Regions Of Copy Number Change Research Articles

Identification of novel mutational drivers reveals oncogene dependencies in multiple myeloma

AbstractUnderstanding the profile of oncogene and tumor suppressor gene mutations with their interactions and impact on the prognosis of multiple myeloma (MM) can improve the definition of disease subsets and identify pathways important in disease pathobiology. Using integrated genomics of 1273 newly diagnosed patients with MM, we identified 63 driver genes, some of which are novel, including IDH1, IDH2, HUWE1, KLHL6, and PTPN11. Oncogene mutations are significantly more clonal than tumor suppressor mutations, indicating they may exert a bigger selective pressure. Patients with more driver gene abnormalities are associated with worse outcomes, as are identified mechanisms of genomic instability. Oncogenic dependencies were identified between mutations in driver genes, common regions of copy number change, and primary translocation and hyperdiploidy events. These dependencies included associations with t(4;14) and mutations in FGFR3, DIS3, and PRKD2; t(11;14) with mutations in CCND1 and IRF4; t(14;16) with mutations in MAF, BRAF, DIS3, and ATM; and hyperdiploidy with gain 11q, mutations in FAM46C, and MYC rearrangements. These associations indicate that the genomic landscape of myeloma is predetermined by the primary events upon which further dependencies are built, giving rise to a nonrandom accumulation of genetic hits. Understanding these dependencies may elucidate potential evolutionary patterns and lead to better treatment regimens.

Genomic Disruption of the Histone Methyltransferase SETD2 in Chronic Lymphocytic Leukemia

Histone methyltransferases (HMTs) are important epigenetic regulators of gene transcription and have been found disrupted at the genomic level in a spectrum of human tumors including hematological malignancies. In CLL, recurring genomic lesions targeting chromatin modifiers are emerging in the literature (Puente 2015, Nature) but their biological and clinical significance remain uncertain. We studied 802 CLL patients, divided into a discovery [n=261, sampled pre-treatment] and two validation cohorts [n=541, 431 pre-treatment and 110 ultra-high risk], using high-resolution SNP-arrays [n=572, Affymetrix SNP6.0 and HumanOmniS-8] and high-throughput re-sequencing [n=320, Haloplex and TruSeq] to identify genomic lesions targeting HMT genes. In our discovery cohort, we identified nine novel regions of copy-number changes; the prime finding was a recurrent deletion of chromosome 3p in 4% of patients between genomic positions 47.12-47.36Mb. This region included SETD2, KIF9 and KLHL18 (Fig 1A), with SETD2 being the most significantly under-expressed gene (p=0.001) in 3p-deleted [n=6] versus non-deleted cases [n=8]. Further validation in two independent cohorts showed that SETD2 deletions were enriched in ultra high-risk CLL and associated with loss of TP53 (p=0.003), genomic complexity (in TP53 wild-type cases, p=0.01) and chromothripsis.Next, we screened for somatic mutations in SETD2 using targeted re-sequencing and identified non-synonymous mutations in four (4.5%) discovery cases (p.D99G, p.W1306X, p.Q1545K, p.E1955Q), and 7/231 (3%) in the pre-treatment validation cases (p.A50T, p.P167L, p.E670K, p.M1742L, p.M1889T (x2), p.I2295M) (Fig 1B). Mutations were somatic in all samples tested [n=5]. To study the clonal nature of the SETD2 deletions, we assigned each genomic CNA with a relative copy-number by normalizing CNA intensity values from array features, and could infer that the 3p deletion was in the dominant clonal population in 14/21 cases with data available for analysis. Employing either the ABSOLUTE algorithm for our discovery cohort or manually correcting for tumor sample purity and local copy-number changes in our validation cohort, we observed that 10/11 SETD2 mutations exhibited a clonal cell fraction. These data strongly imply that SETD2 aberrations represent early clonal events in the pathobiology of CLL. Next, we extended our gene expression analysis to include additional wild-type, deleted, and mutated patients, showing reduced expression both in deleted and mutant cases (p=0.035), thus confirming haplo-insufficiency also for the SETD2-mutant cases. W e reviewed the DNA methylation status of the SETD2 gene body and promoter regions (15 & 9 CpG probes, respectively) from published data, and found no correlation between SETD2 methylation and RNA expression, suggesting that DNA methylation does not play a substantial role in regulating SETD2 expression in CLL.Finally, we analyzed the impact of SETD2 lesions on treatment-free survival (TFS) and overall survival (OS). For TFS and OS, we observed a significantly worse outcome (TFS: 44 vs. 105 months; p=0.004, OS: 85 vs. 199 months; p=0.002) in SETD2 deleted cases that were wild-type for TP53/ATM, compared to cases wild type for TP53/ATM/SETD2 (Fig 1D). Mutant SETD2 cases (wild-type for TP53/ATM) and those wild-type for TP53/ATM/SETD2 exhibited median TFS of 74 and 106 months respectively, differences that did not reach significance (p=0.1). Whilst these data suggest that SETD2 lesions may be clinically relevant, further investigations in larger materials are warranted to understand their full impact on survival.In conclusion, we report for the first time somatic deletions and mutations in SETD2, a gene found disrupted invarious human solid and hematological tumors, in ~7% of CLL patients requiring treatment. These are likely to be early clonal events and associate with TP53 dysfunction, genomic complexity and chromothripsis, with deletions enriched in ultra high-risk CLL. [Display omitted] DisclosuresTausch:Gilead: Other: Travel support. Steele:Portola Pharmaceuticals: Other: Travel bursary to ASH 2015; Janssen: Other: Travel bursary to EHA 2015. Hillmen:Janssen: Consultancy, Honoraria, Research Funding; GSK: Consultancy, Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Celgene: Research Funding; Gilead: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding. Dyer:Gilead: Research Funding; Roche Pharmaceuticals: Speakers Bureau; ONO Pharmaceuticals: Research Funding. Stilgenbauer:AbbVie, Amgen, Boehringer-Ingelheim, Celgene, Genentech, Genzyme, Gilead, GSK, Janssen, Mundipharma, Novartis, Pharmacyclics, Roche: Consultancy, Honoraria, Research Funding. Schuh:Acerta Pharma BV: Research Funding. Strefford:Roche: Research Funding.

