysms (IA) is largely unknown. Also, screening for IA to prevent aneurysmal subarachnoid hemorrhage (aSAH) is inefficient, as disease markers are lacking. We investigated gene expression profiles in blood of previous aSAH patients, who are still at risk for future IA, aiming to gain insight into the pathogenesis of IA and aSAH, and to make a first step towards improvement of aSAH risk prediction.Methods and ResultsWe collected peripheral blood of 119 patients with aSAH at least two years prior, and 118 controls. We determined gene expression profiles using Illumina HumanHT-12v4 BeadChips. After quality control, we divided the dataset in a discovery (2/3) and replication set (1/3), identified differentially expressed genes, and applied (co-)differential co-expression to identify disease-related gene networks. No genes with a significant (false-discovery rate <5%) differential expression were observed. We detected one gene network with significant differential co-expression, but did not find biologically meaningful gene networks related to a history of aSAH. Next, we applied prediction analysis of microarrays to find a gene set that optimally predicts absence or presence of a history of aSAH. We found no gene sets with a correct disease state prediction higher than 40%.ConclusionsNo gene expression differences were present in blood of previous aSAH patients compared to controls, besides one differentially co-expressed gene network without a clear relevant biological function. Our findings suggest that gene expression profiles, as detected in blood of previous aSAH patients, do not reveal the pathogenesis of IA and aSAH, and cannot be used for aSAH risk prediction.

Anti-proliferative, apoptotic and signal transduction effects of hesperidin in non-small cell lung cancer cells.

Hesperidin, a glycoside flavonoid, is thought to act as an anti-cancer agent, since it has been found to exhibit both pro-apoptotic and anti-proliferative effects in several cancer cell types. The mechanisms underlying hesperidin-induced growth arrest and apoptosis are, however, not well understood. Here, we aimed to investigate the anti-proliferative and apoptotic effects of hesperidin on non-small cell lung cancer (NSCLC) cells and to investigate the mechanisms involved. The anti-proliferative and apoptotic effects of hesperidin on two NSCLC-derived cell lines, A549 and NCI-H358, were determined using a WST-1 colorimetric assay, a LDH cytotoxicity assay, a Cell Death Detection assay, an AnnexinV-FITC assay, a caspase-3 assay and a JC-1 assay, respectively, all in a time- and dose-dependent manner. As a control, non-cancerous MRC-5 lung fibroblasts were included. Changes in whole genome gene expression profiles were assessed using an Illumina Human HT-12v4 beadchip microarray platform, and subsequent data analyses were performed using an Illumina Genome Studio and Ingenuity Pathway Analyser (IPA). We found that after hesperidin treatment, A549 and NCI-H358 cells exhibited decreasing cell proliferation and increasing caspase-3 and other apoptosis-related activities, in conjunction with decreasing mitochondrial membrane potential activities, in a dose- and time-dependent manner. Through a GO analysis, by which changes in gene expression profiles were compared, we found that the FGF and NF-κB signal transduction pathways were most significantly affected in the hesperidin treated NCI-H358 and A549 NSCLC cells. Our results indicate that hesperidin elicits an in vitro growth inhibitory effect on NSCLC cells by modulating immune response-related pathways that affect apoptosis. When confirmed in vivo, hesperidin may serve as a novel anti-proliferative agent for non-small cell lung cancer.

940 miRNA and mRNA Profiles From Nondysplastic Mucosa Distinguish UC Patients Harboring Remote Neoplasia and Uncover MiR-4728-3p, a Predicted Regulator of Focal Adhesion, As a Tumor Suppressor in UC

