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Normalization of array-CGH data: influence of copy number imbalances
Johan Staaf, G?ran J?nsson, Markus Ringnér, Johan Vallon-Christersson
BMC Genomics , 2007, DOI: 10.1186/1471-2164-8-382
Abstract: Here we demonstrate that copy number imbalances correlate with intensity in array-CGH data thereby causing problems for conventional normalization methods. We propose a strategy to circumvent these problems by taking copy number imbalances into account during normalization, and we test the proposed strategy using several data sets from the analysis of cancer genomes. In addition, we show how the strategy can be applied to conveniently define adaptive sample-specific boundaries between balanced copy number, losses, and gains to facilitate management of variation in tissue heterogeneity when calling copy number changes.We highlight the importance of considering copy number imbalances during normalization of array-CGH data, and show how failure to do so can deleteriously affect data and hamper interpretation.Microarray-based techniques for genome-wide investigation of copy number aberrations (CNAs) have recently gained much attention. Initially employing arrays developed for gene expression analysis [1], or low-density arrays produced from large-insert genomic clones such as bacterial artificial chromosomes (BACs) [2], the application has evolved rapidly. Currently, specialized high-density arrays with oligonucleotide probes or probes derived from BAC clones are predominately used. Two-channel array-based comparative genomic hybridization (aCGH) is a direct successor to conventional metaphase CGH [3]. In both cases, DNA from two samples are differentially labeled with fluorescent dyes and co-hybridized to immobilized genomic capture probes. By use of aCGH, DNA derived from tumor tissue can be compared with reference DNA, e.g., normal whole blood DNA, and genomic imbalances can effectively be investigated. The main advantage of aCGH over conventional CGH is the increased resolution achieved by microarrays with a large number of individual probes, routinely up to hundreds of thousands, covering the entire genome [4]. The power of aCGH has been demonstrated in tumor studi
Non-coding antisense transcription detected by conventional and single-stranded cDNA microarray
Johan Vallon-Christersson, Johan Staaf, Anders Kvist, Patrik Medstrand, ?ke Borg, Carlos Rovira
BMC Genomics , 2007, DOI: 10.1186/1471-2164-8-295
Abstract: Up to 88% of expressed protein coding loci displayed concurrent expression from the complementary strand. Antisense transcription is cell specific and showed a strong tendency to be positively correlated to the expression of the sense counterparts. Even if their expression is wide-spread, detected antisense signals seem to have a limited distorting effect on sense profiles obtained with double-stranded probes.Antisense transcription in humans can be far more common than previously estimated. However, it has limited influence on expression profiles obtained with conventional cDNA probes. This can be explained by a biological phenomena and a bias of the technique: a) a co-ordinate sense and antisense expression variation and b) a bias for sense-hybridization to occur with more efficiency, presumably due to variable exonic overlap between antisense transcripts.Non-coding RNAs have recently been reported as more common, more diverse, and accredited more important functions than previously anticipated [1-3]. Among the most abundant non-coding transcripts, there is a group called natural antisense transcripts (NATs) that carries regions of perfect complementarity to protein coding (sense) RNAs [4-7]. In silico studies of available transcript sequence data have found that up to 24% of human protein coding loci also encode cis-NATs [8,9]. However, antisense transcripts tend to be poly(A) negative and nuclear localized [10]. If this is true, the abundance of NATs (cis and trans) may be higher yet, since nuclear non-polyadenylated transcripts are underrepresented in transcript sequence databases.This fact may have important implications for researchers, not only because of their potential biological function but they may also turn out to be influential on the interpretation of large experimental data sets. For instance, the cDNA microarray technique has been used in genome-wide expression studies to address basic questions about gene function and in the pursuit of a more prec
Genomic and Transcriptional Alterations in Lung Adenocarcinoma in Relation to EGFR and KRAS Mutation Status
Maria Planck, Karolina Edlund, Johan Botling, Patrick Micke, Sofi Isaksson, Johan Staaf
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0078614
Abstract: Introduction In lung adenocarcinoma, the mutational spectrum is dominated by EGFR and KRAS mutations. Improved knowledge about genomic and transcriptional alterations in and between mutation-defined subgroups may identify genes involved in disease development or progression. Methods Genomic profiles from 457 adenocarcinomas, including 113 EGFR-mutated, 134 KRAS-mutated and 210 EGFR and KRAS-wild type tumors (EGFRwt/KRASwt), and gene expression profiles from 914 adenocarcinomas, including 309 EGFR-mutated, 192 KRAS-mutated, and 413 EGFRwt/KRASwt tumors, were assembled from different repositories. Genomic and transcriptional differences between the three mutational groups were analyzed by both supervised and unsupervised methods. Results EGFR-mutated adenocarcinomas displayed a larger number of copy number alterations and recurrent amplifications, a higher fraction of total loss-of-heterozygosity, higher genomic complexity, and a more distinct expression pattern than EGFR-wild type adenocarcinomas. Several of these differences were also consistent when the three mutational groups were stratified by stage, gender and smoking status. Specific copy number alterations were associated with mutation status, predominantly including regions of gain with the highest frequency in EGFR-mutated tumors. Differential regions included both large and small regions of gain on 1p, 5q34-q35.3, 7p, 7q11.21, 12p12.1, 16p, and 21q, and losses on 6q16.3-q21, 8p, and 9p, with 20-40% frequency differences between the mutational groups. Supervised gene expression analyses identified 96 consistently differentially expressed genes between the mutational groups, and together with unsupervised analyses these analyses highlighted the difficulty in broadly resolving the three mutational groups into distinct transcriptional entities. Conclusions We provide a comprehensive overview of the genomic and transcriptional landscape in lung adenocarcinoma stratified by EGFR and KRAS mutations. Our analyses suggest that the overall genomic and transcriptional landscape of lung adenocarcinoma is affected, but only to a minor extent, by EGFR and KRAS mutation status.
