Copy Number Aberrations Regions Research Articles

CNAnova: a new approach for finding recurrent copy number abnormalities in cancer SNP microarray data

The current generation of single nucleotide polymorphism (SNP) arrays allows measurement of copy number aberrations (CNAs) in cancer at more than one million locations in the genome in hundreds of tumour samples. Most research has focused on single-sample CNA discovery, the so-called segmentation problem. The availability of high-density, large sample-size SNP array datasets makes the identification of recurrent copy number changes in cancer, an important issue that can be addressed using the cross-sample information. We present a novel approach for finding regions of recurrent copy number aberrations, called CNAnova, from Affymetrix SNP 6.0 array data. The method derives its statistical properties from a control dataset composed of normal samples and, in contrast to previous methods, does not require segmentation and permutation steps. For rigorous testing of the algorithm and comparison to existing methods, we developed a simulation scheme that uses the noise distribution present in Affymetrix arrays. Application of the method to 128 acute lymphoblastic leukaemia samples shows that CNAnova achieves lower error rate than a popular alternative approach. We also describe an extension of the CNAnova framework to identify recurrent CNA regions with intra-tumour heterogeneity, present in either primary or relapsed samples from the same patients. The CNAnova package and synthetic datasets are available at http://www.compbio.group.cam.ac.uk/software.html.

Abstract 5258: Genomic characterization and targeted resequencing of high-risk neuroblastoma (the neuroblastoma TARGET)

Abstract Background: Neuroblastoma (NB) is a childhood malignancy of the peripheral nervous system. Although genomic changes (MYCN amplification, 1p LOH, 11q LOH, and hyperdiploidy) are associated with clinical outcome and help define a high-risk group, there are few genes known to be mutated in NB and no genomic aberrations predictive of relapse within this group. The NB TARGET (Therapeutically Applicable Research to Generate Effective Treatments) collaborative research initiative aims to discover novel therapeutic targets and genomic predictors of outcome in an unbiased systematic fashion. Methods: Whole genome copy number (CN) changes were assayed for using the Illumina HumanHap550 SNP array in 631 highly annotated NB specimens, and genes from regions of recurrent aberration were selected for resequencing. Relative CN was derived from absolute CN (OverUnder), and regions recurrently lost or gained were determined using a statistical test for significance across the sample set (GISTIC). Results: Low- and intermediate-risk NB cases showed recurrent whole chromosome (WC) gains of chromosomes 2, 7, 12, 13, and 17. In contrast, high-risk cases showed segmental gains in those same chromosomes (2p, 7q, 12q, 13q, and 17q). A similar pattern was found for CN losses: lower-risk cases had WC losses of 3, 4, 9, 11, 14, 19, and 21 (as well as 16) while high-risk cases had segmental losses of 3p, 4p, 9p, 11q, 14q, 19p, 19q, and 21q (as well as 1p and 5p). Candidate genes from within the regions of CN aberration were prioritized, and 117 genes and microRNAs were resequenced in 188 high-risk NB samples. Analysis to date of over 680,000 traces revealed 841 sequence variants that passed strict filtering criteria. Non-silent sequence variants were found in 79/117 genes (range of cases with variants per gene: 1-19). Ongoing resequencing of matched constitutional DNA revealed that 88% of variants were present in the germline, but putative somatic mutations were discovered in several candidate genes at low frequency (ANAPC5 and IGSF21 in 2/188 cases; ETS1, NCAM1, and NRAS in 1/188 cases), suggesting a low frequency of overall somatic mutation in this gene set. Conclusions: Although the same chromosomes have aberrant CN in both the lower-risk and high-risk groups, the type of aberration (WC vs. segmental) clearly differed, with the high-risk group showing segmental aberrations and the lower-risk group showing WC aberrations for all overlapping regions. This suggests differing mechanisms for genomic rearrangement and reveals potential loci of interest. The low frequency of somatic mutation found by targeted resequencing of genes selected from the large regions of genomic aberration underlines the need for unbiased whole genome sequencing approaches to discover mutated genes in NB. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5258.

