Assessment Of Gene Expression Profiles Research Articles

Differential expression profile of genes involved in the immune response associated to progression of chronic Chagas disease.

Patients with chronic Chagas disease present marked clinical and immunological heterogeneity. During the disease, multiple immune mechanisms are activated to fight the parasite. The purpose of this study was to investigate the expression patterns of genes involved in relevant immunological processes throughout the disease in patients with chronic Chagas disease. High-throughput RT-qPCR with QuantStudio 12K Flex real-time PCR system was used to evaluate the expression of 106 immune-related genes in PBMC from a cohort of cardiac Chagas disease patients (CCC I), asymptomatic patients (IND) and healthy donors (HD) after being stimulated with T. cruzi soluble antigens. Principal component analysis (PCA), cluster analysis and volcano plots were used to identify differentially expressed genes. In addition, gene set enrichment analysis (GSEA) was employed to identify the enriched immunological pathways in which these genes are involved. PCA revealed the existence of a statistically divergent expression profile of the 36 genes correlated with PC1 between CCC I patients and HD (p < 0.0001). Differential gene expression analysis revealed upregulation of 41 genes (expression fold-change > 1.5) and downregulation of 14 genes (expression fold-change < 0.66) (p = 8.4x10-13 to p = 0.007) in CCC I patients versus HD. Furthermore, significant differences in the expression level of specific genes have been identified between CCC I and IND patients (8 up and 1 downregulated). GSEA showed that several upregulated genes in CCC I patients participate in immunological pathways such as antigen-dependent B cell activation, stress induction of HSP regulation, NO2-dependent IL12 pathway in NK cells, cytokines-inflammatory response and IL-10 anti-inflammatory signaling. Cardiac Chagas disease patients show an antigen-specific differential gene expression profile in which several relevant immunological pathways seem to be activated. Assessment of gene expression profiles reveal unique insights into the immune response that occurs along chronic Chagas disease.

Spatially resolved multi-region transcriptomic subtyping and assessment of gene expression profiles associated with long-term benefit from chemo-immunotherapy in patients with extensive-stage small cell lung cancer (ES-SCLC).

8594 Background: Transcriptomic subtyping holds promise for personalized therapy in SCLC, but intratumoral transcriptomic heterogeneity and its clinical significance remain poorly defined. In this study, we aimed to assess transcription factor defined subtypes within multiple regions of intact tissues and identify gen sets associated with long-term chemo-immunotherapy benefit. Methods: We assessed baseline FFPE tumors from 32 ES-SCLC patients enrolled in the IMfirst clinical trial (EudraCT: 2019-002784-10). We used GeoMx DSP to perform transcriptomic analysis from multiple intratumoral regions of interest (ROIs). We used an assay with +1800 genes (GeoMx CTA) plus custom-designed mRNA probes targeting subtype-defining transcription factors not included in the CTA assay (POU2F3, NEUROD1, and YAP1). Custom probes were quantitatively validated using FFPE cell lines. Results: We profiled 175 ROIs from 32 tumors. Cluster analysis based on the expression of ASCL1, NEUROD1, POU2F3, and YAP1 showed that all samples clustered within 4 groups: SCLC-A (76 ROIs [43%]), SCLC-N (31 ROIs [18%]), SCLC-P (18 ROIs [10%]), and SCLC-Y (50 ROIs [29%]). ASCL1 was the most abundantly expressed transcription factor, prevailed in the SCLC-A subtype, and showed inverse correlation with POU2F3 (r=-0.55, p&lt;0.0001) and YAP1(r=-0.70, p&lt;0.0001). YAP1 was expressed at low levels and was more broadly distributed across all 4 subtypes. Differential expression and gene set enrichment analysis (GSEA) using linear mixed models revealed that SCLC-A subtype was enriched in cell cycle and DNA damage repair genes, and showed repression of multiple gene sets associated with anti-tumor immune response. In contrast, SCLC-Y subtype showed the opposite pattern. The SCLC-P subtype was also enriched in genes related to cancer antigens and T-cell checkpoints. Most patients (n=18, 56%) had tumors with coexistence of more than one subtype, not evident through morphological inspection. Combined SCLC-A and SCLC-Y subtypes was the most common mixed phenotype (n=8, 25%). Four patients (13%) had tumors with co-existence of three subtypes. Transcriptional subtypes, subtype-defining transcription factors as single genes, or the presence of subtype heterogeneity, were not associated with outcomes. Gene sets involved in mitochondrial metabolism and MHC class I antigen presentation were the highest enriched pathways in tumors from patients with sustained benefit (time to progression ³ 12 months). Conclusions: This study reveals substantial intratumoral transcriptomic subtype heterogeneity in human SCLC. In this limited sample set, we did not observe outcome association for transcriptional subtypes. Our findings related to long-term chemo-immunotherapy benefit require validation in independent cohorts.

