High-resolution Comparative Genomic Hybridization Research Articles

Abstract 4846: Genomic analysis of high grade bladder cancer: Defining genetic subtypes and therapeutic targets

Abstract Background: Genomic instability, as manifested by copy number alteration (CNA) and mutation, is a hallmark of cancer. We sought to characterize the spectrum of CNAs in 97 high-grade bladder tumors (HGBT) using high-resolution comparative genomic hybridization (CGH). We then performed an integrated analysis of CNA and targeted mutation testing to construct a comprehensive profile of genomic alterations in these tumors and to identify any correlation with clinical outcome. Methods: Genomic DNA from 97 frozen HGBTs (tumor content>70% or greater) and commercially available normal human DNA (Roche) were labeled with cyanine-based fluorescent dyes and hybridized onto a one-million oligonucleotide microarray (Agilent) followed by scanning and image analysis. High-throughput Sanger sequencing of select genes was performed with validation of hotspot oncogene mutations using a mass spectrometry based multiplex Sequenom assay. Results: We found that 47% of HGBT samples possessed a high degree of CNA with widespread amplifications and deletions while 53% contained markedly fewer numbers of events. A non-overlapping pattern of mutations and amplifications was observed when events were grouped within the context of specific signal transduction or cell cycle pathways. Preliminary survival analysis suggests that patients in the high CNA group have a worse prognosis than those in the low CNA group: fifteen out of 46 patients (33%) in the high CNA group have died of their disease vs. 9 of 51 (18%) in the low CNA group. Highly focal amplification events were noted within HER2 (5% of samples), E2F3 (11%), and CCND1 (10%). Immunohistochemistry (IHC) confirmed 3+ overexpression of HER2 in 5 of 5 samples exhibiting genetic amplification. Deletion events were most frequent in CDKN2A (13%). A subset of 11 tumors with small-cell histologic features was included within the samples analyzed and 45% of these tumors possessed E2F3 amplification vs. only 8% within the non-small cell tumor set. Targeted sequencing of select genes revealed that the majority of mutation events occurred within the high-CNA HGBTs. Conclusions: Distinct genetic subsets exist within HGBT and can be classified based on CNA and mutational analysis. The finding of focal amplification events within genes known to drive tumorigenesis in other contexts, such as HER2, underscores the need for global genomic approaches in identifying targets for therapy in bladder cancer. Small-cell bladder cancer may possess a distinct genetic signature characterized by E2F3 amplification. The non-overlapping pattern of CNAs and mutations seen within specific mitogenic pathways suggests that focal driver lesions can be identified for targeted therapies. 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 4846. doi:10.1158/1538-7445.AM2011-4846

Genomic aberrations in diffuse low‐grade gliomas

The current classification of diffuse low-grade gliomas is based mainly on histopathological criteria, which cannot accurately predict the highly variable clinical course observed in patients with such tumors. In an attempt to increase pathogenetic understanding of these tumors, we investigated 38 WHO Grade II astrocytomas, oligodendrogliomas, and oligoastrocytomas using a combination of G-band chromosome analysis and high-resolution comparative genomic hybridization (HR-CGH). Abnormal karyotypes were found in 41% of tumors. Karyotypes of astrocytomas and oligodendrogliomas were near-diploid whereas oligoastrocytomas also displayed near-tetraploid clones. The most common aberrations were losses of chromosomes X, Y, 3, 4, 6, and 11 and gains of chromosomes 8 and 12. The only recurrent structural rearrangement was del(6)(q21). HR-CGH analysis verified karyotyping findings but also revealed frequent losses at 1p, 17q, and 19q and gains of 7q, 10p, 11q, and 20p. Among the tumors were two gemistocytic astrocytomas, a subgroup of diffuse astrocytomas with a particular predisposition for progression but not studied cytogenetically before; one showed a near-diploid, complex karyotype with structural aberrations of chromosomes 1, 3, and 11 whereas both displayed simple aberrations including loss of 11p by HR-CGH. Our findings suggest that within diffuse low-grade gliomas are genetically distinct entities that do not fit the currently used classification. In addition, tumors with complex chromosomal aberrations had a higher tendency for aggressive tumor behavior (shorter progression-free survival) than tumors displaying simple aberrations only (P = 0.07). This could help identify genetic subsets of patients with low-grade glioma who might benefit from early antineoplastic therapy.

