Fold Change Difference Research Articles

A Joint Bayesian Model for Integrating Microarray and RNA Sequencing Transcriptomic Data.

As the sequencing cost continued to drop in the past decade, RNA sequencing (RNA-seq) has replaced microarray to become the standard high-throughput experimental tool to analyze transcriptomic profile. As more and more datasets are generated and accumulated in the public domain, meta-analysis to combine multiple transcriptomic studies to increase statistical power has received increasing popularity. In this article, we propose a Bayesian hierarchical model to jointly integrate microarray and RNA-seq studies. Since systematic fold change differences across RNA-seq and microarray for detecting differentially expressed genes have been previously reported, we replicated this finding in several real datasets and showed that incorporation of a normalization procedure to account for the bias improves the detection accuracy and power. We compared our method with the popular two-stage Fisher's method using simulations and two real applications in a histological subtype (invasive lobular carcinoma) of breast cancer comparing PR+ versus PR- and early-stage versus late-stage patients. The result showed improved detection power and more significant and interpretable pathways enriched in the detected biomarkers from the proposed Bayesian model.

Autophagy Genes of Host Responds to Disruption of Gut Microbial Community by Antibiotics.

Defective autophagic machinery, such as that in Crohn's disease patients homozygous for ATG16L1 risk allele, is associated with alteration of resident gut bacterial communities. However, whether or not host autophagy responds to changes in the resident gut microbial community is not known. Here, we investigated the effect of antibiotic-induced disruption of the gut microbiome (dysbiosis) on autophagy gene expression and the expression of antimicrobial peptides/protein (AMP) over time. To test the hypothesis that antibiotic treatment may cause time-dependent changes in gut bacterial density, autophagy genes, and antimicrobial protein/peptide gene expression. Mice (n=8 per group) were treated with antibiotic cocktail and sacrificed at different intervals of recovery (days 3, 7, 10, 14, 21, 28, 35, and 42) post-antibiotics. DNA and RNA were extracted from small intestinal tissues. Bacterial density, expression of host autophagy genes, and AMP genes were analyzed by relative quantitative PCR. Fold change difference in comparison with untreated control group was calculated using 2-ΔΔCt method. Statistical analysis was performed using nonparametric Mann-Whitney test. Gut bacterial density changed in a time-dependent fashion in response to antibiotic treatment. These changes were concurrent with upregulation of autophagy genes and antimicrobial peptide/protein gene expression. We further showed that an oral gavage of a resident microbe Desulfovibrio, which bloomed in antibiotic-treated animals, induced Atg5 and lysozyme (Lyz) gene expression. Autophagy genes respond to dysbiosis induced by antibiotics. This response may be a host mechanism to detect and possibly correct dysbiosis by activating antimicrobial peptides/proteins that control the microbial load in the gut.

Circular RNAs expressed in chorionic villi are probably involved in the occurrence of recurrent spontaneous abortion

Abstract Recurrent spontaneous abortion (RSA) is an unknown aetiology common disease troubled women all over the world. Recent studies of circular RNAs (circRNAs) in gynecology and obstetrics area have sprung up. Our purpose in this study was to investigate the expression of circRNAs in chorionic villi from patients with RSA versus corresponding healthy gravidas, and to infer the potential role of these circRNAs in RSA occurrence. We selected 35 pairs of RSA patients and healthy control subjects, and analyzed the aberrant circRNA expression in three pairs of villi samples from each group using circRNA microarray. Altogether, including 335 up-regulated and 259 down-regulated circRNAs, 594 aberrant expressed circRNAs (FC ≥ 2.0, P ≤ 0.05) were found. Considering the raw content of each circRNAs, fold change differences and the restriction of the divergent primers, four down-regulated circRNAs (hsa_circRNA_104948, 104547, 101319 and 104938) and four up-regulated (hsa_circRNA_102116, 100709, 102424 and 103102) were selected as validation genes, using real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Paired t-test and receiver operating characteristic (ROC) curves were also established. The result of content verification was accordant exactly with the microarray chip outcome (P ≤ 0.05 in all validation circRNAs). The area under the ROC curves (AUCs) of these eight circRNAs were all greater than 0.5. Further function analysis of circRNAs in RSA patients was performed by paper comparison. Taken together, aberrant villi circRNA expression in RSA played a vital role in its nosogenesis possibly via miRNA sponge function of circRNAs. Deeper analysis of the detailed mechanisms will be worthwhile.