Abstract C33: Integrated analysis of sequence variations and copy number in TCGA colon adenocarcinoma data

Abstract The Cancer Genome Atlas (TCGA) data is available via the TCGA portal and includes copy number data and whole exome sequencing data. Nexus Copy Number 7 was used to evaluate level-1 approved SNP array data from the colon adenocarcinoma (COAD) data set. All samples were subjected to match-paired analysis to evaluate somatic copy number variation. Samples were subjected to extensive pre-processing, including systematic G/C wave correction, ploidy correction, gender correction and quality control. Corresponding level-3 somatic mutation data was used for an integrated analysis of copy number and sequence variation with the COAD data set. Comprehensive copy number analysis identified frequent (>35%) copy number gains of chr7, chr8q, chr13 and chr20, and losses of chr8p, chr17p, and chr18. GISTIC analysis further identified statistically significant focal changes which included loss of FHIT on chr3. Genes KRAS and BRAF were among the most frequently mutated genes. Integrated analysis identified several regions of copy number change that significantly co-occurred with BRAF mutation. Copy number profiles varied significantly between hypermutable and non-hypermutable samples. Potential chromothriptic events were also observed within the COAD dataset. An approach that can integrate structural and sequence variation among samples will be presented. Citation Format: Andrea O'Hara, Raja Keshavan, Louis Culot, Soheil Shams. Integrated analysis of sequence variations and copy number in TCGA colon adenocarcinoma data. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C33.

Abstract 3171: Analysis of copy number and LOH in germline samples from different tumor types in The Cancer Genome Atlas (TCGA) project.

Abstract Using data from The Cancer Genome Atlas (TCGA) project and HapMap consortium, we examined the hypothesis that existing copy number polymorphisms might predispose individuals to cancer and if different common CNVs bias toward different cancer types. This analysis was performed using a subset of the TCGA data, obtained from blood derived “normal” samples of cancer patients. All the data was generated using Affymetrix SNP 6.0 arrays (Affymetrix, Inc.) and processed using Nexus Copy Number ver. 6.1 (BioDiscovery, Inc.). The project contained 451 Glioblastoma (GBM), 353 Colon cancer, and 245 Skin cancer samples in addition to 211 HapMap samples which we used as reference since these individuals were supposed to be healthy. Using Fisher's Exact test, we compared each different cancer types to the HapMap samples to identify regions of copy number change with FDR corrected p-value of less than 0.05 and difference in frequency between each population of at least 10%. Many of the regions found in this process encompass regions identified as common CNV polymorphisms as reported in the Database of Genomic Variance (DGV). For example, a polymorphic region on 8p23.1 containing a number of genes from the Beta defensin family involved in defense response to bacterium were seen significantly gained in germline samples obtained from cancer patients as compared to the HapMap samples (11% in HapMap, 42% in Colon Cancer, 28% in GBM, and 32% in Skin cancer). Additionally, some interesting regions were identified that were not seen as polymorphic in the general population, did not show up in the HapMap samples or reported in DGV, but had high prevalence in the blood derived normal samples from cancer patients. Using gene enrichment analysis on genes discovered in these regions we identified a number of known cancer specific pathways that were highly enriched (e.g. KEGG Glioma and Melanoma pathways being statistically significantly enriched). Looking at the effected genes in these pathways we identified small focal and recurrent events that often only spanned a small portion of a gene covering a few exons. This included genes such as PIK3CA where a focal gain covering exons 10-14 were observed in none of the HapMap samples but in 21% of the germline of patients with Colon cancer, 9% of GBM and 14% of Skin cancer patients. A similar focal and recurrent gain was observed in KIT covering exons 2-7 with no gains in the HapMap samples and 55% in Colon, 15% in GBM, and 50% in Skin cancer patients. Similar observations were also made for other well-known cancer genes such as RAF1 and EGFR but to a lesser extent. We are in a process of comparing matched tumor/normal cases to further investigate possible evolution of these changes from the germline to the surrounding tissue (also available from the TCGA database) to the primary tumor samples. Citation Format: Soheil Shams, Raja Keshavan, Joshua D. Schiffman. Analysis of copy number and LOH in germline samples from different tumor types in The Cancer Genome Atlas (TCGA) project. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3171. doi:10.1158/1538-7445.AM2013-3171

Abstract 3234: Comparative oncogenomics: identifying melanoma drivers from zebrafish to humans.

