Affymetrix Platform Research Articles

Transcriptional responses of PBMC in psychosocially stressed animals indicate an alerting of the immune system in female but not in castrated male pigs.

BackgroundBrain and immune system are linked in a bi-directional manner. To date, it remained largely unknown why immune components become suppressed, enhanced, or remain unaffected in relation to psychosocial stress. Therefore, we mixed unfamiliar pigs with different levels of aggressiveness. We separated castrated male and female pigs into psychosocially high- and low- stressed animals by skin lesions, plasma cortisol level, and creatine kinase activity obtained from agonistic behaviour associated with regrouping. Peripheral blood mononuclear cells (PBMC) were collected post-mortem and differential gene expression was assessed using the Affymetrix platform (n = 16).ResultsRelevant stress-dependent alterations were found only between female samples, but not between castrated male samples. Molecular routes related to TREM 1 signalling, dendritic cell maturation, IL-6 signalling, Toll-like receptor signalling, and IL-8 signalling were increased in high stressed females compared to low stressed females. This indicates a launch of immune effector molecules as a direct response. According to the shifts of transcripts encoding cell surface receptors (e.g. CD14, TLR2, TLR4, TREM1) the study highlights processes acting on pattern recognition, inflammation, and cell-cell communication.ConclusionsThe transcriptional response partly affected the degree of ‘stress responsiveness’, indicating that the high stressed females altered their signal transduction due to potential infections and injuries while fighting.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-967) contains supplementary material, which is available to authorized users.

Abstract B35: Cumulative results from an investigation of transcriptome differences in breast cancer samples from African American and East African triple-negative breast cancer patients

Abstract We report our cumulative results of a multi-year study focusing on the genomic similarities and differences between Triple-negative breast cancer (TNBC) in native African and U.S. African patients. We used a cohort of African-American (AA) TNBC patients from South Florida and an indigenous cohort of Kenyan TNBC patients. Strong U.S. epidemiological data supports breast cancer (BC) as the second leading cause of cancer death among AA women, with a 20% greater mortality rate than that in Caucasian-American women (CA). AA women often present with tumors that are of higher grade and later stage and their relapse-free and overall survival period is significantly lower than CA. Additionally, TNBC has a higher prevalence in AA women. All of these observations lead to a much poorer prognosis for AA BC patients- thus defining the all-encompassing term “disparities in BC in women of African ancestry”. In our study, we used archived formalin fixed paraffin embedded (FFPE) blocks, collected across a stage-matched multi-ethnic U.S. cohort of node-negative TNBC from the University of Miami and similar TNBC samples from the Kijabe Hospital (Kenya). Using 10 μm scrolls from each block, total RNA was isolated, cDNA prepared, and hybridized to a breast cancer enriched gene expression array (Affymetrix Platform BC DSA Research Tool) in collaboration with Almac Diagnostics. Gene expression analysis was conducted using GeneSpring 12.1® analytical software. The raw data, a total of 60,856 gene/probes, was pre-processed using Robust MultiArray Average (RMA) algorithm; background corrected, log2 transformed to the baseline median of all samples and then quintile normalized. The samples that passed final QC were then filtered to remove low-intensity signals (< 20% signal). The final analysis cohort consisted of 10-AA, 13-CA and 21-Kenyan. PCA analysis revealed that the samples clustered well with respect to ethnicity. Unsupervised cluster analysis, based on ethnicity and genes (p value <.05, fold change >2.5), was performed. The resulting dendogram clearly segregated into distinct subgroups based on ethnicity, revealing a pattern of differential gene expression between the cohorts. A list of differentially expressed genes from each cohort (DEG) were selected using ANOVA analysis (fold change > 3.0, p value <.05) followed by the Benjamin and Hochberg method for multiple-testing correction. Finally, the lists of DEG were uploaded into GeneGo MetaCore to identify functionally enriched pathways. These analyses revealed significantly deregulated genes associated with the Wnt/β-catenin pathway in the AA cohort, as compared to the CA, suggesting that this pathway may contribute to the more aggressive phenotype in AA women diagnosed with TNBC. Additionally, significantly deregulated genes associated with the Oncostatin M pathway were discovered in the Kenyan cohort, as compared to the AA and CA tumors. In particular, STAT1 was significantly downregulated in the Kenyan cohort compared to the CA and AA. Thus, our results indicate gene expression differences within several key pathways across these ethnic groups. In summary, this study represents the first direct comparison of gene expression in TNBC specimens across U.S. CA and AA BC patients and Kenyan East Africans. These studies have important implications for further understanding BC ethnic disparities, as well as tailored approaches to prediction, prevention and therapeutic measures. Citation Format: Julie E. Getz, Mary E. Ahearn, Carmen Gomez, Biju Issac, Jennifer Clarke, Mark D. Pegram, Peter Bird, John D. Carpten, Lisa L. Baumbach. Cumulative results from an investigation of transcriptome differences in breast cancer samples from African American and East African triple-negative breast cancer patients. [abstract]. In: Proceedings of the Sixth AACR Conference: The Science of Cancer Health Disparities; Dec 6–9, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2014;23(11 Suppl):Abstract nr B35. doi:10.1158/1538-7755.DISP13-B35