High-Resolution Comparative Genomic Hybridization of Inflammatory Breast Cancer and Identification of Candidate Genes

BackgroundInflammatory breast cancer (IBC) is an aggressive form of BC poorly defined at the molecular level. We compared the molecular portraits of 63 IBC and 134 non-IBC (nIBC) clinical samples.Methodology/FindingsGenomic imbalances of 49 IBCs and 124 nIBCs were determined using high-resolution array-comparative genomic hybridization, and mRNA expression profiles of 197 samples using whole-genome microarrays. Genomic profiles of IBCs were as heterogeneous as those of nIBCs, and globally relatively close. However, IBCs showed more frequent “complex” patterns and a higher percentage of genes with CNAs per sample. The number of altered regions was similar in both types, although some regions were altered more frequently and/or with higher amplitude in IBCs. Many genes were similarly altered in both types; however, more genes displayed recurrent amplifications in IBCs. The percentage of genes whose mRNA expression correlated with CNAs was similar in both types for the gained genes, but ∼7-fold lower in IBCs for the lost genes. Integrated analysis identified 24 potential candidate IBC-specific genes. Their combined expression accurately distinguished IBCs and nIBCS in an independent validation set, and retained an independent prognostic value in a series of 1,781 nIBCs, reinforcing the hypothesis for a link with IBC aggressiveness. Consistent with the hyperproliferative and invasive phenotype of IBC these genes are notably involved in protein translation, cell cycle, RNA processing and transcription, metabolism, and cell migration.ConclusionsOur results suggest a higher genomic instability of IBC. We established the first repertory of DNA copy number alterations in this tumor, and provided a list of genes that may contribute to its aggressiveness and represent novel therapeutic targets.

Human balanced translocation and mouse gene inactivation implicate Basonuclin 2 in distal urethral development

We studied a man with distal hypospadias, partial anomalous pulmonary venous return, mild limb-length inequality and a balanced translocation involving chromosomes 9 and 13. To gain insight into the etiology of his birth defects, we mapped the translocation breakpoints by high-resolution comparative genomic hybridization (CGH), using chromosome 9- and 13-specific tiling arrays to analyze genetic material from a spontaneously aborted fetus with unbalanced segregation of the translocation. The chromosome 13 breakpoint was ∼400 kb away from the nearest gene, but the chromosome 9 breakpoint fell within an intron of Basonuclin 2 (BNC2), a gene that encodes an evolutionarily conserved nuclear zinc-finger protein. The BNC2/Bnc2 gene is abundantly expressed in developing mouse and human periurethral tissues. In all, 6 of 48 unrelated subjects with distal hypospadias had nine novel nonsynonymous substitutions in BNC2, five of which were computationally predicted to be deleterious. In comparison, two of 23 controls with normal penile urethra morphology, each had a novel nonsynonymous substitution in BNC2, one of which was predicted to be deleterious. Bnc2(-/-) mice of both sexes displayed a high frequency of distal urethral defects; heterozygotes showed similar defects with reduced penetrance. The association of BNC2 disruption with distal urethral defects and the gene's expression pattern indicate that it functions in urethral development.

Metabolic and evolutionary insights into the closely-related species Streptomyces coelicolor and Streptomyces lividans deduced from high-resolution comparative genomic hybridization

BackgroundWhilst being closely related to the model actinomycete Streptomyces coelicolor A3(2), S. lividans 66 differs from it in several significant and phenotypically observable ways, including antibiotic production. Previous comparative gene hybridization studies investigating such differences have used low-density (one probe per gene) PCR-based spotted arrays. Here we use new experimentally optimised 104,000 × 60-mer probe arrays to characterize in detail the genomic differences between wild-type S. lividans 66, a derivative industrial strain, TK24, and S. coelicolor M145.ResultsThe high coverage and specificity (detection of three nucleotide differences) of the new microarrays used has highlighted the macroscopic genomic differences between two S. lividans strains and S. coelicolor. In a series of case studies we have validated the microarray and have identified subtle changes in genomic structure which occur in the Asp-activating adenylation domains of CDA non-ribosomal peptide synthetase genes which provides evidence of gene shuffling between these domains. We also identify single nucleotide sequence inter-species differences which exist in the actinorhodin biosynthetic gene cluster. As the glyoxylate bypass is non-functional in both S. lividans strains due to the absence of the gene encoding isocitrate lyase it is likely that the ethylmalonyl-CoA pathway functions as the alternative mechanism for the assimilation of C2 compounds.ConclusionsThis study provides evidence for widespread genetic recombination, rather than it being focussed at 'hotspots', suggesting that the previously proposed 'archipelago model' of genomic differences between S. coelicolor and S. lividans is unduly simplistic. The two S. lividans strains investigated differ considerably in genetic complement, with TK24 lacking 175 more genes than its wild-type parent when compared to S. coelicolor. Additionally, we confirm the presence of bldB in S. lividans and deduce that S. lividans 66 and TK24, both deficient in the glyoxylate bypass, possess an alternative metabolic mechanism for the assimilation of C2 compounds. Given that streptomycetes generally display high genetic instability it is envisaged that these high-density arrays will find application for rapid assessment of genome content (particularly amplifications/deletions) in mutational studies of S. coelicolor and related species.

