Significance Analysis Of Microarrays Software Research Articles

Characterization of immortalized human islet stromal cells reveals a MSC-like profile with pancreatic features

BackgroundMesenchymal stromal cells (MSCs) represent an interesting tool to improve pancreatic islet transplantation. They have immunomodulatory properties and secrete supportive proteins. However, the functional properties of MSCs vary according to many factors such as donor characteristics, tissue origin, or isolation methods. To counteract this heterogeneity, we aimed to immortalize and characterize adherent cells derived from human pancreatic islets (hISCs), using phenotypic, transcriptomic, and functional analysis.MethodsAdherent cells derived from human islets in culture were infected with a hTERT retrovirus vector and then characterized by microarray hybridization, flow cytometry analysis, and immunofluorescence assays. Osteogenic, adipogenic, and chondrogenic differentiation as well as PBMC proliferation suppression assays were used to compare the functional abilities of hISCs and MSCs. Extracellular matrix (ECM) gene expression profile analysis was performed using the SAM (Significance Analysis of Microarrays) software, and protein expression was confirmed by western blotting.ResultshISCs kept an unlimited proliferative potential. They exhibited several properties of MSCs such as CD73, CD90, and CD105 expression and differentiation capacity. From a functional point of view, hISCs inhibited the proliferation of activated peripheral blood mononuclear cells. The transcriptomic profile of hISCs highly clusterized with bone marrow (BM)-MSCs and revealed a differential enrichment of genes involved in the organization of the ECM. Indeed, the expression and secretion profiles of ECM proteins including collagens I, IV, and VI, fibronectin, and laminins, known to be expressed in abundance around and within the islets, were different between hISCs and BM-MSCs.ConclusionWe generated a new human cell line from pancreatic islets, with MSCs properties and retaining some pancreatic specificities related to the production of ECM proteins. hISCs appear as a very promising tool in islet transplantation by their availability (as a source of inexhaustible source of cells) and ability to secrete a supportive “pancreatic” microenvironment.

Systemic release of osteoprotegerin during oxaliplatin-containing induction chemotherapy and favorable systemic outcome of sequential radiotherapy in rectal cancer.

In colorectal cancer, immune effectors may be determinative for disease outcome. Following curatively intended combined-modality therapy in locally advanced rectal cancer metastatic disease still remains a dominant cause of failure. Here, we investigated whether circulating immune factors might correlate with outcome. An antibody array was applied to assay changes of approximately 500 proteins in serial serum samples collected from patients during oxaliplatin-containing induction chemotherapy and sequential chemoradiotherapy before final pelvic surgery. Array data was analyzed by the Significance Analysis of Microarrays software and indicated significant alterations in serum osteoprotegerin (TNFRSF11B) during the treatment course, which were confirmed by osteoprotegerin measures using a single-parameter immunoassay. Patients experiencing increase in circulating osteoprotegerin during the chemotherapy had significantly better 5-year progression-free survival than those without increase (78% versus 48%; P = 0.009 by log-rank test). Hence, systemic release of this soluble tumor necrosis factor decoy receptor following the induction phase of neoadjuvant therapy was associated with favorable long-term outcome in patients given curatively intended chemoradiotherapy and surgery but with metastatic disease as the main adverse event. This finding suggests that osteoprotegerin may mediate or reflect systemic anti-tumor immunity invoked by combined-modality therapy in locally advanced rectal cancer.

