32 research outputs found
DNA copy number changes in high-grade malignant peripheral nerve sheath tumors by array CGH
<p>Abstract</p> <p>Background</p> <p>Malignant peripheral nerve sheath tumors (MPNSTs) are rare and highly aggressive soft tissue tumors showing complex chromosomal aberrations. In order to identify recurrent chromosomal regions of gain and loss, and thereby novel gene targets of potential importance for MPNST development and/or progression, we have analyzed DNA copy number changes in seven high-grade MPNSTs using microarray-based comparative genomic hybridization (array CGH).</p> <p>Results</p> <p>Considerable more gains than losses were observed, and the most frequent minimal recurrent regions of gain included 1q24.1-q24.2, 1q24.3-q25.1, 8p23.1-p12, 9q34.11-q34.13 and 17q23.2-q25.3, all gained in five of seven samples. The 17q23.2-q25.3 region was gained in all five patients with poor outcome and not in the two patients with disease-free survival. cDNA microarray analysis and quantitative real-time reverse transcription PCR were used to investigate expression of genes located within these regions. The gene lysyl oxidase-like 2 (<it>LOXL2</it>) was identified as a candidate target for the 8p23.1-p12 gain. Within 17q, the genes topoisomerase II-α (<it>TOP2A</it>), ets variant gene 4 (E1A enhancer binding protein, <it>E1AF</it>) (<it>ETV4</it>) and baculoviral IAP repeat-containing 5 (survivin) (<it>BIRC5</it>) showed increased expression in all samples compared to two benign tumors. Increased expression of these genes has previously been associated with poor survival in other malignancies, and for <it>TOP2A</it>, in MPNSTs as well. In addition, we have analyzed the expression of five micro RNAs located within the 17q23.2-q25.3 region, but none of them showed high expression levels compared to the benign tumors.</p> <p>Conclusion</p> <p>Our study shows the potential of using DNA copy number changes obtained by array CGH to predict the prognosis of MPNST patients. Although no clear correlations between the expression level and patient outcome were observed, the genes <it>TOP2A</it>, <it>ETV4 </it>and <it>BIRC5 </it>are interesting candidate targets for the 17q gain associated with poor survival.</p
Global Gene Expression Profiling of Human Osteosarcomas Reveals Metastasis-Associated Chemokine Pattern
Global gene expression analysis was performed on a panel of 23 osteosarcoma samples of primary and metastatic origin using the Applied Biosystems Gene Expression Array System. When comparing the primary tumours with the metastases, we found a significantly increased expression of genes involved in immunological processes, for example coding for cytokines and chemokines, in the metastatic samples. In addition, a comparison of the gene expression in primary samples from patients with or without metastases demonstrated that patients who later developed metastases had high expression of the chemokine (C-X-C motif) receptor 4 (CXCR4), similar to the metastatic samples, suggesting that these signal molecules play an important role in promoting metastasis. Increased knowledge of mechanisms and interactions between specified molecular signalling pathways in osteosarcomas could lead to a more rational strategy for development of targeted therapy
Evaluation of commercial DNA and RNA extraction methods for high-throughput sequencing of FFPE samples
<div><p>Nucleic acid material of adequate quality is crucial for successful high-throughput sequencing (HTS) analysis. DNA and RNA isolated from archival FFPE material are frequently degraded and not readily amplifiable due to chemical damage introduced during fixation. To identify optimal nucleic acid extraction kits, DNA and RNA quantity, quality and performance in HTS applications were evaluated. DNA and RNA were isolated from five sarcoma archival FFPE blocks, using eight extraction protocols from seven kits from three different commercial vendors. For DNA extraction, the truXTRAC FFPE DNA kit from Covaris gave higher yields and better amplifiable DNA, but all protocols gave comparable HTS library yields using Agilent SureSelect XT and performed well in downstream variant calling. For RNA extraction, all protocols gave comparable yields and amplifiable RNA. However, for fusion gene detection using the Archer FusionPlex Sarcoma Assay, the truXTRAC FFPE RNA kit from Covaris and Agencourt FormaPure kit from Beckman Coulter showed the highest percentage of unique read-pairs, providing higher complexity of HTS data and more frequent detection of recurrent fusion genes. truXTRAC simultaneous DNA and RNA extraction gave similar outputs as individual protocols. These findings show that although successful HTS libraries could be generated in most cases, the different protocols gave variable quantity and quality for FFPE nucleic acid extraction. Selecting the optimal procedure is highly valuable and may generate results in borderline quality specimens.</p></div
Variants identified in the samples extracted with different DNA extraction methods.