Terminal heat stress-responsive genes analysis in heat susceptible HDR77 genotype of wheat (Triticum aestivum L.) by using semi-quantative RTPCR

Abiotic stresses particularly heat stress constitutes a serious threat to the world food production in bread wheat. In the present study, candidate gene expression analysis of HDR77 genotype (which is a susceptible genotype based on electrolyte leakage experiment) was carried out and only those candidate genes which were showing two fold change differences were selected from previous research papers These genes were run over cDNA of HDR 77genotype used as template and intensity of bands was checked by semi-quantative RT PCR. Few genes like HSP 80 gene, MADS WP12 gene, HSP 26.6B gene, WRKY53b gene, WRKY72-a gene, HSP26 gene and WRKY 72b gene were upregulated whereas TaVRT- MADS gene, NAC gene and WRKY 10 gene were down-regulated . The heat stress responsive genes will increase our understanding of molecular basis for heat tolerance in different wheat genotypes and future improvement of heat tolerance in wheat and other crops.

Circulating microRNAs Predict the Initiation of NHL in a Novel In Vivo Model: Impact of Age and Sex Via a Systems Biology Approach

Background: Extensive epidemiological data have demonstrated an exponential rise in the incidence of NHL associated with increasing age. The molecular etiology of this remains largely unknown. Additionally, recent data have suggested that older females with diffuse large B cell lymphoma (DLBCL) have improved survival when treated with rituximab-based therapy. We recently reported that there were significant global transcriptomic differences in DLBCL cases in the TCGA based on patient age and sex (Beheshti et al Cancer Inform. 2015). The hypotheses of the current studies were that age and sex-dependent circulating microRNA (miRNA) signatures exist and predict NHL initiation and progression. Leveraging a comprehensive systems biology approach in combination with a novel in vivo spontaneous DLBCL model, we sought to identify circulating miRNA signatures that drive initiation and progression.Methods: We utilized a novel murine model for spontaneous DLBCL initiation (Smurf2-deficient mice with C57BL/6 background, Nat Commun. 2013) at two age groups: 2 and 15 months old. All Smurf2-deficient mice develop visible DLBCL starting at 15 months of age. Total miRNA was isolated from serum collected for all age groups for both Smurf2-deficient mice and wild-type C57BL/6 mice as controls. Using innovative technology with Droplet Digital and real time PCR (ddPCR), we quantified the 10 circulated miRNAs in the serum. Predictions on how miRNA signature impacts cancer risk were obtained using previously established methods (Beheshti et al. Cancer Res. 2015) by relating impact of miRNA on DLBCL and determined a cancer risk score based on fold-change differences compared with control. Additional effect of how miRNAs impact the transcriptome globally was completed via CluePedia Cytoscape plugin and functional significance on cancer was determined through Ingenuity Pathway Analysis (IPA).Results: Using systems biology techniques, we identified 10 common circulating miRNAs that were significantly expressed in the spleen and bone marrow of Smurf2-deficient male and female mice at 2 months of age compared with wild-type mice. Furthermore, we predicted the impact it had on DLBCL initiation and progression from this miRNA signature (see Figure). For young Smurf2-deficient mice compared with wild-type mice, there was a predicted risk of cancer promotion due to the miRNA signature detection at 12 months prior to tumor formation. This miRNA signature was further modulated at older ages associated with the beginning of DLBCL formation. Additionally, this miRNA signature was more prominent in males and ongoing work is examining distinct miRNA associated with females. When determining the impact of the miRNA signature on DLBCL biology, we identified that these miRNAs have the most prominent impact on oncogenes, MYC and JUN with global impact on cancer related functions starting several months before actual histologic DLBCL formation.Conclusions: It was determined that there is a key miRNA signature circulating throughout a host prior to the formation of a tumor starting at 2 months old which becomes modulated by age and tumor formation. This age based circulating miRNA signature can be used to predict DLBCL development at a young age before actual tumor formation. Furthermore, this can potentially be used as a simple biomarker at a young age to predict future lymphoma development and allow for advanced novel therapeutic strategies to prevent lymphomagenesis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Xenobiotic metabolism in differentiated human bronchial epithelial cells