Abstract CN07-03: Natural history of bronchial preneoplasia

Abstract Preinvasive bronchial lesions defined as dysplasia and carcinoma in-situ (CIS) have been considered as precursors of squamous cell carcinoma of the lung. The risk and rate of progression of preinvasive lesions to invasive squamous cell carcinoma as well as the mechanism of progression or regression are incompletely understood. The evidence for the multistage, stepwise progression model is weak with relatively few documented lesions that progress through normal to hyperplasia, metaplasia and various grades of dysplasia to CIS and then to invasive carcinoma. Collective data from longitudinal studies using autofluorescence bronchoscopy directed sequential bronchial biopsies from the Netherlands, Canada, France, and Japan as well as published data suggest that the time to development of CIS or invasive squamous cell carcinoma from precursor lesions does not follow an anticipated trend from a stepwise progression model. Aside from severe dysplasia that has the shortest progression time, there is no pattern between the baseline pathology and the time of development of CIS or invasive cancer. The presence of dyspalsia or CIS is a field cancerization risk marker for lung cancer both in the central airways and peripheral lung. Specific genetic alterations and host factors such as the inflammatory load and levels of anti-inflammatory proteins in the lung likely influence the progression or regression of pre-invasive lesions. Recently, using integrative genomic approach, combining high-resolution comparative genomic hybridization and gene expression microarray profiles, the focal amplification of an oncogene -BRF2 in chromosome region 8p12 was found to be specific for development of lung squamous cell carcinoma through the increase of Pol III mediated transcription. BRF2 was frequently activated in carcinoma in situ and dysplasia (PLoS Med 7(7): e1000315. doi:10.1371/journal.pmed.1000315). Molecular analysis of rare lesions that progress from normal, hyperplasia, metaplasia or dysplasia to CIS or invasive cancer will shed further light on the key molecular determinants driving development to an invasive phenotype versus those associated with tobacco smoke damage. Citation Information: Cancer Prev Res 2010;3(12 Suppl):CN07-03.

Molecular cytogenetic characterization of an inherited maternal duplication 20p11.21p13 associated with a small 20p11.21 deletion

Partial trisomy for the short arm of chromosome 20 is a relatively rare chromosomal abnormality. Most cases are derived from reciprocal translocations with the presence of partial monosomy of other chromosomes, and thus it has been difficult to delineate a specific phenotype [review in Chaabouni et al., 2007]. The molecular cytogenetic characterization of chromosome 20p rearrangements has been described in a few cases, which has allowed not only a more accurate genotype–phenotype correlation, but also the investigation of its underlying mechanisms. Sidwell et al. [2000] reported on a case of pure trisomy 20p arisen from de novo isochromosome formation, and associated with a non-reciprocal rearrangement involving the translocation of 20q11.1qter sequences onto chromosome 4p. Ravel et al. [2003] described a de novo isolated interstitial tandem duplication of chromosome 20p12 with no evidence of repeated sequences mediating this rearrangement. Ardalan et al. [2005] reported on a derivative with 20pter duplication and 20qter deletion originated from a familial pericentric intrachromosomal insertion following homologous pairing and crossover at meiosis. Chaabouni et al. [2007] described a case of de novo duplication spanning almost the whole short arm of chromosome 20p and associated with a small distal 20q deletion. Recently, Leclercq et al. [2009] reported on the first case of inversion duplication deletion involving the short arm of chromosome 20p (inv dup del 20p), which likely formed through a U-type exchange. High-resolution oligonucleotide CGH array revealed an 18.16 Mb duplication of 20p11.22p13 in association with a 20p13 terminal deletion sizing 2.02 Mb. Herein, we report on a case of trisomy 20p of maternal origin initially detected by conventional cytogenetic investigation. Molecular cytogenetic studies with fluorescent in situ hybridization (FISH) and single-nucleotide polymorphism (SNP) array revealed a direct tandem duplication of 20p11.21p13 associated with a contiguous 20p11.21 proximal microdeletion. The clinically normal mother had the same chromosome abnormality detected in about 15% of her lymphocyte cells. We have compared the phenotype of our patient to similar cases from the literature in order to further delineate trisomy 20p syndrome. The patient, a 21-year-old woman (Fig. 1), was the first child of non-consanguineous normal parents, aged 23 (mother) and 22 years (father). The mother had two miscarriages at 10 weeks of gestation and a normal son was subsequently delivered. Patient’s pregnancy was uncomplicated and she was delivered at term by caesarean. Apgar scores were 6 and 8 at 1 and 5 min, respectively. Birth weight was 2,960 g ( 10th centile) and birth length 49 cm (25th centile). The sucking reflex was present. At 2 months she had surgery for bilateral inguinal and umbilical hernia. By the age of 9 months she had little control over her head and neck and could not sit without support. She started walking at the age of 18 months with frequent falls. Independent walk was achieved at 2 years of age. On examination at 21 years of age, occipital-frontal circumference (OFC) was 54 cm (2–50th centile), height 1.70 m (90th centile) and weight 65 kg (75–90th centile). She had a high forehead, hypertelorism, downslanted palpebral fissures, deep-set eyes, thick