237 (PB-061) Poster - Gene expression profile at week 2 of neoadjuvant therapy course predicts outcome in HER2-positive breast cancer patients: an explorative analysis from NeoALTTO

Background: NeoALTTO showed increased pathological complete response (pCR) with paclitaxel combined with dual over single anti-HER2 blockade. The trial included six initial weeks of treatment with lapatinib (L), trastuzumab (T) or their combination (L+T) followed by chemotherapy (CHT). A tumor biopsy was planned during the CHT-free window at day14 ± 2. Herein, we tested the hypothesis that prognostication of clinical outcome is feasible through assessment of gene expression profile (GEP) following two weeks of anti-HER2 therapy.

Positive effects of Phycocyanobilin on gene expression in glutamate-induced excitotoxicity in SH-SY5Y cells and animal models of multiple sclerosis and cerebral ischemia

BackgroundOxidative stress has a predominant role in the pathogenesis of neurodegenerative diseases and therefore the modulation of genes and the identification of biological pathways associated with antioxidant therapies, have an impact on its treatment. ObjectiveThe objective of this study was the comparison of 2 methods for the analysis of real-time PCR (qPCR) data, through the use of the evaluation of genes that mediate the effect of Phycocyanobilin (PCB) and its validation in animal models. MethodsWe evaluated the effect of PCB:” in vitro” on gene modulation through qPCR analyzed by parametric ANOVA and multivariate principal component analysis (PCA) in a model of glutamate-induced excitotoxicity in the SH-SY5Y cell line and” in vivo”; in animal models of multiple sclerosis (MS) and cerebral ischemia (CI). ResultsThe results showed that PCA is a robust and powerful method that allows the assessment of gene expression profiles. We detected the significant down-regulation of the CYBB (NOX2), and HMOX1 by the action of PCB in SH-5YSH cell line insulted with Glutamate. The decrease in pro-inflammatory cytokines and markers related to apoptosis and innate immune response, mediated the effect of PCB in the animal models of MS and CI, respectively. ConclusionWe concluded that the mechanisms by which PCB protected cells included the reduction of oxidative stress damage, which could contribute to its clinical efficacy for the treatment of neurodegenerative diseases.

Support of acute lymphoblastic leukemia cells by nonmalignant bone marrow stromal cells.

The present report describes work examining the manner in which nonmalignant bone marrow stromal cells prevent acute lymphoblastic leukemia (ALL) cell death. The initial focus was on the role of stromal cell-derived C-X-C motif chemokine 12 (CXCL12). Interference with CXCL12 production by stroma or blockade of its interactions with ALL by plerixafor did increase ALL cell death and in sensitive ALLs there was synergistic effect with conventional chemotherapy drugs. However, in contrast to most reports, there was considerable heterogeneity regarding the effect between 7 unique primary ALLs, with several exhibiting no sensitivity to CXCL12 blockade. The diversity in effect was not explained by differences in the expression of ALL cell surface receptors for CXCL12. The modest and variable effects of interference with CXCL12 on ALL led to the assessment of gene expression profiles of stromal cells and ALL cells. Gene set enrichment analysis identified pathways associated with metabolism and redox reactions as potentially important in the stromal cell: leukemia cell interaction. Exploratory imaging studies demonstrated bidirectional transfer of intracellular calcien-labelled molecules and also bidirectional transfer of mitochondria between stromal cells and ALL cells, providing potential means of metabolic interdependence of stromal cells and ALL cells.

