Genome Profiling To Identify Disease Genes

s of the 1 MC-GARD Meeting, 3–5 May 2007: Thursday 3 May 104 identified a set of genes that lie on genomic regions that are commonly amplified or deleted in both murine and human Brca1 or Brca2 tumors. We are currently investigating which of these genes could be driving the selection of these different amplicons or deletions. O2 09.45 – 10.00 COMPREHENSIVE CHARACTERIZATION OF GENOMIC ABERRATIONS IN GANGLIOGLIOMAS BY COMPARATIVE GENOMIC HYBRIDIZATION (CGH), ARRAYBASED CGH AND INTERPHASE-FISH Alexander Hoischen; Marion Ehrler; Jana Fassunke; Christina Landwehr; Bernhard Radlwimmer; Peter Lichter; Johannes Schramm; Albert J Becker; Ruthild G Weber. Rheinische Friedrich-Wilhelms-University, Germany E-mail: [email protected] Introduction. Gangliogliomas (GG) are generally benign (WHO-grade I or II) neuroepithelial tumors frequently associated with epilepsy. They are composed of dysplastic neuronal and neoplastic glial elements.Because little is known about their molecular pathogenesis, we aimed to identify genomic aberrations involved in GG tumorigenesis. Methods and Materials. Fifty-two GGs (47 WHO-grade I, 5 WHO-grade II) were screened for chromosomal imbalances by CGH. Twenty-one GGs with available high molecular weight DNA were analyzed by highresolution array-CGH to an 8k-BAC-array. InterphaseFISH to tumor tissue-sections was performed. Results. Genomic aberrations were detected by CGH in 60% of GGs with an average of 1.85±0.34 (mean±SEM) alterations per tumor (range: 0-13). Recurrent gains were identified on chromosomes 7 (17% of tumors), 5 (13%), 8 (12%), Y (8% of tumors from male patients), 20 and X (8% each), 12 and 19 (6% each), 4, 9q and 17 (4% each). Recurrent losses were found on chromosomes 22q (17%), 9 (10%), Y (8% of tumors from male patients), 16 (8%), 17, 18, 20 and 21q (6% each), 10q, 13q and 19 (4% each). Combined gains of chromosomes 7 and 8 were detected in six cases. Array-CGH confirmed the aberration pattern and additionally detected four cases with smaller imbalances (dim10q21.1-q26, dim10q22.3q25.3, dim15q11.2-q22.2, enh12q13.3-q14.1). By interphase-FISH, a subpopulation of glial cells was found to contain the chromosomal imbalances detected, whereas in the dysplastic neuronal cells no aberrations were found. Two GGs had recurred as malignant glioblastomas (GBM, WHO-grade IV). These GBMs contained a loss of CDKN2A/CDKN2B in one case and an amplification of CDK4 in the other case. Interestingly, the loss of CDKN2A/CDKN2B and a gain of CDK4 were already detectable in the primary GGs of WHOgrade I and II.Conclusions. Our study provides the first comprehensive overview of genomic alterations in a large series of gangliogliomas. O3 10.00 – 10.15 BAC TO INFINIUM: A COMPARISON OF NEARTILING PATH BAC ARRAYS AND ILLUMINA HAP300 SNP ARRAYS FOR MOLECULAR GENETIC PROFILING OF BREAST CANCER Rachael Natrajan; Socorro Maria Rodríguez Pinilla; Maryo B. K. Lambros; Aleksandra Bielen; Alan Mackay; Kerry Fenwick; Narinder Tamber; Chris Jones; Alan Ashworth. Breakthrough Breast Cancer Centre (Institute of Cancer Research), UK E-mail:{rachael.natrajan, aleksandra.bielen}@icr.ac.uk Introduction. Array-based comparative genomic hybridisation (aCGH) has proven to be a powerful tool to characterise the molecular genetic profiles of cell lines and human tumours, to fine map specific amplicons and to identify the likeliest 'amplicon drivers'. Whilst providing accurate copy number alterations within tumours, bacterial artificial chromosome (BAC) arrays do not provide information regarding copy neutral events (e.g., endoreduplication and mitotic recombination). On the other hand, profiles obtained with SNP platforms are reported to show a greater variation for regions with no copy number alterations. Our aims were to define whether Infinium SNP analysis accurately identifies single copy number gains and whether copy neutral events could be reliably identified. Methods and Materials. We compared the molecular genetic profiles of a series of grade III breast carcinomas of distinct subtypes. Tumour samples were microdissected to ensure >90% of neoplastic cells and profiled with both platforms. Normalised and smoothed Log2 ratios were converted into categorical variables (i.e., homozygous deletions, losses, no change, gains and amplifications) according to previously defined and FISH-validated thresholds. Results of BAC array analysis were directly compared to the copy number scores as generated by Illumina's proprietary software 'Bead studio v2' using the log R ratios and B allele frequencies. Amplifications were confirmed by means of in situ hybridisation. Abstracts of the 1 MC-GARD Meeting, 3–5 May 2007: Thursday 3 May 105s of the 1 MC-GARD Meeting, 3–5 May 2007: Thursday 3 May 105 Results. In general there was good agreement between whole arm gains and losses and high-level amplifications, however the Bead studio software failed to identify a number of low level gains and deletions detected by BAC arrays. Surprisingly, no significant increase in the resolution of the detected copy number changes was found with Infinium arrays. On the other hand, Illumina arrays identified regions with copy