High-resolution Genomic Array Research Articles

Genetic Analysis of Multiple Myeloma Identifies Cytogenetic Alterations Implicated in Disease Complexity and Progression.

Simple SummaryMultiple myeloma (MM) is the second most common hematological neoplasia with a high incidence in elderly populations. The disease is characterized by a severe chaos of genomic abnormality. Comprehensive examinations of myeloma cytogenetics are needed for better understanding of MM and potential application to the development of novel therapeutic regiments. Here we utilized gene expression profiling and CytoScan HD genomic arrays to investigate molecular alterations in myeloma leading to disease progression and poor clinical outcomes. We demonstrates that genetic abnormalities within MM patients exhibit unique protein network signatures that can be exploited for implementation of existing therapies targeting key pathways and the development of novel therapeutics.Multiple myeloma (MM) is a genetically heterogeneous disease characterized by genomic chaos making it difficult to distinguish driver from passenger mutations. In this study, we integrated data from whole genome gene expression profiling (GEP) microarrays and CytoScan HD high-resolution genomic arrays to integrate GEP with copy number variations (CNV) to more precisely define molecular alterations in MM important for disease initiation, progression and poor clinical outcome. We utilized gene expression arrays from 351 MM samples and CytoScan HD arrays from 97 MM samples to identify eight CNV events that represent possible MM drivers. By integrating GEP and CNV data we divided the MM into eight unique subgroups and demonstrated that patients within one of the eight distinct subgroups exhibited common and unique protein network signatures that can be utilized to identify new therapeutic interventions based on pathway dysregulation. Data also point to the central role of 1q gains and the upregulated expression of ANP32E, DTL, IFI16, UBE2Q1, and UBE2T as potential drivers of MM aggressiveness. The data presented here utilized a novel approach to identify potential driver CNV events in MM, the creation of an improved definition of the molecular basis of MM and the identification of potential new points of therapeutic intervention.

Exon-focused targeted oligonucleotide microarray design increases detection of clinically relevant variants across multiple NHS genomic centres

In recent years, chromosomal microarrays have been widely adopted by clinical diagnostic laboratories for postnatal constitutional genome analysis and have been recommended as the first-line test for patients with intellectual disability, developmental delay, autism and/or congenital abnormalities. Traditionally, array platforms have been designed with probes evenly spaced throughout the genome and increased probe density in regions associated with specific disorders with a resolution at the level of whole genes or multiple exons. However, this level of resolution often cannot detect pathogenic intragenic deletions or duplications, which represent a significant disease-causing mechanism. Therefore, new high-resolution oligonucleotide comparative genomic hybridisation arrays (oligo-array CGH) have been developed with probes targeting single exons of disease relevant genes. Here we present a retrospective study on 27,756 patient samples from a consortium of state-funded diagnostic UK genomic centres assayed by either oligo-array CGH of a traditional design (Cytosure ISCA v2) or by an oligo-array CGH with enhanced exon-level coverage of genes associated with developmental disorders (CytoSure Constitutional v3). The new targeted design used in Cytosure v3 array has been designed to capture intragenic aberrations that would have been missed on the v2 array. To assess the relative performance of the two array designs, data on a subset of samples (n = 19,675), generated only by laboratories using both array designs, were compared. Our results demonstrate that the new high-density exon-focused targeted array design that uses updated information from large scale genomic studies is a powerful tool for detection of intragenic deletions and duplications that leads to a significant improvement in diagnostic yield.

Prognostic Stratification of Acute Myeloid Leukemia and Mylodysplastic Syndrome Patients on the Basis of Genetic Variations

