Oligonucleotide Arrays Research Articles

MicroRNA‐148a is downregulated in gastric cancer, targets MMP7, and indicates tumor invasiveness and poor prognosis

Gastric cancer (GC) develops through deregulation of gene expression and accumulation of epigenetic abnormalities, leading to tumor cell acquisition of malignant features. MicroRNAs (miRNAs) play a critical role in cancer development where they can act as oncogenes or oncosuppressors. To identify miRNAs that are associated with some clinicopathologic features of GC and/or participate in tumor progression, miRNA expression in 20 GC tissues and five corresponding non-neoplastic gastric mucosa was examined by miRNA microarray. Oligonucleotide array analysis was carried out for miRNA target prediction. The functions of candidate miRNAs and their target genes were also analyzed by quantitative RT-PCR, Western blotting, reporter gene assay, and cell invasion assay. Comparison of miRNA expression profiles revealed that downregulation of miR-148a was identified in most of the GC tissues. Downregulation of miR-148a was significantly correlated with an advanced clinical stage, lymph node metastasis, and poor clinical outcome. Custom oligonucleotide array analysis revealed that MMP7 expression was markedly downregulated in miR-148a-overexpressing GC cells; MMP7 was found to be a direct and functional target of miR-148a, participating in cell invasion. These results suggest that miR-148a contributes to the maintenance of homeostasis in normal stomach tissue and plays an important role in GC invasion by regulating MMP7 expression.

Pigeons: A Novel GUI Software for Analysing and Parsing High Density Heterologous Oligonucleotide Microarray Probe Level Data.

Genomic DNA-based probe selection by using high density oligonucleotide arrays has recently been applied to heterologous species (Xspecies). With the advent of this new approach, researchers are able to study the genome and transcriptome of a non-model or an underutilised crop species through current state-of-the-art microarray platforms. However, a software package with a graphical user interface (GUI) to analyse and parse the oligonucleotide probe pair level data is still lacking when an experiment is designed on the basis of this cross species approach. A novel computer program called Pigeons has been developed for customised array data analysis to allow the user to import and analyse Affymetrix GeneChip® probe level data through XSpecies. One can determine empirical boundaries for removing poor probes based on genomic hybridisation of the test species to the Xspecies array, followed by making a species-specific Chip Description File (CDF) file for transcriptomics in the heterologous species, or Pigeons can be used to examine an experimental design to identify potential Single-Feature Polymorphisms (SFPs) at the DNA or RNA level. Pigeons is also focused around visualization and interactive analysis of the datasets. The software with its manual (the current release number version 1.2.1) is freely available at the website of the Nottingham Arabidopsis Stock Centre (NASC).

The Effect of Antineoplastons A10 and AS2-1 and Metabolites of Sodium Phenylbutyrate on Gene Expression in Glioblastoma Multiforme

Antineoplastons are peptide and amino acid derivatives that occur naturally in the human body. They inhibit the growth of neoplastic cells without growth inhibition of normal cells. Phenylacetylglutaminate (PG) is an active ingredient of antineoplastons A10 and AS2-1 (ANP) and is also a metabolic by-product of phenylbutyrate (PB). The formulation of antineoplaston AS2-1 is a 4:1 mixture of phenylacetate (PN) and PG. Antineoplaston A10 is a 4:1 mixture of PG and isoPG. This study investigates the molecular mechanism of action of PG and PN. The Human U87 glioblastoma (GBM) cell line was used as the model system in this study. A total human gene array screen using the Affymetrix Human Genome plus 2.0 oligonucleotide arrays was performed using mRNA derived from U87 cells exposed to PG and PN. Pathway analysis was performed to allow the visualization of effect on metabolic pathways and gene interaction networks. Our preliminary results indicate that PG and PN interrupt signal transduction in RAS/MAPK/ERK and PI3K/AKT/PTEN pathways, interfere with cell cycle, decrease metabolism and promote apoptosis in human U87 GBM cells. The effect on multiple cellular pathways and targets, suggests that ANP and PB are promising candidates for clinical studies in GBM.

Genome-wide and gene-specific epigenomic platforms for hepatocellular carcinoma biomarker development trials.