Abstract The elucidation of core networks driving tumorigenesis is greatly enhanced by studying tumors from multiple species. A comparison of tumors between evolutionarily distant organisms such as humans and fish offers a particularly powerful method to identify such core functionalities. In the past several years, the zebrafish has emerged as an important vertebrate model of cancer because it develops tumors that are histologically and functionally similar to the human disease. Overexpression of the BRAFV600E oncogene under the melanocyte-specific mitf promoter leads to a 100% penetrant melanoma in the context of p53 loss of function. We have previously shown that these zebrafish tumors exhibit characteristics of neural crest progenitors, rather than differentiated melanocytes, similar to many human melanomas which also overexpress the neural crest markers sox10 and ednrb. To identify the genomic lesions that are common to both species, we are performing RNA and DNA-sequencing of the zebrafish tumors and comparing them to human melanoma resequencing samples to identify common driver events. To facilitate this, we have developed technologies for both short and long term culture of zebrafish melanoma cell lines, along with normal tissue from each individual animal. One such melanoma line, ZMEL1, has been comprehensively characterized. RNA-seq demonstrated upregulation of neural crest genes such as ednrb, and pathway analysis indicated enrichment for ERK/MAP kinase and PI3K/AKT signaling pathways, and these tumors are sensitive to inhibition by the BRAF inhibitor PLX4720. Whole-genome sequencing (WGS) was performed on tumor cell line and matched normal tissue to a depth of 39X coverage on the Illumina platform. A total of 5193 single basepair mutations were identified, of which 2% were found in the coding region, 41% in intronic regions and 56% in the intergenic DNA. Within the coding region, 25% were G>C and 18% T>C substitutions, with approximately equal number of non-synonymous to silent mutations. Coding mutations in several genes such as rps6kb1b and plb1 are of particular interest because of their reported mutation in human cancer as well. 35 regions of copy number changes were found, comprising over 600 genes, and these include amplification of known oncogenes such as SETDB1. Functional analysis of the genomic changes common to both zebrafish and human tumors is now being undertaken using in vitro and in vivo melanoma models. In summary, comparative oncogenomics between zebrafish and human melanoma provides a powerful method for filtering the vast number of changes down to a list of manageable, testable hypotheses. This method can be broadly applied to other tumors types. Citation Format: Richard M. White, Erin Langdon, Charles Kaufman, Ellen van Rooijen, Jennifer Yen, Peter Van Loo, Leonard Zon. Comparative oncogenomics: identifying melanoma drivers from zebrafish to humans. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3234. doi:10.1158/1538-7445.AM2013-3234 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract LB-270: Exploration of TCGA generated DNA copy number data in ovarian cancer points to interesting proapaptotic gene for survival prediction

Abstract Exploration of TCGA generated DNA copy number data in ovarian cancer points to interesting proapaptotic gene for survival prediction Using data generated by The Cancer Genome Atlas (TCGA) project, we investigated the use of copy number changes in prediction of both response to therapy as well as identification of possible prognostic survival bio-markers. The analysis was performed using an initial data set of 557 arrays (68 Affymetrix 6.0 and 489 Agilent 244K arrays). The raw data was processed using Nexus Copy Number version 5.1 software (BioDiscovery, El Segundo CA) and regions of copy number change were identified using the built-in FASST and SNP-FASST algorithms. As previously reported, using only the Agilent data set, we had identified a number of regions with statistically significant differences across between sample groups that responded to therapy as compared to those with progressive disease1. Performing gene enrichment analysis on these regions indicated a relationship with apoptosis. In our current study, we have also considered survival and have identified FAM105A located on the short arm of chromosome 5 as a possible component of Ovarian Cancer. A number of studies have identified the small 728Kb region between TRIO and ANKH gained in various cancers and hypothesized that these genes are possible oncognes2–3. Using Kaplan-Mier survival analysis, we have in fact been able to show that the FAM105A gene located between TRIO and ANKH is highly predictive of survival (p=0.00024) with the deletion causing a much poorer prognosis. This result was validated using the Affymetrix data on a different set of samples. Very little is known about this gene except the fact that using functional screening, it has demonstrated strong proapaptotic behavior which is in concordance with our findings here4. We are in the process of expanding the validation data set to several hundred samples. 2 Judith N Kloth, Jan Oosting, Tom van Wezel, Karoly Szuhai, Jeroen Knijnenburg, Arko Gorter, Gemma G Kenter, Gert Jan Fleuren, and Ekaterina S Jordanova, Combined array-comparative genomic hybridization and single-nucleotide polymorphism-loss of heterozygosity analysis reveals complex genetic alterations in cervical cancer, BMC Genomics. 2007; 8: 53. Published online 2007 February 20. doi: 10.1186/1471–2164–8-53. 3 Coe BP, Henderson LJ, Garnis C, Tsao MS, Gazdar AF, Minna J, Lam S, Macaulay C, Lam WL., High-resolution chromosome arm 5p array CGH analysis of small cell lung carcinoma cell lines, Genes Chromosomes Cancer. 2005 Mar;42(3):308–13. 4 Otto Mannherz, Daniel Mertens, Meinhard Hahn, Peter Lichter, Functional screening for proapoptotic genes by reverse transfection cell array technology, Genomics, Volume 87, Issue 5, May 2006, Pages 665–672, ISSN 0888–7543, DOI: 10.1016/j.ygeno.2005.12.009. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-270. doi:10.1158/1538-7445.AM2011-LB-270