High-Resolution Comparative Genomic Hybridization of Mirna Genes In Therapy-Related AML Identifies a Somatic Deletion of MiR-223

AbstractAbstract 2759MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and diverse cellular processes. Expression profiling of miRNAs has identified dysregulated miRNAs in many cancers, including acute myeloid leukemia (AML). However, the mechanism for altered miRNA gene expression and the frequency of miRNA gene mutations in AML is largely unknown. We performed next-generation sequencing and high-resolution comparative genomic hybridization (CGH) to determine the frequency of miRNA gene mutations in 30 patients with therapy-related AML (t-AML). The sequencing is completed, and final analysis is underway. Herein, we report the results of the CGH. We designed custom CGH arrays for all 835 miRNA genes in miRBase (version 14) and 44 genes involved in miRNA processing. Average probe spacing was 30 bp for miRNA genes and 80 bp for miRNA processing genes and 10 kb flanking regions were interrogated for all genes. In each case, genomic DNA from leukemic blasts was compared with DNA from a skin biopsy from that patient. The median age of the 30 patients with t-AML was 49.2 years (25-77) with M:F ratio 13:17. The mean blast % was 81% (31-100). Consistent with previous reports, many of these patients had an abnormal karyotype with abnormalities of chromosome 5 and 7, 13% and 17% respectively. As expected, CGH analysis confirmed copy number alterations already identified by cytogenetics. In addition, we identified a single t-AML sample (from a male patient) that carried a small (435 kb) hemizygous deletion of miR-223 on chromosome × that was not apparent by cytogenetics. Quantitative PCR of genomic DNA confirmed the loss of the miR-223 gene, and real time RT-PCR demonstrated loss of miR-223 expression. Of note, miR-223 has been implicated in the regulation of granulopoiesis, and mice lacking miR-223 display a myeloproliferative phenotype (Johnnidis, Nature 2008). We screened an additional 27 AML patients for miR-223 expression and identified 3 other samples with miR-223 expression 2 standard deviations below the normal (based on CD34+ cells from healthy donors). No copy number alterations in miR-223 were detected in these patients, suggesting epigenetic silencing of miR-223. Consistent with this possibility, one of these patients carried a t(8;21), which has been shown to epigenetically silence miR-223 (Fazi, Cancer Cell 2007). The mechanism by which miR-223 is silenced in the other two AML samples is under investigation. In summary, cytogenetically silent deletions of miRNA genes are uncommon in t-AML. Loss of miR-223 expression can occur through somatic mutation or epigenetic silencing and is likely to contribute to leukemic transformation in a subset of patients with AML. Disclosures:No relevant conflicts of interest to declare.

C20orf94 deletion Is Strongly Associated with TEL/AML1 Rearrangement and Links Illegitimate V(D)J Recombination with Gender Bias In Childhood Acute Lymphoblastic Leukemia