Screening for characteristic microRNAs between pre-invasive and invasive stages of cervical cancer

The aim of the present study was to investigate the characteristic microRNAs (miRNAs) expressed during the pre-invasive and invasive stages of cervical cancer. A gene expression profile (GSE7803) containing 21 invasive squamous cell cervical carcinoma samples, 10 normal squamous cervical epithelium samples and seven high-grade squamous intraepithelial cervical lesion samples, was obtained from the Gene Expression Omnibus. Differentially expressed genes (DEGs) were identified using significance analysis of microarray software, and a Gene Ontology (GO) enrichment analysis was conducted using the Database for Annotation, Visualization and Integrated Discovery. The miRNAs that interacted with the identified DEGs were selected, based on the TarBase v5.0 database. Regulatory networks were constructed from these selected miRNAs along with their corresponding target genes among the DEGs. The regulatory networks were visualized using Cytoscape. A total of 1,160 and 756 DEGs were identified in the pre-invasive and invasive stages of cervical cancer, respectively. The results of the GO enrichment demonstrated that the DEGs were predominantly involved in the immune response and the cell cycle, in the pre-invasive and invasive stages, respectively. Furthermore, a total of 18 and 26 characteristic miRNAs were screened in the pre-invasive and invasive stages, respectively. These miRNAs may be potential biomarkers and targets for the diagnosis and treatment of the different stages of cervical cancer.

Comparative transcript profiling of gene expression of fresh and frozen–thawed bull sperm

Although frozen semen is widely used commercially in the cattle breeding industry, the resultant pregnancy rate is lower than that produced using fresh semen. Cryodamage is a major problem in semen cryopreservation; it causes changes to sperm transcripts that may influence sperm function and motility. We used suppression subtractive hybridization technology to establish a complementary DNA subtractive library, and combined microarray technology and sequence homology analysis to screen and analyze differentially expressed genes in the library, comparing fresh sperm with the frozen–thawed sperm of nine bulls. Overall, 19 positive differentially expressed unigenes were identified using microarray data and Significance Analysis of Microarrays software (|score (d)| ≥ 2, fold change > 1, and false discovery rate < 0.05). Of 15 differentially expressed unigenes exhibited high sequence homology (E-value ≤ 1 × 10−3), 12 were upregulated in frozen–thawed sperm, the remaining 3 were upregulated in fresh sperm, and 4 other clones were identified as unknown because of incomplete sequences or because there was no significant sequence homology (E-value > 1E−03) and were considered novel genes. The expression of five of these genes—RPL31, PRKCE, PAPSS2, PLP1, and R1G7—was verified by quantitative real-time reverse transcription–polymerase chain reaction. There was a significant differential expression of the RPL31 gene (P < 0.05). Our preliminary results provide an overview of differentially expressed transcripts between fresh and frozen–thawed sperm of Holstein bulls.

Identification and characterization of genes differentially expressed in X and Y sperm using suppression subtractive hybridization and cDNA microarray.

Differential expression of genes leads to variations in the phenotypes of X and Y sperm, although some differentially expressed gene products are shared through intercellular bridges. Genes differentially expressed in bovine X and Y sperm were identified by a combination of suppression subtractive hybridization (SSH), cDNA microarray, and sequence-homology analysis. Microarray data and Significance Analysis of Microarrays software were used to identify 31 differentially expressed genes, only four of which were previously identified. These genes are involved in fundamental life processes of mature sperm, and may be associated with the differences between X and Y sperm since 27 versus 4 were upregulated in X versus Y sperm, respectively. The levels of expression of seven genes-including the known genes UTY, DPH3, CYTB, and ISCU, and the unknown genes X + Y contig 41, X + Y contig 18, and Y + X contig 16-were validated by quantitative real-time PCR, and some genes were clearly differentially expressed by X and Y sperm, despite the presence of intercellular bridges among spermatids. These results provide a theoretical basis for research on gene expression during sperm development, as well as on sex control at the level of sperm.

Comparative transcriptome pairwise analysis of spontaneously transformed multipotent stromal cells from human adipose tissue

Potential markers and the mechanism of the spontaneous transformation of multipotent stromal cells (MSC) with perivascular immunophenotype have been determined. A transcriptome comparative study was performed involving six paired specimens of normal and spontaneously transformed MSC with perivascular immunophenotype, obtained in the first passages following the isolation of adipose tissue of six healthy donors. According to the results obtained using the microarray Illumina HT-12 v4 with the Significance Analysis of Microarrays software, differentially expressed transcripts were revealed and a statistical analysis using Gene Ontology and Molecular Signatures Databases was conducted. The association of the spontaneous transformation of isolated MSC with perivascular immunophenotype with previously identified oncogenic cell transformation pathways (E2F, ATR/ATM, RAS, and RHOA) is suggested and further aims for more detailed study are set. Potential transformation markers, including largely unknown genes and those previously not associated with cancer genes (HSPB6, PLAC9, FEZ1, DTWD1, APH1A, and ATP5L) are described.