Differentiated human bronchial epithelial cells in air liquid interface cultures (ALI-PBEC) represent a promising alternative for inhalation studies with rodents as these 3D airway epithelial tissue cultures recapitulate the human airway in multiple aspects, including morphology, cell type composition, gene expression and xenobiotic metabolism. We performed a detailed longitudinal gene expression analysis during the differentiation of submerged primary human bronchial epithelial cells into ALI-PBEC to assess the reproducibility and inter-individual variability of changes in transcriptional activity during this process. We generated ALI-PBEC cultures from four donors and focussed our analysis on the expression levels of 362 genes involved in biotransformation, which are of primary importance for toxicological studies. Expression of various of these genes (e.g., GSTA1, ADH1C, ALDH1A1, CYP2B6, CYP2F1, CYP4B1, CYP4X1 and CYP4Z1) was elevated following the mucociliary differentiation of airway epithelial cells into a pseudo-stratified epithelial layer. Although a substantial number of genes were differentially expressed between donors, the differences in fold changes were generally small. Metabolic activity measurements applying a variety of different cytochrome p450 substrates indicated that epithelial cultures at the early stages of differentiation are incapable of biotransformation. In contrast, mature ALI-PBEC cultures were proficient in the metabolic conversion of a variety of substrates albeit with considerable variation between donors. In summary, our data indicate a distinct increase in biotransformation capacity during differentiation of PBECs at the air–liquid interface and that the generation of biotransformation competent ALI-PBEC cultures is a reproducible process with little variability between cultures derived from four different donors.

Antioxidant-rich leaf extract of Barringtonia racemosa significantly alters the in vitro expression of genes encoding enzymes that are involved in methylglyoxal degradation III.

BackgroundBarringtonia racemosa is a medicinal plant belonging to the Lecythidaceae family. The water extract of B. racemosa leaf (BLE) has been shown to be rich in polyphenols. Despite the diverse medicinal properties of B. racemosa, information on its major biological effects and the underlying molecular mechanisms are still lacking.MethodsIn this study, the effect of the antioxidant-rich BLE on gene expression in HepG2 cells was investigated using microarray analysis in order to shed more light on the molecular mechanism associated with the medicinal properties of the plant.ResultsMicroarray analysis showed that a total of 138 genes were significantly altered in response to BLE treatment (p < 0.05) with a fold change difference of at least 1.5. SERPINE1 was the most significantly up-regulated gene at 2.8-fold while HAMP was the most significantly down-regulated gene at 6.5-fold. Ingenuity Pathways Analysis (IPA) revealed that “Cancer, cell death and survival, cellular movement” was the top network affected by the BLE with a score of 44. The top five canonical pathways associated with BLE were Methylglyoxal Degradation III followed by VDR/RXR activation, TR/RXR activation, PXR/RXR activation and gluconeogenesis. The expression of genes that encode for enzymes involved in methylglyoxal degradation (ADH4, AKR1B10 and AKR1C2) and glycolytic process (ENO3, ALDOC and SLC2A1) was significantly regulated. Owing to the Warburg effect, aerobic glycolysis in cancer cells may increase the level of methylglyoxal, a cytotoxic compound.ConclusionsBLE has the potential to be developed into a novel chemopreventive agent provided that the cytotoxic effects related to methylglyoxal accumulation are minimized in normal cells that rely on aerobic glycolysis for energy supply.

Abstract 1840: Differential expression and mechanistic pathways of prostate cancer identified from FFPE tissue using surrogate or whole transcriptome TempO-Seq targeted gene expression assays

Abstract Highly sensitive and quantitative whole transcriptome and surrogate transcriptome targeted sequencing TempO-Seq assays were used to profile gene expression from 1 mm sq focal areas of FFPE sections (5 μm thick) of normal and cancerous tissue within the same prostate. The whole transcriptome assay directly measures changes within the whole transcriptome. The surrogate assay targets fewer genes yet permits in silico extrapolation to identify changes across the whole transcriptome. Both approaches identified differentiating biomarkers and mechanistic pathways in prostate cancer, both corroborating what has been reported and adding new mechanistic insights which will be discussed. These new observations likely result from the sensitivity of the TempO-Seq assays and their robust performance measuring gene expression from focal areas of FFPE, which in turn permit improved isolation of histology. Benefits derived from the TempO-Seq platform which will be discussed are: 1) Insensitivity to RNA degradation (measurements from intact RNA RIN = 9.1 correlate to measurements from degraded RNA RIN = 3.0, R2 = 0.97). 2) Does not require RNA extraction. Measures total RNA from FFPE samples, both soluble RNA in the lysate and crosslinked insoluble RNA. 3) Simple capture-free ligation-based assay protocol, without 3’ or 5’ bias to the probes used to measure each gene. No requirement for poly-adenylation or capture of the RNA (typically required in ligation-based assays). 4) Single base specificity derived from the specificity of probe ligation. 5) Misligation rates &amp;lt;0.01% (unlike other ligation-based assays where misligation can be as great as 30%) enable no sample background controls to be run with typical counts of 0 for most genes and 1 to 3 counts for a few genes. 6) High signal-to-noise provides single cell sensitivity and ability to measure differential gene expression from small focal areas of tissue. 7) Excellent reproducibility (average ∼5% CV) between biological replicates across all expressed genes results in high significance of even small fold change differences. 8) Sequencing only the ligated probes complementary to a 50-base sequence of each gene eliminates the need for bioinformatics to analyze the sequencing data. Pooling of samples into a single sequencing run permits 42 or 865 surrogate samples and 8 or 153 whole transcriptome samples to be sequenced at the same time on the MiSeq or NextSeq, respectively, at an average of 250 counts/expressed gene. In combination, this results in low sequencing cost/sample without sacrifice of information or quality. 9) Simple, robust protocol requires only a PCR or qPCR instrument, and access to a sequencer. Citation Format: Milos Babic, Peter Shepard, Joanne Yeakley, Ruchir Shah, Deepak Mav, Raymond B. Nagle, Bruce Seligmann. Differential expression and mechanistic pathways of prostate cancer identified from FFPE tissue using surrogate or whole transcriptome TempO-Seq targeted gene expression assays. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1840.