Introduction: In prior studies, we identified RNAs in non-dysplastic mucosa that distinguished patients with ulcerative colitis (UC) and concomitant neoplastic lesions from those without neoplasia. We sought to validate these findings in a larger independent cohort and identify miRNAs regulating these genes to discover biomarkers and elucidate neoplastic pathways in this high-risk population. Methods: Colonoscopic or surgical biopsies of noninflamed, non-dysplastic rectosigmoid mucosa were obtained from subjects with long-standing UC with (n=19) or without remote neoplasia (n=23). mRNAs were analyzed with the Illumina HumanHT-12 v4 BeadChip microarray, and miRNAs were analyzed by the miRCURY LNA microRNA array 7th gen (Exiqon). Following pre-processing, two-group comparison was performed using R/Bioconductor package ‘Limma'. Real-time PCR was performed for miR-4728-3p. HT-29 and HCT-116 were transfected with mature miR-4728-3p or scrambled controls and predicted target proteins were assessed by Westerns. Results: By unsupervised hierarchical clustering, UC subjects with neoplasia clustered by both miRNA and mRNA profiles compared to those without neoplasia. There were 35 significantly downregulated and 47 up-regulated miRNAs (adj. p 0.5, adj. p<0.01). Hierarchical clustering was used to assemble genes into modules, which were assessed for enrichment in Gene Ontology (GO) annotations. Genes with enriched functional GO annotations that were in the same module as predicted miRNA regulators were analyzed for their locations in KEGG regulatory pathways to assess their potential roles in regulatory sub-networks. miR-47283p was identified as a regulator of three genes (CAV1, THBS2, COL1A2) involved in in focal adhesion signaling, the most enriched pathway in UC subjects with neoplasia (p=2.2 x 10-4). miR-4728-3p was down-regulated (LogFC=-0.42, p=0.003) as confirmed by qPCR in preliminary studies, whereas all three target genes were significantly up-regulated in UCassociated neoplasia [CAV1 (LogFC=1.06, adj p=2.9 x 10-17), THSB2 (LogFC=1.33, adj. p= 1.7 x 10-18) and COL1A2 (LogFC=1.01 adj = 1.4 x 10-9)]. miR-4728-3p transfection into colon cancer cells down-regulated CAV1 as well as COL1A2 and study of its affects on THBS2 are in progress. We are also examining miR-4728-3p-induced changes on cell invasion, migration, and focal adhesion disassembly. Conclusion: Patients with long-standing ulcerative colitis who harbor remote neoplasia can be identified based on miRNA and mRNA profiles in non-dysplastic tissue. Using a newly developed algorithm to analyze miRNA and mRNA expression from the same tissues, we identified miR-4728-3p as a key regulator of focal adhesion and likely an important tumor-suppressor in UC-associated colon carcinogenesis.