Gene Expression Profile of Blood Cells for the Prediction of Delayed Cerebral Ischemia after Intracranial Aneurysm Rupture: A Pilot Study in Humans

Background: Delayed cerebral ischemia (DCI) is a potentially devastating complication after intracranial aneurysm rupture and its mechanisms remain poorly elucidated. Early identification of the patients prone to developing DCI after rupture may represent a major breakthrough in its prevention and treatment. The single gene approach of DCI has demonstrated interest in humans. We hypothesized that whole genome expression profile of blood cells may be useful for better comprehension and prediction of aneurysmal DCI. Methods: Over a 35-month period, 218 patients with aneurysm rupture were included in this study. DCI was defined as the occurrence of a new delayed neurological deficit occurring within 2 weeks after aneurysm rupture with evidence of ischemia either on perfusion-diffusion MRI, CT angiography or CT perfusion imaging, or with cerebral angiography. DCI patients were matched against controls based on 4 out of 5 criteria (age, sex, Fisher grade, aneurysm location and smoking status). Genome-wide expression analysis of blood cells obtained at admission was performed by microarrays. Transcriptomic analysis was performed using long oligonucleotide microarrays representing 25,000 genes. Quantitative PCR: 1 µg of total RNA extracted was reverse-transcribed, and the resulting cDNA was diluted 10-fold before performing quantitative PCR. Microarray data were first analyzed by ‘Significance Analysis of Microarrays' software which includes the Benjamini correction for multiple testing. In a second step, microarray data fold change was compared using a two-tailed, paired t test. Analysis of receiver-operating characteristic (ROC) curves and the area under the ROC curves were used for prediction analysis. Logistic regression models were used to investigate the additive value of multiple biomarkers. Results: A total of 16 patients demonstrated DCI. Significance Analysis of Microarrays software failed to retrieve significant genes, most probably because of the heterogeneity of the patients included in the microarray experiments and the small size of the DCI population sample. Standard two-tailed paired t test and C-statistic revealed significant associations between gene expression and the occurrence of DCI: in particular, the expression of neuroregulin 1 was 1.6-fold upregulated in patients with DCI (p = 0.01) and predicted DCI with an area under the ROC curve of 0.96. Logistic regression analyses revealed a significant association between neuroregulin 1 and DCI (odds ratio 1.46, 95% confidence interval 1.02-2.09, p = 0.02). Conclusions: This pilot study suggests that blood cells may be a reservoir of prognostic biomarkers of DCI in patients with intracranial aneurysm rupture. Despite an evident lack of power, this study elicited neuroregulin 1, a vasoreactivity-, inflammation- and angiogenesis-related gene, as a possible candidate predictor of DCI. Larger cohort studies are needed but genome-wide microarray-based studies are promising research tools for the understanding of DCI after intracranial aneurysm rupture.

Gene expression-based prediction of myeloma cell sensitivity to histone deacetylase inhibitors

Background:Multiple myeloma (MM) is still a fatal plasma cell cancer. Novel compounds are currently clinically tested as a single agent in relapsing patients, but in best cases with partial response of a fraction of patients, emphasising the need to design tools predicting drug efficacy. Histone deacetylase inhibitors (HDACi) are anticancer agents targeting epigenetic regulation of gene expression and are in clinical development in MM.Methods:To create a score predicting HDACi efficacy, five MM cell lines were treated with trichostatin A (TSA) and gene expression profiles were determined.Results:The expression of 95 genes was found to be upregulated by TSA, using paired supervised analysis with Significance Analysis of Microarrays software. Thirty-seven of these 95 genes had prognostic value for overall survival in a cohort of 206 newly diagnosed MM patients and their prognostic information was summed up in a histone acetylation score (HA Score); patients with the highest HA Score had the shorter overall survival. It is worth noting that MM cell lines or patients' primary MM cells with a high HA Score had a significant higher sensitivity to TSA, valproic acid, panobinostat or vorinostat.Conclusion:In conclusion, the HA Score allows identification of MM patients with poor survival, who could benefit from HDACi treatment.