Resistance Exercise Sustains p70S6K1 Activity in Response to Protein Ingestion at 4 hours

The additive effect of protein feeding and resistance exercise on the stimulation of muscle protein synthesis is well established. However, it is unclear whether the activity of the signalling protein p70S6K1 is enhanced when protein feeding is combined with resistance exercise compared with protein feeding alone. PURPOSE:To directly compare the response of p70S6K1 activity to protein feeding alone vs protein feeding combined with resistance exercise. METHODS:Retrospective signalling analysis was carried out on muscle tissue from a previously published study. Twenty-three resistance trained males consumed a high protein breakfast before resting for 3 h. Following a bout of unilateral resistance exercise (8 × 10 leg press and leg extension exercises; 80% 1 RM) participants consumed a whey protein isolate drink (containing 10, 20 or 40 g protein). The activity of p70S6K1 was measured at 0 and 4 h in rested and exercised legs using a [γ-32P] ATP kinase assay. Statistical analysis of the fold change (0-4 h) in p70S6K1 activity between rested and exercised legs was conducted using a paired samples t-test (protein doses were pooled). Linear regression analysis was performed on the difference in fold change of p70S6K1 between rested and exercised (x-axis) against the previously published myofibrillar muscle protein synthesis data (y-axis). Effect sizes were calculated and reported as Cohen’s d with confidence intervals (CI). RESULTS:The fold change in p70S6K1 activity in response to protein ingestion from 0-4 h was 62% higher (p=0.004; d=0.61; CI=0.14 to 1.08) in the exercised leg (1.8 ± 1.3 fold; mean ± SD) compared with the rested leg (1.1 ± 0.8 fold). Regression analysis revealed a significant linear relationship between the difference in p70S6K1 activity fold change between rested and exercised legs and previously published myofibrillar muscle protein synthesis data (p=0.031; r2=0.204; y=1.28+0.11*x). CONCLUSIONS: Resistance exercise enhances the response of p70S6K1 activity to protein feeding and likely contributes to the enhanced response of myofibrillar muscle protein synthesis when protein feeding and resistance exercise are combined. Partially funded by GSK Consumer Healthcare.

Trait changes induced by species interactions in two phenotypically distinct strains of a marine dinoflagellate.

Populations of the toxigenic marine dinoflagellate Alexandrium are composed of multiple genotypes that display phenotypic variation for traits known to influence top-down processes, such as the ability to lyse co-occurring competitors and prospective grazers. We performed a detailed molecular analysis of species interactions to determine how different genotypes perceive and respond to other species. In a controlled laboratory culture study, we exposed two A. fundyense strains that differ in their capacity to produce lytic compounds to the dinoflagellate grazer Polykrikos kofoidii, and analyzed transcriptomic changes during this interaction. Approximately 5% of all analyzed genes were differentially expressed between the two Alexandrium strains under control conditions (without grazer presence) with fold-change differences that were proportionally higher than those observed in grazer treatments. Species interactions led to the genotype-specific expression of genes involved in endocytotic processes, cell cycle control and outer membrane properties, and signal transduction and gene expression regulatory processes followed similar patterns for both genotypes. The genotype-specific trait changes observed in this study exemplify the complex responses to chemically mediated species interactions within the plankton and their regulation at the gene level.