MP31-15 GENE EXPRESSION PROFILING REVEALS MOLECULAR SUBTYPES OF BENIGN PROSTATIC HYPERPLASIA

You have accessJournal of UrologyBenign Prostatic Hyperplasia: Basic Research1 Apr 2015MP31-15 GENE EXPRESSION PROFILING REVEALS MOLECULAR SUBTYPES OF BENIGN PROSTATIC HYPERPLASIA Keyan Salari, Seth Bechis, Rongbin Ge, Jian Hong, Aleksander Otsetov, Zongwei Wang, Shahin Tabatabaei, and Aria Olumi Keyan SalariKeyan Salari More articles by this author , Seth BechisSeth Bechis More articles by this author , Rongbin GeRongbin Ge More articles by this author , Jian HongJian Hong More articles by this author , Aleksander OtsetovAleksander Otsetov More articles by this author , Zongwei WangZongwei Wang More articles by this author , Shahin TabatabaeiShahin Tabatabaei More articles by this author , and Aria OlumiAria Olumi More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.1370AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Benign prostatic hyperplasia (BPH) affects over 80% of men by age 70 and poses a substantial burden on healthcare. At least 25-30% of men do not respond to 5-alpha reductase (5-AR) inhibitors, raising the hypothesis that there may be molecular heterogeneity in BPH underlying this variability in clinical response. In previous work, we showed that some men carry epigenetic modifications via methylation of the 5-AR promoter region that modulate gene expression and protein levels, thereby suggesting a possible mechanism of resistance to 5-AR inhibitors. In this study, we performed genome-wide gene expression profiling of prostatic tissue from patients with BPH to identify molecular subtypes of the disease with potential clinical relevance. METHODS Prostatic tissue specimens were obtained at the time of surgery from 22 patients with symptomatic BPH undergoing prostate debulking surgery. Each patient's tissue was analyzed for the presence of 5-AR type 2 protein expression by immunohistochemistry and 5-AR2 gene promoter methylation status by PCR-based methylation analysis. Total RNA was extracted from each specimen for gene expression profiling. Whole-genome gene expression profiling was performed using Illumina Human HT-12 v4 BeadChip Arrays. Gene expression microarray data was analyzed by unsupervised hierarchical clustering and Gene Set Enrichment Analysis (GSEA) using the GenePattern software platform. RESULTS Unsupervised hierarchical clustering analysis of whole-genome gene expression profiles identified two subtypes of patients with distinct gene expression signatures. Among the most differentially expressed genes were several notable androgen-regulated genes, including TMPRSS2, NKX3-1, AZGP1, KLK3 (encoding the PSA protein) and multiple other members of the kallikrein family of serine proteases (all P <0.001, FDR <0.02). By GSEA, multiple gene sets composed of androgen-regulated genes and androgen receptor target genes were found to be significantly enriched in one of the two subtypes. Interestingly, patients from this Androgen-Up subtype were also noted to have a higher frequency of methylation of the 5AR-2 gene promoter (though not statistically significant). CONCLUSIONS Patients with BPH exhibit variable prostatic gene expression patterns. Our findings suggest the presence of distinct molecular subtypes of BPH, driven by differences in androgen gene regulation. These differences may partially explain the variable response of patients to 5-AR inhibitors and thereby inform clinical strategies for devising personalized therapy for BPH patients. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e361 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Keyan Salari More articles by this author Seth Bechis More articles by this author Rongbin Ge More articles by this author Jian Hong More articles by this author Aleksander Otsetov More articles by this author Zongwei Wang More articles by this author Shahin Tabatabaei More articles by this author Aria Olumi More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Evolutionary forward genomics reveals novel insights into the genes and pathways dysregulated in recurrent early pregnancy loss.

Are the genes that gained novel expression in the endometria of Eutherian (placental) mammals more likely to be dysregulated in patients with endometrial-associated recurrent early pregnancy loss (REPL)? There was a significant enrichment of genes dysregulated in REPL patients among the Eutherian-specific endometrial genes. Pregnancy loss is the most common complication of human pregnancy. REPL has multiple etiologies, including dysregulation of endometrial function, leading to 'suboptimal' implantation. Although the implantation process is tightly regulated in Eutherian (placental) mammals, the molecular factors contributing to dysregulated endometrial gene expression patterns in women with REPL are largely unknown. Endometrial biopsies were obtained from 32 REPL patients during the mid-luteal phase, and evaluated for glandular development arrest based on elevated nuclear cyclin E levels in gland cells, and for out-of-phase endometrial development based on histology. Gene expression levels were measured using Illumina Human HT-12v4 BeadChip arrays. Differentially expressed genes were identified between patients with (i) out-of-phase (n = 10) versus normal (n = 22) histological dating and (ii) abnormally elevated (n = 9) versus normal (n = 23) cyclin E levels in the nuclei of endometrial glands, using a likelihood ratio test. Enrichment of dysregulated genes in REPL endometria among Eutherian-specific genes was tested by permutation. Gene ontology and pathway enrichment analyses were carried out for the dysregulated genes. Fifty-eight and eighty-one genes were identified as differentially expressed at P < 0.001 in women with out-of-phase histological dating and abnormally elevated glandular cyclin E levels, respectively. Genes that were recruited into endometrial expression during the evolution of pregnancy in Eutherian mammals were significantly enriched for dysregulated genes (P = 0.002 for histology, P = 0.021 for cyclin E), as well as for genes involved in immune response and signaling pathways with essential roles in implantation and endometrial biology. Small sample size limits the statistical power to detect dysregulated genes, and the lack of non-REPL control women does not allow us to test for the contribution of these genes to overall risk of REPL. Enrichment of functional gene categories, as well as genes gained expression in the Eutherian endometria, help to identify molecular etiologies that contribute to normal functioning of the endometrium. These pathways are also strong candidates for successful pregnancy outcomes. Using the evolutionary history of mammalian gene expression in the endometrial tissue may be a promising approach to discover genes involved in female reproductive disorders. This work is supported by National Institutes of Health (NIH) grant R01 HD21244 to C.O. Authors declare no competing interests.