Acute Myeloid Leukemia (AML) is a heterogeneous disorder characterized by the accumulation of acquired genetic alterations in hematopoietic progenitor cells that alter normal mechanisms of self-renewal, and differentiation. Once a rapidly and life fatal disease, the prognosis of AML has improved in the past decades. Recurrent cytogenetic and molecular genetic abnormalities are proven to be of diagnostic and therapeutic value. All cases of AML cannot be explained by the low resolution cytogenetic techniques or biased molecular genetic approaches. Recent advancements in high resolution genomic techniques has allowed detection of additional submicroscopic abnormalities in the AML genome. This study set out to determine the copy number alterations (CNAs) in bone marrow and oral epithelial samples from 08 AML cases by whole genome Single Nucleotide Polymorphism (SNP) genotyping using Illumina OmniExpress-12 v1-1 Bead Chips containing 700,000 SNPs. Additionally, using a targeted approach with TaqMan real-time quantitative polymerase reaction (RT-qPCR), we tested for previously reported SNP (rs11554137) in a group of 47 AML patients.The group of patients comprised of AML-M1 (n=1), M2 (n=5), M4 (n=1) and M5 (n=1), SNP genotyping data analysis identified 13 acquired CNAs in 05 out 08 AML patients. Deletions were more common than amplifications. These CNAs were distributed across all FAB subtypes except AML-M3.AML-M4 subtypes were found to have more CNAs compared to other subtypes. Notably, in three AML-M2 cases4q24 microdeletion was identified. This region spans TET2 gene which is has been shown to cause microdeletions and copy-neutral loss of heterozygosity (LOH) in a patient with diverse myeloid malignancies. Oral epithelial cells from the same patients were negative for the CNAs. Moreover, the 47 patients tested for known SNPs, 4 patients (8.5%) were found positive for rs11554137. SNP (rs11554137) has been reported to be of prognostic importance for overall survival (OS).The results from this study shows CNAs in regions implicated in myeloid malignancies. The use of high-resolution genomic array identified significant number of CNAs and SNPs which were not previously documented in AML patients in Pakistani population. Further studies dissecting out their role in AML pathogenesis are required. DisclosuresNo relevant conflicts of interest to declare.

Chromosomal instability analysis and regional tumor heterogeneity in colon cancer

Chromosomal instability (CIN) is classically defined as an increase in the rate at which numerical or structural chromosomal aberrations are acquired in a cancer cell. The number of somatic copy number abnormalities (CNAs) revealed by high resolution genomic array can be considered as a surrogate marker for CIN, but several points, related to sample processing and data analysis, need to be standardized. In this work we analyzed 51 CRC samples and matched normal mucosae by whole genome SNP arrays and compared different bioinformatics tools in order to identify broad (>25% of a chromosomal arm) and focal somatic copy number abnormalities (BCNAs and FCNAs respectively). In 15 tumors, two samples, separated by at least 1 cm, were taken from the same tumor mass (double-sampling pairs) in order to evaluate differences in detection of chromosomal abnormalities between distant regions of the same tumor and their influence on CIN quantitative and qualitative analysis. Our data show a high degree of correlation of the quantitative CIN index (somatic BCNA number) between distant tumor regions. On the contrary, a lower correlation is observed in terms of chromosomal distribution of BCNAs, as summarized by a simplified cytogenetic table. Quantitative or qualitative analysis of FCNAs, including homozygous deletions and high level amplifications, did not add further information on the CIN status. The use of the index "somatic BCNA number" can be proposed for a robust classification of tumors as CIN positive or negative even in the presence of a significant tumor regional heterogeneity.

DECIPHER: web-based, community resource for clinical interpretation of rare variants in developmental disorders

Patients with developmental disorders often harbour sub-microscopic deletions or duplications that lead to a disruption of normal gene expression or perturbation in the copy number of dosage-sensitive genes. Clinical interpretation for such patients in isolation is hindered by the rarity and novelty of such disorders. The DECIPHER project (https://decipher.sanger.ac.uk) was established in 2004 as an accessible online repository of genomic and associated phenotypic data with the primary goal of aiding the clinical interpretation of rare copy-number variants (CNVs). DECIPHER integrates information from a variety of bioinformatics resources and uses visualization tools to identify potential disease genes within a CNV. A two-tier access system permits clinicians and clinical scientists to maintain confidential linked anonymous records of phenotypes and CNVs for their patients that, with informed consent, can subsequently be shared with the wider clinical genetics and research communities. Advances in next-generation sequencing technologies are making it practical and affordable to sequence the whole exome/genome of patients who display features suggestive of a genetic disorder. This approach enables the identification of smaller intragenic mutations including single-nucleotide variants that are not accessible even with high-resolution genomic array analysis. This article briefly summarizes the current status and achievements of the DECIPHER project and looks ahead to the opportunities and challenges of jointly analysing structural and sequence variation in the human genome.