Abstract 2256: Novel prognostic targets identified using integrated analysis of copy number and expression data in esophageal adenocarcinoma

Abstract Introduction: The incidence of esophageal adenocarcinoma (EAC) has increased 6-fold in the west over the last 30 years, with a dismal survival rate of less than 20%. Understanding the genomic aberrations and biology of this cancer may enhance disease interventions. Aims: i) To identify novel molecular targets with clinical relevance using an integrated approach in analyzing genome-wide copy number and expression data of EAC. ii) To correlate reduced expression of molecular targets with promoter hypermethylation in EAC. Methods: 30K oligonucleotide array comparative genomic hybridization (CGH) and gene expression microarray profiling were performed on 56 chemo-naive EAC resection samples with long-term clinical follow-up data (median follow up: 20 months, range 0.5-137) to identify regions of copy number changes leading to differential expression levels. Fluorescence in situ hybridization (FISH) assays were carried out on touch-imprint slides of frozen tumor samples to confirm amplification status. Immunohistochemistry (IHC) assays on tissuemicroarrays (TMAs) on the same (n=56) and an independent (n=371; median follow-up: 17 months, range 0.5-193) cohort of EAC samples were used for subsequent validation at the protein level. Kaplan-Meier survival analyses were performed based on IHC data to determine prognostic significance of novel molecular targets. An Illumina Infinium Array was used to investigate the methylation status of 24 independent EAC tumors, compared with 24 premalignant Barrett's Esophagus (BE) samples. Results: Integrated array CGH data with matched gene expression microarray data highlighted 4 genes with copy number gains (log2 ratio>0.32) and increased expression (fold-change>6.5): EGFR, WT1, NEIL2 and MTMR9. FISH assays confirmed amplifications of these genes (more than 6 copies/centromere) in 11% of EAC samples and IHC assays (n=371 independent cases) demonstrated protein over-expression (IHC scores above 2): EGFR (10%), WT1 (20%), NEIL2 (14%) and MTMR9 (25%), corroborating findings from the integrated analysis. Each of these targets individually had prognostic significance in our cohort (P<0.060). Interestingly, reduced expressions of WT1 (P=0.037) and NEIL2 (P=0.051) were associated with poorer prognosis in EAC. In order to investigate the mechanism of reduced expression of these targets which might have contributed to a worse prognosis, methylation array data was utilized from an independent dataset. This showed increased methylation of WT1 in EAC (63%) compared to BE (55%; t-test P=0.028) and normal controls (50%), but no difference in methylation status of NEIL2 between disease states. Conclusion: Integration of array CGH and gene expression data identified novel molecular targrets in EAC and reduced expression of WT1 may be secondary to methylation. These genes have prognostic potential and require further study. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2256. doi:10.1158/1538-7445.AM2011-2256

Abstract LB-266: Clonal analysis of aneuploid pancreatic ductal adenocarcinoma genomes in patient biopsies using Agilent oligonucleotide technologies and Pacific Biosciences SMRT™ sequencing system

Abstract One of the challenges for the application of emerging genome technologies to the study of human cancer in patients in vivo is the presence of admixtures of normal cells and stroma in patient samples. Furthermore, tumor samples frequently contain multiple neoplastic populations even in small clinical biopsies. Consequently, it is difficult to accurately detect the multiple genomic aberrations typically present in a biopsy of interest and to distinguish whether they arise concurrently or are present in distinct cell populations in the same sample. Recent advances in flow cytometry technology provide high throughput flow rates and the detection of relatively rare events in dilute samples. Coupling this technology with robust methods to label cells and advanced data acquisition and analyses methods enables the application of flow cytometry to high definition cancer genomics in patients. In our current studies of pancreatic ductal adenocarcinoma (PDA) we developed methods that enable whole genome analyses of clonal populations of sorted aneuploid and diploid nuclei from patient biopsies with as little as 1–5% tumor cell content. Our aCGH studies using Agilent 400k oligonucleotide arrays and flow sorted samples identified 19q13.2 as a region that is recurrently targeted by gene amplification in PDA genomes. Significantly, we identified this amplicon in two distinct aneuploid populations representing 19.8% and 17.4% of the total cellular fraction of a PDA biopsy. We mapped the amplicon to an interval spanning from 43,526,781-cen to 46,171,091-tel, a region targeting 2,644,310 bases in each of the aneuploid populations. This region of interest is relatively gene rich and contains a series of cancer associated genes of interest (e.g. PAK4, AKT2, HIPK4, MAP3K10, RAB4B) to PDA. In addition, our data suggests this region contains variable internal regions of copy number changes suggesting structural rearrangements within the amplicon and its immediate boundaries. To further study this clonally selected region we used the Agilent Sure Select Target Enrichment System to design capture oligonucleotides to sample the 19q13.2 amplicon and its boundaries in the aneuploid PDA genome. These sequences were then interrogated with the PacBio RS system. The integration of these data provides a high resolution clonal analysis of genomic aberrations including point mutations, inversions and insertions that are present in the 19q13.2 amplicon, its immediate boundaries, and the genomes in which it arose. These data provide a highly unique and valuable proof of concept for the application of our integrated clonal genomics methods to the advancement of personalized medicine for patients with PDA and other malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-266. doi:10.1158/1538-7445.AM2011-LB-266

Novel prognostic targets identified using integrated analysis of copy number and expression data in oesophageal adenocarcinoma

IntroductionThe incidence of oesophageal adenocarcinoma (OAC) has increased sixfold in the west over the last 30 years, with a dismal survival rate of less than 20%. Understanding the genomic aberrations...