The majority of the epigenomic reports in hepatocellular carcinoma have focused on identifying novel differentially methylated drivers or passengers of the oncogenic process. Few reports have considered the technologies in place for clinical translation of newly identified biomarkers. The aim of this study was to identify epigenomic technologies that need only a small number of samples to discriminate HCC from non-HCC tissue, a basic requirement for biomarker development trials. To assess that potential, we used quantitative Methylation Specific PCR, oligonucleotide tiling arrays, and Methylation BeadChip assays. Concurrent global DNA hypomethylation, gene-specific hypermethylation, and chromatin alterations were observed as a hallmark of HCC. A global loss of promoter methylation was observed in HCC with the Illumina BeadChip assays and the Nimblegen oligonucleotide arrays. HCC samples had lower median methylation peak scores and a reduced number of significant promoter-wide methylated probes. Promoter hypermethylation of RASSF1A, SSBP2, and B4GALT1 quantified by qMSP had a sensitivity ranging from 38% to 52%, a specificity of 100%, and an AUC from 0.58 to 0.75. A panel combining these genes with HCC risk factors had a sensitivity of 87%, a specificity of 100%, and an AUC of 0.91.

Chromosome 15q11-q13 copy number gain detected by array-CGH in two cases with a maternal methylation pattern.

BackgroundThe 15q11-q13 region contains many low copy repeats and is well known for its genomic instability. Several syndromes are associated with genomic imbalance or copy-number-neutral uniparental disomy. We report on two patients: Patient 1 is a boy with developmental delay and autism; and Patient 2 is a girl with developmental delay, hypotonia and dysmorphism. We performed analyses to delineate their dosage in the 15q region, determine whether the patients’ dosage correlates with phenotypic severity, and whether genes in the amplified regions are significantly associated with identified functional networks.ResultsFor the proximal region of 15q, molecular cytogenetic analysis with Agilent oligonucleotide array showed a copy number of 3 for Patient 1 and a copy number of 4 for Patient 2. Fluorescent in situ hybridization analysis of Patient 2 showed two different populations of cells with different marker chromosomes. Methylation analysis of the amplified region showed that the extra copies of small nuclear ribonucleoprotein polypeptide N gene were of maternal origin. Phenotypic severity did not correlate with the size and dosage of 15q, or whether the amplification is interstitial or in the form of a supernumerary marker. Pathway analysis showed that in Patient 2, the main functional networks that are affected by the genes from the duplicated/triplicated regions are developmental disorder, neurological disease and hereditary disease.ConclusionsThe 15q11-q13 gains that were found in both patients could explain their phenotypic presentations. This report expands the cohort of patients for which 15q11-q13 duplications are molecularly characterized.

Large Scale Explorative Oligonucleotide Probe Selection for Thousands of Genetic Groups on a Computing Grid: Application to Phylogenetic Probe Design Using a Curated Small Subunit Ribosomal RNA Gene Database

Phylogenetic Oligonucleotide Arrays (POAs) were recently adapted for studying the huge microbial communities in a flexible and easy-to-use way. POA coupled with the use of explorative probes to detect the unknown part is now one of the most powerful approaches for a better understanding of microbial community functioning. However, the selection of probes remains a very difficult task. The rapid growth of environmental databases has led to an exponential increase of data to be managed for an efficient design. Consequently, the use of high performance computing facilities is mandatory. In this paper, we present an efficient parallelization method to select known and explorative oligonucleotide probes at large scale using computing grids. We implemented a software that generates and monitors thousands of jobs over the European Computing Grid Infrastructure (EGI). We also developed a new algorithm for the construction of a high-quality curated phylogenetic database to avoid erroneous design due to bad sequence affiliation. We present here the performance and statistics of our method on real biological datasets based on a phylogenetic prokaryotic database at the genus level and a complete design of about 20,000 probes for 2,069 genera of prokaryotes.

Species and gene divergence in Littorina snails detected by array comparative genomic hybridization.