A case for preART-adjusted endpoints in HIV therapeutic vaccine trials

BackgroundIn a randomized, double-blind, placebo-controlled phase 2 clinical trial of Vacc-4x, a peptide-based therapeutic HIV-1 p24Gag vaccine candidate, 135 HIV-infected participants (vaccine:placebo=92:43) received a series of six immunizations while on combination antiretroviral therapy (cART). At week 28, all participants underwent an analytical treatment interruption (ATI) for up to 24 weeks. preART VL appeared to be higher among Vacc-4x recipients. Based on a previous analysis, during ATI viral load (VL) appeared to be lower in Vacc-4x recipients, but no difference in CD4 level was observed between Vacc-4x and placebo groups. We propose fold-change-based endpoints and report comparative analyses accounting for imbalanced preART VL and missing data. MethodsAll analyses included per-protocol (PP) participants who received the full immunization and underwent ATI. Linear regression models were used to identify predictors of study endpoints and to estimate the vaccine effect based on fold changes in CD4 counts or VL over preART values at week 40 or at set-point (geometric mean over weeks 48 and 52 values). We adjusted for potential baseline factors and used a multiple imputation approach to account for missing endpoints due to cART resumption or dropout. P-values were adjusted for multiple comparisons using q-values. ResultspreART VL and CD4 count were significant predictors of study endpoints. The vaccine recipients had a higher fold change in week 40 CD4 counts (vaccine vs. placebo mean fold-change difference=0.08; p=0.02; q=0.03), a higher fold change in CD4 count set-point (0.06; p=0.06; q=0.07), a lower fold change in week 40 VL (−0.47; p=0.03; q=0.05), and a lower fold change in VL set-point (−0.50; p=0.02; q=0.03). ConclusionsThese exploratory analyses consistently suggested that Vacc-4x provided positive effects on both CD4 counts and VL. Future HIV therapeutic vaccine studies may adopt similar preART-adjusted endpoints and missing data imputation methods in vaccine effect evaluations.

Epidermal growth factor–containing fibulin-like extracellular matrix protein 1 expression and regulation in uterine leiomyoma

To determine the presence, differential expression, and regulation of epidermal growth factor-containing fibulin-like extracellular matrix protein 1 (EFEMP1) in uterine leiomyomas. Laboratory invivo and invitro study with the use of human leiomyoma and myometrial tissue and primary cells. Academic medical center. Leiomyoma and myometrial tissue samples and cultured cells. 5-Aza-2'-deoxycytidine (5-aza-dC) treatment. Fold-change difference between EFEMP1 and fibulin-3 expression in leiomyoma tissue and cells compared with matched myometrial samples, and fold-change difference in EFEMP1 expression with 5-Aza-dC treatment. Invivo, EFEMP1 expression was 3.19-fold higher in myometrial tissue than in leiomyoma tissue. EFEMP1 expression invitro was 5.03-fold higher in myometrial cells than in leiomyoma cells. Western blot and immunohistochemistry staining of tissue and cells confirmed similar findings in protein expression. Treatment of leiomyoma cells with 5-Aza-dC resulted in increased expression of EFEMP1 invitro. The EFEMP1 gene and its protein product, fibulin-3, are both significantly down-regulated in leiomyoma compared with myometrium when studied both invivo and invitro. The increase in EFEMP1 expression in leiomyoma cells with 5-Aza-dC treatment suggest that differential methylation is responsible, in part, for the differences seen in gene expression.

Circulating microRNA 132-3p and 324-3p Profiles in Patients after Acute Aneurysmal Subarachnoid Hemorrhage

BackgroundAneurysmal subarachnoid hemorrhage (SAH) is a highly morbid and fatal condition with high rate of cognitive impairment and negative impact in quality of life among survivors. Delayed cerebral infarction (DCI) is one the major factors for these negative outcomes. In this study we compared the circulating microRNA profiles of SAH patients and healthy individuals, and the circulating microRNA profiles of SAH patients with and without DCI.MethodsPeripheral blood samples on Day 7 after the onset of SAH were subjected to microarray analysis with Affymetrix miRNA 3.0 array and quantitative PCR analysis. SAH patients with (N = 20) and without DCI (N = 20) and Healthy controls (N = 20) were included for analyses.ResultsWe demonstrated that 99 miRNAs were found to be dysregulated in the SAH patient group with DCI. 81 miRNAs were upregulated and 18 were downregulated. Findings from KEGG pathway analysis showed that miRNAs and target genes for axon guidance and TGF-beta signaling were involved, implying that the resulted differential miRNA expression pattern reflect the results of SAH instead of etiology of the disease. miR-132-3p and miR-324-3p showed distinctive upregulations in qPCR [miR-132: 9.5 fold (95%CI: 2.3 to 16.7) in DCI group and 3.4 fold (95%CI: 1.0 to 5.8) in Non-DCI group; miR-324: 4924 fold (95%CI: 2620 to 7228) in DCI group and 4545 fold (95%CI: 2408 to 6683) in non-DCI group]. However, there were no significant differences in fold changes between SAH patients with and without DCI [fold change ratios (mean+/-SD): 2.7+/-4.2 and 1.1+/-1.1 for miRNA-132 and miRNA-324].ConclusionOur study demonstrated that as compared to healthy control, miR-132 and miR-324 showed a upregulation in both SAH DCI and Non-DCI groups. However, the differences between the SAH DCI and non-DCI groups were not statistically significant.