B15 Dysregulation of Gene Expression in the Striatum of Bachd Rats and Abnormalities Involved on Molecular and Protein Levels

Background Huntington disease (HD) is an autosomal dominantly inherited neurodegenerative disorder caused by an expansion of CAG repeats in the gene coding for the huntingtin protein (htt). Mutant htt has been proposed to cause neuronal dysfunction and neuronal loss through multiple mechanisms. Transcriptional changes may be a core pathogenic feature of HD. Methods Utilising the Affymetrix platform we performed a genome-wide RNA expression analysis in two BACHD transgenic rat lines (TG5 and TG9) at 12 months of age, which carry full-length human mutant htt with different expression levels. Results Applying a threshold of significance of 0.01, we found 1608 genes and 871 genes differently expressed in TG5 and TG9 rats compared to wild type littermates, respectively. We only chose highly up/down regulated genes for further analysis by setting an additional threshold of 1.5 for fold change. Comparison with data from human HD brains revealed a high concordance in involved functional and IPA (Ingenuity Pathway Analysis) canonical pathways relevant in HD. In addition, we investigated the causes leading to gene expression changes on molecular and protein levels in BACHD rats including the amount of wild type htt, interaction of mhtt with polyQ containing transcription factors as well as chromatin structure. Conclusions Results of this study demonstrate that this BACHD rat model recapitulates accurately gene expression changes of the human disease and therefore supports its application in preclinical research. Analysis of causative events in BACHD rats confirmed some findings in in vitro test, and also provides a novel insight into initiators of perturbed gene transcription in HD.

Abstract 313: Elucidation Of The Fibrotic Transcription Factors Gene Network Leading To Chagas Heart Disease

Background: Trypanosoma cruzi the causative agent of Chagas heart disease (CHD) remains incurable. The major pathology induced by the parasite is cardiac fibrosis leading to heart failure followed by death. The mechanisms of T. cruzi induced cardio-pathology remains largely unknown. We hypothesize that T. cruzi infection regulates the expression of profibrotic genes in human cardiac myocytes (HCM), tilting the heart towards a profibrotic phenotype seen in CHD patients. Methods and Results: To elucidate the molecular mechanisms of T. cruzi induced cardiac fibrosis, we challenged primary HCM with T. cruzi for two hours and purified total RNA for microarray. We investigated changes at the whole transcriptome level on an affymetrix platform. The arrays were done in triplicates at different time points; changes in gene expression greater than 2-fold and having a Benjamini and Hochburg false discovery rate corrected p-value <0.05 were considered significant. The microarray data was validated using real-time PCR followed by PCR array and immunoblotting, to evaluate changes in the protein expression levels of fibrotic transcription factors. Protein expression levels were evaluated in triplicate and analyzed by ANOVA. The fibrotic interactome induced by T. cruzi in HCM was elucidated using Cytoscape. Our results indicate that exposure of HCM to T. cruzi upregulates the transcript levels of two transcription factors associated with fibrosis, SNAI1 (more than 2 fold up-regulated) and Early Growth Response protein 1, EGR1, (about four fold up-regulated). SNAI1 and EGR1 were increased at the protein level. Furthermore, we identified the first interactome regulating fibrosis in primary HCM induced by T. cruzi . Conclusions: This is the first report showing that T. cruzi upregulates the expression of profibrotic transcription factors in HCM early during the process of cellular infection and the operational fibrotic interactome. Thus, abnormal sustained expression of SNAI1 and EGR1 upregulate the expression of genes essential for conversion of HCM towards a profibrotic phenotype in CHD. Elucidation of the molecular mechanisms by which T. cruzi induces cardiac fibrosis will lead to the identification of new therapeutic targets for CHD.

DECIPHERING INTRINSIC AND ADAPTIVE RESISTANCE TO PI3K PATHWAY INHIBITORS IN GLIOMA

BACKGROUND: Glioblastoma represents a compelling disease for signal transduction targeted therapy. Despite the evidence defining the PI3K network as a high quality target, regulatory feedback loops as well as cross talk with other networks make targeting the PI3K network a challenging task. Complete control of GBM is dependent on the elucidation of the escape mechanisms and the rational design of combination therapies. A major objective in our laboratory is to develop robust predictive molecular and genetic markers/signature that will lead to selection precision therapy. These predictive markers will allow us to determine rational combinatorial approaches to target aberrations in the PI3K network and at the same time prevent deleterious effects of feedback loops and cross talk. METHODS: We have utilized a large panel of glioma stem cell (GSC) lines to perform drug sensitivity/resistance screens. Reponses profiles of GSCs using multiple PI3K inhibitors in cytotoxicity assay were generated. Molecular targets/pathways contributing to alternate signaling pathway activation by inhibitors were identified using gene expression profiling and protein expression/activity profiling. Top gene targets were subjected to loss of function studies using RNAi. Synergistic effect of knocking down the target along with PI3K inhibitors on cell proliferation, and tumorigenicity were assessed. RESULTS: We have utilized several system approaches to identify targets in compensatory or parallel pathways after PI3K inhibition. Our results suggest that different molecular profiles in GSC dictate the differences in viability observed after drug treatment, despite a common blockade of PI3K pathway. Resistant and sensitive cell lines were profiled using the Affymetrix and RPPA platforms to identify genes that are differentially expressed in sensitive and resistant GSCs responsible for intrinsic resistance. Using gene expression profiling, we also identified adaptive response gene targets in pre- and post-treatment resistant GSC. This approach has uncovered alternate pathways and shutting down these pathways would synergize the effect on cell growth when combined with PI3K pathway inhibitors. We have selected the top 10 candidate genes and two targets are being studied for rational combination with PI3K inhibitors CONCLUSIONS: Failure of PI3K pathway inhibitors is due to escape through overlapping or parallel signaling pathways, and that complete control of GBM depend on the elucidation of these escape mechanisms and we show that therapies to target essential pathways overcome or circumvent treatment resistance and can provide the next generation of improved therapeutic options. SECONDARY CATEGORY: Tumor Biology.