BackgroundArray comparative genomic hybridization (aCGH) is commonly used to screen different types of genetic variation in humans and model species. Here, we performed aCGH using an oligonucleotide gene-expression array for a non-model species, the intertidal snail Littorina saxatilis. First, we tested what types of genetic variation can be detected by this method using direct re-sequencing and comparison to the Littorina genome draft. Secondly, we performed a genome-wide comparison of four closely related Littorina species: L. fabalis, L. compressa, L. arcana and L. saxatilis and of populations of L. saxatilis found in Spain, Britain and Sweden. Finally, we tested whether we could identify genetic variation underlying “Crab” and “Wave” ecotypes of L. saxatilis.ResultsWe could reliably detect copy number variations, deletions and high sequence divergence (i.e. above 3%), but not single nucleotide polymorphisms. The overall hybridization pattern and number of significantly diverged genes were in close agreement with earlier phylogenetic reconstructions based on single genes. The trichotomy of L. arcana, L. compressa and L. saxatilis could not be resolved and we argue that these divergence events have occurred recently and very close in time. We found evidence for high levels of segmental duplication in the Littorina genome (10% of the transcripts represented on the array and up to 23% of the analyzed genomic fragments); duplicated genes and regions were mostly the same in all analyzed species. Finally, this method discriminated geographically distant populations of L. saxatilis, but we did not detect any significant genome divergence associated with ecotypes of L. saxatilis.ConclusionsThe present study provides new information on the sensitivity and the potential use of oligonucleotide arrays for genotyping of non-model organisms. Applying this method to Littorina species yields insights into genome evolution following the recent species radiation and supports earlier single-gene based phylogenies. Genetic differentiation of L. saxatilis ecotypes was not detected in this study, despite pronounced innate phenotypic differences. The reason may be that these differences are due to single-nucleotide polymorphisms.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-687) contains supplementary material, which is available to authorized users.

Systematic review of accuracy of prenatal diagnosis for abnormal chromosome diseases by microarray technology.

The accuracy of prenatal diagnosis for abnormal chromosome diseases by chromosome microarray technology and karyotyping were compared. A literature search was carried out in the MEDLINE database with the keywords "chromosome" and "karyotype" and "genetic testing" and "prenatal diagnosis" and "oligonucleotide array sequence". The studies obtained were filtered by using the QUADAS tool, and studies conforming to the quality standard were fully analyzed. There was one paper conforming to the QUADAS standards including 4406 gravidas with adaptability syndromes of prenatal diagnosis including elderly parturient women, abnormal structure by type-B ultrasound, and other abnormalities. Microarray technology yielded successful diagnoses in 4340 cases (98.8%), and there was no need for tissue culture in 87.9% of the samples. All aneuploids and non-parallel translocations in 4282 cases of non-chimera identified by karyotyping could be detected using microarray analysis technology, whereas parallel translocations and fetal triploids could not be detected by microarray analysis technology. In the samples with normal karyotyping results, type-B ultrasound showed that 6% of chromosomal deficiencies or chromosome duplications could be detected by microarray technology, and the same abnormal chromosomes were detected in 1.7% of elderly parturient women and samples with positive serology screening results. In the prenatal diagnosis test, compared with karyotyping, microarray technology could identify the extra cell genetic information with clinical significance, aneuploids, and non-parallel translocations; however, its disadvantage is that it could not identify parallel translocations and triploids.

Potential Roles of mTOR and Protein Degradation Pathways in the Phenotypic Expression of Feed Efficiency in Broilers

The cost of feed represents as much as 70% of the total cost of raising a meat producing animal to market weight. Thus, feed efficiency (FE; g gain: g feed) is a very important genetic trait in animal agriculture. We have observed that a hallmark of low feed efficiency in a highly selected male broiler (meat chicken) line was extensive protein oxidation that probably resulted from increased reactive oxygen species being produced by the mitochondria. Repair or resynthesis of damaged proteins would therefore represent a considerable energetic drain and contribute to the phenotypic expression of low feed efficiency. In the present study, a software program (Ingenuity Pathway Analysis, IPA) facilitated the analysis and interpretation of data from a 4 x 44k chicken oligo array on breast muscle along with data from previous studies obtained from broilers individually phenotyped for FE. The findings support a hypothesis that differential expression of genes associated with the Akt/mTOR, protein ubiquitination, and proteasome pathways through modulation of transcription and protein turnover could play an important role in the phenotypic expression of feed efficiency. Confirmation of this hypothesis will require a thorough assessment of protein expression as well as protein and enzyme activity measurements associated with these pathways in the low and high FE broiler phenotypes.