Recurrent copy number abnormalities and copy neutral-loss of heterozygosity in colorectal cancer identified by high-density SNP array

Two forms of genetic instability have been observed in colorectal cancer (CRC): chromosomal instability, that is present in 85% cases and is characterized by structural and numerical chromosomal abnormalities (aneuploidy); and microsatellite instability (MSI), characterized by a deficiency of the mismatch repair system and associated to a normal or quasi-normal karyotype. Recent advances in molecular cytogenetics techniques are likely to introduce a revolution in the field. In the present study we analysed a series of 57 CRC samples and their related normal mucosae by high resolution genomic arrays (Affymetrix SNP 6.0 arrays) and set up a series of bioinformatics tools that allow the generation of a rapid report on broad (>25% of a chromosomal arm) and small somatic copy number abnormalities (CNAs), somatic homozygous deletions, focal high level amplifications, and regions of loss of heterozygosity (LOH). MSI tumors represented 14% of our CRC series and showed a median values of somatic broad CNAs of 3 (range 1-6), while microsatellite stable (MSS) tumors showed a median value of 12 (range 0-24). Therefore all MSI tumors were below a threshold of 6 CNAs, while 16.2 % of MSS were below this threshold, representing a group of microsatellite and chromosomal stable tumors. No correlation was observed between the number of tumor associated CNAs and the number of somatic copy neutral-LOH regions, suggesting that different mechanisms underlie such chromosomal abnormalities. Analysis of recurrent CNAs identified somatic homozygous deletions that cannot be ascribed to regions of inherent fragility.

High-Resolution genomic arrays identify CNVs that phenocopy the chromosome 22q11.2 deletion syndrome

The 22q11 Deletion Syndrome includes the overlapping phenotypes of DiGeorge/Velocardiofacial Syndromes, characterized by conotruncal heart defects, cleft palate, thymus, and parathyroid gland dysplasia. The majority (90%) of patients harbor detectable chr22q11.2 deletions, but a genetic etiology for the remainder of patients without a deletion can remain undefined despite major birth defects. We analyzed DNA from eight patients with normal 22q11 FISH studies by high-density single nucleotide polymorphism (SNP) arrays and identified potentially pathogenic copy number variants (CNVs) in four of eight patients. Two patients showed large CNVs in regions of known genomic disorders: one a deletion of distal chr22q11.2 and the other a duplication of chr5q35. A 3-Mb deletion of chr19p13.3 that includes a gene associated with conotruncal heart defects was found in a third patient. Two potentially pathogenic CNVs were found in a fourth patient: a large heterozygous deletion of chr6p24 and a smaller duplication of chr9p24. Our findings support a recent consensus statement advocating chromosomal microarray analysis as a first-line diagnostic approach for patients with multiple congenital anomalies. In patients with phenotypes suggestive of the 22q11.2 syndrome spectrum and normal FISH, microarray analysis can uncover the molecular basis of other genomic disorders whose features overlap those of 22q11.2 deletions.

Sequential SNP6.0 Microarray Analysis In Rai Stage 0 CLL Patients with Either Stable or Progressive Identifies Genomic Evolution In Both Cohorts