Genetic and cytokine changes associated with symptomatic stages of CLL

The pathogenesis and drug resistance of symptomatic CLL patients involves genetic changes associated with the CLL clone as well as changes within the microenvironment. To further understand these processes, we compared early stage CLL to symptomatic late stage using gene expression and serum cytokine profiling to gain insight of the genetic and microenvironment changes associated with the most severe form of the disease. Patients were classified into low stage (Rai stage 0/I/II) and high stage (Rai stage III/IV). Gene expression profiles were obtained on pretreatment samples using the HG-U133A 2.0 Affymetrix platform. A comparison of low versus high stage CLL revealed a set of 21 genes differentially expressed genes. 15 genes were up regulated in the high stage compared to low stage while 6 genes were down regulated. Analysis of GO molecular function revealed 9 of 21 genes were involved in transcription factor activity. Serum cytokine profiles showed six cytokines to be significantly different in high stage patients. Two chemokines, SDF-1/CXCL12 and uPAR known to be involved in stem cell mobilization and homing were increased in serum of high stage patients. This study has identified therapeutic targets for symptomatic CLL patients.

The role of transforming growth factor β signaling in fibroblast-like synoviocytes from patients with oligoarticular juvenile idiopathic arthritis: dysregulation of transforming growth factor β signaling, including overexpression of bone morphogenetic protein 4, may lead to a chondrocyte phenotype and may contribute to bony hypertrophy.

This study was designed to investigate the pathogenic contributions of fibroblast-like synoviocytes (FLS) to juvenile idiopathic arthritis (JIA) by identifying pathways with dysregulated gene expression in FLS from patients with oligoarticular JIA. FLS were derived from synovial fluid obtained by arthrocentesis from patients with JIA undergoing intraarticular steroid injections and from orthopedic control patients. Gene expression profiles of the JIA and control FLS were obtained using the Affymetrix platform, with application of Ingenuity Pathway Analysis and Gene Set Enrichment Analysis software to define gene sets in dysregulated pathways and networks of potential pathologic relevance in this disease. Biologically relevant differentially expressed genes were confirmed by RNA and protein analysis. Exploration of global gene expression profiles of the JIA FLS revealed important dysregulated pathways, including the transforming growth factor β (TGFβ) signaling, as well as endochondral bone formation, cartilage formation, and β-catenin networks. Importantly, bone morphogenetic protein 4 (BMP-4) was significantly overexpressed in the JIA FLS. FLS from patients with oligoarticular JIA exhibit a chondrocyte phenotype, as evidenced by expression of type II collagen and aggrecan. Dysregulation of the pathways involved in the pathogenesis of oligoarticular JIA were revealed through gene expression profiling. JIA FLS displayed dysregulated TGFβ signaling and exhibited a hypertrophic chondrocyte phenotype. These characteristics, along with contributions from the β-catenin network may have implications for endochondral bone formation and local growth disturbances in oligoarticular JIA. Overexpression of BMP-4 in FLS from patients with oligoarticular JIA in particular may play an important role in disease pathogenesis, with a direct effect on functional outcome and with implications for future treatment.

Abstract P255: Genetic Variation in Chromosome 8p23 is Associated With Sedentary Behavior in Humans