AbstractAbstract 1718Childhood acute lymphoblastic leukemia (ALL) is characterized by recurrent structural and/or numeric genetic aberrations and has been consistently reported to occur with a 20% higher incidence in males relative to females. We performed 100k Affymetrix SNP-array analysis in a selected set of 20 ALL samples. In five out of these 20 patients, a previously described deletion of the 5‘ region of C20orf94 – a gene coding for an uncharacterized DNA repair-associated protein – was observed. The results were validated by high-resolution custom-made CGH array analysis. As the breakpoints within C20orf94 were defined to recurrent positions, we next applied a PCR assay to screen diagnostic leukemic specimens of a representative cohort of 513 patients enroled into the German multicenter trial ALL-BFM 2000 on treatment of childhood ALL. Here, C20orf94 deletions were detected in 164 patients (32.0%). None of 134 available remission samples exhibited the deletion as well as none of 145 healthy blood donors. Sequencing analysis in 40 patients revealed specific breakpoint junctions with typical characteristics of illegitimate V(D)J recombination in all samples. When analyzing the association of C20orf94 deletion with clinical characteristics in the entire screening cohort, the most significant interrelationships were observed for male gender (75.6% of positives; P < 0.001) and TEL/AML1-rearranged ALL (43.3% of positives; P < 0.001). A negative association was observed for a DNA index of ≥1.16 (3.0% of positives; P < 0.001). In contrast, C20orf94 deletion did not show any effect on treatment outcome. Additional screening of an independent cohort of 232 TEL/AML1-rearranged ALLs identified 145 positive samples and not only confirmed the high incidence of C20orf94 deletion in this subgroup, but also allowed validation of its specific association with male gender (55.9% of positives; P = 0.049). In 21 of 22 (17 TEL/AML1-positive and 5 other pre B cell ALLs) paired initial and relapse leukemic samples C20orf94 deletions were maintained at relapse. Breakpoint sequencing of paired initial and relapse leukemic samples revealed a stable monoclonal breakpoint sequence in 9 patients indicating stability of C20orf94 deletions during disease progression. The remaining samples either developed heterogeneity of C20orf94 breakpoints at relapse after monoclonality at initial diagnosis or initially already showed a polyclonal pattern. Backtracking of C20orf94 deletions to birth in seven TEL/AML1-positive patients using material derived from Guthrie cards did not yield any positive results. In conclusion, we describe the frequent and uniform deletion of the 5‘ part of C20orf94 in childhood ALL - particularly in males and the TEL/AML1-rearranged subtype. These findings suggest a potential role for C20orf94 deletion in the pathogenesis of childhood ALL and point to differences in illegitimate V(D)J recombination as one potential explanation for the observed and currently only poorly understood gender bias in childhood ALL. Disclosures:No relevant conflicts of interest to declare.

Archival Fine-Needle Aspiration Cytopathology (FNAC) Samples: Untapped Resource for Clinical Molecular Profiling

Microarray technologies provide high-resolution maps of chromosome imbalances and epigenomic aberrations in the cancer cell genome. Such assays are often sensitive to sample DNA integrity, voiding the utility of many archival pathology specimens and necessitating the special handling of prospective clinical specimens. We have identified the remarkable preservation of higher-molecular weight DNA in archival fine-needle aspiration cytopathology specimens from patients greater than 10 years of age. We further demonstrate the outstanding technical performance of 57 fine-needle aspiration cytopathology samples for aberration detection on high-resolution comparative genomic hybridization array, DNA methylation, and single nucleotide polymorphism genotyping platforms. Forty-four of 46 malignant aspirates in this study manifested unequivocal genomic aberrations. Importantly, matched Papanicolaou and Diff-Quik fine-needle aspiration cytopathology samples showed critical differences in DNA preservation and DNA integrity. Overall, this study identifies a largely untapped reserve of human pathology specimens for molecular profiling studies, with ramifications for the prospective collection of clinical biospecimens.

Stromal and epithelial cells of endometriosis from different anatomical sites share the same genomic imbalances, suggesting a common origin

Endometriosis (EDT) is a prevalent and chronic gynecological disease characterized by the presence of endometrial-like tissue outside the uterus. Although EDT is considered a benign disease, it presents some characteristics similar to tumors, such as genetic alterations deregulating cell proliferation, invasiveness, metastasis, apoptosis and immune response evasion. High Resolution Comparative Genomic Hybridization (HR-CGH) is a tool for screening of genomic imbalances in distinct cell components and requires a minimal amount of tissue and DNA, such as occurs in EDT.

Identification of candidate genes of autism on the basis of molecular cytogenetic and in silico studies of the genome organization of chromosomal regions involved in unbalanced rearrangements

Autism is one of the most widely spread mental diseases among children. Different genetic anomalies make a considerable contribution to the etiology of this disease; therefore, the identification of candidate genes of autism can be regarded as a topical task of modern medical genetics. The molecular cytogenetic examination of children with autism was carried out using high-resolution comparative genome hybridization and subsequent in silico analysis of chromosomal regions involved in unbalanced rearrangements. Five of 126 (4%) children with autism had unbalanced rearrangements of chromosomes 5, 17, 21 (deletions) and chromosomes 4 and 22 (duplications). The following candidate genes were identified in children with autism by in silico analysis: SCARB2, TPPP, PDCD6, SEPT5, GP1BB, PI4KA, NPTX1, STCH, NRIP1, and CXADR. These methods also allowed us to find a possible association between gene clusterization and the formation of the described chromosomal rearrangements. Thus, this study demonstrates that the molecular cytogenetic and bioinformatic methods can be successfully used to search for candidate genes of different diseases and analyze the genome organization.