CellExpress: a comprehensive microarray-based cancer cell line and clinical sample gene expression analysis online system.

With the advancement of high-throughput technologies, gene expression profiles in cell lines and clinical samples are widely available in the public domain for research. However, a challenge arises when trying to perform a systematic and comprehensive analysis across independent datasets. To address this issue, we developed a web-based system, CellExpress, for analyzing the gene expression levels in more than 4000 cancer cell lines and clinical samples obtained from public datasets and user-submitted data. First, a normalization algorithm can be utilized to reduce the systematic biases across independent datasets. Next, a similarity assessment of gene expression profiles can be achieved through a dynamic dot plot, along with a distance matrix obtained from principal component analysis. Subsequently, differentially expressed genes can be visualized using hierarchical clustering. Several statistical tests and analytical algorithms are implemented in the system for dissecting gene expression changes based on the groupings defined by users. Lastly, users are able to upload their own microarray and/or next-generation sequencing data to perform a comparison of their gene expression patterns, which can help classify user data, such as stem cells, into different tissue types. In conclusion, CellExpress is a user-friendly tool that provides a comprehensive analysis of gene expression levels in both cell lines and clinical samples. The website is freely available at http://cellexpress.cgm.ntu.edu.tw/. Source code is available at https://github.com/LeeYiFang/Carkinos under the MIT License. Database URL: http://cellexpress.cgm.ntu.edu.tw/

Standardisation of minimal residual disease in multiple myeloma.

The assessment of the effectiveness of chemotherapy in oncology cannot disregard the concept of minimal residual disease (MRD). In fact, the efforts of numerous scientific groups all over the world are currently focusing on this issue, with the sole purpose of defining sensitive, effective assessment criteria that are, above all, able to give acceptable, easily repeatable results worldwide. Regarding this issue, especially with the advent of new drugs, multiple myeloma is one of the haematologic malignancies for which a consensus has not yet been reached. In this review, we analyse various techniques that have been used to improve the sensitivity of response, aimed at reducing the cut-off values previously allowed, as well as serological values like serum-free light chain, or immunophenotypic tools on bone marrow or peripheral blood, like multi-parameter flow cytometry, or molecular ones such as allele-specific oligonucleotide (ASO)-qPCR and next-generation/high-throughput sequencing technologies (NGS). Moreover, our discussion makes a brief reference to promising techniques, such as mass spectrometry for identifying Ig light chain (LC) in peripheral blood, and the assessment of gene expression profile not only in defining prognostic risk at the diagnosis but also as a tool for evaluation of response.

Assessment of gene expression profiles of peripheral blood mononuclear cells from patients with a history of carbamazepine hypersensitivity