Sedentary behavior, usually quantified as sitting time, has been shown to be associated with several adverse health outcomes, even among individuals who meet the current recommendations for physical activity. Both twin and family studies have shown that sedentary behavior aggregates in families and is a heritable trait. However, little is known about genes and DNA sequence variants that contribute to propensity to be sedentary. The purpose of our study was to conduct a genome-wide association study to identify genomic regions and DNA sequence variants associated with sedentary time. A total of 67,530 Caucasians (56% female, mean age 62.1 years [SD 12.5]) from the Kaiser Permanente Research Program on Genes, Environment, and Health (RPGEH) cohort were used for the discovery analyses. Other RPGEH ethnicities (African-Americans: N=2,500; East Asians: N=6,300; Hispanics: N=8,800) were used for replication studies. Information on daily sitting time outside of work was obtained by a questionnaire. A custom panel of 657,184 autosomal single nucleotide polymorphisms (SNPs) was genotyped using Affymetrix platform. Associations between sedentary time (dichotomized; cases 3 hours or more, controls less than 3 hours per day) and the SNPs were tested with PLINK logistic model using age, sex, BMI and admixture principal components as covariates. The strongest evidence of association was detected on chromosome 8p23.1. A total of 24 SNPs within a 1.34 Mb region were associated with sedentary time at genome-wide significance level (p<5x10 -8 ), with SNP rs11774552 showing the peak association (OR=0.924, p= 1.7x10 -12 ). Sex-specific analyses confirmed that the associations were present both in men and women. The same SNPs were not associated with sedentary time in other ethnicities. However, SNP rs28754712, which is located in the immediate vicinity (+456 bp) of rs11774552, showed a significant association with sedentary time (OR=0.795, p=1.2x10 -3 ) in African-Americans. In summary, our results provide the first evidence of genomic region and DNA sequence variants associated with sedentary behavior in humans. If confirmed in additional replication studies, these findings will advance our understanding of biological mechanisms contributing to propensity to be sedentary.

Molecular Profiles of Parvalbumin-Immunoreactive Neurons in the Superior Temporal Cortex in Schizophrenia

Dysregulation of pyramidal cell network function by the soma- and axon-targeting inhibitory neurons that contain the calcium-binding protein parvalbumin (PV) represents a core pathophysiological feature of schizophrenia. In order to gain insight into the molecular basis of their functional impairment, we used laser capture microdissection (LCM) to isolate PV-immunolabeled neurons from layer 3 of Brodmann's area 42 of the superior temporal gyrus (STG) from postmortem schizophrenia and normal control brains. We then extracted ribonucleic acid (RNA) from these neurons and determined their messenger RNA (mRNA) expression profile using the Affymetrix platform of microarray technology. Seven hundred thirty-nine mRNA transcripts were found to be differentially expressed in PV neurons in subjects with schizophrenia, including genes associated with WNT (wingless-type), NOTCH, and PGE2 (prostaglandin E2) signaling, in addition to genes that regulate cell cycle and apoptosis. Of these 739 genes, only 89 (12%) were also differentially expressed in pyramidal neurons, as described in the accompanying paper, suggesting that the molecular pathophysiology of schizophrenia appears to be predominantly neuronal type specific. In addition, we identified 15 microRNAs (miRNAs) that were differentially expressed in schizophrenia; enrichment analysis of the predicted targets of these miRNAs included the signaling pathways found by microarray to be dysregulated in schizophrenia. Taken together, findings of this study provide a neurobiological framework within which hypotheses of the molecular mechanisms that underlie the dysfunction of PV neurons in schizophrenia can be generated and experimentally explored and, as such, may ultimately inform the conceptualization of rational targeted molecular intervention for this debilitating disorder.

Accurate Data Processing Improves the Reliability of Affymetrix Gene Expression Profiles from FFPE Samples

Formalin fixed paraffin-embedded (FFPE) tumor specimens are the conventionally archived material in clinical practice, representing an invaluable tissue source for biomarkers development, validation and routine implementation. For many prospective clinical trials, this material has been collected allowing for a prospective-retrospective study design which represents a successful strategy to define clinical utility for candidate markers. Gene expression data can be obtained even from FFPE specimens with the broadly used Affymetrix HG-U133 Plus 2.0 microarray platform. Nevertheless, important major discrepancies remain in expression data obtained from FFPE compared to fresh-frozen samples, prompting the need for appropriate data processing which could help to obtain more consistent results in downstream analyses. In a publicly available dataset of matched frozen and FFPE expression data, the performances of different normalization methods and specifically designed Chip Description Files (CDFs) were compared. The use of an alternative CDFs together with fRMA normalization significantly improved frozen-FFPE sample correlations, frozen-FFPE probeset correlations and agreement of differential analysis between different tumor subtypes. The relevance of our optimized data processing was assessed and validated using two independent datasets. In this study we demonstrated that an appropriate data processing can significantly improve the reliability of gene expression data derived from FFPE tissues using the standard Affymetrix platform. Tools for the implementation of our data processing algorithm are made publicly available at http://www.biocut.unito.it/cdf-ffpe/.