EIF2C Is Overexpressed and Amplified in Head and Neck Squamous Cell Carcinoma

Aim: To discover putative oncogenes in head and neck squamous cell carcinoma (HNSCC) by integrating data from whole-genome comparison of array-based comparative genomic hybridization (CGH) and expression microarray analysis of HNSCC. Methods: We integrated published data defining regions of loss/gain identified from the profiling of 21 HNSCC using high-resolution (<1 Mb) CGH arrays and data from an mRNA expression microarray (approx. 12,000 genes) comparing 6 normal tissues and 8 HNSCC tumor tissues. Eukaryotic translation initiation factor 2C subunit 2 (EIF2C2) was found to be the most significantly overexpressed gene by mRNA expression array, and corresponded to the most common region of amplification found by the CGH array described by Sparano et al. We validated EIF2C2 overexpression in primary tissue, overexpression and amplification in HNSCC lines (JHU-011, JHU-012, FADU) relative to a minimally transformed oral keratinocyte cell line (OKF6) and performed knockdown experiments. Results: The tumor tissues had an average mRNA expression level of 123 (SD = 49) compared to the normal tissues (18.6, SD = 10) (p = 0.0005) by expression array. Quantitative RT-PCR validation of our expression arrays found that normal tissues had an average expression of 0.76 (SE = 0.08) and tumor tissues of 2.1 (SE = 0.35) (p = 0.0008). EIF2C2 was found to be amplified and overexpressed in 3 HNSCC cell lines. Knockdown of EIF2C2 in cell lines (JHU-012 and JHU-011) inhibited proliferation. Conclusion:EIF2C2 is amplified and overexpressed in HNSCC cell lines and primary tumors and functionally significant in cell lines.

Integration of transcript expression, copy number and LOH analysis of infiltrating ductal carcinoma of the breast

BackgroundA major challenge in the interpretation of genomic profiling data generated from breast cancer samples is the identification of driver genes as distinct from bystander genes which do not impact tumorigenesis. One way to assess the relative importance of alterations in the transcriptome profile is to combine parallel analyses that assess changes in the copy number alterations (CNAs). This integrated analysis permits the identification of genes with altered expression that map within specific chromosomal regions which demonstrate copy number alterations, providing a mechanistic approach to identify the 'driver genes'.MethodsWe have performed whole genome analysis of CNAs using the Affymetrix 250K Mapping array on 22 infiltrating ductal carcinoma samples (IDCs). Analysis of transcript expression alterations was performed using the Affymetrix U133 Plus2.0 array on 16 IDC samples. Fourteen IDC samples were analyzed using both platforms and the data integrated. We also incorporated data from loss of heterozygosity (LOH) analysis to identify genes showing altered expression in LOH regions.ResultsCommon chromosome gains and amplifications were identified at 1q21.3, 6p21.3, 7p11.2-p12.1, 8q21.11 and 8q24.3. A novel amplicon was identified at 5p15.33. Frequent losses were found at 1p36.22, 8q23.3, 11p13, 11q23, and 22q13. Over 130 genes were identified with concurrent increases or decreases in expression that mapped to these regions of copy number alterations. LOH analysis revealed three tumors with whole chromosome or p arm allelic loss of chromosome 17. Genes were identified that mapped to copy neutral LOH regions. LOH with accompanying copy loss was detected on Xp24 and Xp25 and genes mapping to these regions with decreased expression were identified. Gene expression data highlighted the PPARα/RXRα Activation Pathway as down-regulated in the tumor samples.ConclusionWe have demonstrated the utility of the application of integrated analysis using high resolution CGH and whole genome transcript analysis for detecting driver genes in IDC. The high resolution platform allowed a refined demarcation of CNAs and gene expression profiling provided a mechanism to detect genes directly impacted by the CNA. This is the first report of LOH integrated with gene expression in IDC using a high resolution platform.

Combined microdeletions and CHD7 mutation causing severe CHARGE/DiGeorge syndrome: clinical presentation and molecular investigation by array-CGH

Phenotypic variation in CHARGE syndrome remains unexplained. A subcategory of CHARGE patients show overlapping phenotypic characteristics with DiGeorge syndrome (thymic hypo/aplasia, hypocalcemia, T-cell immunodeficiency). Very few have been tested or reported to carry a mutation of the CHD7 (chromodomain helicase DNA-binding domain) gene detected in two-thirds of CHARGE patients. In an attempt to explore the genetic background of a severe CHARGE/DiGeorge phenotype, we performed comparative genomic array hybridization in an infant carrier of a CHD7 mutation. The high-resolution comparative genomic array hybridization revealed interesting findings, including a deletion distal to the DiGeorge region and disruptions in other chromosomal regions of genes implicated in immunological and other functions possibly contributing to the patient's severe phenotype and early death.