CMDS: a population-based method for identifying recurrent DNA copy number aberrations in cancer from high-resolution data

DNA copy number aberration (CNA) is a hallmark of genomic abnormality in tumor cells. Recurrent CNA (RCNA) occurs in multiple cancer samples across the same chromosomal region and has greater implication in tumorigenesis. Current commonly used methods for RCNA identification require CNA calling for individual samples before cross-sample analysis. This two-step strategy may result in a heavy computational burden, as well as a loss of the overall statistical power due to segmentation and discretization of individual sample's data. We propose a population-based approach for RCNA detection with no need of single-sample analysis, which is statistically powerful, computationally efficient and particularly suitable for high-resolution and large-population studies. Our approach, correlation matrix diagonal segmentation (CMDS), identifies RCNAs based on a between-chromosomal-site correlation analysis. Directly using the raw intensity ratio data from all samples and adopting a diagonal transformation strategy, CMDS substantially reduces computational burden and can obtain results very quickly from large datasets. Our simulation indicates that the statistical power of CMDS is higher than that of single-sample CNA calling based two-step approaches. We applied CMDS to two real datasets of lung cancer and brain cancer from Affymetrix and Illumina array platforms, respectively, and successfully identified known regions of CNA associated with EGFR, KRAS and other important oncogenes. CMDS provides a fast, powerful and easily implemented tool for the RCNA analysis of large-scale data from cancer genomes.

Pattern-Selection Based Power Analysis and Discrimination of Low- and High-Grade Myelodysplastic Syndromes Study Using SNP Arrays

Copy Number Aberration (CNA) in myelodysplastic syndromes (MDS) study using single nucleotide polymorphism (SNP) arrays have been received increasingly attentions in the recent years. In the current study, a new Constraint Moving Average (CMA) algorithm is adopted to determine the regions of CNA regions first. In addition to large regions of CNA, using the proposed CMA algorithm, small regions of CNA can also be detected. Real-time Polymerase Chain Reaction (qPCR) results prove that the CMA algorithm presents an insightful discovery of both large and subtle regions. Based on the results of CMA, two independent applications are studied. The first one is power analysis for sample estimation. An accurate estimation of sample size needed for the desired purpose of an experiment will be important for effort-efficiency and cost-effectiveness. The power analysis is performed to determine the minimum sample size required for ensuring at least () detected regions statistically different from normal references. As expected, power increase with increasing sample size for a fixed significance level. The second application is the distinguishment of high-grade MDS patients from low-grade ones. We propose to calculate the General Variant Level (GVL) score to integrate the general information of each patient at genotype level, and use it as the unified measurement for the classification. Traditional MDS classifications usually refer to cell morphology and The International Prognostic Scoring System (IPSS), which belongs to the classification at the phenotype level. The proposed GVL score integrates the information of CNA region, the number of abnormal chromosomes and the total number of the altered SNPs at the genotype level. Statistical tests indicate that the high and low grade MDS patients can be well separated by GVL score, which appears to correlate better with clinical outcome than the traditional classification approaches using morphology and IPSS sore at the phenotype level.

Using Combined High Density SNP/CNV Arrays as a Clinical Karyotyping Tool in Myeloid Malignancies.