Comparison of RNA-Seq and Microarray in Transcriptome Profiling of Activated T Cells

To demonstrate the benefits of RNA-Seq over microarray in transcriptome profiling, both RNA-Seq and microarray analyses were performed on RNA samples from a human T cell activation experiment. In contrast to other reports, our analyses focused on the difference, rather than similarity, between RNA-Seq and microarray technologies in transcriptome profiling. A comparison of data sets derived from RNA-Seq and Affymetrix platforms using the same set of samples showed a high correlation between gene expression profiles generated by the two platforms. However, it also demonstrated that RNA-Seq was superior in detecting low abundance transcripts, differentiating biologically critical isoforms, and allowing the identification of genetic variants. RNA-Seq also demonstrated a broader dynamic range than microarray, which allowed for the detection of more differentially expressed genes with higher fold-change. Analysis of the two datasets also showed the benefit derived from avoidance of technical issues inherent to microarray probe performance such as cross-hybridization, non-specific hybridization and limited detection range of individual probes. Because RNA-Seq does not rely on a pre-designed complement sequence detection probe, it is devoid of issues associated with probe redundancy and annotation, which simplified interpretation of the data. Despite the superior benefits of RNA-Seq, microarrays are still the more common choice of researchers when conducting transcriptional profiling experiments. This is likely because RNA-Seq sequencing technology is new to most researchers, more expensive than microarray, data storage is more challenging and analysis is more complex. We expect that once these barriers are overcome, the RNA-Seq platform will become the predominant tool for transcriptome analysis.

Effect of allele frequencies, effect sizes and number of markers on prediction of quantitative traits in chickens

The objective was to assess goodness of fit and predictive ability of subsets of single nucleotide polymorphism (SNP) markers constructed based on minor allele frequency (MAF), effect sizes and varying marker density. Target traits were body weight (BW), ultrasound measurement of breast muscle (BM) and hen house egg production (HHP) in broiler chickens. We used a 600K Affymetrix platform with 1352 birds genotyped. The prediction method was genomic best linear unbiased prediction (GBLUP) with 354564 single nucleotide polymorphisms (SNPs) used to derive a genomic relationship matrix (G). Predictive ability was assessed as the correlation between predicted genomic values and corrected phenotypes from a threefold cross-validation. Predictive ability was 0.27±0.002 for BW, 0.33±0.001 for BM and 0.20±0.002 for HHP. For the three traits studied, predictive ability decreased when SNPs with a higher MAF were used to construct G. Selection of the 20% SNPs with the largest absolute effect sizes induced a predictive ability equal to that from fitting all markers together. When density of markers increased from 5K to 20K, predictive ability enhanced slightly. These results provide evidence that designing a low-density chip using low-frequency markers with large effect sizes may be useful for commercial usage.

Illuminaio: An open source IDAT parsing tool for Illumina microarrays

The IDAT file format is used to store BeadArray data from the myriad of genomewide profiling platforms on offer from Illumina Inc. This proprietary format is output directly from the scanner and stores summary intensities for each probe-type on an array in a compact manner. A lack of open source tools to process IDAT files has hampered their uptake by the research community beyond the standard step of using the vendor’s software to extract the data they contain in a human readable text format. To fill this void, we have developed the illuminaio package that parses IDAT files from any BeadArray platform, including the decryption of files from Illumina’s gene expression arrays. illuminaio provides the first open-source package for this task, and will promote wider uptake of the IDAT format as a standard for sharing Illumina BeadArray data in public databases, in the same way that the CEL file serves as the standard for the Affymetrix platform.

Mitochondrial genome variations among arsenic exposed individuals and potential correlation with apoptotic parameters

Exposure to arsenic (As) causes serious health hazards. Therefore, there is a sustained effort to understand the molecular basis of the risk posed by the toxicant. It has been reported that apoptotic changes ensue on exposure to As. To investigate the molecular basis of such changes, we sequenced the entire mitochondrial (mt) genome from PBMC of a subset of these individuals (As-exposed=16 and unexposed=18) using Affymetrix platform. Our analysis revealed that As exposure does not induce large-scale mt-DNA variations, but that specific deleterious changes could induce mt dysfunction. A Glu115Ter mutation as well as 17 other in silico predicted deleterious variants were identified exclusively in exposed individuals. The number of variants in mt Complex I in As-exposed individuals was positively correlated with their respective intracellular ROS level. In addition, the extent of potentially damaging variants in As-exposed individuals had significant positive correlation to the degree of G0 /G1 cell cycle arrest.

Identification Of Chromosomal Abnormalities By Microarray-Based Genomic Profiling As Compared To Karyotyping In Patients With Low and Intermediate-1 Risk Myelodysplastic Syndromes Within a Prospective Clinical Trial: A Study On Behalf Of The HOVON89 Study Group