Transcriptomic Analysis of the Developing and Adult Mouse Cochlear Sensory Epithelia

The adult mammalian cochlea lacks regenerative ability and the irreversible degeneration of cochlear sensory hair cells leads to permanent hearing loss. Previous data show that early postnatal cochlea harbors stem/progenitor-like cells and shows a limited regenerative/repair capacity. These properties are progressively lost later during the postnatal development. Little is known about the genes and pathways that are potentially involved in this difference of the regenerative/repair potentialities between early postnatal and adult mammalian cochlear sensory epithelia (CSE). The goal of our study is to investigate the transcriptomic profiles of these two stages. We used Mouse Genome 430 2.0 microarray to perform an extensive analysis of the genes expressed in mouse postnatal day-3 (P3) and adult CSE. Statistical analysis of microarray data was performed using SAM (Significance Analysis of Microarrays) software. We identified 5644 statistically significant differentially expressed transcripts with a fold change (FC) >2 and a False Discovery Rate (FDR) ≤0.05. The P3 CSE signature included 3,102 transcripts, among which were known genes in the cochlea, but also new transcripts such as, Hmga2 (high mobility group AT-hook 2) and Nrarp (Notch-regulated ankyrin repeat protein). The adult CSE overexpressed 2,542 transcripts including new transcripts, such as Prl (Prolactin) and Ar (Androgen receptor), that previously were not known to be expressed in the adult cochlea. Our comparative study revealed important genes and pathways differentially expressed between the developing and adult CSE. The identification of new candidate genes would be useful as potential markers of the maintenance or the loss of stem cells and regenerative/repair ability during mammalian cochlear development.

Molecular differences in triple negative breast cancer between race/ethnicities.

Abstract Abstract #2089 Background: A disparity in prognosis of triple negative (TN) breast cancer (BC) has been observed between African American (AA) and Caucasian (CAU) race/ethnicities afflicted with this aggressive and invasive BC subtype. Etiological understanding of these differences involves accounting for several factors associated with phenotype and genotype. Here, we address the latter using the Illumina DASL (cDNA mediated, Annealing, Selection, Extension, and Ligation) assay to quantify mRNA expression of 512 breast cancer related genes in a cohort of 24 CAU and 56 AA TN BC tissues sourced from formalin-fixed, paraffin-embedded (FFPE) blocks.&amp;#x2028; Material and Methods: The DASL assay was used to measure mRNA expression levels from FFPE sourced tissues in both cohorts of self-identified patients. CAU BC patients were obtained from St. Mary's Hospital, Montreal, Quebec and AA BC patients were obtained from Grady Hospital, Atlanta, Georgia. RNA extraction used the RNA High Pure Kit (Roche) and was taken from archival FFPE tissues either 5µm tissue sections or cores. Differential mRNA regulation was identified by Significance Analysis of Microarrays (SAM) software using a false discover rate (FDR) less than 1% and a two fold-change criteria to determine differential regulation.&amp;#x2028; Results: In all, 33 genes were found differentially expressed between AA and CAU TN BC tumor samples, 32 of which were upregulated in the AA cohort, only 1 of which was upregulated in the CAU group. The upregulated gene in CAU TN BC was TFF1. Upregulated genes in the AA cohort (order of statistical significance according to SAM software) were KIF20A, EP300, AURKB, FGF4, C14orf155, USP22, EPOR, ZNF668, SCNN1G, MAPT, FLNB, EP400, LTA, ACOT11, RBP3, CSF3, E2F2, TGFB1, CCNE1, L1CAM, NDP, VWF, RHOB, FEN1, BIN1, KRT17, CDC42EP4, SERPINF1, CHI3L2, NES, BCL2, and RERG.&amp;#x2028; Discussion: TFF1 upregulated in the CAU population, has been indicated as biomarker of favorable prognosis in endocrine therapy in clinical studies which is consistent with race/ethnicity disparities. The remaining genes upregulated in the AA cohort include transcription factors E2F2 and RBP3/E2F1 both with cyclin binding domains which may interact with CCNE1, extracellular and adhesion related genes KRT17, L1CAM and FGF4, genes associated with cell cycle AURKB, EP400, and EP300 (activator of HIF-1A). Several RAS related genes were also found differentially expressed in the AA cohort including RHOB, RERG, BIN1, and EPOR. Moreover, it is worth mentioning that BCL2 which is expressed in the aggressive mammary cancer cell line MCF-7 was also found upregulated in the AA cohort. These initial findings suggest that several differentially regulated genes between AA and CAU race/ethnicities may account for the disparity in outcomes resultant in these populations. These initial data warrant further investigation which is currently ongoing. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2089.