BackgroundCarbamazepine (CBZ) therapy is associated with hypersensitivity reactions, which can be multisystemic and sometimes fatal. The pathogenesis of these reactions is incompletely understood but heterologous immunity generated to viral antigens has been proposed as a potential mechanism. Our aim was to investigate changes in gene expression profiles of peripheral blood mononuclear cells (PBMCs) from patients with CBZ hypersensitivity to characterise the pathways involved in immune activation. MethodsPBMCs were isolated from five individuals with a history of hypersensitivity to CBZ. The PBMCs were incubated for 24 h with CBZ, carbamazepine 10,11-epoxide (CBZE), cell culture medium, or tetanus toxoid. Expression profiles for mRNA and microRNA were generated with microarrays (Affymetrix, Santa Clara, CA, USA). Differential gene expression was undertaken by limma analysis in the R statistical software. Ingenuity pathway analysis (IPA) was used to define the molecular mechanisms in development of CBZ hypersensitivity. FindingsIncubation of PBMCs with CBZ, CBZE, and tetanus toxoid led to significant differential expression (log2-fold change >1 or <–1, p<0·05) of multiple mRNA (n=204) and microRNA (n=148) transcripts with little overlap between the different incubation conditions. The top differentially expressed genes for CBZ-treated and CBZE-treated cells identified in IPA analysis were COX8A, IFI35, IFIT3, PARP9, PARP12, PTGES, RSAD2, USP18, USP41, miR433, miR455, miR3194, miR4723, and miR345. These genes were highly interconnected in functional networks and defined five top functional categories—namely, viral infection, psoriasis, inflammation of body region or organ, and antiviral response. InterpretationIncubation of PBMCs from CBZ hypersensitive individuals with CBZ or CBZE led to treatment-specific changes in gene expression associated with activation of antiviral and inflammatory pathways. These CBZ-specific and CBZE-specific mRNA and microRNA changes could potentially act as novel diagnostic biomarkers and treatment targets for CBZ hypersensitivity reactions, but further validation and confirmation of specificity in larger numbers of patients are needed. FundingVY was supported by the North West England Medical Research Council Fellowship Scheme (clinical pharmacology and therapeutics), which is funded by the Medical Research Council (grant number G1000417/94909), ICON, GlaxoSmithKline, AstraZeneca, and the Medical Evaluation Unit.

Development and validation of a harmonized TaqMan-based triplex real-time RT-PCR protocol for the quantitative detection of normalized gene expression profiles of seven porcine cytokines.

Dysregulation of cytokine responses plays a major role in the pathogenesis of severe and life-threatening infectious diseases like septicemia or viral hemorrhagic fevers. In pigs, diseases like African and classical swine fever are known to show exaggerated cytokine releases. To study these responses and their impact on disease severity and outcome in detail, reliable, highly specific and sensitive methods are needed. For cytokine research on the molecular level, real-time RT-PCRs have been proven to be suitable. Yet, the currently available and most commonly used SYBR Green I assays or heterogeneous gel-based RT-PCRs for swine show a significant lack of specificity and sensitivity. The latter is however absolutely essential for an accurate quantification of rare cytokine transcripts as well as for detection of small changes in gene expressions. For this reason, a harmonized TaqMan-based triplex real-time RT-PCR protocol for the quantitative detection of normalized gene expression profiles of seven porcine cytokines was designed and validated within the presented study. Cytokines were chosen to represent different immunological pathways and targets known to be involved in the pathogenesis of the above mentioned porcine diseases, namely interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-8, tumor necrosis factor (TNF)-α and interferon (IFN)-α. Beta-Actin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as reference genes for normalization. For absolute quantification a synthetic standard plasmid was constructed comprising all target cytokines and reference genes within a single molecule allowing the generation of positive control RNA. The standard as well as positive RNAs from samples, and additionally more than 400 clinical samples, which were collected from animal trials, were included in the validation process to assess analytical sensitivity and applicability under routine conditions. The resulting assay allows the reliable assessment of gene expression profiles and provides a broad applicability to any kind of immunological research in swine.

Assessment of gene expression profiles in primary and secondary zoospores of Plasmodiophora brassicae by dot blot and real-time PCR

Information on the molecular basis of pathogenicity of the clubroot pathogen Plasmodiophora brassicae is very limited. Although the sequences of more than 100 P. brassicae genes are available in GenBank, their expression and regulation are largely unknown. In this study, specific primers were designed and used to amplify genomic fragments of 118 P. brassicae genes that represent all database-available proteins and ESTs. The PCR products were blotted on membranes and hybridized with digoxigenin-labeled double-stranded cDNA, derived from either primary or secondary zoospores of P. brassicae. The same primers were also used in real-time PCR against the single-stranded cDNA synthesized from the two types of zoospores. Both dot blot and real-time PCR identified up- and down-regulated genes and the correlation between these two techniques was confirmed. Real-time PCR indicated that 58 genes were up-regulated in the secondary zoospores relative to the primary zoospores, whereas 55 were down-regulated. These data suggest that different mechanisms are utilized by the pathogen in causing primary and secondary infections. The expression patterns of genes with known or putative functions suggest the relative importance of these genes during pathogenesis. In contrast, highly expressed or regulated genes with unknown function can be further studied in identifying pathogenicity factors.