Differentially Expressed and Prognostically Significant Lincrnas May Impact Immune System and Tumor Progression in Multiple Myeloma (MM)

Long intergenic non-coding RNA (lincRNA) are transcripts longer than 200 nucleotides which have a diverse sets of regulatory functions but do not get translated into protein. lincRNAs are located between the protein coding genes and do not overlap exons of either protein-coding or other non-lincRNA. However precise role of individual lincRNA in disease biology remains unclear. Here, we have evaluated the lincRNA expression and their potential biological functions in MM.We performed RNA-seq on CD138+ MM cells from 296 newly diagnosed patients and 16 normal bone marrow plasma cells (NBM) and analyzed for lincRNA expression. Data from paired-end RNAseq reads were mapped to the latest human genome, differentially expressed lincRNAs were identified and for each expressed lincRNA event free survival was examined with univariate cox regression model and support vector machine. Finally, we identified protein coding genes that are strongly correlated (cor > 0.5) with lincRNAs with significant altered expression in MM and impact on EFS to identify their biological role.lincRNA and protein coding genes that have more than 10 reads/million reads for at least 15 normal samples or 62 MM samples (20% all MM samples) were included in the analysis. We identified 60 differentially expressed lincRNA (adj p value <0.05), 51 of those had at least 1.5 fold change difference. The differentially expressed lncRNAs were in close proximity of Ig-related genes, genome stability related genes, hosting miRNAs such as mir222 and mir22 and previously reported for other cancers (PVT and TTY15). We evaluated relation of these lincRNAs with event free survival (EFS) and observed 6 lincRNAs associated with shorter EFS. We have developed multivariate signature model to predict EFS by using these 6 lincRNAs. We divided our dataset into training (n=99) and test (n=156) dataset and we utilized support vector machine classification to divide samples into 2 groups using six lincRNAs. This model was able to predict good and poor survival groups in training dataset (p val < 0.001) as well as test dataset (p val = 0.002) (Figure).We examined genome wide correlation between these six differentially expressed and prognostically significant lincRNAs to expressed protein coding genes to identify their biological functions in MM. Four of these lincRNAs strongly correlated with 47 to 504 genes (abs(cor) > 0.5), affecting immune system pathways and pathways in cancer including Jak-STAT signaling pathway. We also found that these lincRNAs are also highly correlated with tumor development genes such as TNFRSF1B,FGR,TP53BP2,TNF and T or B cells related genes PIK3CD, BCL6. In addition, two of these lincRNAs (LINC00936 and CTB-61M7.2) were found highly correlated with their protein coding neighbor genes ATP2B1(cor = 0.45) and FCAR (cor = 0.95) respectively and MIR22HG was host gene for mir22 which may indicate lincRNAs are using different machinery in MM to regulate protein coding genes.In summary, we report that lincRNA is differentially expressed and prognostically significant in myeloma and may function through their impact on immune system and tumor progression. Our ongoing integrative approach will provide further evidence of their regulatory role in MM with potential therapeutic application. [Display omitted] DisclosuresAnderson:acetylon pharmaceuticals: Equity Ownership; Celgene Corporation: Consultancy; Gilead: Consultancy; Oncocorp: Equity Ownership; Millennium: Consultancy; BMS: Consultancy. Munshi:onyx: Membership on an entity's Board of Directors or advisory committees; celgene: Membership on an entity's Board of Directors or advisory committees; novartis: Membership on an entity's Board of Directors or advisory committees; millenium: Membership on an entity's Board of Directors or advisory committees.

Genomic Landscape Predictive of Minimal Residual Disease (MRD) in Multiple Myeloma (MM)