Obesity is associated with more activated neutrophils in African American male youth.

BackgroundThere is emerging evidence suggesting the role of peripheral blood leukocytes in the pathogenesis of obesity and related diseases. However, few studies have taken a genome wide approach to investigating gene expression profiles in peripheral leukocytes between obese and lean individuals with the consideration of obesity related shifts in leukocyte types.MethodWe conducted this study in 95 African Americans of both genders (age 14-20, 46 lean and 49 obese). Complete blood count with differential test (CBC) was performed in whole blood. Genome wide gene expression analysis was obtained using Illumina HumanHT-12 V4 Beadchip with RNA extracted from peripheral leukocytes. Out of the 95 participants, 64 had neutrophils stored. The validation study was based on Real-time polymerase chain reaction with RNA extracted from purified neutrophils.ResultsCBC test suggested that in males, obesity was associated with increased neutrophil percentage (p=0.03). Genome wide gene expression analysis showed that in males, the majority of the most differentially expressed genes were related to neutrophil activation. Validation of the gene expression levels of ELANE (neutrophil elastase) and MPO (myeloperoxidase) in purified neutrophils demonstrated that the expression of these two genes – important biomarkers of neutrophils activation – were significantly elevated in obese males (p=0.01 and p=0.02, respectively).ConclusionThe identification of increased neutrophil percentage and activation in obese African American males suggests that neutrophils play an essential role in the pathogenesis of obesity related disease. Further functional and mechanistic studies on neutrophils may contribute to the development of novel intervention strategies reducing the burden associated with obesity-related health problems.

Abstract 4800: Heterogeneity of cancer associated fibroblasts in non-small cell lung cancer is defined by the level of collagen gel contraction activity

Abstract Background: Cancer associated fibroblasts (CAFs) are well known to strongly influence tumor development, progression and metastasis. Their characteristics and prognostic gene signature in non-small cell lung cancer (NSCLC) patients have been recognized. However, the functional heterogeneity of CAFs between patients and their genetic basis were far less known. The results revealed two functionally and also genetic distinct subgroups of CAFs which were closely related to the degree of tumor desmoplasia and also prognostic of survival in NSCLC patients. Methods: 28 independent lung cancer resection specimens were used to establish primary cultures of CAFs and noncancer fibroblasts from the corresponding nonneoplastic lung parenchyma. We applied collagen gel contraction and xCELLigence Real-Time Cell Analysis of proliferation and in vivo tumorigenicity for measuring the CAF activity. To assess the grading of desmoplasia, the percentage of desmoplasia in total tumor stroma was used to define high desmoplasia (HD) versus low desmoplasia (LD). Microarray data on RNA extracted from contracted gels following 8 hours incubation was performed using Illumina Human HT-12v4 Bead Chips array and was preprocessed and normalized using RMA and values were log2 transformed. Results: We have identified that CAF activity depends on higher ability to contract collagen gel, increased cell proliferation and tumor growth. Using microarray gene expression analysis of the 24 CAF cell lines (12 CAFs-HD versus 12 CAFs-LD), we observed high correlation of differential gene expression in extreme CAF-HD to tumor stroma while we found a high correlation between differential gene expression in extreme CAF-LD to normal lung. We were also able to validate our previous published subset of 11 genes (13 probe sets) in CAFs-HD versus CAFs-LD. To study the degree of desmoplasia and outcome, we used MedBio data set containing 181 records. We observed that desmoplasia appears to be associated with the time to relapse in univariable analysis. The association was far stronger in the adenocarcioma group with significance for both univariable and multivariable analysis. Conclusion: In this study, we provided the direct and robust evidence of functional heterogeneity of CAFs in NSCLC patients and first showed that CAFs from tumor with severe desmoplasia had higher gel contraction activity, proliferation rate and enhanced tumor growth. Furthermore, we developed desmoplasia-specific gene signature that could subgroup CAFs and contribute to their functional heterogeneity which would also allow us to discriminate among patients who would more likely benefit from tumor stroma targeting therapies in the future. Citation Format: Roya Navab, Jing Hao, Shingo Sakashita, Dennis Wang, Melania Pintilier, Yuhui Wang, Chang-Qi Zhu, Kalpana Venkat, Igor Jurisica, Ming-Sound Tsao. Heterogeneity of cancer associated fibroblasts in non-small cell lung cancer is defined by the level of collagen gel contraction activity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4800. doi:10.1158/1538-7445.AM2014-4800