AbstractAbstract 1696Sequential karyotypic and/or FISH analysis in patients with CLL show evidence of genomic evolution in 10 – 20% of cases. Common secondary abnormalities include loss of the second allele of 13q14, additional trisomies or translocations involving the immunoglobulin gene loci in patients with trisomy 12 and complex karyotypic abnormalities in patients with loss and/or mutation of the p53 and ATM genes. However the clinical significance of genomic evolution in patients presenting with early stage disease remains uncertain. Nor is it clear whether the use of high resolution genomic arrays can detect additional clinically relevant abnormalities in this cohort of patients. To address these issues we performed sequential genomic studies using the affymetrix SNP 6.0 platform (Affymetrix, Santa Clara, CA), G-banding and FISH in 29 patients presenting with either stage A0 CLL or MBL in whom IGVH gene mutational status and CD38 and ZAP 70 expression were known. In each case, Partek Genomic Suite (Partek Incorporated, St Louis, MS) was used to identify acquired copy number alterations (CNAs) and copy number neutral LOH events (CNNLOH) by comparing the disease presentation (PT) and subsequent (FU) SNP tumor profiles to each other and to matched germ-line DNA extracted from buccal cells. Patients were subdivided into those that subsequently exhibited stable disease for a minimum of 5 years (n=9) with a median time between sampling of 8 years (range 2 – 15) or those that developed progressive disease (n=20) with a median time between sampling of 4 years (range 1 – 10). 8 patients with progressive disease were treated prior to the follow up sample. All stable cases and 13/20 with progressive disease had mutated IGHV genes. The concordance between G-banding, FISH and SNP array data was >99%. In each case, a more complex genome was present at FU. The acquisition of additional CNAs was not associated with IgVH mutation status, ZAP-70, CD38 positivity or whether a patient had been treated. When these data were stratified for whether a patient subsequently exhibited stable or progressive disease, the following observations were made. Firstly, there was no difference in the acquisition of small CNAs (<1Mb) between stable or progressive patients. However, the mean number of additional large CNAs (>1Mb) per patient and the total genomic size of these additional changes were greater in progressive cases; stable and progressive cases exhibited a mean of 0.67 and 1.1 additional CNAs at FU and a mean total imbalance of 2.33Mb and 16.9Mb, respectively. In this limited series these differences did not reach statistical significance. Interestingly, the frequency of copy number variations (CNV, annotated in the Database of Genomic Variation; http://projects.tcag.ca/variation/) was higher in cases that went on to develop progressive disease (P=0.002). The size of established recurrent CNAs also changed between time-points. Most notably, several cases exhibited larger 13q14 deletions at FU. Two patients with deletion or CNNLOH of 17p displayed a more complex genome at FU, characterized by multiple CNAs larger than 1Mb. One patient acquired CNNLOH on 17p at FU in addition to five additional CNAs (>1Mb). Conversely, the degree of complexity did not increase in a patient with an ATM deletion. Six cases without an apparent abnormality of ATM or TP53 showed complex genomes at PT and additional CNAs (mean 2.8) at FU. A series of novel, recurrent CNAs were observed exclusively in FU samples, including duplications of 13q and 15q observed in 38% and 53% of patients, respectively. In conclusion, using high resolution microarray analysis we show genomic evolution in early stage patients with predominantly good risk biomarkers who pursued either an indolent or progressive course. However, our data suggests that larger acquired CNAs may be more prognostically relevant than smaller regions. We show preliminary data implicating CNVs in the pathogenesis of CLL and we have also identified a series of novel acquired regions of CNAs that may harbor cancer genes which it is hoped will contribute to our understanding of the pathophysiology of CLL and/or have clinical relevance. Disclosures:No relevant conflicts of interest to declare.

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.

Clonal selection of 11q CN-LOH and CBL gene mutation in a serially studied patient during MDS progression to AML

By conventional metaphase and SNP array cytogenetics we serially studied a patient affected by high-risk myelodysplastic syndrome (MDS), documenting the conversion from partial trisomy 8q to trisomy 8 and partial tetrasomy 8q during progression to acute myeloid leukemia (AML). Moreover, the serial application of high resolution genomic array analysis at different disease stages allowed the description of cryptic abnormalities and the demonstration of their enrichment in the AML phase. In particular the detection and quantification of a copy-neutral loss of heterozygosity region located in chromosome 11q guided the search for point mutations in the CBL gene, thus allowing the escription of the novel missense mutation K382E and the demonstration of its selection during progression to secondary AML.

Discovering Tumor Suppressor Genes Through Genome-Wide Copy Number Analysis

Classical tumor suppressor gene discovery has largely involved linkage analysis and loss-of-heterozygosity (LOH) screens, followed by detailed mapping of relatively large chromosomal regions. Subsequent efforts made use of genome-wide PCR-based methods to detect rare homozygous deletions. More recently, high-resolution genomic arrays have been applied to cancer gene discovery. However, accurate characterization of regions of genomic loss is particularly challenging due to sample heterogeneity, the small size of deleted regions and the high frequency of germline copy number polymorphisms. Here, we review the application of genome-wide copy number analysis to the specific problem of identifying tumor suppressor genes.

Abstract 5757: A genome-wide screen for microdeletions reveals disruption of polarity complex genes in diverse human cancers

Abstract In a genome-wide screen of 684 cancer cell lines, we identified homozygous intragenic microdeletions involving genes encoding components of the apical-basal cell polarity complexes. Among these, PARD3 is disrupted in cell lines and primary tumors from squamous carcinomas and glioblastomas. Reconstituting PARD3 expression in both cell types restores tight junctions and retards contactdependent proliferation. Searching specifically for small intragenic microdeletions using high resolution genomic arrays may be complementary to other genomic deletion screens and resequencing efforts in identifying new tumor suppressor genes. 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 5757.