Assessment of Gene Expression Profiles in Peripheral Occlusive Arterial Disease

BackgroundMolecular events responsible for the onset and progression of peripheral occlusive arterial disease (POAD) are incompletely understood. Gene expression profiling may point out relevant features of the disease. MethodsTissue samples were collected as operatory waste from a total of 36 patients with (n = 18) and without (n = 18) POAD. The tissues were histologically evaluated, and the patients with POAD were classified according to Leriche-Fontaine (LF) classification: 11% with stage IIB, 22% with stage III, and 67% with stage IV. Total RNA was isolated from all samples and hybridized onto Agilent 4×44K Oligo microarray slides. The bioinformatic analysis identified genes differentially expressed between control and pathologic tissues. Ten genes with a fold change ≥ 2 (1 with a fold change ≥ 1.8) were selected for quantitative polymerase chain reaction validation (GPC3, CFD, GDF10, ITLN1, TSPAN8, MMP28, NNMT, SERPINA5, LUM, and FDXR). C-reactive protein (CRP) was assessed with a specific assay, while nicotinamide N-methyltransferase (NNMT) was evaluated in the patient serum by enzyme-linked immunosorbent assay. ResultsA multiple regression analysis showed that the level of CRP in the serum is correlated with the POAD LF stages (r2 = 0.22, P = 0.046) and that serum NNMT is higher in IV LF POAD patients (P = 0.005). The mRNA gene expression of LUM is correlated with the LF stage (r2 = 0.45, P = 0.009), and the mRNA level of ITLN1 is correlated with the ankle-brachial index (r2 = 0.42, P = 0.008). ConclusionsOur analysis shows that NNMT, ITLN1, LUM, CFD, and TSPAN8 in combination with other known markers, such as CRP, could be evaluated as a panel of biomarkers of POAD.

Molecular diagnostics of families with long-QT syndrome

The Department of Pediatric Cardiology, Medical University of Silesia in Katowice-Ligota, studied 24 patients with clinically diagnosed (using ECG) long-QT syndrome (LQTS) in 18 cases. Nine patients were diagnosed with LQT1 and nine with LQT2. The other six individuals were healthy, with no symptoms characteristic for prolonged QT syndrome, but came from families with confirmed disease occurrence. The study was conducted on members of four families. In order to search for mutations (using mSSCP and sequencing), genomic DNA was obtained from patients to determine the expression levels of the genes KCNQ1 and KCNH2 (HERG), involved in the occurrence of clinical signs of disease. Total RNA was extracted from peripheral blood. Consent to the use of blood samples of patients had been given by the Bioethics Commission of the Medical University of Silesia. mSSCP analysis and sequencing did not confirm the occurrence of mutations in KCNQ1 and HERG associated with the occurrence of LQTS. Analysis of gene expression profile of KCNQ1 and HERG confirmed the presence of disease in people with a known clinical diagnosis. Overexpression, as well as reduced expression, was observed for the examined genes. KCNQ1 was inhibited in two families, whereas HERG was reduced in one and overexpressed in the other. Gene expression profile analysis showed abnormal expressions of KCNQ1 and HERG in healthy subjects, which may be a sign of predisposition to develop the disease. The novelty of our study involved the use of total mRNA isolated from human peripheral blood, and the very limited evidence in the literature to date regarding the assessment of gene expression profile of HERG and KCNQ1 in relation to the presence of prolonged QT syndrome.