Molecular Mechanisms of Quercitrin-induced Apoptosis in Non-small Cell Lung Cancer

Quercitrin (QR; quercetin-3-O-rhamnoside) has been used previously as an antibacterial agent and has been shown to inhibit the oxidation of low-density lipoproteins and prevent an allergic reaction. Furthermore, it was demonstrated that quercitrin exerts protective effects against H2O2-induced dysfunction in lung fibroblast cells. However, the mechanisms of quercitrin effects on cancer cell proliferation and apoptosis is not well understood. The aim of this study is to investigate the cytotoxic and apoptotic effects of quercitrin and the molecular mechanisms of quercitrin-induced apoptosis in non-small cell lung cancer (NSCLC) cell lines. Time- and dose-dependent antiproliferative and apoptotic effects of quercitrin determined by WST-1cell proliferation assay, lactate dehydrogenase (LDH) cytotoxicity assay, determination of nucleosome enrichment factor, changes in caspase-3 activity, loss of mitochondrial membrane potential (MMP) and also the localization of phosphatidylserine in the plasma membrane. Changes in whole genome gene expression levels were examined by Illumina Human HT-12v4 beadchip microarrays. There were significant increases in caspase-3 activity, loss of MMP, and increases in apoptotic cell population in response to quercitrin in A549 and NCI-H358 NSCLC cells in a time- and dose-dependent manner. Our results demonstrated that genes involved in leukocyte transendothelial migration, cell adhesion and phosphatidylinositol signaling system pathways were the most statistically significant pathways in NCI-H358 and A549cells. These results revealed that quercitrin has antiproliferative and apoptotic effects on lung cancer cells by modulating the immune response. After confirming its anticarcinogenic effects invivo, quercitrin could be a novel and strong anticancer agent against NSCLC.

THU0487 Identification of 131 Differential Gene Expression Profiles in Good Responders in the First Few Weeks after MTX Commencement: Results from the Rheumatoid Arthritis Methotrexate Study (RAMS)

BackgroundMethotrexate (MTX) is the DMARD of first choice in most patients with rheumatoid arthritis (RA). However, a significant minority (30-40%) of patients do not respond to MTX therapy. Gene expression...

Abstract B20: Prediction of lung cancer survival by genes in the PAM50 breast cancer panel.