A Genome-Wide Screen for Microdeletions Reveals Disruption of Polarity Complex Genes in Diverse Human Cancers

In a genome-wide screen of 684 cancer cell lines, we identified homozygous intragenic microdeletions involving genes encoding components of the apical-basal cell polarity complexes. Among these, PARD3 is disrupted in cell lines and primary tumors from squamous carcinomas and glioblastomas. Reconstituting PARD3 expression in both cell types restores tight junctions and retards contact-dependent proliferation. Searching specifically for small intragenic microdeletions using high-resolution genomic arrays may be complementary to other genomic deletion screens and resequencing efforts in identifying new tumor suppressor genes.

New flow cytometry in hematologic malignancies

The complex and heterogeneous biology of acute lymphoblastic leukemia is unfolding as more experimental approaches such as expression profiling, DNA sequencing, and high resolution genomic arrays continue to unveil new recurrent alterations. In this perspective article, Drs. Cools and Vandenberghe examine new diagnostic applications of flow cytometry. See related paper on page 1767.

Identification of Novel Recurrent Copy Number Variations and Regions of Copy-Neutral Loss of Heterozygosity by High Resolution Genomic Array in Pre-Treatment and Relapsed B-CLL.

Abstract 1098Poster Board I-120 BackgroundB-cell chronic lymphocytic leukaemia (B-CLL) is the most common form of adult leukaemia in the Western World. It is a heterogeneous disease and important biological and clinical differences have been identified. However, the molecular mechanisms underlying emergence and maintenance of B-CLL after treatment remain elusive.Array based comparative genomic hybridization (aCGH) has revolutionized our ability to perform genome wide analyses of copy number variation (CNV) within cancer genomes. Single Nucleotide Polymorphism arrays (aSNP) provide genotyping and copy number variation data and detect regions of copy neutral Loss of Heterozygosity (cnLOH) with the potential to indicate genes involved in leukaemia pathogenesis.Both technologies are evolving rapidly and emerging platforms are thought to allow high resolution (HR) of abnormalities down to a single gene level. AimThe aim of the current study was therefore to test a HR-aCGH and a HR-aSNP platform for their ability to detect large and small CNVs and regions of cnLOH in B-CLL. More specifically, we wanted to:•describe recurrent CNVs in B-CLL•correlate them with results known from fluorescent in situ hybridization (FISH)•compare CNVs in pre-treatment samples and at relapse using paired samples from the same patient•identify regions of cnLOH MethodWe used a high resolution 244K aCGH platform and a 1Mio SNP array in parallel to test and characterize enriched B-CLL peripheral blood samples (>80% CD19+;CD5+) from 44 clinically annotated patients collected at our institution. To distinguish CNVs seen commonly in the general population the results were compared with ‘in house' control data sets and the Database of Genomic Variants (http://projects.tcag.ca/variation/). ResultsOur results show that large abnormalities, already noted by FISH, were reliably identified and the boundaries of abnormalities at 11q22.3, 13q14.2 and 17p could be defined more precisely. In addition, novel and recurrent CNVs within the sample set were identified (1p33; 3p24.3; 3p14.2; 4q12; 4q13.3; 6q21; 6q27; 8p22; 10q24; 11p15.4; 11q12; 11q13.4; 11q14.1; 11q22.1; 11q23.3; 13q14.11; 14q21.1; 15q15.1; 15q25.3; 17p13.3; 17q22; 18p11.32; 18p23; 19p13.13; 19p13.12; 19p13.32; 22q11.21; 22q11.22). Interestingly, some of these abnormalities contain single gene alterations involving oncogenes, chemokine receptors, kinases and transcription factors important in B cell development and differentiation. Assessment of smaller CNVs (less then 10 consecutive oligonucleotides) also revealed recurrent CNVs involving single genes that were clustered according to function and pathways.Comparison of paired pre-treatment and relapse samples showed differences in large CNVs in 6 out of the 14 pairs with the majority being losses within the relapse sample. In particular, relapse samples contained new losses within 2q33.1-2q37.1; 4q13.2-4q13.3; 5q31.3-5q34; 7q36.3; 10q23.1-10q25.1 11q12.3 and multiple losses within 13q14.1-13q14.3. Taken together, these data indicates that genomic instability plays a role in clonal evolution and selection after treatment in at least some patients. Analysis of a bigger cohort of matched pre-treatment and relapse samples is on-going.The importance of copy neutral LOH in B-CLL has been a subject of debate. Using the 1Mio HR-aSNP, we were able to detect multiple regions of cnLOH throughout the genome. Examination of the four regions that are known to have prognostic significance when deleted identified cnLOH involving 13q11-13q34(ter) and cnLOH of 13q21.1-q34(ter) outside the FISH region.Deletions of the 17p13.1 locus including the p53 gene confer poor prognosis in B-CLL and direct treatment decisions. Interestingly, we were able to identify cnLOH involving this region in 5% of samples. In addition, we also noticed cnLOH in 17p13.2 containing genes previously implicated in cancer. The exact pathogenetic and prognostic implications of these findings remain to be established. ConclusionUsing HR-aCGH and HR-aSNP we have identified novel recurrent CNVs and regions of cnLOH in patients with B-CLL. Sequential analysis of the same patients over time suggests that at least in some patients, clonal complexity and dynamics are driven by genomic instability. DisclosuresNo relevant conflicts of interest to declare.