Differentiation-Promoting Culture of Competent and Noncompetent Keratinocytes Identifies Biomarkers for Head and Neck Cancer

Aberrant contact-inhibited proliferation and differentiation induction couple with tumor severity, albeit with an imprecise association with prognosis. Assessment of contact inhibition and differentiation-promoting culture in this study of normal and immortalized oral keratinocytes (NOK and SVpgC2a, respectively) demonstrated elevated cloning ability and saturation density in the immortalized versus normal state, including consistent absence of differentiated morphological features. Transcriptomic analysis implicated 48 gene ontology categories, 8 molecular networks, and 10 key regulator genes in confluency-induced differentiation of NOK, all of which remained nonregulated in SVpgC2a. The SVpgC2a versus NOK transcriptome enriched 52 gene ontology categories altogether, 18 molecular networks, and 39 key regulator genes, several of which were associated with epithelial-mesenchymal transition. Assessment of the previously described gene sets relative to training data sets of head and neck squamous cell carcinoma samples, one including data on tumor differentiation and patient outcome and one present in the Human Gene Expression Map, identified four genes with association to poor survival (COX7A1, MFAP5, MPDU1, and POLD1). This gene set predicted poor outcome in an independent data set of 71 head and neck squamous cell carcinomas. The present study defines, for the first time to our knowledge, the broad gene spectrum that couples to induction, and loss, of oral keratinocyte differentiation. Bioinformatics assessments of the results relative to clinical data generated novel differentiation-related tumor biomarkers relevant to patient outcome.

Development of a Multiplex Assay for Rapid Assessment of Gene Expression Profiling of Rho GTPases Family Genes In MDS Patients

AbstractAbstract 2907We have previously identified a panel of genes by gene expression profiling which differentially expressed between primary bone marrow CD34+ cell of MDS patients and the normal control cells. The expression pattern of these genes were also confirmed individually by qRT-PCR in expanded cohort of MDS patients. To further explore the feasibility of using proper number of these genes as biomarkers and evaluate the clinical significance in the diagnosis and prognostic prediction of MDS patients, a convenient, robust, sensitive, specific and less expensive method need to be developed. Therefore, endeavors have been made to establish a bead-based flow-cytometric multiplex assay.To establish this method, a technique called Multiplex ligation-dependent probe amplification (MLPA) was employed to amplify several targeted cDNAs by using single pair of identical primers. Each MLPA probe consists of two short synthetic oligonucleotide, and the tag which was coupled chemically to the fluorescent beads was complementary to one probe. Just as a pilot study to simultaneously detect the expression of four genes of the Rho GTPase family, five beads with different fluorescence intensity coupled to RAC2, RhoBTB3, SPA-1, Rap1GAP and GAPDH were designed. Biotinylated PCR amplicons were then hybridized to the complementary tag on each bead set. Bound amplicons were then detected by flow cytometry using a streptavidin-linked reporter dye, PE. 111 BM specimens were analyzed in total, consisting of RA(22), RAEB(22), RAEBt(9), AML(33), and control group (22, including hyperplastic anemia, iron deficiency anemia, aplastic anemia etc). The difference in the transcriptional level of RAC2, RhoBTB3, SPA-1 and Rap1GAP relative to GAPDH were analyzed using wilcoxon non-parametric test and SNK method among different groups. The results were confirmed by qRT-PCR.The bead-based flow-cytometric array had a excellent sensitivity and a wide linear range, could get a positive signal for PCR product from 0.0025 to 0.1umol, the fine specificity was proved by no cross-hybridization signals presented among different bead set, and the reproducibility were also good enough(P<0.001). the expression profiling of RAC2, RhoBTB3, SPA-1,Rap1GAP and GAPDH detected by this liquid bead-based flow-cytometric array were thus obtained. A significant difference among five groups for the expression level of RAC2, RhoBTB3, SPA-1 and Rap1GAP relative to GAPDH was found (P<0.0001, P=0.0491, P=0.0206 and P=0.0046 respectively). For RAC2, no difference existed between AML and control groups, while both these two groups demonstrated significant difference compared with RA, RAEB and REABt groups. The RA group had the highest expression level, AML the lowest; and RAEB in between. For RhoBTB3, a significant difference could be seen between RAEBt and other groups, RAEBt had the highest level of RhoBTB3. There was no difference in SPA-1 expression level determined between each two groups. However, an increasing tendency from control group to RA, RAEB, and REABt could be observed, whereas the expression of SPA-1 dramatically dropped to the lowest level in AML. For Rap1GAP, differences were found between AML and other group; These results were validated by qRT-PCR, and the data obtained by each method had close linear correlation, the Pearson correlation coefficient was 0.930, 0.946, 0.945 and 0.921 for RAC2, RhoBTB3, SPA-1 and Rap1GAP respectively (P<0.001 for all four).Based on these preliminary results, A liquid bead-based flow-cytometric multiplex assay for a rapid assessment of gene expression profile had been successfully developed and validated by qRT-PCR. This method will facilitates the clinical usage of a certain group of Rho GTPase family genes as well as other appropriate genes as useful biomarkers in the diagnosis of MDS patients and provide interesting clues to further explore the pathogenesis of MDS. Disclosures:No relevant conflicts of interest to declare.