Validation of a custom 512-gene breast cancer panel on the illumina-based DASL mRNA expression platform: focus on triple negative breast tumors using RNA from stored formalin-fixed, paraffin blocks.

Abstract Abstract #2027 Background: DASL (cDNA-mediated Annealing, Selection, extension Ligation) is a recently described, RT-PCR-based, multiplex gene expression assay developed by Illumina specifically for use with fragmented RNA prepared from formalin-fixed paraffin-embedded (FFPE) samples. For use with this assay, we have designed a custom 512-gene breast cancer panel, validated this panel with FFPE breast tumor samples, and identified novel genes associated with the triple negative (TN) subtype.&amp;#x2028; Material and Methods: The DASL assay was used to measure mRNA levels from RNA isolated from 5 um FFPE breast tumor sections using the Roche High Pure Kit. Samples that passed our quality control tests were run in the assay. Arrays were imaged using a BeadArray Reader and data was analyzed using BeadStudio software. Differential mRNA regulation was identified by Significance Analysis of Microarrays software using a false discover rate (q-value &amp;lt; 0.01) or Bonferroni correction of the two-tailed Student's t-test. IHC concordance was measured by mRNA expression fold-change for positive versus negative IHC categories.&amp;#x2028; Results: To validate our custom breast cancer panel, we performed the DASL assay on 187 samples from 87 patients with differing receptor status as previously determined by IHC (ER, PR, HER2) and/or FISH (HER2). Correlation between 152 common genes on Illumina's 502-gene standard cancer panel and our custom panel was very high; R2 value of 0.88. Concordance between IHC/FISH and DASL data for ESR1, PGR and ERBB2 was &amp;gt; 90%. We identified 73 genes differentially expressed between TN and the other tumor samples. Of these, 20 were in common between the standard and custom panels, 53 were uniquely expressed on the custom panel. These included genes associated with the TN/Basal subtype identified in previous microarray studies; CXCL1, CDH3, ANXA8, KRT5, TRIM29, KRT17, MFGE8, CX3CL1, FZD7, CHI3L2, B3GNT5 (PNAS 100:8418, 2003) and CDH3, CRABP1, CX3CL1, FOXC1, MMP7 (BMC Genomics 7:96, 2006) further validating both DASL assay and custom panel. In addition, we identified genes not previously linked to the TN subtype, highlighting the potential utility of the custom panel for profiling breast tumor FFPE specimens in the DASL assay.&amp;#x2028; Discussion: We have designed and validated a custom 512-gene breast cancer panel for expression profiling studies using RNA prepared from FFPE tumor specimens on the DASL platform. Thus far, we have identified a 73-gene set with the custom panel (significantly more genes than with the standard panel) that can discriminate TN from other receptor expressing subtypes. We are now applying this platform to samples from clinical trials in order to identify both prognostic and predictive breast tumor gene sets. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2027.