Bursts of retrotransposition reproduced in Arabidopsis

Retrotransposons, which proliferate by reverse transcription of RNA intermediates, comprise a major portion of plant genomes. Plants often change the genome size and organization during evolution by rapid proliferation and deletion of long terminal repeat (LTR) retrotransposons. Precise transposon sequences throughout the Arabidopsis thaliana genome and the trans-acting mutations affecting epigenetic states make it an ideal model organism with which to study transposon dynamics. Here we report the mobilization of various families of endogenous A. thaliana LTR retrotransposons identified through genetic and genomic approaches with high-resolution genomic tiling arrays and mutants in the chromatin-remodelling gene DDM1 (DECREASE IN DNA METHYLATION 1). Using multiple lines of self-pollinated ddm1 mutant, we detected an increase in copy number, and verified this for various retrotransposons in a gypsy family (ATGP3) and copia families (ATCOPIA13, ATCOPIA21, ATCOPIA93), and also for a DNA transposon of a Mutator family, VANDAL21. A burst of retrotransposition occurred stochastically and independently for each element, suggesting an additional autocatalytic process. Furthermore, comparison of the identified LTR retrotransposons in related Arabidopsis species revealed that a lineage-specific burst of retrotransposition of these elements did indeed occur in natural Arabidopsis populations. The recent burst of retrotransposition in natural population is targeted to centromeric repeats, which is presumably less harmful than insertion into genes. The ddm1-induced retrotransposon proliferations and genome rearrangements mimic the transposon-mediated genome dynamics during evolution and provide experimental systems with which to investigate the controlling molecular factors directly.

Chromosomal changes in aggressive breast cancers with basal-like features

Using high-resolution oligonucleotide comparative genomic hybridization arrays, we evaluated chromosomal copy number changes in a series of 16 breast cancers, selected on the basis of highly similar pathologic and molecular features characteristic of the “basal-like” phenotype. Each of these cancers showed numerous gains and losses, reflecting multiple chromosomal rearrangements during the development of these high-grade cancers. Chromosomal losses were particularly prevalent on chromosomal arms 5q, 8p, 9q, 12q, 17p, 19p, and Xq, and gains were commonly seen on chromosomal arms 1q, 8q, and 17q. The regions of high-level amplification (copy number change more than eightfold) on 4q12, 8q23.3, 19p12, and 19q13.2 were particularly remarkable. These regions included candidate oncogenes cKIT, JUND, and AKT2, and immunohistochemistry confirmed that these particular genes were highly expressed in the cancers harboring the specific amplifications. Each of these amplifications was observed only in individual cases, however, and no particular chromosomal alteration appeared to generally characterize this group of cancers. Thus, genomic changes among breast cancers with basal-like features appear to be very heterogeneous. Distinct high-level amplifications may provide new targets for treating some of these cancers, but copy number changes do not reveal a distinctive genomic fingerprint for this proposed class of breast cancers.