IMAGe syndrome: clinical and genetic implications based on investigations in three Japanese patients

Arboleda et al. have recently shown that IMAGe (intra-uterine growth restriction, metaphyseal dysplasia, adrenal hypoplasia congenita and genital abnormalities) syndrome is caused by gain-of-function mutations of maternally expressed gene CDKN1C on chromosome 11p15.5. However, there is no other report describing clinical findings in patients with molecularly studied IMAGe syndrome. Here, we report clinical and molecular findings in Japanese patients. We studied a 46,XX patient aged 8·5 years (case 1) and two 46,XY patients aged 16·5 and 15·0 years (cases 2 and 3). Clinical studies revealed not only IMAGe syndrome-compatible phenotypes in cases 1-3, but also hitherto undescribed findings including relative macrocephaly and apparently normal pituitary-gonadal endocrine function in cases 1-3, familial glucocorticoid deficiency (FGD)-like adrenal phenotype and the history of oligohydramnios in case 2, and arachnodactyly in case 3. Sequence analysis of CDKN1C, pyrosequencing-based methylation analysis of KvDMR1 and high-density oligonucleotide array comparative genome hybridization analysis for chromosome 11p15.5 were performed, showing an identical de novo and maternally inherited CDKN1C gain-of-function mutation (p.Asp274Asn) in cases 1 and 2, respectively, and no demonstrable abnormality in case 3. The results of cases 1 and 2 with CDKN1C mutation would argue the following: [1] relative macrocephaly is consistent with maternal expression of CDKN1C in most tissues and biparental expression of CDKN1C in the foetal brain; [2] FGD-like phenotype can result from CDKN1C mutation; and [3] genital abnormalities may primarily be ascribed to placental dysfunction. Furthermore, lack of CDKN1C mutation in case 3 implies genetic heterogeneity in IMAGe syndrome.

A new microarray platform for whole-genome expression profiling of Mycobacterium tuberculosis

Microarrays have allowed gene expression profiling to progress from the gene level to the genome level, and oligonucleotide microarrays have become the platform of choice for large-scale, targeted gene expression studies. cDNA arrays and spotted oligonucleotide arrays have gradually given way to in situ synthesized oligonucleotide-based DNA microarrays for whole-genome expression profiling. With the identification of new coding and regulatory sequences, it is imperative that microarrays be updated to enable more complete expression profiling of genomes. We report here a new in situ synthesized oligonucleotide-based microarray platform for Mycobacterium tuberculosis that has been updated for the latest genome information and incorporates hitherto unannotated genes with described biological functions. This microarray has greater coverage of mycobacterial genes than any other array reported to date. We have also evaluated different labeled-target preparation methods and hybridization conditions for this new microarray to obtain high quality data and reproducible results. The new design has been rigorously validated for its specificity and performance using samples isolated from mycobacteria grown under different environment conditions. Further, the quality of the generated data has been compared with published data and is superior to that obtained using spotted oligonucleotide microarrays.

Screening of the different TNM stage-associated genes in lung adenocarcinoma by genomewide gene expression profile analysis in the Chinese population

Objective To screen for the differentially expressed genes associated with various TNM stages in lung adenocarcinoma of Chinese by comparing the expression profiles in tumor samples of different TNM stages. Methods This study was a case-case study.Lung adenocarcinoma specimens were collected from total of 240 patients receiving thoracic surgery in our hospital from May of 2008 to October of 2011.There were 90 samples (30 each for stageⅠ,ⅡandⅢA) for the gene array,and 150 samples (50 each for stageⅠ,ⅡandⅢA) for the further validation by qRT-PCR.A 35K Oligo gene array,covering about 25,100 genes,was used to screen the differentially expressed genes among 90 tumor samples of lung adenocarcinoma at various TNM stages.To verify the data from gene array,three genes of Zimp7,GINS2 and NAG-1 were confirmed by real-time RT-PCRin a different set of samples from the gene array.The expression levels of tested genes in different TNM stages were compared using ANOVA statistic method. Results Five hundreds and ninty-two differentially expressed genes in lung adenocarcinomas compared to the surrounding normal lung tissues were obtained,among which 264 genes were upregulated and 328 downregulated.From the 592 candidates,10 differentially expressed genes were identified among different TNM stages (StageⅠ,ⅡandⅢA),of which Zimp7,GINS2 and NAG-1 genes were first reported to be present at a high level in lung adenocarcinoma.The results of qRT-PCR for the three genes were consistent with those from the gene array. Conclusion Ten candidate genes associated with different TNM stages in lung adenocarcinoma were identified in the Chinese population,which would provides new insight into the molecular basis underlying the development of lung adenocarcinoma and may offer new targets for the diagnosis,therapy and prognosis prediction of lung adenocarcinoma.(Chin J Lab Med,2013,36:1104-1108) Key words: Lung neoplasms; Adenocarcinoma; Neoplasm staging; Gene expression profiling; Oligonucleotide array sequence analysis