Integrative Genomic Analyses Identify BRF2 as a Novel Lineage-Specific Oncogene in Lung Squamous Cell Carcinoma

Traditionally, non-small cell lung cancer is treated as a single disease entity in terms of systemic therapy. Emerging evidence suggests the major subtypes--adenocarcinoma (AC) and squamous cell carcinoma (SqCC)--respond differently to therapy. Identification of the molecular differences between these tumor types will have a significant impact in designing novel therapies that can improve the treatment outcome. We used an integrative genomics approach, combing high-resolution comparative genomic hybridization and gene expression microarray profiles, to compare AC and SqCC tumors in order to uncover alterations at the DNA level, with corresponding gene transcription changes, which are selected for during development of lung cancer subtypes. Through the analysis of multiple independent cohorts of clinical tumor samples (>330), normal lung tissues and bronchial epithelial cells obtained by bronchial brushing in smokers without lung cancer, we identified the overexpression of BRF2, a gene on Chromosome 8p12, which is specific for development of SqCC of lung. Genetic activation of BRF2, which encodes a RNA polymerase III (Pol III) transcription initiation factor, was found to be associated with increased expression of small nuclear RNAs (snRNAs) that are involved in processes essential for cell growth, such as RNA splicing. Ectopic expression of BRF2 in human bronchial epithelial cells induced a transformed phenotype and demonstrates downstream oncogenic effects, whereas RNA interference (RNAi)-mediated knockdown suppressed growth and colony formation of SqCC cells overexpressing BRF2, but not AC cells. Frequent activation of BRF2 in >35% preinvasive bronchial carcinoma in situ, as well as in dysplastic lesions, provides evidence that BRF2 expression is an early event in cancer development of this cell lineage. This is the first study, to our knowledge, to show that the focal amplification of a gene in Chromosome 8p12, plays a key role in squamous cell lineage specificity of the disease. Our data suggest that genetic activation of BRF2 represents a unique mechanism of SqCC lung tumorigenesis through the increase of Pol III-mediated transcription. It can serve as a marker for lung SqCC and may provide a novel target for therapy. Please see later in the article for the Editors' Summary.

Abstract B12: Complete characterization of the “microRNAome” of a human acute myeloid leukemia

Abstract MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression and have been implicated in the pathogenesis of human cancer. Most current studies utilize array-based or quantitative reverse-transcription-polymerase chain reaction (RT-PCR) approaches to measure miRNA expression. However, these approaches do not interrogate all known (or predicted) miRNAs and are unable to detect mutations in miRNAs. Herein, we use next-generation sequencing approaches to comprehensively assess miRNA expression, to identify genetic variants of all miRNA genes and miRNA binding sites in a patient with AML. Methods: This patient (AML1) was a female in her 50s. Routine cytogenetics revealed a normal 46 XX karyotype, and highresolution comparative genomic hybridization studies revealed no somatic copy number alterations at a resolution of ~5kb. We previously reported the sequence of genic regions in the cancer genome of this patient (Nature 456:66, 2008). Massively parallel sequencing of small RNAs isolated from the myeloblasts of AML1 was performed using the ABI SOLiD sequencing platform. Pooled RNA isolated from CD34+ bone marrow cells of 4 healthy volunteers (CD34) was used as control. To detect genetic variants of miRNA genes, we used 454-based sequencing of all 695 miRNA genes in the Sanger miR database (version 12.0). Finally, we analyzed the previously generated whole genome sequence for AML1 for genetic variants in the 3'-untranslated regions (3'-UTR) of all coding genes. Results: 28×10 and 20×10 small RNA sequence reads were obtained from AML1 and CD34 respectively. 8 novel miRNAs were identified from sequences that mapped to unannotated regions of human genome. Expression of 498 known miRNAs was detected with miR-233 being the most highly expressed miRNA in both AML1 and CD34; remarkably, it represented 47.3% of all miRNA reads in AML1. MiRNA gene sequencing of AML1 leukemic blast identified several single nucleotide variants. The whole genome sequence of AML1 skin DNA was used to differentiate germline polymorphism (SNPs) from somatic mutations. 13 novel SNPs and no somatic mutation were detected. Analysis of the 3'UTR of all coding genes in leukemic blasts and skin of AML1 revealed a single somatic mutation in the 3'-UTR of TNFAIP2. This mutation results in suppression of TNFAIP2 protein expression in a miRNA dependent fashion possibly by creating a new miRNA binding site. However, no recurrent mutations in the 3'-UTR of TNFAIP2 were detected in an additional 180 patients with AML. Conclusions: These data demonstrate the feasibility of ‘next generation’ sequencing technologies to identify novel miRNAs, accurately measure mature miRNA expression, and identify both somatic and germline genetic variants of miRNA genes and miRNA binding sites in primary cancer. Using this platform, studies are underway to comprehensively characterize miRNAs in additional human AML samples. Citation Information: Clin Cancer Res 2010;16(14 Suppl):B12.