Myelodysplastic syndromes (MDS) represent a heterogeneous group of clonal hematopoietic disorders, characterized by ineffective hematopoiesis resulting in cytopenias and a highly variable clinical course. Cytogenetics are routinely used as one of the diagnostic, prognostic and therapeutic markers in the clinical management of MDS (Greenberg et al. Blood 120:2454,2012). Although karyotyping is generally considered as the gold standard in the cytogenetic characterization of MDS, 40-60% of the patients exhibit a normal karyotype. Intrinsically, the resolution of karyotyping is limited by its capacity to detect only those copy number changes that are microscopically visible (5-10 Mb in size). In contrast, microarray-based genomic profiling analyses allow a genome-wide detection of copy number alterations (CNAs), down to 100 kb in size, and regions of copy neutral loss of heterozygosity (CNLOH). Such analyses also overcome some of the other major limitations of karyotyping such as low success rate due to inadequate metaphase yield and/or poor banding quality. We have compared karyotyping and fluorescence in situ hybridization (FISH) with microarray-based genomic profiling with respect to the detection yield for genetic abnormalities in bone marrow samples from lower risk MDS patients.We used the HOVON89 study-cohort, a prospective phase II randomized multicenter study to assess the efficacy of lenalidomide with or without erythropoietin and granulocyte-colony stimulating factor in patients with low/intermediate-1 risk MDS; www.trialregister.nl; NTR1825; EudraCT nr.: 2008-002195-10. Inclusion target of the study is 200 low/intermediate-1 risk MDS patients (134 enrolled, inclusion ongoing). Data regarding cytogenetics, FISH and microarray were obtained in a fully blinded fashion for 68 MDS patients. For microarray-based genomic profiling we used the recently launched high resolution CytoScan HD Array (Affymetrix) platform. The following interpretation criteria were applied: (i) the threshold for CNAs was set at >5 Mb, (ii) inclusion of <5 Mb CNAs segments that coincide with known cancer genes as reported on www.sanger.ac.uk and (iii) the threshold for CNLOH was set at >10 Mb and to telomere. Karyotyping and interphase FISH were performed using standard cytogenetic methods. In all 4 patients where karyotyping revealed no metaphases interphase FISH was performed.Thirty-six of the 68 (53%) MDS patients had an abnormal microarray profile. Of interest, in 13 of these 36 patients no abnormalities were observed by karyotyping and/or FISH. All these abnormalities observed by microarray only, involved focal <5 Mb CNAs (containing e.g. the TET2, RUNX1 and DNMT3A genes) and regions of CNLOH (coinciding with 1p, 2p, 4q, 7q, 11p, 11q, 12q, 14q and 18q), which are all out of the scope of karyotyping and FISH. All CNAs identified by karyotyping and FISH were also observed by microarray-based genomic profiling, including a case with loss of 5q in 5% of the cells and loss of 7q in 9% of the cells as observed by interphase FISH and another case with loss of 5q in 3 of 20 the analyzed metaphases by karyotyping. These observations demonstrate the high sensitivity of the CytoScan HD array platform for the identification of CNAs in (small) subclones. As expected balanced translocations such as t(3;3)(q21;q26) and t(2;14)(q37;q22) present in 2 of the patients in our cohort were not identified by microarray-based genomic profiling. This study will be extended to all patients to be included in the HOVON89 trial.In conclusion, we demonstrate that in the present cohort of 68 patients, microarray-based genomic profiling allows the identification of almost all copy number abnormalities also observed by karyotyping and FISH. In addition, we show that microarray-based genomic profiling allows the detection of potential prognostic relevant abnormalities (focal CNAs and CNLOH) which would have remained undetected by karyotyping and FISH. The predictive and/or prognostic value of these novel CNAs and CNLOH will be evaluated within the ongoing prospective clinical HOVON89 trial in lower risk MDS. Disclosures:No relevant conflicts of interest to declare.

Expression Level Of A Coagulation Factor Gene, F13A1 Defines Characteristic Subpopulations Of Childhood Acute Lymphoblastic Leukemia

Using multiparameter flow cytometry, Western blot, ELISA and laser scanning microscopy, leukemic B-cell progenitor lymphoblasts were identified as a novel expression site of coagulation factor XIII subunit A (FXIIIA) (Kiss et al Thromb Haemost 2006). Three-year overall survival (OS) of children with FXIIIA-positive acute lymphoblastic leukemia (ALL) was significantly higher (87%) than 3-yr OS of patients with FXIIIA-negative ALL (65%). Here we report on gene expression profiles (GEP) associated with FXIIIA-positive vs. FXIIIA–negative childhood (ch)ALL. Finnally, the expression levels of F13A1 gene in cytogenetic subgroups were investigated.GEP of 11 FXIIIA-positive and 9 FXIIIA-negative samples of patients treated at the Department of Hematology-Oncology, University of Debrecen was investigated using HG-U 133 plus 2.0 Affymetrix Platform Array. Expression sequence tags (EST) characterized by expression levels with at least 2 logs difference among FXIIIA-positive vs. FXIIIA-negative samples were selected and validated with real-time quantitative PCR. Extending the analysis, we have searched the database containing GEPs of 317 children with ALL treated at the Division of Hemato-Oncology, Department of Women's and Children's Health, University of Padova, using R package and Partek Genomic Suite softwares.FXIIIA-positive and FXIIIA-negative samples exhibited a characteristically different GEP. In the FXIIIA-positive samples one of the overexpressed genes was F13A1. FXIIIA-negative samples contained two characteristic groups of overexpressed genes. One of these consisted of genes participating in B-cell development: EBF1, IKZF1 and PAX5. The second set consisted of genes encoding for tyrosine kinases: JAK1, JAK3, NC07523, NC08002, NC06523, NC08345 and for serine-threonine kinase NC00027. In contrast, FXIIIA-positive samples contained only two overexpressed tyrosine kinases: C-KIT and JAK2.A wide variation of F13A1 expression levels of the 317 Padova patients was observed. Since expression level of FXIIIA protein of these patients has not been determined, we have arbitrarily defined F13A1 gene expression levels below 106 as “low” and gene expression levels exceeding 109 as “high”. Considering these two groups, we have investigated the distribution of F13A1 expression among the known cytogenetic subgroups of ALL. Low F13A1 expression was prevalent among “B-other” samples, high F13A1 expression was associated with t(1;19).The pattern of overexpressed ESTs, accumulation of low F13A1 expressing samples in the “B-other” cytogenetic group of ALL and the unfavorable disease outcome of FXIIIA-negative cases may suggest a possible overlap between FXIIIA-negative ALL and BCR-ABL1-like ALL identified recently in the “B-other” group. The nature and extent of this overlap will be investigated prospectively in the BFM ALL-IC 2009 clinical trial. In contrast, high expression levels of F13A1 accumulated preferentially within the t(1;19) genetic group, associated with a good prognosis. Detection of FXIIIA expression by flow cytometry may offer an easy and non-expensive tool for defining new prognostic subpopulations of ALL. Grant supportTÁMOP 4.2.2.A-11/1/KONV-2012-0025 project (CK, KG, HZ, IG, IS and JK). The project is co-financed by the European Union and the European Social Fund and the AIRC (Associazione Italiana Ricerca su Cancro; SB) project. Disclosures:No relevant conflicts of interest to declare.