The Genome of Chronic Lymphocytic Leukemia

AbstractAbstract 51Chronic lymphocytic leukemia (CLL) is a malignancy of CD5+ mature B cells that includes two distinct subtypes marked by the differential presence of immunoglobulin gene mutations and a distinct clinical course. The pathogenesis of CLL is largely unknown: in contrast to other types of B cell malignancies, CLL is not associated with recurrent, balanced chromosomal translocations, nor genes have been found that are specifically targeted by somatic mutations, with the exception of ATM and p53 in 6–12% of cases. Instead, more than 80% of CLL patients carry genomic deletions of chromosomal regions 13q14, 11q22–23, and 17p13, or trisomy 12. Of these, the 13q14.3 deletion, encompassing the DLEU2/miR-15a/miR-16-1 cluster (Calin and Croce, Nat Rev Cancer 2006), has been recently shown to promote the development of CLL in mice (Klein et al., Cancer Cell 2010), suggesting its pathogenetic role in the human disease. To determine the extent of somatic genetic lesions (mutations and gene copy number changes) that are present in the CLL genome, we have integrated next generation whole-exome sequencing analysis (Nimblegen/Roche FLX454) and genome-wide high-density single nucleotide polymorphism array analysis (Affymetrix SNP 6.0) in 5 cases representative of the two CLL immunogenetic subgroups and their paired normal DNAs. Candidate tumor-specific nonsynonymous mutations were verified by Sanger sequencing in the same tumor/normal pairs, and all genes validated as mutated were then screened in an independent panel of 48 CLL DNAs by PCR amplification/direct sequencing of their entire coding region. The whole-exome sequencing approach revealed a total of 36 mutations, corresponding to 36 distinct genes at an average of 7 mutations/case (range, 5–9 mutations/case). The majority of these events were represented by single base pair substitutions (N=34, of which 30 missense mutations and 4 nonsense mutations), while frameshift insertion/deletions were rare (N=2 deletions of 4 and 32 base pairs, respectively; 5.5%). Analysis of the mutation features showed a prevalence of transitions versus transversions (64% vs 36%) and an elevated G+C over A+T ratio (66% vs 44%), analogous to the mutation spectrum observed in the genome of epithelial tumors such as colorectal, pancreatic and brain cancer. SNP array analysis in 4 of the 5 “discovery” cases confirmed the presence of the 13q14 deletion in 2 samples and identified 25 additional regions of copy number changes, corresponding to ∼7 lesions/case (range: 3 to 10) and mostly represented by deletions (N=16/27, ∼60%). When screened in the extended CLL panel, several of the 36 genes identified through the whole exome sequencing approach appeared to be mutated in at least one additional patient. Overall, these data indicate that the coding genome of CLL contains on average ∼14 somatic gene alterations per case. When classified based on functional annotation, most of these lesions appeared to converge on discrete signaling pathways, which likely represent important pathogenetic and possibly therapeutic targets in CLL. Disclosures:No relevant conflicts of interest to declare.

High-density single nucleotide polymorphism array analysis and ASXL1 gene mutation screening in chronic myeloid leukemia during disease progression

We have undertaken a genome-wide single nucleotide polymorphism (SNP) array analysis of 41 chronic myeloid leukemia (CML) patients. In total, 44 regions of uniparental disomy (UPD) >3 Mb were identified in 24 of 32 patients in chronic phase (CP), and 21 regions of UPD >3 Mb were identified in 13 of 21 patients in blast crisis (BC). Chromosome 8 had the highest frequency of UPD regions in both CP and BC samples. Eight recurrent regions of UPD were observed among the 41 patients, with chromosome 8 showing the highest frequency. Ten regions of copy number change (CNC) >3 Mb were observed in 4 of 21 patients in BC, whereas none were observed in CP. We have identified several recurrent regions of UPD and CNC in CML that may be of pathogenetic importance. Overrepresentation of genomic aberrations (UPD and copy number gain) mapping to chromosome 8 was observed. Selected candidate genes mapping within the aberrant genomic regions were sequenced and mutation of the TP53 gene was observed in one case in BC and of the ASXL1 gene in 6 of 41 cases in CP or BC. Mutation of ASXL1 represents an important new molecular abnormality in CML.

Abstract 2141: Analysis of DNA copy number changes in ovarian cancer data reveals differences in progressive vs. complete response samples

Abstract Data from The Cancer Genome Atlas (TCGA) project, related to Ovarian Cancer was obtained from the TCGA web site. The data comprised a total of 489 samples hybridized on 2-color oligo-arrays with 244K probes per array. Raw log-ratio data representing DNA copy number values relative to a DNA pool of normal samples were used in the analysis. The raw data was processed using Nexus Copy Number version 5 software (BioDiscovery, El Segundo CA) and regions of copy number change were identified using the built-in FASST algorithm with significance threshold of 5.0 E-6. After applying a QC filter to remove hybridizations with excessive noise, 463 samples remained for further analysis. Of these, there were 214 solid tumors and 242 normal samples (either surrounding tissue or blood from same patient). In our analysis we compared copy number differences from samples with Progressive disease (n=29) and those with Complete response to therapy (n=245). Using a Fisher's Exact test and a maximum p-value of 0.05 we identified significant regions of copy number change covering a genome area composed of a total of 1049 genes. Included were regions of significant increase in the number of DNA copy number gains in the for the Complete response samples as compared to Progressive cases. The most significant of these were in the 6p21.1 − 6p12.1 and 5p14.1 − 5p13.3 regions. Other regions include a number of well-know genes related to apoptosis, such as TNF (on 6p21.33) and RHOB (on 2p24.1) that were also significantly gained in Complete response but showed DNA loss in Progressive samples. 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 2141.