Abstract Accumulating evidence shows that lung tumors are responsive to the steroid hormones β-estradiol and progesterone, and variables related to hormone receptor status have been reported to affect lung cancer survival. These findings suggest that there may be similarities in how hormonal pathways control tumor progression between lung cancer and breast cancer. We examined the relationship between disease-free survival (DFS) and expression of genes included in three published breast cancer survival signatures in a cohort of 104 early-stage (IA and IB) non-small cell lung cancers. Cases were selected from the University of Pittsburgh Lung Cancer SPORE Tissue Bank based on the following criteria: tumor tissue originated from a completely resected primary T1N0 or T2N0 adenocarinoma or squamous cell lung cancer; fresh-frozen lung cancer tissue was available; no neo-adjuvant therapy given; clinical variables (smoking history, age at diagnosis, sex, size of tumor, pleural invasion, site of recurrence) and outcome were known. RNA isolated from banked frozen tumor tissue was used to assess mRNA expression with the Illumina Human HT-12 v4 BeadChip; all mRNA analyzed had passed quality checks. Data were also subjected to background subtraction and quantile normalization. The Illumina BeadChip contains 16 out of 16 published Oncotype DX genes, 61 out of 66 MammaPrint genes, and 49 of 50 PAM50 genes. Supervised Principal Component Analysis was conducted to evaluate the ability to predict disease-free survival (DFS) for each gene panel as a group and also by using the top genes from the gene panels that showed differential expression in the lung cancer cohort. All results were subjected to 10-fold cross-validation. Genes from the Oncotype DX panel showed no differential expression in the cohort and had no ability to separate cases based on DFS. For the MammaPrint genes, three probes (ALDH4A1, FLT1, RECQL5) showed significant differences in expression in the cohort (p &amp;lt; 0.05), and the gene panel showed a relationship to DFS (HR 1.72, p = 0.07). Examination of the top differentially expressed genes did not improve prediction for the MammaPrint genes. For the PAM50 gene panel, three probes (CXXC5, FGFR4, FOXC1) showed significant differential expression (p &amp;lt; 0.05) and 23 probes showed differential expression at p &amp;lt; 0.1. The PAM50 gene panel as a group was able to separate lung cancer cases based on DFS (HR 1.9, p = 0.03). The PAM50 genes with differential expression at p &amp;lt; 0.1 also separated the cohort into four prognosis groups (p = 0.008 for trend). The worst prognosis group had a HR of 4.76 (p = 0.002). These genes were then analyzed in a subset of 44 cases in the Stage I cohort for whom ERβ protein expression status was known from immunohistochemistry (IHC) analysis. Comparing ERβ high (Allred score of &amp;gt;4, N= 20) and ERβ low (Allred score of 4 or less, N = 24) cases, the top PAM50 genes were more informative in ERβ high cases (HR 11.7, p = 0.0007) compared to ERβ low cases (HR 3.38, p = 0.045). For ERβ high cases, Kaplan-Meier survival curves showed that 50% of the high-risk group relapsed by 22 months, while 50% relapse was not reached in the low-risk group at 60 months. Similar analyses in the MammaPrint and Oncotype DX gene panels did not improve DFS prediction. We next validated the top PAM50 genes in a different cohort of 64 NSCLC cases, which included all stages and histologies, and had available ERβ IHC data, expression data from the Illumina Human HT-12 v4 BeadChip, and survival information. The top PAM50 genes were also able to predict DFS in this cohort: HR 2.19, p = 0.034 in all cases and HR 3.24, p = 0.042 in ERβ high cases. Pathway analysis indicated that the informative PAM50 genes describe a network that contains the estrogen and progesterone receptors, HER2, and cyclin E. The top canonical pathways identified were HER2/HER3 signaling and estrogen signaling. These results suggest that genes involved in interactions between hormonal and HER signaling may be predictive of lung cancer survival, especially in early-stage lung cancer, and provide further evidence for the importance of hormonal pathways in the biology of lung cancer. Supported by NCI SPORE in Lung Cancer P50 040990 and by the University of Pittsburgh Medical Center Genomics Initiative. Citation Format: Jill M. Siegfried, Yan Lin, Sanja Dacic, Brenda Diergaarde, Laura P. Stabile, Hui-Min Lin, Marjorie Romkes. Prediction of lung cancer survival by genes in the PAM50 breast cancer panel. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr B20.

Abstract 2633: Gene signatures derived from in vitro isolated migratory and invasive breast cancer cells indicate early metastatic relapse.