A Cross-Platform Comparison of Affymetrix and Agilent Microarrays Reveals Discordant miRNA Expression in Lung Tumors of c-Raf Transgenic Mice

Non-coding RNAs play major roles in the translational control of gene expression. In order to identify disease-associated miRNAs in precursor lesions of lung cancer, RNA extracts from lungs of either c-Raf transgenic or wild-type (WT) mice were hybridized to the Agilent and Affymetrix miRNA microarray platforms, respectively. This resulted in the detection of a range of miRNAs varying between 111 and 267, depending on the presence or absence of the transgene, on the gender, and on the platform used. Importantly, when the two platforms were compared, only 11–16% of the 586 overlapping genes were commonly detected. With the Agilent microarray, seven miRNAs were identified as significantly regulated, of which three were selectively up-regulated in male transgenic mice. Much to our surprise, when the same samples were analyzed with the Affymetrix platform, only two miRNAs were identified as significantly regulated. Quantitative PCR performed with lung RNA extracts from WT and transgenic mice confirmed only partially the differential expression of significant regulated miRNAs and established that the Agilent platform failed to detect miR-433. Finally, bioinformatic analyses predicted a total of 152 mouse genes as targets of the regulated miRNAs of which 4 and 11 genes were significantly regulated at the mRNA level, respectively in laser micro-dissected lung dysplasia and lung adenocarcinomas of c-Raf transgenic mice. Furthermore, for many of the predicted mouse target genes expression of the coded protein was also repressed in human lung cancer when the publically available database of the Human Protein Atlas was analyzed, thus supporting the clinical significance of our findings. In conclusion, a significant difference in a cross-platform comparison was observed that will have important implications for research into miRNAs.

Impact of gestational chronodisruption on fetal cardiac genomics

We recently reported that gestational chronodisruption induces fetal growth restriction and marked effects on fetal adrenal physiology. Here, whole-transcriptome profiling was used to test whether gestational chronodisruption modifies gene expression in the fetal heart, potentially altering cardiac development. At day 10 of gestation (E10), pregnant rats were randomized in two groups: constant light (LL) and control 12h light/12h dark photoperiod (LD). RNA isolated from E18 heart was subjected to microarray analysis (Affymetrix platform for 28,000 genes). Integrated transcriptional changes were assessed by gene ontology and pathway analysis. Significant differential expression was found for 383 transcripts in LL relative to LD fetal heart (280 up-regulated and 103 down-regulated); with 42 of them displaying a 1.5-fold or greater change in gene expression. Deregulated markers of cardiovascular disease accounted for alteration of diverse gene networks in LL fetal heart, including local steroidogenesis and vascular calcification, as well as cardiac hypertrophy, stenosis and necrosis/cell death. DNA integrity was also overrepresented, including a 2.1-fold increase of Hmga1 mRNA, which encodes for a profuse architectural transcription factor. microRNA analysis revealed up-regulation of miRNAs 218-1 and 501 and concurrent down-regulation of their validated target genes. In addition, persistent down-regulation of Kcnip2 mRNA and hypertrophy of the left ventricle were found in the heart from 90days-old offspring from LL mothers. The dysregulation of a relevant fraction of the fetal cardiac transcriptome, together with the diversity and complexity of the gene networks altered by gestational chronodisruption, suggest enduring molecular changes which may shape the hypertrophy observed in the left ventricle of adult LL offspring.

Abstract B62: Cell cycle alterations in paired diagnosis-relapse childhood acute lymphoblastic leukemia.