Application of array comparative genomic hybridization in 256 patients with developmental delay or intellectual disability

We used whole-genome exon-targeted oligonucleotide array comparative genomic hybridization (array CGH) in a cohort of 256 patients with developmental delay (DD)/intellectual disability (ID) with or without dysmorphic features, additional neurodevelopmental abnormalities, and/or congenital malformations. In 69 patients, we identified 84 non-polymorphic copy-number variants, among which 41 are known to be clinically relevant, including two recently described deletions, 4q21.21q21.22 and 17q24.2. Chromosomal microarray analysis revealed also 15 potentially pathogenic changes, including three rare deletions, 5q35.3, 10q21.3, and 13q12.11. Additionally, we found 28 copy-number variants of unknown clinical significance. Our results further support the notion that copy-number variants significantly contribute to the genetic etiology of DD/ID and emphasize the efficacy of the detection of novel candidate genes for neurodevelopmental disorders by whole-genome array CGH.Electronic supplementary materialThe online version of this article (doi:10.1007/s13353-013-0181-x) contains supplementary material, which is available to authorized users.

Susceptibility to Acute Rheumatic Fever Based on Differential Expression of Genes Involved in Cytotoxicity, Chemotaxis, and Apoptosis

It is unknown why only some individuals are susceptible to acute rheumatic fever (ARF). We investigated whether there are differences in the immune response, detectable by gene expression, between individuals who are susceptible to ARF and those who are not. Peripheral blood mononuclear cells (PBMCs) from 15 ARF-susceptible and 10 nonsusceptible (control) adults were stimulated with rheumatogenic (Rh+) group A streptococci (GAS) or nonrheumatogenic (Rh-) GAS. RNA from stimulated PBMCs from each subject was cohybridized with RNA from unstimulated PBMCs on oligonucleotide arrays to compare gene expression. Thirty-four genes were significantly differentially expressed between ARF-susceptible and control groups after stimulation with Rh+ GAS. A total of 982 genes were differentially expressed between Rh+ GAS- and Rh- GAS-stimulated samples from ARF-susceptible individuals. Thirteen genes were differentially expressed in the same direction (predominantly decreased) between the two study groups and between the two stimulation conditions, giving a strong indication of their involvement. Seven of these were immune response genes involved in cytotoxicity, chemotaxis, and apoptosis. There was variability in the degree of expression change between individuals. The high proportion of differentially expressed apoptotic and immune response genes supports the current model of autoimmune and cytokine dysregulation in ARF. This study also raises the possibility that a "failed" immune response, involving decreased expression of cytotoxic and apoptotic genes, contributes to the immunopathogenesis of ARF.

Application of Array-Based Comparative Genomic Hybridization to Pediatric Neurologic Diseases

PurposeArray comparative genomic hybridization (array-CGH) is a technique used to analyze quantitative increase or decrease of chromosomes by competitive DNA hybridization of patients and controls. This study aimed to evaluate the benefits and yield of array-CGH in comparison with conventional karyotyping in pediatric neurology patients.Materials and MethodsWe included 87 patients from the pediatric neurology clinic with at least one of the following features: developmental delay, mental retardation, dysmorphic face, or epilepsy. DNA extracted from patients and controls was hybridized on the Roche NimbleGen 135K oligonucleotide array and compared with G-band karyotyping. The results were analyzed with findings reported in recent publications and internet databases.ResultsChromosome imbalances, including 9 cases detected also by G-band karyotyping, were found in 28 patients (32.2%), and at least 19 of them seemed to be causally related to the abnormal phenotypes. Regarding each clinical symptom, 26.2% of 42 developmental delay patients, 44.4% of 18 mental retardation patients, 42.9% of 28 dysmorphic face patients, and 34.6% of 26 epilepsy patients showed abnormal array results.ConclusionAlthough there were relatively small number of tests in patients with pediatric neurologic disease, this study demonstrated that array-CGH is a very useful tool for clinical diagnosis of unknown genome abnormalities performed in pediatric neurology clinics.