Metal-proteinase ADAM12, kinesin 14 and checkpoint suppressor 1 as new molecular markers of laryngeal carcinoma

The assessment of gene expression profile in laryngeal cancer allows implementation of molecular biology methods in diagnostics, as well as in prognosticating the course of disease, thus allowing taking most optimal decisions as regards the method of treatment, scope of surgical procedure, or the necessity of adding complementary radiotherapy. The aim of the project was to analyze the gene expression profile in laryngeal cancer using oligonucleotide microarrays, having in mind searching new molecular markers for that carcinoma. The study comprised a group of 43 patients (38 males and 5 females) suffering from squamous cell laryngeal carcinoma, diagnosed and surgically treated in the years 2005-2007 in the ENT Department of the Silesian Medical University in Katowice, Poland. RNA was isolated from frozen tissue fragments, with the use of columns RNeasy Midi and Mini Kit (Qiagen). For the examination of gene expression profile, oligonucleotide microarrays of high density were used, provided by Affymetrix (U 133 2.0 PLUS) containing over 54,000 probes for over 47,000 transcripts. Four genes previously not examined in that respect in laryngeal carcinoma, occurred to be good markers of the neoplasm. They are: metal-proteinase ADAM12, cyclin-dependent kinase 2-CDK2, kinesin 14-KIF14, suppressor 1 of checkpoint-CHES1. The analysis of gene expression profile allows, in laryngeal carcinoma, to point out to new genes, which in future may become molecular markers of the carcinoma.

Microscale culture of human liver cells for drug development

Tissue function depends on hierarchical structures extending from single cells ( approximately 10 microm) to functional subunits (100 microm-1 mm) that coordinate organ functions. Conventional cell culture disperses tissues into single cells while neglecting higher-order processes. The application of semiconductor-driven microtechnology in the biomedical arena now allows fabrication of microscale tissue subunits that may be functionally improved and have the advantages of miniaturization. Here we present a miniaturized, multiwell culture system for human liver cells with optimized microscale architecture that maintains phenotypic functions for several weeks. The need for such models is underscored by the high rate of pre-launch and post-market attrition of pharmaceuticals due to liver toxicity. We demonstrate utility through assessment of gene expression profiles, phase I/II metabolism, canalicular transport, secretion of liver-specific products and susceptibility to hepatotoxins. The combination of microtechnology and tissue engineering may enable development of integrated tissue models in the so-called 'human on a chip'.

Genomic and proteomic profiling II: Comparative assessment of gene expression profiles in leiomyomas, keloids, and surgically-induced scars