GOBO: Gene Expression-Based Outcome for Breast Cancer Online
Markus Ringnér,Erik Fredlund,Jari H?kkinen,?ke Borg,Johan Staaf
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0017911
Abstract: Microarray-based gene expression analysis holds promise of improving prognostication and treatment decisions for breast cancer patients. However, the heterogeneity of breast cancer emphasizes the need for validation of prognostic gene signatures in larger sample sets stratified into relevant subgroups. Here, we describe a multifunctional user-friendly online tool, GOBO (http://co.bmc.lu.se/gobo), allowing a range of different analyses to be performed in an 1881-sample breast tumor data set, and a 51-sample breast cancer cell line set, both generated on Affymetrix U133A microarrays. GOBO supports a wide range of applications including: 1) rapid assessment of gene expression levels in subgroups of breast tumors and cell lines, 2) identification of co-expressed genes for creation of potential metagenes, 3) association with outcome for gene expression levels of single genes, sets of genes, or gene signatures in multiple subgroups of the 1881-sample breast cancer data set. The design and implementation of GOBO facilitate easy incorporation of additional query functions and applications, as well as additional data sets irrespective of tumor type and array platform.
Landscape of somatic allelic imbalances and copy number alterations in HER2-amplified breast cancer
Johan Staaf, G?ran J?nsson, Markus Ringnér, Bo Baldetorp, ?ke Borg
Breast Cancer Research , 2011, DOI: 10.1186/bcr3075
Abstract: High-density whole genome array-based comparative genomic hybridization (aCGH) and single nucleotide polymorphism (SNP) array data from 260 HER2-amplified breast tumors or cell lines, and 346 HER2-negative breast cancers with molecular subtype information were assembled from different repositories. Copy number alteration (CNA), loss-of-heterozygosity (LOH), copy number neutral allelic imbalance (CNN-AI), subclonal CNA and patterns of tumor DNA ploidy were analyzed using bioinformatical methods such as genomic identification of significant targets in cancer (GISTIC) and genome alteration print (GAP). The patterns of tumor ploidy were confirmed in 338 unrelated breast cancers analyzed by DNA flow cytometry with concurrent BAC aCGH and gene expression data.A core set of 36 genomic regions commonly affected by copy number gain or loss was identified by integrating results with a previous study, together comprising > 400 HER2-amplified tumors. While CNN-AI frequency appeared evenly distributed over chromosomes in HER2-amplified tumors, not targeting specific regions and often < 20% in frequency, the occurrence of LOH was strongly associated with regions of copy number loss. HER2-amplified and HER2-negative tumors stratified by molecular subtypes displayed different patterns of LOH and CNN-AI, with basal-like tumors showing highest frequencies followed by HER2-amplified and luminal B cases. Tumor aneuploidy was strongly associated with increasing levels of LOH, CNN-AI, CNAs and occurrence of subclonal copy number events, irrespective of subtype. Finally, SNP data from individual tumors indicated that genomic amplification in general appears as monoallelic, that is, it preferentially targets one parental chromosome in HER2-amplified tumors.We have delineated the genomic landscape of CNAs, amplifications, LOH, and CNN-AI in HER2-amplified breast cancer, but also demonstrated a strong association between different types of genomic aberrations and tumor aneuploidy irrespectiv
The gene expression landscape of breast cancer is shaped by tumor protein p53 status and epithelial-mesenchymal transition
Erik Fredlund, Johan Staaf, Juha K Rantala, Olli Kallioniemi, ?ke Borg, Markus Ringnér
Breast Cancer Research , 2012, DOI: 10.1186/bcr3236
Abstract: Modules of highly connected genes were extracted from a gene co-expression network that was constructed based on Pearson correlation, and module activities were then calculated using a pathway activity score. Functional annotations of modules were experimentally validated with an siRNA cell spot microarray system using the KPL-4 breast cancer cell line, and by using gene expression data from functional studies. Modules were derived using gene expression data representing 1,608 breast cancer samples and validated in data sets representing 971 independent breast cancer samples as well as 1,231 samples from other cancer forms.The initial co-expression network analysis resulted in the characterization of eight tightly regulated gene modules. Cell cycle genes were divided into two transcriptional programs, and experimental validation using an siRNA screen showed different functional roles for these programs during proliferation. The division of the two programs was found to act as a marker for tumor protein p53 (TP53) gene status in luminal breast cancer, with the two programs being separated only in luminal tumors with functional p53 (encoded by