We have evaluated various types of single nucleotide polymorphism arrays (SNP-A) as a karyotyping platform in over 600 cases of bone marrow failure and various myeloid disorders including MDS, MDS/MPD, and AML and in over 360 controls. We have shown that SNP-A not only reliably confirms chromosome gains and losses identified by metaphase cytogenetics (MC) but also allows for detection of previously cryptic chromosomal lesions. Moreover, through the ability of combining copy number measurement with genotyping, SNP-A also enables detection of copy-neutral loss of heterozygosity (LOH), a lesion that cannot be recognized using traditional MC. We have previously shown that this type of lesion occurs frequently in myeloid disorders. Recently, ultra-high density SNP-A were introduced (Affymetrix 6.0 array platform) with modified chemistry and a combination of SNP (>906,600) and copy number variant probes (CNV >946,000) to allow for precise delineation of the location and size of submicroscopic chromosomal defects, copy-neutral LOH and germline CNVs. This technology will likely supplant previous SNP-A platforms in performance and opens a new era in clinical cytogenetic diagnostics. In this pilot trial, we applied the Affymetrix SNP 6.0 array to samples obtained at diagnosis from 193 patients (59 AML, 115 MDS, 19 MDS/MPD). For 95 samples, both Affymetrix 250K and 6.0 arrays were applied; 6.0 SNP-A allowed for more identification of 33 regions of copy number aberrations and 11 copy-neutral LOH, suggesting a higher resolution of this technology over 250K arrays. By MC, 43% (71/167) of patients had chromosomal aberrations including a complex karyotype; 48% (81/167) had a normal karyotype and 9% (15/167) showed no growth. By 6.0 SNP-A, aberrations were detected in 78% (150/193) of patients. Significantly, among the samples with either a normal karyotype or non-informative results, aberrations were found by SNP-A in 57% and 73%, respectively. In an illustrative case with a balanced translocation by MC, copy number changes were found at the junctions, indicating that the translocation breakpoints contained small deletions. Comparison of 6.0 SNP-A results with MC showed concordance for detection of MC-defined lesions. However, 9% (15/167) of defects were only detected by MC and could not be detected by SNP 6.0 array in patients with a low proportion of abnormal metaphases. Gains and losses detected by SNP-A 6.0 required a sensitivity threshold of 20% clonal cells. We identified recurrent lesions not previously reported as being CNV. These comprised 238 regions of loss, 257 regions of gain, and 97 regions of copy-neutral LOH. When feasible (or if indicated to resolve a discordance with MC or to exclude CNV), DNA from sorted CD3+ cells was used as a reference. Recurrent losses were located in 37%(71/193) patients at 5q, 7p, 7q, 13q, 17p, 20q, 1p, 12p, 3q and 4q. Recurrent gains in 28%(55/193) patients included 13q, 11p, 11q, 21q, 3p, 8p, 8q, 9q, 19p and 5p. Somatic copy-neutral LOH was identified in 20%(39/193) patients at 13q, 1p, 5q, 6p, 7q, 11q, 17q, 3p, 4q, and 12q. Homozygous deletion was observed in one patient with a 6 Mb loss at 5q34 (not visible on MC) within a heterozygous 5q11.2–5q35.3 deletion Copy neutral LOH was found in 29% (56/193) patients overall. AML patients appeared to have more frequent copy neutral LOH(34%) than MDS (24%), but not significant (P=0.2). The 5%(9/193) of patients had more than 2 chromosomes with copy-neutral LOH. Among the samples showing a normal karyotype, 20% (16/81) of patients showed copy -neutral LOH as the sole aberration without additional copy number aberrations. To our knowledge, this is the first systematic application of SNP-A 6.0 in a large cohort of patients with myeloid malignancies. Our results demonstrate that this technology complements, but does not replace conventional MC for identification of abnormalities in myeloid neoplasia.

Cytogenetic characterization and gene expression profiling of the trastuzumab-resistant breast cancer cell line JIMT-1

Resistance to the HER-2 targeting drug trastuzumab can be observed clinically, but the lack of suitable experimental models hampers studies of resistance mechanisms. We characterized a HER-2–positive carcinoma cell line (JIMT-1) derived from a 62-year-old breast cancer patient which was clinically resistant to trastuzumab. Multicolor fluorescence in situ hybridization revealed a complex hyperdiploid karyotype with numerous marker chromosomes and unbalanced translocations. Comparative genomic hybridization (CGH) revealed numerous regions of copy number aberration (CNA). Further analysis by array CGH identified 27 regions of CNA (16 amplified, 11 deleted). Thirty-eight percent of the genes in the amplified regions were overexpressed, compared to only 9% in regions of normal copy number ratios (CNR). Accordingly, 26% of the genes in the deleted regions were underexpressed, compared to 10% in regions of normal CNR. Most amplified and overexpressed genes were located on chromosome 1 as well as on 8q, 12q14.1, 17q11∼q21, and 20q13. In 17q11∼q21, we identified two separate amplicons, the HER-2 amplicon and a previously unreported amplicon at 17q21.31. Several aberrant genes are implicated in cancer development (e.g., JUN, CDK4, and SLUG protooncogenes, as well as the drug/hormone–metabolizing genes GSTM1 and CYP24). We conclude that cytogenetic and expression profiling of JIMT-1 revealed several new features that need further characterization and may shed light on trastuzumab resistance.

Prognostic value of genomic alterations in head and neck squamous cell carcinoma detected by comparative genomic hybridisation.

A total of 45 primary head and neck squamous cell carcinomas were analysed by comparative genomic hybridisation to identify regions of chromosomal deletion and gain. Multiple regions of copy number aberration were identified including gains affecting chromosomes 3q, 8q, 5p, 7q, 12p and 11q and deletion of material from chromosomes 3p, 11q, 4p, 5q, 8p, 10q, 13q and 21. Kaplan–Meier survival analysis revealed significant correlations between gain of 3q25–27 and deletion of 22q with reduced disease-specific survival. In addition, gain of 17q and 20q, deletion of 19p and 22q and amplification of 11q13 were significantly associated with reduced disease-free survival. A Cox proportional hazards regression model identified deletion of 22q as an independent prognostic marker. The data presented here provide further evidence that the creation of a genetically based tumour classification system will soon be possible, complementing current histopathological characterisation.