Progress in the treatment of multiple myeloma (MM) has increased extent and frequency of response, as well as prolonged progression-free (PFS) and overall survival (OS). Today complete remission (CR) rates up to 70% are achieved with new drug combinations. This has lead to development of sensitive next generation sequencing (NGS) -based methods to predict deeper responses that may more accurately predict survival outcomes in MM. Our large recent study has confirmed the clinical impact of achieving MRD- status in MM. Here we are evaluating the genomic alterations that may predict attainment of MRD negative status in MM.MRD status was evaluted in 279 patients from IFM/DFCI 2009 trial. We obtained gene expression by RNA-seq, and copy number profile by cytoScan HD array to evaluate genomic differences between MRD negative and MRD positive groups. We generated copy number data for 175 / 279 patients (72 MRD- and 103 MRD+) with Affymetrix Cytoscan HD array and compared genome wide copy number alterations. We observed statistically significant copy number alterations in chromosome 1p, 2, 4q, 11q, 13, 14 and 20 between MRD- and MRD+ patients. However, the extent of alterations in these regions is limited. The largest difference was on chromosome 11q arm where MRD- patients had 2.2 copies on average and MRD+ had 2.4 (p value < 0.001).Similarly, we generated gene expression profiles with RNAseq for 69 MRD- patients and 92 MRD+ patients to study gene expression alterations that may predict attainment of MRD negative status and to examine possible biological pathways. Although first two component of principle component analysis (PCA) showed that two groups have similar expression profile, we were able to identify 586 differentially expressed genes; 333 of those were up and 253 were down regulated in MRD+ compared to MRD- groups. We found that seven oncogenes (CCND1, CD79B, IDH1, PATZ1, PAX5, POU2AF1, RUNX1) were significantly high in MRD+ and two (CCND2 and MYCN) were high in MRD-. Additional genes that were high in MRD+ samples were enriched in genes regulated by NF-kB in response to TNF, P53 pathway, KRAS signaling and genes down-regulated in response to ultraviolet (UV) radiation. Genes that were high in MRD- compared to MRD+ were also enriched in genes up-regulated by STAT5 in response to IL2 stimulation, p53 pathways and networks, and genes up-regulated in response to ultraviolet (UV) radiation pathways.Finally, we have created a signature to predict MRD+ and MRD- in MM samples from differentially expressed genes. We used 40 genes that has at least 2 fold change difference between MRD+ and MRD- groups as a predictor and we randomly separated 161 RNAseq samples into train (n=99) and test group (n=62). We developed our classifiers with diagonal discriminant analysis and we achieved 0.79 classifier performance on test dataset. Then we tested our signature against 1000 random signature and it was significantly different than random signatures (Figure).In conclusion, we here report a first genomic landscape predictive of minimal residual disease (MRD) in Multiple Myeloma (MM). This analysis will help understand genomic and molecular correlates of achieving minimal residula disease and confirms feasibility of using RNAseq data from diagnosis sample to predict MRD status. The ongoing integration of other genomic correlates such as copy number status as well as alternate splicing may allow further improvement in the performance of prediction. [Display omitted] DisclosuresAnderson:Gilead: Consultancy; acetylon pharmaceuticals: Equity Ownership; Oncocorp: Equity Ownership; Celgene Corporation: Consultancy; BMS: Consultancy; Millennium: Consultancy. Attal:jansen: Honoraria; celgene: Membership on an entity's Board of Directors or advisory committees. Munshi:onyx: Membership on an entity's Board of Directors or advisory committees; celgene: Membership on an entity's Board of Directors or advisory committees; millenium: Membership on an entity's Board of Directors or advisory committees; novartis: Membership on an entity's Board of Directors or advisory committees.

Abstract A25: Predictive biomarkers for bevacizumab response in recurrent glioblastoma patients

Abstract BACKGROUND: Bevacizumab (BEV) plus chemotherapy has shown high response rates in recurrent glioblastoma (GBM) and patients who achieve response have an improved overall survival as well as quality of life. Recent retrospective analysis of the randomized phase III trial, AVAglio, indicate that patients with the proneural GBM subtype have a survival benefit when treated with BEV in combination with standard treatment. However, no validated biomarkers able to predict BEV response have been identified and the biology reflecting a clinical BEV response is poorly understood. The primary objective of this study was to evaluate the predictive and prognostic value of GBM subtypes in recurrent GBM patients treated with BEV therapy. The secondary objective was to identify biomarkers able to predict response to BEV therapy in recurrent GBM patients. METHODS: A total of 90 recurrent GBM patients treated with BEV combination treatment according to a previously published treatment protocol were included. Inclusion criteria: BEV plus irinotecan treatment in the period between May 2005-2011; available GBM tissue (according to WHO); response evaluable (RANO). RNA from tumor tissue was analyzed by the NanoString platform covering 800 genes. Raw data was assigned to molecular subtypes for each of the samples using the PAMR classifier model, previously trained on the AVAglio dataset. In order to identify novel candidate biomarkers able to predict response, differentially expressed genes (fold-change difference &amp;gt; 1.5) between patients responding versus progressing on BEV were identified using a t-test. Biomarkers significantly (P&amp;lt;0.05) associated with response in multivariate logistic regression analysis adjusted for recently validated clinical prognostic factors were selected for the final predictive model. RESULTS: Molecular subtypes were not associated with response or overall survival. However, two novel independent predictive biomarkers (gene1 down-regulated and gene2 up-regulated in responders) of BEV response and overall survival were identified. Results will be presented. Citation Format: Thomas Urup, Signe Regner Michaelsen, Lars Rønn Olsen, Anders Toft, Ib Jarle Christensen, Kirsten Grunnet, Helle Broholm, Michael Kosteljanetz, Shohreh Issazadeh-Navikas, Hans Skovgaard Poulsen, Ulriik Lassen. Predictive biomarkers for bevacizumab response in recurrent glioblastoma patients. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A25.