Expression Analysis of Juvenile Pilocytic Astrocytomas by Oligonucleotide Microarray Reveals Two Potential Subgroups

Juvenile pilocytic astrocytoma (JPA) is one of the most common brain tumors in children. The expression profiles of 21 JPAs, determined using Affymetrix GeneChip U133A, were compared with subjects with normal cerebella. The genes involved in neurogenesis, cell adhesion, synaptic transmission, central nervous system development, potassium ion transport, protein dephosphorylation, and cell differentiation were found to be significantly deregulated in JPA. These 21 JPAs were further clustered into two major groups by unsupervised hierarchical clustering using a set of 848 genes with high covariance (0.5-10). Supervised analysis with Significance Analysis of Microarrays software between these two potential subgroups identified a list of significant differentially expressed genes involved in cell adhesion, regulation of cell growth, cell motility, nerve ensheathment, and angiogenesis. Immunostaining of myelin basic protein on paraffin sections derived from 18 incompletely resected JPAs suggests that JPA without myelin basic protein-positively stained tumor cells may have a higher tendency to progress.

Increased expression of metallothionein is associated with irinotecan resistance in gastric cancer.

To gain insight into clinically relevant mechanisms of irinotecan resistance, we undertook oligonucleotide microarray analyses on paired malignant effusion samples obtained from eight gastric cancer patients treated with weekly irinotecan. Pretreatment and posttreatment (48 h) effusion samples were obtained for each patient, and the change in expression profile was compared between clinical responders and nonresponders. When differences in the expression of genes were examined using SAM (Significance Analysis of Microarrays) software, five isoforms of the metallothionein family were identified to have significantly higher signal log ratios in five nonresponders, compared with three responders. Compared with control cells, metallothionein 1X (MT1X)-transfected AGS cells showed a 1.4-fold higher irinotecan IC(50) by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and tended to form more colonies. These findings collectively suggest that irinotecan-induced up-regulation of metallothionein might be associated with irinotecan resistance in patients with gastric cancer, although it remains to be confirmed in a larger data set.

Transcriptional profiling of nasal epithelium during natural colds in asthmatic subjects

Rationale Although cold viruses precipitate most asthma exacerbations, the mechanisms leading to exacerbations rather than just colds are poorly understood. Transcriptional profiling of nasal mucosa may help identify genes related to exacerbations. Methods To determine the feasibility of measuring expression of a large number of genes in nasal epithelium, we scraped the nasal mucosa with Rhinoprobe during acute colds and at least 6 weeks later (baseline) in 25 episodes of colds (21 subjects, 17 with asthma). RNA was extracted using RNeasy kit and expression of 384 genes measured by two-step multiplex real-time RT-PCR. Gene expression levels were normalized to several housekeeping genes and analyzed using SAM (Significance Analysis of Microarrays) software. False discovery rate (FDR) was set at 5% or less, and magnitude of expression change was set to 2-fold or greater. Results Median FDR was 2.75%. We found 25 genes expressed more during acute colds than at baseline including ICAM-1, GM-CSF, G-CSF, IL-10, IL-10R, CCR1, CCR2, CCR5, eotaxin, and GRO1-3. The gene most significantly increased was endothelial cell-specific molecule-1 (ESM-1), a proteoglycan secreted by endothelial cells that inhibits LFA-1/ICAM-1 binding and may regulate lymphocyte migration. In addition, we found increase in genes related to generation of reactive oxygen species (NADPH oxidase p91 subunit, and glutaredoxin), co-stimulatory molecules (CD80 and ICOS), and ion channels (CLCA3 and SLC8A1, a Ca/Na exchanger). Conclusion Transcriptional signatures for a large number of genes in nasal mucosal samples can be used to monitor disease progression and to identify new genes related to virus-induced nasal inflammation and possibly asthma exacerbations.