Abstract LB-61: Comparative study of different diagnostic techniques (AI, MLPA, FISH) in breast cancer patients with 1q25.3 alterations

Abstract Background: Breast cancer is the most frequently diagnosed malignancy among women and is the leading cause of cancer related death worldwide. Molecular genetic studies have revealed many subgroups of breast cancer within which the genomic alterations affecting chromosome arm 1q are considered to be an early event in breast carcinogenesis and are correlated with favorable prognosis for the patients. We have found a high percentage of concordance between the AI and MLPA assays pointing towards gain of the 1q25.3 region in breast cancer. Aims: Our main objective was to compare the sensitivity and specificity of AI and synthetic probe-based MLPA with other cytogenetic method (FISH) in breast cancer patients with or without previously confirmed alterations within 1q25.3. Methods: FISH was performed on formalin-fixed paraffin-embedded tumor material in order to verify previous findings and assess the level of genetic alterations of 1q25.3 in breast cancer. The 1q25.3 test and 1p35 reference probes labeled with different fluorochromes were utilized for analysis. Results: A total of 70 nuclei from each breast cancer case were examined and scored for the percentage of 1q25.3 alterations. The non-tumorigenic nuclei obtained from healthy individuals served as adequate cut-off for the 1q25.3-specific changes. The overall FISH results are consistent with results obtained from previous analysis in the majority of analysed cases. Furthermore, FISH resolved the level of 1q25.3 alterations in few cases that were uncertain by AI and MLPA analysis. Conclusion: This study shows that both AI and MLPA assays are able to map regions of copy number changes in the cancer genome and are in concordance with molecular cytogenetics. These three techniques proved to be an efficient tool for diagnosis of 1q25.3 alterations, however, confusing results in AI and MLPA analysis might be obtained while using non-microdissected tumor DNA as a template. 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 LB-61.

Global Methylation Array Analysis of Multiple Myeloma Samples Indicate An Alteration of Epigenetics During the Transition From MGUS to Myeloma and An Increased Frequency of Gene Methylation in t(4;14) Myeloma.

Abstract 121The methylation status of genes in myeloma can change as the disease progresses and as such identifying genes deregulated by methylation that mediate disease aetiology and progression may offer epigenetically relevant therapeutic targets. We have analyzed 153 presenting myeloma samples for methylation differences using the Illumina Infinium humanmethylation27 array, which interrogates 27,578 highly informative CpG sites per sample at the single-nucleotide resolution using bisulfite converted DNA. Data are presented as an average beta-score where 1.0 is fully methylated and 0 is fully unmethylated. Samples were analyzed using Illumina GenomeStudio and the custom differential methylation algorithm. Initially, we compared global methylation of genes between MGUS, myeloma (n=153) and relapsed myeloma (n=18) in order to determine the effect of clinical stage on the general methylation state of the genome. There were 267 probesets showing an increase in methylation between presenting and relapsed myeloma. However, the largest changes in DNA methylation were between MGUS and myeloma with 4209 probesets showing a decrease in methylation and 879 probesets showing an increase in methylation as the pre-malignant stage progresses to myeloma. In order to address the potential for differential methylation between cytogenetic subtypes of myeloma we compared the translocation groups (t(4;14) n=14; t(11;14) n=32; t(14;16) n=7; t(14;20) n=3) and samples with no split IgH locus (n=66). When average beta-scores for each translocation are compared using a 1.5 fold-change cut-off we identified 8.7% of probes differentially methylated in t(4;14), 5.1% in t(14;20), 3.3% in t(14;16), and 2% in t(11;14), indicating that the t(4;14) translocation has the largest effect on genome methylation, and in addition there are significant methylation effects associated with deregulation of the MAF transcription factors. The t(4;14) translocation in myeloma results in the over-expression of two genes, MMSET and FGFR3, of which MMSET has histone methyltransferase properties and it has been shown that methylation of chromatin is associated with DNA methylation at CpG islands resulting in transcriptional repression. In this analysis the t(4;14) samples had a greater than 1.5-fold increase in methylation in 2410 probesets, corresponding to 1685 unique genes, when compared with non-translocation samples. On average the remaining translocation groups had only 746 probes with differential methylation, and with the exception of the t(14;20) group most were hypomethylated. Identifying the genes affected by these methylation changes is important. The gene with the largest fold-change in methylation in t(4;14) samples was APC. Clinically relevant changes in methylation may be characterised by associated changes in gene expression and when methylation and expression array data from the same samples are compared there are 23 genes with decreased expression and increased methylation in t(4;14) samples compared with non-translocation samples. These include potential tumor suppressor genes GLTSCR2 and NME4, as well as SEPTIN9. We also looked for differential methylation between common cytogenetic subgroups including hyperdiploidy (HRD n=64 vs. normal n=67), 1q+ (n=44 vs. n=83), del(1p32.3) (n=20 vs. n=104), del(13q) (n=66 vs. n=68), del(16q) (n=36 vs. n=96), and del(17p) (n=8 vs. n=126) but were unable to show that any gross differences in global methylation between samples with and without the abnormality. However, there were a limited number of genes that had a greater than 1.5-fold change in methylation between the analysis groups, indicating that there are genes of potential interest. We are also mapping the methylation of genes within these regions of copy number change. In summary, we have identified that the major influences on epigenetics occur at the transition between MGUS and myeloma. t(4;14) myeloma, characterised by deregulation of MMSET, along with the translocations that deregulate the transcription factor MAF have a higher frequency of genome methylation than the cases lacking these events. These analyses enable us to identify targets which may be sensitive to modulation by epigenetic therapies in vivo. Disclosures:No relevant conflicts of interest to declare.