Patterns of gene expression among murine models of hemorrhagic shock/trauma and sepsis.

Controversy remains whether the leukocyte genomic response to trauma or sepsis is dependent upon the initiating stimulus. Previous work illustrated poor correlations between historical models of murine trauma and sepsis (i.e., trauma-hemorrhage and lipopolysaccharide injection, respectively). The aim of this study is to examine the early genomic response in improved murine models of sepsis [cecal ligation and puncture (CLP)] and trauma [polytrauma (PT)] with and without pneumonia (PT+Pp). Groups of naïve, CLP, PT, and PT+Pp mice were killed at 2 h, 1 or 3 days. Total leukocytes were isolated for genome-wide expression analysis, and genes that were found to differ from control (false discovery rate adjusted P < 0.001) were assessed for fold-change differences. Spearman correlations were also performed. For all time points combined (CLP, PT, PT+Pp), there were 10,426 total genes that were found to significantly differ from naïve controls. At 2 h, the transcriptomic changes between CLP and PT showed a positive correlation (rs) of 0.446 (P < 0.0001) but were less positive thereafter. Correlations were significantly improved when we limited the analysis to common genes whose expression differed by a 1.5 fold-change. Both pathway and upstream analyses revealed the activation of genes known to be associated with pathogen-associated and damage-associated molecular pattern signaling, and early activation patterns of expression were very similar between polytrauma and sepsis at the earliest time points. This study demonstrates that the early leukocyte genomic response to sepsis and trauma are very similar in mice.

A genome-wide study of DNA methylation modified by epigallocatechin-3-gallate in the CAL-27 cell line.

In order to gain greater understanding of the mechanisms underlying the effect of epigallocatechin-3-gallate (EGCG) on DNA methylation and its chemopreventative action in oral squamous cell carcinoma (OSCC), a genome‑wide methylation and mRNA expression screen was performed in the CAL‑27 cell line with and without EGCG (100 µM) treatment. A total of 761 differentially methylated gene loci were identified following treatment with EGCG. Comparison of gene expression profiling in OSCC samples revealed 184 transcripts with a significant difference (P<0.05) and a fold change difference >2 compared with controls. Gene ontology analysis of differentially methylated loci and functional annotation of the differentially expressed genes indicated that the main pathways involved were metabolic, mitogen‑activated protein kinase (MAPK), wnt, and cell cycle pathways. In conclusion, the present study indicates that EGCG can affect the methylation status and gene expression in the CAL‑27 cell line. Additionally, the changes in several important signaling pathways may reveal the antitumor mechanism of EGCG.

Abstracts fromThe 33rd AnnualNational Neurotrauma SymposiumJune 28–July 1, 2015Santa Fe, New Mexico

Abstracts fromThe 33^rd AnnualNational Neurotrauma SymposiumJune 28–July 1, 2015Santa Fe, New Mexico

Development of a protein marker panel for characterization of human induced pluripotent stem cells (hiPSCs) using global quantitative proteome analysis

The emergence of new methods for reprogramming of adult somatic cells into induced pluripotent stem cells (iPSC) led to the development of new approaches in drug discovery and regenerative medicine. Investigation of the molecular mechanisms underlying the self-renewal, expansion and differentiation of human iPSC (hiPSC) should lead to improvements in the manufacture of safe and reliable cell therapy products. The goal of our study was qualitative and quantitative proteomic characterizations of hiPSC by means of electrospray ionization (ESI)-MSe and MALDI-TOF/TOF mass spectrometry (MS). Proteomes of hiPSCs of different somatic origins: fibroblasts and peripheral blood CD34+ cells, reprogrammed by the same technique, were compared with the original somatic cells and hESC. Quantitative proteomic comparison revealed approximately 220 proteins commonly up-regulated in all three pluripotent stem cell lines compared to the primary cells. Expression of 21 proteins previously reported as pluripotency markers was up-regulated in both hiPSCs (8 were confirmed by Western blot). A number of novel candidate marker proteins with the highest fold-change difference between hiPSCs/hESC and somatic cells discovered by MS were confirmed by Western blot. A panel of 22 candidate marker proteins of hiPSC was developed and expression of these proteins was confirmed in 8 additional hiPSC lines.