Molecular Characterization of Breast Cancer with High-Resolution Oligonucleotide Comparative Genomic Hybridization Array

We used high-resolution oligonucleotide comparative genomic hybridization (CGH) arrays and matching gene expression array data to identify dysregulated genes and to classify breast cancers according to gene copy number anomalies. DNA was extracted from 106 pretreatment fine needle aspirations of stage II-III breast cancers that received preoperative chemotherapy. CGH was done using Agilent Human 4 x 44K arrays. Gene expression data generated with Affymetrix U133A gene chips was also available on 103 patients. All P values were adjusted for multiple comparisons. The average number of copy number abnormalities in individual tumors was 76 (range 1-318). Eleven and 37 distinct minimal common regions were gained or lost in >20% of samples, respectively. Several potential therapeutic targets were identified, including FGFR1 that showed high-level amplification in 10% of cases. Close correlation between DNA copy number and mRNA expression levels was detected. Nonnegative matrix factorization (NMF) clustering of DNA copy number aberrations revealed three distinct molecular classes in this data set. NMF class I was characterized by a high rate of triple-negative cancers (64%) and gains of 6p21. VEGFA, E2F3, and NOTCH4 were also gained in 29% to 34% of triple-negative tumors. A gain of ERBB2 gene was observed in 52% of NMF class II and class III was characterized by a high rate of estrogen receptor-positive tumors (73%) and a low rate of pathologic complete response to preoperative chemotherapy (3%). The present study identified dysregulated genes that could classify breast cancer and may represent novel therapeutic targets for molecular subsets of cancers.

Complex balanced translocation t(1;5;7)(p32.1;q14.3;p21.3) and two microdeletions del(1)(p31.1p31.1) and del(7)(p14.1p14.1) in a patient with features of Greig cephalopolysyndactyly and mental retardation

Complex chromosome rearrangements (CCRs) are rare structural abnormalities that involve at least two chromosomes and more than two breakpoints and are often associated with developmental delay, mental retardation, and congenital anomalies. We report on a de novo, apparently balanced translocation t(1;5;7)(p32.1;q14.3;p21.3) involving three chromosomes in a 7-year-old boy with severe psychomotor retardation, neonatal muscular hypertonia, congenital heart defect, polysyndactyly of hands and feet, and dysmorphic features resembling Greig cephalopolysyndactyly syndrome. Analysis of the chromosome breakpoints using fluorescence in situ hybridization (FISH) with locus-specific BAC clones and long-range PCR products did not identify chromosome imbalance at any of the interrogated regions. High-resolution comparative genomic hybridization (HR-CGH) and array CGH (aCGH) revealed two additional cryptic de novo deletions, del(1)(p31.1p31.1) and del(7)(p14.1p14.1), respectively, that are not associated with the translocation breakpoints. FISH and polymorphic marker analyses showed that the deletion on derivative chromosome 1 is between 4.2 and 6.1 Mb, and the deletion on derivative chromosome 7 is approximately 5.1 Mb, and that both are paternal in origin. The deletion on chromosome 7p encompasses the GLI3 gene that is causative for the Greig cephalopolysyndactyly, Pallister-Hall and some cases of Acrocallosal syndromes. We discuss the potential mechanisms of formation of the described CCR.

Whole‐genome array‐CGH identifies novel contiguous gene deletions and duplications associated with developmental delay, mental retardation, and dysmorphic features

Cytogenetic imbalances are the most frequently identified cause of developmental delay or mental retardation, which affect 1-3% of children and are often seen in conjunction with growth retardation, dysmorphic features, and various congenital anomalies. A substantial number of patients with developmental delay or mental retardation are predicted to have cytogenetic imbalances, but conventional methods for identifying these imbalances yield positive results in only a small fraction of these patients. We used microarray-based comparative genomic hybridization (aCGH) to study a panel of 20 patients predicted to have chromosomal aberrations based on clinical presentation of developmental delay or mental retardation, growth delay, dysmorphic features, and/or congenital anomalies. Previous G-banded karyotypes and fluorescence in situ hybridization results were normal for all of these patients. Using both oligonucleotide-based and bacterial artificial chromosome (BAC)-based arrays on the same panel of patients, we identified 10 unique deletions and duplications ranging in size from 280 kb to 8.3 Mb. The whole-genome oligonucleotide arrays identified nearly twice as many imbalances as did the lower-resolution whole-genome BAC arrays. This has implications for using aCGH in a clinical setting. Analysis of parental DNA samples indicated that most of the imbalances had occurred de novo. Moreover, seven of the 10 imbalances represented novel disorders, adding to an increasing number of conditions caused by large-scale deletions or duplications. These results underscore the strength of high-resolution genomic arrays in diagnosing cases of unknown genetic etiology and suggest that contiguous genomic alterations are the underlying pathogenic cause of a significant number of cases of developmental delay.