TP53). Moreover, a module containing fibroblast and stroma-related genes was highly expressed in fibroblasts, but was also up-regulated by overexpression of epithelial-mesenchymal transition factors such as transforming growth factor beta 1 (TGF-beta1) and Snail in immortalized human mammary epithelial cells. Strikingly, the stroma transcriptional program related to less malignant tumors for luminal disease and aggressive lymph node positive disease among basal-like tumors.We have derived a robust gene expression landscape of breast cancer that reflects known subtypes as well as heterogeneity within these subtypes. By applying the modules to TP53-mutated samples we shed light on the biological consequences of non-functional p53 in otherwise low-proliferating luminal breast cancer. Furthermore, as in the case of the stroma module
Tumor Genome Wide DNA Alterations Assessed by Array CGH in Patients with Poor and Excellent Survival Following Operation for Colorectal Cancer
Kristina K. Lagerstedt,Johan Staaf,G?ran J?nsson,Elisabeth Hansson
Cancer Informatics , 2007,
Abstract: Genome wide DNA alterations were evaluated by array CGH in addition to RNA expression profiling in colorectal cancer from patients with excellent and poor survival following primary operations.DNA was used for CGH in BAC and cDNA arrays. Global RNA expression was determined by 44K arrays. DNA and RNA from tumor and normal colon were used from cancer patients grouped according to death, survival or Dukes A, B, C and D tumor stage. Confi rmed DNA alterations in all Dukes A – D were judged relevant for carcinogenesis, while changes in Dukes C and D only were regarded relevant for tumor progression.Copy number gain was more common than loss in tumor tissue (p 0.01). Major tumor DNA alterations occurred in chromosome 8, 13, 18 and 20, where short survival included gain in 8q and loss in 8p. Copy number gains related to tumor progression were most common on chromosome 7, 8, 19, 20, while corresponding major losses appeared in chromosome 8. Losses at chromosome 18 occurred in all Dukes stages. Normal colon tissue from cancer patients displayed gains in chromosome 19 and 20. Mathematical Vector analysis implied a number of BAC-clones in tumor DNA with genes of potential importance for death or survival. The genomic variation in colorectal cancer cells is tremendous and emphasizes that BAC array CGH is presently more powerful than available statistical models to discriminate DNA sequence information related to outcome. Present results suggest that a majority of DNA alterations observed in colorectal cancer are secondary to tumor progression. Therefore, it would require an immense work to distinguish primary from secondary DNA alterations behind colorectal cancer.
Detailed Analysis of Focal Chromosome Arm 1q and 6p Amplifications in Urothelial Carcinoma Reveals Complex Genomic Events on 1q, and SOX4 as a Possible Auxiliary Target on 6p
Pontus Eriksson, Mattias Aine, Gottfrid Sj?dahl, Johan Staaf, David Lindgren, Mattias H?glund
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0067222
Abstract: Background Urothelial carcinoma shows frequent amplifications at 6p22 and 1q21–24. The main target gene at 6p22 is believed to be E2F3, frequently co-amplified with CDKAL1 and SOX4. There are however reports on 6p22 amplifications that do not include E2F3. Previous analyses have identified frequent aberrations occurring at 1q21–24. However, due to complex rearrangements it has been difficult to identify specific 1q21–24 target regions and genes. Methods We selected 29 cases with 6p and 37 cases with 1q focal genomic amplifications from 261 cases of urothelial carcinoma analyzed by array-CGH for high resolution zoom-in oligonucleotide array analyses. Genomic analyses were combined with gene expression data and genomic sequence analyses to characterize and fine map 6p22 and 1q21–24 amplifications. Results We show that the most frequently amplified gene at 6p22 is SOX4 and that SOX4 can be amplified and overexpressed without the E2F3 or CDKAL1 genes being included in the amplicon. Hence, our data point to SOX4 as an auxiliary amplification target at 6p22. We further show that at least three amplified regions are observed at 1q21–24. Copy number data, combined with gene expression data, highlighted BCL9 and CHD1L as possible targets in the most proximal region and MCL1, SETDB1, and HIF1B as putative targets in the middle region, whereas no obvious targets could be determined in the most distal amplicon. We highlight enrichment of G4 quadruplex sequence motifs and a high number of intraregional sequence duplications, both known to contribute to genomic instability, as prominent features of the 1q21–24 region. Conclusions Our detailed analyses of the 6p22 amplicon suggest SOX4 as an auxiliary target gene for amplification. We further demonstrate three separate target regions for amplification at 1q21–24 and identified BCL9, CHD1L, and MCL1, SETDB1, and HIF1B as putative target genes within these regions.