Abstract Background Cancer cell motility and tissue invasion are cornerstones of metastatic dissemination. Adequate molecular profiling of chemotaxis-driven tumor cells is impeded by the transient and stochastic nature of motility occurring in only a small subset of tumor cells. To unveil molecular pathways implicated in breast cancer cell invasion and migration, an in vitro set-up was designed to extract total RNA for microarray hybridisation from isolated migratory and invasive breast cancer cells at an early and a late time point. These target populations were compared to corresponding non-migratory and non-invasive cells for migration- and invasion-related expression profiling to assess potential prognostic relevance. Methods and results A 24-well Transwell set-up was used to perform both migration and invasion experiments on MDA-MB-231 breast cancer cells after 24h serum starvation (400000 cells/insert). A Matrigel coating (20%/15μL) was applied to the bottom-side of insert membranes in both settings. For invasion experiments, a layer of Matrigel (20%/20μL) was added on top of the membranes. Total RNA was isolated at early and late timepoints from both migratory / invasive and reference cells and hybridized onto Illumina HumanHT-12 v4 BeadChips in biological triplicates. Normalization and differential gene expression analysis were performed using the Limma-package in BioConductor. Lists of differentially expressed genes were analyzed using Ingenuity software. Using the nearest shrunken centroid algorithm gene signatures characteristic for early or late invasive or migratory cells were generated. The resulting models were applied onto six distinct gene expression data sets of pre-treatment samples from patients with mostly lymph node negative breast cancer. Survival analysis was performed using distant metastasis-free or disease-free survival as endpoint. Although all signatures were associated with survival in at least one data set, the gene signature associated with late invasion was associated with survival (range HR: 1.403 - 1.929; all P&amp;lt;0.05) in all but two data sets. Of note, gene signatures associated with the late timepoints were prognostically more relevant (significance obtained for 66.7% when using the models for the late timepoint vs. 16.7% and 33.3% when using the models for the early timepoint). Conclusions Gene expression characteristics of invasive and migratory breast cancer cells indicate poor prognosis on several publicly available breast cancer expression data sets. For both migration and invasion, the strongest prognostic relevance was associated with the late time points, possibly by extended differences caused by early events. The role of identified target genes is being functionally validated and additionally, these data can be subjected to multivariate regression analysis to study association with the molecular subtypes. Citation Format: Ridha Limame, Ken Op de Beeck, Steven Van Laere, Guy Van Camp, Marc Peeters, Luc Dirix, Filip Lardon, Olivier De Wever, Patrick Pauwels. Gene signatures derived from in vitro isolated migratory and invasive breast cancer cells indicate early metastatic relapse. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2633. doi:10.1158/1538-7445.AM2013-2633

Blood Transcriptions in Clinical Islet Cell Transplantation with T Cell Depletion and Antiinflammatory Protocol