Genomic profile of a secretory breast cancer with an ETV6–NTRK3 duplication

Background:Secretory breast cancer (SBC) is a rare entity characterised by indolent clinical behaviour, distinctive histological features and the presence of a recurrent chromosomal translocation t(12;15)(p13;q25), leading to the formation of...

Genomic analysis using high-density single nucleotide polymorphism-based oligonucleotide arrays and multiplex ligation-dependent probe amplification provides a comprehensive analysis of INI1/SMARCB1 in malignant rhabdoid tumors.

A high-resolution genomic profiling and comprehensive targeted analysis of INI1/SMARCB1 of a large series of pediatric rhabdoid tumors was done. The aim was to identify regions of copy number change and loss of heterozygosity (LOH) that might pinpoint additional loci involved in the development or progression of rhabdoid tumors and define the spectrum of genomic alterations of INI1 in this malignancy. A multiplatform approach using Illumina single nucleotide polymorphism-based oligonucleotide arrays, multiplex ligation-dependent probe amplification, fluorescence in situ hybridization, and coding sequence analysis was used to characterize genome-wide copy number changes, LOH, and genomic alterations of INI1/SMARCB1 in a series of pediatric rhabdoid tumors. The biallelic alterations of INI1 that led to inactivation were elucidated in 50 of 51 tumors. INI1 inactivation was shown by a variety of mechanisms, including deletions, mutations, and LOH. The results from the array studies highlighted the complexity of rearrangements of chromosome 22 compared with the low frequency of alterations involving the other chromosomes. The results from the genome-wide single nucleotide polymorphism array analysis suggest that INI1 is the primary tumor suppressor gene involved in the development of rhabdoid tumors with no second locus identified. In addition, we did not identify hotspots for the breakpoints in sporadic tumors with deletions of chromosome 22q11.2. By employing a multimodality approach, the wide spectrum of alterations of INI1 can be identified in the majority of patients, which increases the clinical utility of molecular diagnostic testing.

Genome-wide profiling of follicular lymphoma by array comparative genomic hybridization reveals prognostically significant DNA copy number imbalances

AbstractThe secondary genetic events associated with follicular lymphoma (FL) progression are not well defined. We applied genome-wide BAC array comparative genomic hybridization to 106 diagnostic biopsies of FL to characterize regional genomic imbalances. Using an analytical approach that defined regions of copy number change as intersections between visual annotations and a Hidden Markov model–based algorithm, we identified 71 regional alterations that were recurrent in at least 10% of cases. These ranged in size from approximately 200 kb to 44 Mb, affecting chromosomes 1, 5, 6, 7, 8, 10, 12, 17, 18, 19, and 22. We also demonstrated by cluster analysis that 46.2% of the 106 cases could be sub-grouped based on the presence of +1q, +6p/6q−, +7, or +18. Survival analysis showed that 21 of the 71 regions correlated significantly with inferior overall survival (OS). Of these 21 regions, 16 were independent predictors of OS using a multivariate Cox model that included the international prognostic index (IPI) score. Two of these 16 regions (1p36.22-p36.33 and 6q21-q24.3) were also predictors of transformation risk and independent of IPI. These prognostic features may be useful to identify high-risk patients as candidates for risk-adapted therapies.

TRAF3 and TNFAIP3, Two Negative Regulators of NF-Kb Signaling Pathways, Are Mutated in Waldenström's Macroglobulinemia