BackgroundLeiomyoma have often been compared to keloids because of their fibrotic characteristic and higher rate of occurrence among African Americans as compared to other ethnic groups. To evaluate such a correlation at molecular level this study comparatively analyzed leiomyomas with keloids, surgical scars and peritoneal adhesions to identify genes that are either commonly and/or individually distinguish these fibrotic disorders despite differences in the nature of their development and growth.MethodsMicroarray gene expression profiling and realtime PCR.ResultsThe analysis identified 3 to 12% of the genes on the arrays as differentially expressed among these tissues based on P ranking at greater than or equal to 0.005 followed by 2-fold cutoff change selection. Of these genes about 400 genes were identified as differentially expressed in leiomyomas as compared to keloids/incisional scars, and 85 genes as compared to peritoneal adhesions (greater than or equal to 0.01). Functional analysis indicated that the majority of these genes serve as regulators of cell growth (cell cycle/apoptosis), tissue turnover, transcription factors and signal transduction. Of these genes the expression of E2F1, RUNX3, EGR3, TBPIP, ECM-2, ESM1, THBS1, GAS1, ADAM17, CST6, FBLN5, and COL18A was confirmed in these tissues using quantitative realtime PCR based on low-density arrays.Conclusionthe results indicated that the molecular feature of leiomyomas is comparable but may be under different tissue-specific regulatory control to those of keloids and differ at the levels rather than tissue-specific expression of selected number of genes functionally regulating cell growth and apoptosis, inflammation, angiogenesis and tissue turnover.

Toxicogenomic analysis of gene expression changes in human liver cells after exposure to environmentally friendly lubricants

AbstractA lubricant based on synthetic esters and a mixture of this lubricant with additives and metals simulating the intake caused by usage were investigated with various ecotoxicological and genotoxicological tests. Tests with algae, daphnids and bacteria demonstrated an influence of the mixture on ecotoxicity. The genotoxicity tests, however, showed no effects for both samples. In addition, genetic effects were examined in detail by using gene expression profiles of HepG2 cells. Comparison of the set of regulated genes for early and late time points indicated a certain concordance between the genes up‐regulated after 6 and 24 hours of exposure. The correlation for the corresponding down‐regulated gene groups is slightly lower. Generally, the number of regulated genes is low (3–6%) demonstrating a marginal influence. The results indicate that the assessment of gene expression profiles, in addition to the quantification of toxic effects, may give important information on ways of toxic action of a substance. These data can be used in the development of new chemicals or products in order to minimize toxic effects. Copyright © 2007 John Wiley &amp; Sons, Ltd.

Adding Confidence to Gene Expression Clustering

It has been well established that gene expression data contain large amounts of random variation that affects both the analysis and the results of microarray experiments. Typically, microarray data are either tested for differential expression between conditions or grouped on the basis of profiles that are assessed temporally or across genetic or environmental conditions. While testing differential expression relies on levels of certainty to evaluate the relative worth of various analyses, cluster analysis is exploratory in nature and has not had the benefit of any judgment of statistical inference. By using a novel dissimilarity function to ascertain gene expression clusters and conditional randomization of the data space to illuminate distinctions between statistically significant clusters of gene expression patterns, we aim to provide a level of confidence to inferred clusters of gene expression data. We apply both permutation and convex hull approaches for randomization of the data space and show that both methods can provide an effective assessment of gene expression profiles whose coregulation is statistically different from that expected by random chance alone.

PREDICTING PROSTATE CANCER BEHAVIOR USING TRANSCRIPT PROFILES

Prostate cancer represents a disease with diverse clinical outcomes. Treatment strategies that optimize benefit and minimize morbidities depend on accurate estimates of disease status and likelihood of progression. Emerging technologies capable of qualitatively and quantitatively profiling genes expressed by neoplastic tissues may provide insights into tumor behavior. This review discusses the use of microarray based transcript expression profiling to stratify human cancers into risk categories. MEDLINE was used to perform a comprehensive literature review of reports describing the assessment of gene expression profiles in malignant diseases. Particular emphasis was placed on studies developing models using individual genes or gene cohorts as predictors of prostate cancer outcome. Alterations in the expression of individual genes identified by microarray analyses have been used in studies of outcome in cancers of the prostate and other tissue types. Profiles of expressed genes have been used to develop prediction models that stratify cancers into prognostic categories based on relapse rates or responses to therapy. Gene expression profiles offer an opportunity for acquiring molecular determinants correlating with clinical outcome. With rare exceptions these profiles have yet to be validated or used in prospective studies. Future research will benefit from assessments of intratumor heterogeneity and host factors such as the immune response and hormonal milieu. The prospective validation of predictive models will serve to prove usefulness in the clinical arena.