Abstract Approximately 25% of children with acute lymphoblastic leukemia (ALL) relapse. After relapse, the overall cure rate remains low, despite intensified chemotherapy and stem cell transplantation. The Laboratory of Pediatric Hemato-Oncology of the University of Padua is the AIEOP (Italian Association for Pediatric Hemato-Oncology) referring laboratory and up to 5,000 pediatric leukemia samples are processed every year. For all the ALL diagnosis and relapse samples DNA index is analysed for the risk-treatment stratification of the patients. The analysis evaluates both DNA ploidy and the proliferative state through the determination of the cell population in the S-phase of the cell cycle. Cell cycle analysis of childhood ALL, performed in our laboratory, reveal that S-phase is significant increased in bone marrow from relapsed patients compared to bone marrow of the same patients at the diagnosis. The study of the molecular mechanisms involved in S-phase increase at relapse will help us to improve our knowledge about relapse mechanism and to design new therapeutic strategy exploiting these changes in cell cycle control. We studied 180 patients affected by B-ALL enrolled in AIEOP protocols with more that 60% of blast cells in the bone marrow, both at diagnosis and relapse; extramedullary relapses were excluded. Cell cycles were analysed by flow citometry. To evaluate changes in cell cycle distribution at relapse in respect to diagnosis, for each patients we calculate ΔS as the difference between S-phase fraction at relapse and S-phase fraction at diagnosis. Gene expression profiling were performed using Human Genome U133 Plus 2.0 array (Affymetrix platform). Gene expression analysis, real time PCR and western blot were carry out to compare higher and lower ΔS patients. Preliminary gene expression analysis, on higher ΔS vs lower ΔS samples of relapsed patients, identified 58 differentially expressed probe sets corresponding to genes involved in proliferation, DNA damage and cell migration (LFDR&amp;lt;0.05). Among these genes, we found some E2F1 targets such as MAD2L1, CIP2a and RFC3. Western blot and real time PCR revealed increased cyclinA2 expression, another E2F1 target, in samples from relapsed patients with higher ΔS. We observed that higher [[Unable to Display Character: &amp;#8710;]]S correlate to very early time to relapse (within 18 month from diagnosis) and these patients are classified as high risk patients that barely respond to treatments after relapse. Very early relapse more frequently happens during maintenance therapy based on methotrexate and 6-mercaptopurine, and methotrexate resistance could be mediated by increased levels of E2F1 transcriptional activity. At the present stage we identified S-phase increase as a molecular determinant of high risk in B-ALL relapse. Further studies of these alterations of cell cycle control and of E2F1 activation may help the development of more specific and less toxic therapies that overcome resistance to currently used chemotherapeutics. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B62. Citation Format: Roberta Bortolozzi, Giampietro Viola, Maddalena Paganin, Silvia Bresolin, Silvia Disarò, Elena Porcù, Valentino Conter, Franco Locatelli, Barbara Buldini, Giuseppe Basso. Cell cycle alterations in paired diagnosis-relapse childhood acute lymphoblastic leukemia. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B62.

Optimised laser microdissection of the human ocular surface epithelial regions for microarray studies

BackgroundThe most important challenge of performing insitu transcriptional profiling of the human ocular surface epithelial regions is obtaining samples in sufficient amounts, without contamination from adjacent tissue, as the region of interest is microscopic and closely apposed to other tissues regions. We have effectively collected ocular surface (OS) epithelial tissue samples from the Limbal Epithelial Crypt (LEC), limbus, cornea and conjunctiva of post-mortem cadaver eyes with laser microdissection (LMD) technique for gene expression studies with spotted oligonucleotide microarrays and Gene 1.0 ST arrays.MethodsHuman donor eyes (4 pairs for spotted oligonucleotide microarrays, 3 pairs for Gene 1.0 ST arrays) consented for research were included in this study with due ethical approval of the Nottingham Research Ethics Committee. Eye retrieval was performed within 36 hours of post-mortem period. The dissected corneoscleral buttons were immersed in OCT media and frozen in liquid nitrogen and stored at −80°C till further use. Microscopic tissue sections of interest were taken on PALM slides and stained with Toluidine Blue for laser microdissection with PALM microbeam systems. Optimisation of the laser microdissection technique was crucial for efficient and cost effective sample collection.ResultsThe starting concentration of RNA as stipulated by the protocol of microarray platforms was taken as the cut-off concentration of RNA samples in our studies. The area of LMD tissue processed for spotted oligonucleotide microarray study ranged from 86,253 μm2 in LEC to 392,887 μm2 in LEC stroma. The RNA concentration of the LMD samples ranged from 22 to 92 pg/μl. The recommended starting concentration of the RNA samples used for Gene 1.0 ST arrays was 6 ng/5 μl. To achieve the desired RNA concentration the area of ocular surface epithelial tissue sample processed for the Gene 1.0 ST array experiments was approximately 100,0000 μm2 to 130,0000 μm2. RNA concentration of these samples ranged from 10.88 ng/12 μl to 25.8 ng/12 μl, with the RNA integrity numbers (RIN) for these samples from 3.3 to 7.9. RNA samples with RIN values below 2, that had failed to amplify satisfactorily were discarded.ConclusionsThe optimised protocol for sample collection and laser microdissection improved the RNA yield of the insitu ocular surface epithelial regions for effective microarray studies on spotted oligonucleotide and affymetrix platforms.