Detection of signaling pathways related to nuclear factor-κB in patients with psoriasis vulgaris using PCR-array analysis

Objective To detect the expression of 84 signaling molecules associated with nuclear factor-κB in lesions of Uygur patients with psoriasis.Methods Skin specimens were obtained from the lesional and paralesional skin of eight Uygur patients with psoriasis.Total RNA was extracted from the skin specimens and reversely transcribed into cDNA.PCR-array analysis was carried out to quantify the expressions of 84 signaling molecules related to nuclear factor-κB.Genes with a fold-change ＞ or =2 were defined as differentially expressed.Results Among the 84 tested genes,22 showed upregulated expression,7 downregulated expression,and the remaining 54 genes showed no significant changes in psoriatic lesions compared with the normal skin.The strongest upregulation was observed in the gene expressions of Caspase recruitment domain family 11 (CARD11) and granulocyte-macrophage colony-stimulating factor 2 (CSF2),and the most significant downregulation in the gene expression of interleukin 10 (IL-10),tumor necrosis factor superfamily member 5 (CD40) and nuclear factor of kappa light polypeptide gene enhancer in B-cell inhibitor,epsilon (NFκBIE).Conclusion Multiple molecules involved in the NF-κB signaling pathway might be activated or inhibited in lesions of patients with psoriasis. Key words: Psoriasis; Oligonucleotide array sequence analysis; NF-kappaB; Granulocyte-macrophage colony-stimulating factor; CARD11

Novel Fusion Transcripts PVT1-NSMCE2 and BF104016-NSMCE2 Resulting In Depletion Of NSMCE2 Protein In Acute Myeloid Leukemia Harboring 8q24 Amplicons

Little is known about the molecular pathogenesis of acute myeloid leukemia (AML) with high-level DNA amplifications commonly defined as the presence of double minutes (dmins) and/or homogeneously staining regions (hsr), although dmins and hsr are found in nearly 1% of karyotypically abnormal AML and myelodysplastic syndromes (MDS). It is sometimes difficult to precisely assign dmins, hsr, and marker chromosomes (mars) to specific chromosomal bands, while most of them consist of an amplified segment from chromosome band 8q24 or 11q23. In AML, the 8q24 amplicon has been previously identified in a commonly involved segment of about 4 Mb, which contains several genes such as TRIB1 (C8FW), NSMCE2 (non-SMC element 2), PVT1, and MYC. To further define the pathogenetic lesions of 8q24 amplicons in AML, we performed comprehensive molecular studies on leukemic cells from a patient with AML (patient 1) with two mars and leukemic cell lines HL60 and K562, identifying two novel chimeric transcripts, PVT1-NSMCE2 and BF104016-NSMCE2. Regarding PVT1-NSMCE2 fusion transcripts, PVT1 exon 1a fused to NSMCE2 exon 3 in patient 1, PVT1 exon 3a to NSMCE2 exon 4 in patient 2, and PVT1 exon 4b to NSMCE2 exon 4 in K562. Patient 2 was identified from the screening of an additional 50 patients with AML or MDS by RT-PCR. As for BF104016-NSMCE2 fusion transcript, BF104016 exon 1 fused to NSMCE2 exon 6 in HL60 harboring hsr and dmins. BF104016 is located inside the CCDC26 gene, sharing the same exon of CCDC26. In patient 1, fluorescence in situ hybridization identified the amplification of 5'PVT1 on the mars and dmins, and the amplification of NSMCE2 only on the mars. In HL60, PVT1 and NSMCE2 were amplified on dmins. Although real-time quantitative PCR showed the amplification of the aberrant NSMCE2 chimeric transcripts, western blot analysis demonstrated the depletion of NSMCE2 protein. High-resolution oligonucleotide array analysis demonstrated two amplicons at 8q24 commonly found in patient 1 and HL60. Furthermore, the coding directions of these three fusion genes were different at the 8q24; NSMCE2 and PVT1 are transcribed from centromere toward telomere, opposite to the direction of transcription of the CCDC26 gene. These results suggest that the chimeric formation of these genes were caused by chromothripsis. PVT1 and CCDC26 were known as large intervening non-coding RNAs, both of which were considered to be associated with the oncogenesis. NSMCE2 is known as a small ubiquitin-like modifier (SUMO) E3 ligase and is required for DNA repair. Functional analysis of NSMCE2 chimeric transcripts with PVT1 or CCDC26 will contribute to understanding of the leukemogenesis in AML harboring 8q24 amplicons. Disclosures:Taniwaki:Novartis: Honoraria.