Introduction: Allogeneic islet cell transplantation (ICT) is a promising treatment for patients with brittle type 1 diabetes. Recent reports have demonstrated that T cell depletiing immunosuppression protocol could improve islet engraftment as well as prolong graft function. We have implemented the T cell depletion plus antiinflammatory (TCD-AI) protocol using antithymocyte globulin (ATG) and double blockage of IL-1β and TNF-α. Global investigation of peripheral blood transcriptions is helpful for understanding systemic responses as well as discovering diagnostic biomarkers. Modular level analysis for cDNA microarray allows effective interpretation on the background determination with multiple immunologic disease populations (1). In this study, peripheral blood transcriptions were measured to explore systemic biological response for early phase of ICT with TCD-AI protocol. Methods: Five islet recipients received TCD-AI protocol with ATG for induction, along with anakinra and etanercept. Peripheral blood samples were collected on the screening day (baseline), post-transplant days 4, 7, 14, 28 and 42. Isolated RNA were hybridized on to Illumina HumanHT-12 v4 BeadChip. Probe-level differential gene expression analysis was conducted using mixed model adjusting for correlation for longitudinal data to compare between baseline and post-transplant samples. A false discovery rate of 0.1 was used to account for multiple testing. Modular analysis of gene expressions were employed with previously determined sets of genes (1). Results: Four patients (80%) achieved insulin independence after single islet infusion and the median duration of insulin independence is 17.5 months (range: 4-36 months) as of the analysis. The number of genes that were expressed with significant difference (p< 0.05) are 866, 465, 492, 160, 353 and 80 on day 4, 7, 14, 21, 28 and 42 after transplant when compare to baseline. Modular analysis of cDNA microarray on day 4 revealed upregulated modules annotated as interferon responses (M3.4), inflammation (M4.2), myeloid lineage (M4.6) and cell cycle (M6.13), and downregulated modules as coagulation cascade (M3.6), T-cells (M4.1 and M6.15), lymphoid lineage (M4.7 and M6.9) and cytotoxicity (M4.15) (Figure 1). Among these modules, T-cells (M4.1 and M6.19) and cytotoxicity (M4.15) modules were downregulated throughout the study period.[Figure 1]Figure 1. Blood transcriptional fingerprints with islet recipients receiving TCD-AI protocol. Relative changes in whole blood gene expressions with islet recipients for day 4 to 42, compared to baseline. Colored spots represent the percentage of significantly upregulated (red) or downregulated (blue) transcripts (p< 0.002). Conclusion: Gene expression modules annotated as T-cells and cytotoxicity were downregulated in early phase of ICT, suggesting that effective control of cellular immune response as the background mechanism of improved outcome of TCD-AI protocol. (1) Chaussabel D, et al. Immunity 2008;29:150-164.

Abstract 4319: Gene expression profiling of isolated small migratory and invasive breast cancer cells at different times during chemotaxis

Abstract Background Chemotactic cell motility and tissue invasion are key processes initiating cancer cell dissemination and metastasis. Adequate molecular profiling of chemotaxis-driven tumor cells is impeded by the transient and stochastic nature of motility occurring in only a small subset of tumor cells. To unveil molecular pathways implicated in breast cancer cell invasion and migration, an in vitro set-up was designed to isolate total RNA for microarray hybridisation from sequestered migratory and invasive breast cancer cell subpopulations at two predefined incubation stages. These target populations were compared to non-migratory and non-invasive cells to obtain a molecular picture of migratory and invasive breast cancer cell behavior. Methods and results A 24-well Transwell set-up was used to perform both migration and Matrigel invasion experiments on MDA-MB-231 breast cancer cells after 24h serum starvation (4x105 cells/insert). A Matrigel coating (20%/15μL) was applied to the bottom-side of insert membranes in both settings. For invasion experiments, a layer of Matrigel (20%/20μL) was added to the top side of the membranes. Initially, an “early” and a “late” timepoint for RNA-extraction have been selected by monitoring migration and invasion using the xCELLigence system (Roche Applied Science). High-quality total RNA was isolated at these predefined timepoints from both migratory / invasive and reference cells and hybridized onto Illumina HumanHT-12 v4 BeadChips in biological triplicates. Differential gene expression analysis and pathway analysis were done using BioConductor in R and Ingenuity Pathway Analysis respectively, considering only FDR-corrected p-values &amp;lt; 0.1 as significant. First, a time-independent comparison was performed between all grouped migratory and invasive samples, revealing a total of 988 differentially expressed genes, of which the majority occurring in the late phase. Further analysis focused on early expression changes as these may be causative to more extended late events. Pathway analysis of differential genes associated with early migratory and early invasive cells in comparison with their reference counterparts identified NF-κB-related (p=0.0034) and TGFβ-related (p=0.04) expression to be closely associated with migration and invasion respectively. Conclusions This work has aimed to determine specific gene expression characteristics of solely migrating and invading breast cancer cells. Preliminary findings suggest strong involvement of NF-κB-based pathways early in migration, but not in invasion. On the other hand, TGFβ-signaling changes were more closely associated with early invasive behaviour. Moreover, a global comparison identified significant expression differences between both states of cell motility. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4319. doi:1538-7445.AM2012-4319