<p>(A-D) Venn diagram showing the distribution of variants between DNA extraction methods in four different FFPE samples, SARC1-4. Variants were detected by MuTect and Strelka, using an artificial control as normal. The data was filtered on exonic variants with allele frequency > 5% and coverage > 100x, being present at < 1% in the normal population (ExAC database).</p
Yield of final DNA HTS libraries obtained using DNA extracted with different extraction kits.
<p>Yield of final DNA HTS libraries obtained using DNA extracted with different extraction kits.</p
Modulation of the osteosarcoma expression phenotype by microRNAs.
BACKGROUND: Osteosarcomas are the most common primary malignant tumors of bone and show multiple and complex genomic aberrations. miRNAs are non-coding RNAs capable of regulating gene expression at the post transcriptional level, and miRNAs and their target genes may represent novel therapeutic targets or biomarkers for osteosarcoma. In order to investigate the involvement of miRNAs in osteosarcoma development, global microarray analyses of a panel of 19 human osteosarcoma cell lines was performed. PRINCIPAL FINDINGS: We identified 177 miRNAs that were differentially expressed in osteosarcoma cell lines relative to normal bone. Among these, miR-126/miR-126*, miR-142-3p, miR-150, miR-223, miR-486-5p and members of the miR-1/miR-133a, miR-144/miR-451, miR-195/miR-497 and miR-206/miR-133b clusters were found to be downregulated in osteosarcoma cell lines. All miRNAs in the paralogous clusters miR-17-92, miR-106b-25 and miR-106a-92 were overexpressed. Furthermore, the upregulated miRNAs included miR-9/miR-9*, miR-21*, miR-31/miR-31*, miR-196a/miR-196b, miR-374a and members of the miR-29 and miR-130/301 families. The most interesting inversely correlated miRNA/mRNA pairs in osteosarcoma cell lines included miR-9/TGFBR2 and miR-29/p85α regulatory subunit of PI3K. PTEN mRNA correlated inversely with miR-92a and members of the miR-17 and miR-130/301 families. Expression profiles of selected miRNAs were confirmed in clinical samples. A set of miRNAs, miR-1, miR-18a, miR-18b, miR-19b, miR-31, miR-126, miR-142-3p, miR-133b, miR-144, miR-195, miR-223, miR-451 and miR-497 was identified with an intermediate expression level in osteosarcoma clinical samples compared to osteoblasts and bone, which may reflect the differentiation level of osteosarcoma relative to the undifferentiated osteoblast and fully differentiated normal bone. SIGNIFICANCE: This study provides an integrated analysis of miRNA and mRNA in osteosarcoma, and gives new insight into the complex genetic mechanisms of osteosarcoma development and progression
Overview of nucleic acids extracted with the different extraction kits from commercial vendors.
<p>Overview of nucleic acids extracted with the different extraction kits from commercial vendors.</p
Yield and amplifiability of extracted DNA and RNA.
<p>(A) Average total amount of DNA. (B) Average total amount of RNA. (C) Amplifiable DNA quantified with the FFPE QC kit from Illumina. (D) Amplifiable RNA quantified with the PreSeq QC assay from ArcherDx. The average total amount and average delta Ct values for the different samples and extraction methods are shown. The standard deviation is shown as vertical bars. Methods with significant differences in yield are marked as connected with horizontal bars (p < 0.05). Samples with Ct values > 45 were censored giving a delta Ct of 21, shown as #.</p
Yield of Archer FusionPlex Sarcoma Assay libraries obtained using RNA extracted with different extraction kits.
<p>Yield of Archer FusionPlex Sarcoma Assay libraries obtained using RNA extracted with different extraction kits.</p