Genetic Mechanisms Of Cytarabine, Etoposide and Daunorubicin Resistance In Pediatric Acute Leukemias

IntroductionDrug resistance is a major problem in chemotherapy of leukemia. Several mechanisms of this phenomenon have been identified, but the underlying genomic changes are still poorly understood. Lack of drug sensitivity arises from a complex range of molecular events, which ultimately result in the blasts escaping death. Analysis of the gene expression profiles of cancer cells in correlation with in vitro cytotoxicity assay may define mechanisms of sensitivity and resistance to specific drugs. OBJECTIVE: To define and compare whole-genome responses to cytarabine (Ara-C), etoposide (VP16) and daunorubicin (DNR) in pediatric patients diagnosed with acute leukemias, and to explain in vitro chemoresistance phenotype of leukemic blasts. MethodsIn order to determine the ex vivo drug resistance profile, MTT cytotoxicity assay was performed on mononuclear cells obtained from 51 patients with ALL and 16 patients with AML. Gene expression profiles were prepared on the basis of cRNA hybridization to oligonucleotide arrays of the human genome (Affymetrix). Hierarchical clustering, assignment location and biological function were performed during the correlation analysis for identified probe sets. Verification of the relative expression level of genes (EGR1, GATA2, RUFY3, LDHA, DUSP2, BIN2, ICAM3, TTC28) was carried out by RT-qPCR in the study group and in an independent group of 53 patients. ResultsGenetic expression profiles were identified, including those appropriate for ALL and AML: 181 and 106 genes for Ara-C, 274 and 314 for VP16, 146 and 495 for the DNR. For each of the drugs, a characteristic group of genes or processes that are responsible for the lack of sensitivity, were identified: (1) for Ara-C: overexpression of genes responsible for removing the drug from the cells, as well as changes in the nucleic acid metabolic process, especially transcription from RNA polymerase II promoter (eg. ZBTB16, FOSB, NFATC1, ZNF518B, PHF20L1 and RUFY); (2) for VP16: changes in expression level of genes involved in the regulation of mRNA transcription and DNA metabolism genes, including those controlling replication as well as those belonging to the double helix damage repair enzymes and genes participating in post-transcriptional mRNA splicing (eg. TOP2B, CSNK1E, BRIP1, ATR, MSH3 and MSH6); (3) for DNR: differentiated expression of factors involved in the replication and transcription processes, increased expression of kinases and intensification of the DNA repair processes (eg. ATF2, GATA2, TOX, RUNX3, MNDA, ST18, TFDP1, NFE2,SOX11 and PAX5). For each profile several common genes, such as: AGAP1, PRKCH, RAB31, BCL2A1, GCA, HLA-DRA, HLA-DPA1, IL8, RGS10, CEBPD, CLEC2, ANXA1, PLEK, S100A8, SLC, CXCL2, SOX, BTG, DEFA4 and TPD52, were identified. Pathway and functional gene ontology analysis showed that several features, independent of the initial type of leukemia cells and pattern of resistance include: overexpression of chemokines and hydrolases, increase in the expression of genes responsible for the maintenance of chromatin architecture, overexpression of anti-apoptotic genes, decrease in expression level of genes that promote apoptosis, decrease in gene expression of Wnt signaling pathway, down regulation of the expression of transcription factors, changes in the expression level of genes associated with the activation of B and T lymphocytes, differences in expression of genes responsible for the cell surface receptor linked signal transduction and intracellular signaling cascade. ConclusionsThis analysis showed that the mechanism of response to drugs is significantly genetically determined. Predictive sets of marker genes and functional groups for simultaneous assessment of the sensitivity to these 3 drugs, were identified. Many of these changes converge and ultimately lead to avoidance of apoptosis and further over-proliferation of cancer cells. This could also suggest that targeting these pathways as potential pharmacogenetics and therapeutic candidates may be useful for improving treatment outcomes in pediatric acute leukemias. This could also suggest that targeting these pathways as potential pharmacogenetics and therapeutic candidates may be useful for improving treatment outcomes. Acknowledgments:This study was supported by Grant from the National Science Centre No. DEC-2011/03/D/NZ5/05749. Disclosures:No relevant conflicts of interest to declare.