Waldenström's macroglobulinemia (WM) is an incurable low-grade lymphoproliferative disorder characterized by bone marrow (BM) infiltration of a clonal population of small B-lymphocytes, plasmacytoid lymphocytes and plasma cells that secrete monoclonal IgM antibody. Because it is difficult to obtain tumor metaphases for karyotype studies, few recurrent chromosomal abnormalities have been reported and the genetic basis of the disease remains poorly defined. Therefore, we performed a comprehensive high-resolution study to identify genomic abnormalities involved in WM pathogenesis. Fifty-seven WM patients were analyzed using a combination of array-based comparative genomic hybridization (aCGH)(N=42); gene expression profiling (GEP)(N=22), DNA sequencing (N=24) and cIgM-FISH (N=57). Agilent 244A and Affymetrix U133 platforms were used in the aCGH and GEP experiments, respectively. An NF-κB index was calculated from GEP data, as a surrogate marker of NF-κB transcription activity. NFKB2 sub cellular localization was determined by immunofluorescence staining. Overall, 83% of samples have aCGH defined chromosomal abnormalities, with a median of 3 abnormalities per patient (range 0 to 27). We identified 16 recurrent regions of copy number change found in >5% (3 or more patients); 10 deleted and 6 amplified regions. The most common abnormality was the entire or partial deletion of 6q, identified in 40% of cases. Four nonoverlapped minimal deleted regions were identified on 6q (MDR1 to MDR4), each of them being present in at least 14 of 17 patients with the abnormality. Gain of 6p was the second most common abnormality (17%) and its presence was always concomitant with 6q loss. Other recurrent deletions were 13q14 (10%) and 7q22, 8p, 11q22-q23, 11q23-q24 and 17p11-p13 (7% each). Copy gains were identified as partial or entire gains of chromosomes 18 (17%), 4 (12%) and 3 (10%), 8q (10%) and Xq27.1-q28 (10%). Three patients had either homozygous deletions or LOH with mutations affecting both alleles of TRAF3 on 14q32. TRAF3 inactivation was correlated with an increased NF-kB transcriptional signature and it was associated with activation of the non-canonical NFkB pathway. Additionally, we identified the inactivation of TNFAIP3, another negative regulator of the NF-kB pathways, in one of 24 patients. Finally, interstitial deletions identified in 4 patients at 13q14 identified a 1.1 Mb MDR, including MIRN15A and MIRN16-1. These findings confirm that focal 13q14 deletion is not restrict to B-CLL and that the MIRN15A/MIRN16-1 abnormalities might be common to several indolent B-cell diseases. Overall, we identified TRAF3 and TNFAIP3 inactivation in 5.3% and 2.4%, respectively. To our knowledge, this is the first study showing the inactivation of bona fide tumor suppressor genes in WM patients. Furthermore, these genes are negative regulators of NF-kB signaling pathway, highlighting its biologic importance in sustaining and limiting growth in WM. Mutational activation of this pathway, which is normally activated by ligand-receptor interactions within the BM microenvironment, suggest a therapeutic role for inhibitors of NF-KB pathway activation in the treatment of WM.

Utility of Oligonucleotide Array–Based Comparative Genomic Hybridization for Detection of Target Gene Deletions

direct DNA sequencing is the primary clinical technique for identifying mutations in human disease, but sequencing often does not detect intragenic or whole-gene deletions. Oligonucleotide array-based comparative genomic hybridization (CGH) is currently in clinical use to detect major changes in chromosomal copy number. a custom oligonucleotide-based microarray was constructed to provide high-density coverage of an initial set of 130 nuclear genes involved in the pathogenesis of metabolic and mitochondrial disorders. Standard array CGH procedures were used to test patient DNA samples for regions of copy number change. Sequencing of regions of predicted breakpoints in genomic DNA and PCR analysis were used to confirm oligonucleotide array CGH data. oligonucleotide array CGH identified intragenic exonic deletions in 2 cases: a heterozygous single-exon deletion of 4.5 kb in the SLC25A13 gene [solute carrier family 25, member 13 (citrin)] in an individual with citrin deficiency and a homozygous 10.5-kb deletion of exons 13-17 in the ABCB11 gene [PFIC2, ATP-binding cassette, sub-family B (MDR/TAP), member 11] in a patient with progressive familial intrahepatic cholestasis. In 2 females with OTC deficiency, we also found 2 large heterozygous deletions of approximately 7.4 Mb and 9 Mb on the short arm of the X chromosome extending from sequences telomeric to the DMD gene [dystrophin (muscular dystrophy, Duchenne and Becker types)] to sequences within or centromeric to the OTC gene (ornithine carbamoyltransferase). these examples illustrate the successful use of custom oligonucleotide arrays to detect either whole-gene deletions or intragenic exonic deletions. This technology may be particularly useful as a complementary diagnostic test in the context of a recessive disease when only one mutant allele is found by sequencing.

High-Resolution Array-Based Comparative Genomic Hybridization of Bladder Cancers IdentifiesMouse Double Minute 4(MDM4) as an Amplification Target Exclusive ofMDM2andTP53

Loss of p53 function in urothelial cell carcinoma (UCC) by mutation or inactivation disrupts normal cell cycle checkpoints, generating a favorable milieu for genomic instability, a hallmark of UCC. The aim of this study was to characterize novel DNA copy number changes to identify putative therapeutic targets. We report our findings using array comparative genomic hybridization on a whole-genome BAC/PAC/cosmid array with a median clone interval of 0.97 Mb to study a series of UCC cases. TP53 status was determined by direct sequencing, and an in-house tissue microarray was constructed to identify protein expression of target genes. Array comparative genomic hybridization allowed identification of novel regions of copy number changes in addition to those already known from previous studies. A novel amplification previously unreported in UCC was identified at 1q32. A chromosome 1 tile path array was used to analyze tumors that showed gains and amplification; the mouse double minute 4 (MDM4) homologue was identified as the amplified gene. MDM4 mRNA expression correlated with copy number and tumor grade. Copy number changes of MDM4 and MDM2 occurred exclusively in tumors with wild-type p53. Overexpression of MDM4 corresponded to disruption of p53 transcriptional activity. Immunohistochemistry on an independent series by tissue microarray identified an inverse relationship between Mdm4 and Mdm2, with Mdm4 expression highest in invasive UCC. The data indicate that gain/amplification and overexpression of MDM4 is a novel molecular mechanism by which a subset of UCC escapes p53-dependent growth control, thus providing new avenues for therapeutic intervention.