Normalization of Illumina Infinium whole-genome SNP data improves copy number estimates and allelic intensity ratios
Johan Staaf, Johan Vallon-Christersson, David Lindgren, Gunnar Juliusson, Richard Rosenquist, Mattias H?glund, ?ke Borg, Markus Ringnér
BMC Bioinformatics , 2008, DOI: 10.1186/1471-2105-9-409
Abstract: We demonstrate an asymmetry in the detection of the two alleles for each SNP, which deleteriously influences both allelic proportions and copy number estimates. The asymmetry is caused by a remaining bias between the two dyes used in the Infinium II assay after using the normalization method in Illumina's proprietary software (BeadStudio). We propose a quantile normalization strategy for correction of this dye bias. We tested the normalization strategy using 535 individual hybridizations from 10 data sets from the analysis of cancer genomes and normal blood samples generated on Illumina Infinium II 300 k version 1 and 2, 370 k and 550 k BeadChips. We show that the proposed normalization strategy successfully removes asymmetry in estimates of both allelic proportions and copy numbers. Additionally, the normalization strategy reduces the technical variation for copy number estimates while retaining the response to copy number alterations.The proposed normalization strategy represents a valuable tool that improves the quality of data obtained from Illumina Infinium arrays, in particular when used for LOH and copy number variation studies.Genomic copy number alterations (CNA) and allelic imbalances are common events in the development of cancer and certain genetic disorders [1,2]. The introduction of whole genome genotyping (WGG) arrays based on single nucleotide polymorphism (SNP) genotyping [3,4] allows for combined DNA copy number (SNP-CGH) and loss-of-heterozygosity (LOH) analysis at high resolution [5]. Currently, two major SNP array platforms are in use, Affymetrix GeneChip arrays [6] and Illumina BeadChips [7]. The Infinium assay for Illumina BeadChips is based on allele-specific hybridization coupled with primer extension of genomic DNA using primers directly surrounding the SNP on randomly ordered bead arrays [4]. The Infinium assay has been further developed into allele-specific single base extension using two color labeling with the Cy3 and Cy5 fluorescent dy
Segmentation-based detection of allelic imbalance and loss-of-heterozygosity in cancer cells using whole genome SNP arrays
Johan Staaf, David Lindgren, Johan Vallon-Christersson, Anders Isaksson, Hanna G?ransson, Gunnar Juliusson, Richard Rosenquist, Mattias H?glund, ?ke Borg, Markus Ringnér
Genome Biology , 2008, DOI: 10.1186/gb-2008-9-9-r136
Abstract: Cancer development involves genomic aberrations such as gene copy number gains or losses and allele-specific imbalances [1]. Array-based comparative genomic hybridization (aCGH) [2] has, since its introduction, become a widely adopted tool for identification and quantification of DNA copy number alterations (CNAs) in tumor genomes [3]. The introduction of whole genome genotyping (WGG) arrays based on single nucleotide polymorphism (SNP) genotyping [4,5] allows for combined DNA copy number (SNP-CGH) and loss-of-heterozygosity (LOH) analysis at high resolution [6]. Current SNP arrays can genotype several hundreds of thousands of SNPs simultaneously. LOH analysis has in the past been a vital tool for the discovery of chromosomal regions harboring tumor-suppressor genes when inactivated by the classic mechanism of allelic loss [7]. LOH occurs as a consequence of reduction in copy number in a diploid genome but it may also appear as copy number-neutral LOH resulting from uniparental disomy or mitotic recombination events. The latter type of changes is not detectable by conventional aCGH platforms. Moreover, increases in copy number due to, for example, mono-allelic amplification may falsely be detected as LOH [8]. Therefore, by combining LOH and copy number analysis, regions of LOH derived from either copy number loss or neutral events may be identified. Conventional LOH studies compare the genotype of a tumor to its matched constitutional genotype. Current generations of WGG arrays have been reported to provide sufficiently high marker density to infer regions of LOH by the absence of heterozygous loci without the use of a matched control [9]. However, the increased marker density disqualifies the assumption of independence between allele calls of adjacent SNPs due to linkage disequilibrium. This may lead to detection of non-tumor specific homozygous regions based solely on the marker density. In the absence of a matched normal, haplotype correction methods may be requi
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