Array-Based Comparative Genomic Hybridization (aCGH) In Myelofibrosis Reveals Divergent Genetic Copy Number Alterations Among Patients With Post-Essential Thrombocythemia-Myelofibrosis/Post-Polycythemia Rubra Vera-Myelofibrosis (PET-MF and PPV-MF) and Patients With Idiopathic Myelofibrosis

BackgroundMyelofibrosis (MF) is a myeloid neoplasm marked by clonal proliferation of myeloid cells with marrow fibrosis and subsequent clinical findings. Often, MF is noted on original bone marrow biopsy, and is termed idiopathic myelofibrosis (iMF). Many patients with ET and PV later develop MF, and this is termed, PET-MF, or PPV-MF. The JAK2V617F mutation is seen in most PV patients (95%) and no more than 50% of patients with ET and MF. There are neither existing published data clearly heralding the JAK2V617F mutation frequency in PET/PPV-MF, nor is there clear evidence that PET/PPV-MF and iMF can be distinguished via mutations within the JAK-STAT pathway, or other frequently seen mutations in MF (eg ASLX1, TET2, EZH2, etc). MethodsArray-based comparative genomic hybridization (aCGH) was performed on genomic DNA extracted from marrow aspirate using an Agilent 180K oligonucleotide array platform in order to discover recurrent and cooperating genetic aberrations, and gain insight into the hierarchy of molecular pathogenesis of fibrosis. BM aspirate from 11 pts with symptomatic MF were analyzed. Copy number (CN) alterations were compared to a reference set and mapped to functional genes. ResultsaCGH yielded CN losses in a divergent set of genes among patients with PET/PPV-MF (8 total patients) and iMF (3 total patients). CN losses were detected in RET (4/8), CIC (5/8), CD79a (5/8), NFKB (6/8), and MLLT (3/8), and CN gain of TRIP11 (2/8) more commonly in PET/PPV-MF, and only rarely in iMF. ASXL1, IGF2, INS, MAFB, and TOP1 were aberrated with CN loss in iMF in 67% of patients, and completely absent in PET/PPV-MF. In addition, there was suggestion of differential CN alteration between patients with clear history of PET-MF or PPV-MF. Aberrancy within CRTC1, ELL, RECQL4 was seen exclusively in PET-MF, and not seen in PPV-MF or iMF; ELN aberrancy was seen exclusively in PPV-MF. The cohort will be updated with additional patients at the meeting. ConclusionsMyelofibrosis is a complicated disorder characterized by a wide amalgam of pathologic and clinical findings. Mutational analysis, as of yet, has not clearly provided a means to differentiate or subclassify myelofibrosis. While CN variations seen in this small cohort occurred in some genes thought to be involved in the pathogenesis of fibrosis (eg ELN, IGF2, INS), implicated in tumorigenesis (eg RECQL4, CD79a, ASXL1, MLLT etc), or associated with hematopoiesis (eg MAFB, NFKB), the relationship between the abnormalities seen in this analysis and the biology of these cases of MF is not clear. Nonetheless, this preliminary analysis provides some insight into the possible use of aCGH as a tool to help molecularly classify MF. Disclosures:Hockett:CombiMatrix Mol Diagn: Employment.

Gene expressions changes in bronchial epithelial cells: Markers for respiratory sensitizers and exploration of the NRF2 pathway

For the classification of respiratory sensitizing chemicals, no validated in vivo nor in vitro tests are currently available. In this study, we evaluated whether respiratory sensitizers trigger specific signals in human bronchial epithelial (BEAS-2B) cells at the level of the transcriptome. The cells were exposed during 6, 10, and 24h to 4 respiratory sensitizers and 6 non-respiratory sensitizers (3 skin sensitizers and 3 respiratory irritants) at a concentration inducing 20% cell viability loss after 24h. Changes in gene expression were evaluated using Agilent Whole Human Genome 4×44K oligonucleotide arrays. A limited number of 11 transcripts could be identified as potential biomarkers to identify respiratory sensitizers. Three of these transcripts are associated to immune system processes (HSPA5, UPP1, and SEPRINE1). In addition, the transcriptome was screened for transcripts that are differentially expressed compared to vehicle control for each chemical. The results show that the NRF2-mediated oxidative stress response is activated in the cell line after stimulation with all of the chemicals that were selected in our study, and that – at the level of gene expression – this pathway shows no potential to discriminate between any of the three compound groups: respiratory sensitizers, skin sensitizers, or electrophilic respiratory irritants.