Array CGH as a potential predictor of radiocurability in intermediate risk prostate cancer

Prostate cancer is the most common male cancer and up to one fifth of diagnosed patients will die of their disease. Current prognostic variables including T-category (of the TNM staging), the absolute or kinetics of prostatic specific antigen (PSA) and the pathologic Gleason score (GS) are utilized to place men in low, intermediate and high-risk prostate cancer risk groupings. There is great heterogeneity within the non-indolent intermediate risk group with respect to clinical response. It is therefore imperative that further genetic and other prognostic factors be identified to better individualize treatment. Somatic alterations in prostate cancer. Herein, we review the potential for somatic alterations in tumor-associated genes (based on comparative genomic hybridization (CGH) in prostate cancers to be novel prognostic, and possibly predictive, factors for prostate cancer radiotherapy response. Intermediate risk prostate cancers show alterations in a number of genes thought to be involved in radiosensitivity, DNA repair, cell death and stem cell renewal. These include deletions at 21q (TMPRSS2: ERG), 13q (RB1), 10q (PTEN), 8p (NKX3.1), additions at 8q21 (containing c-Myc)) and haplo-insufficiency for p53, PARP1, ATM and DNA-PKcs. Conclusions. The use of high-resolution CGH for fine-mapping of deletions and amplifications in pre-radiotherapy prostate cancer biopsies is feasible. Genetic alterations may delineate localized prostate cancer from systemic disease and be used as a predictive factor in that patients would be individually triaged to local (surgery versus radiotherapy) and/or adjuvant (adjuvant androgen ablation or post-operative radiotherapy) therapies in a prospective fashion to improve outcome. The knowledge of abnormal DNA repair pathways within in a given patient could allow for the judicious use of targeted agents (PARP/ATM inhibitors) as personalized medicine.

Structural and numerical abnormalities resolved in one-step analysis: the most common chromosomal rearrangements detected by comparative genomic hybridization in childhood acute lymphoblastic leukemia

Comparative genomic hybridization (CGH) is a technique that permits detection of chromosomal imbalances. This method allows the detection of gains and losses of genetic material at a resolution lower than 5 Mb. The limitations of conventional cytogenetic studies, such as morphologically insufficient quality of metaphases or the mitotic index, can be eliminated by use of CGH. It is particularly important in the diagnosis of leukemias, and CGH could be a useful tool enabling more precise cytogenetic analysis of leukemic cells. A group of 89 children with acute lymphoblastic leukemia was studied by means of CGH using bone marrow obtained from all consecutive pediatric patients. CGH experiments were performed according to the manufacturer's instruction with minor modifications. In addition, each sample was examined with standard GTG technique and fluorescence in situ hybridization. The conventional cytogenetics failed in 12 patients (13.5%), and 22 patients (24.7%) had a normal karyotype. Structural and numerical changes were found in 55 cases (61.8%) displaying a different abnormalities including deletions, trisomies, tetrasomies, isochromosomes, and markers with unknown origin. However, all samples were successfully analyzed by CGH. We have shown that high-resolution comparative genomic hybridization analysis is a reliable and relatively quick one-step method to identify main aberrations that would not be detected by either conventional G banding or by conventional fluorescence in situ hybridization. It should be considered to establish CGH as a routine analysis for screening patients with acute lymphoblastic leukemia.

Posterior Simulation in Countable Mixture Models for Large Datasets

Mixture models, or convex combinations of a countable number of probability distributions, offer an elegant framework for inference when the population of interest can be subdivided into latent clusters having random characteristics that are heterogeneous between, but homogeneous within, the clusters. Traditionally, the different kinds of mixture models have been motivated and analyzed from very different perspectives, and their common characteristics have not been fully appreciated. The inferential techniques developed for these models usually necessitate heavy computational burdens that make them difficult, if not impossible, to apply to the massive data sets increasingly encountered in real world studies. This paper introduces a flexible class of models called generalized Pólya urn (GPU) processes. Many common mixture models, including finite mixtures, hidden Markov models, and Dirichlet processes, are obtained as special cases of GPU processes. Other important special cases include finite-dimensional Dirichlet priors, infinite hidden Markov models, analysis of densities models, nested Chinese restaurant processes, hierarchical DP models, nonparametric density models, spatial Dirichlet processes, weighted mixtures of DP priors, and nested Dirichlet processes. An investigation of the theoretical properties of GPU processes offers new insight into asymptotics that form the basis of cost-effective Markov chain Monte Carlo (MCMC) strategies for large datasets. These MCMC techniques have the advantage of providing inferences from the posterior of interest, rather than an approximation, and are applicable to different mixture models. The versatility and impressive gains of the methodology are demonstrated by simulation studies and by a semiparametric Bayesian analysis of high-resolution comparative genomic hybridization data on lung cancer. The appendixes are available online as supplemental material.