number silent LOH not detected by aCGH in 83% of samples. Identification of such events will enhance the understanding of the genetic evolution of the tumour and provide a greater insight into the mechanisms involved with inactivation of tumour suppressor genes. By comparing the log R ratios from the SNP chip analysis with B allele frequency, one can more readily discern copy number variation from the norm. However, in this study, it was observed that this approach is hindered by the inclusion of as little as 10% of normal cells within the tumour sample, making its application to micro-dissected tumour samples challenging. Conclusions. Based on our analysis so far, it is apparent that the thresholds for determining copy number alterations are sub-optimal with the Beadstudio software v2. Our results also demonstrate that BAC arrays and SNP chips can be used in a complementary fashion. Deconvolution of the amplicons and identification of regions harbouring putative tumour suppressor genes in high-grade breast cancer samples using both highresolution techniques is currently being undertaken. O4 10.15 – 10.30 GENOME-WIDE SCREENING OF HEAD AND NECK SQUAMOUS CELL CARCINOMA (HNSCC) USING SINGLE NUCLEOTIDE POLYMORPHIC MARKER (SNP)-ARRAYS Frederike Dijk; Boudewijn J. M. Braakhuis; Thijs Krugers; Ruud H. Brakenhoff. VU University Medical Center, Amsterdam, The Netherlands E-mail: [email protected] Introduction. Genetic profiling of HNSCC can be performed using microarray comparative genomic hybridization (maCGH) that detects numerical chromosomal aberrations, but it underestimates allelic losses because of copy neutral events. Allelic loss is relatively frequent and occurs early in carcinogenesis. Moreover, established maCGH platforms do not always allow to use DNA from archival formalin-fixed paraffin embedded (FFPE) tissue. A recently introduced SNP array platform allows for simultaneous detection of numerical and allelic aberrations. This is carried out on short (40 bp) segments of genomic DNA around the SNPs of interest, suggesting application for archival DNA. We aim to evaluate the performance of the Illumina SNP platform for FFPE HNSCC tissue specimen. We ultimately aim to elucidate whether the metastatic phenotype can be predicted based on a genetic profile of the primary HNSCC. Methods and Materials. In total, 12 HNSCC and 12 corresponding normal DNA FFPE samples were analyzed. samples of primary HNSCC were from patients with more than three tumour-positive lymph nodes, who have a 50% risk to develop distant metastases. To evaluate the accuracy of the Illumina platform, samples were included from which the genetic changes were determined previously using established platforms. Three fresh-frozen samples were included for comparison. Results. Analysis of SNPs showed a call frequency of 0.9931 for FFPE vs 0.9960 for frozen samples. Comparison of archival material to frozen material and to results obtained from frozen material of the same tumours with maCGH using BAC arrays or allelic loss determination by microsatellite PCR, shows that the genetic data obtained are practically identical. Conclusions. These findings indicate that genome-wide SNP screening can be performed on FFPE-HNSCC material with high accuracy using the Illumina platform. Analysis of the results with respect to prediction of the metastatic phenotype on the basis of a genetic profile using archival HNSCC samples is currently in progress. 10.30 – 11.00 TEA AND COFFEE O5 11.00 – 11.15 INTEGRATED GENOMIC AND TRANSCRIPTIONAL PROFILING YIELDS PUTATIVE MARKER GENES FOR CERVICAL CANCER Saskia M Wilting, Jillian de Wilde, Chris JLM Meijer, Johannes Berkhof, Yajun Yi, Wessel N van Wieringen, Boudewijn JM Braakhuis, Bauke Ylstra, Peter JF Snijders, Renske DM Steenbergen Department of Pathology, Department of Clinical Epidemiology and Biostatistics, Department of Mathematics, Department of Otolaryngology/Head and Neck Surgery, VU University, Amsterdam, The Netherlands; Division of Genetic Medicine, VanderbiltAbstracts of the 1 MC-GARD Meeting, 3–5 May 2007: Thursday 3 May 106s of the 1 MC-GARD Meeting, 3–5 May 2007: Thursday 3 May 106 Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, Tenessee, USA E-mail: [email protected] A better understanding of the consequences of recurrent (epi)genetic alterations that occur during cervical carcinogenesis is essential in the search for novel biomarkers. In this study we determined genome-wide expression profiles of 10 squamous cell carcinomas (SCCs), 5 adenocarcinomas (AdCAs) and 6 normal epithelial samples. Expression patterns were subsequently combined with genome-wide chromosomal profiles in the same carcinomas. Differential gene expression analysis identified 76 genes with altered expression in carcinomas compared to normal epithelium. Microarray results for a subset of these genes were validated by real-time RT-PCR. Among the differentially expressed genes a relative overrepresentation of genes located at chromosome 3q, one of the most frequently gained areas in SCCs, was observed (false discovery rate (FDR)<0.005). To further investigate the relationship between gene expression and chromosomal alterations 2 statistical approaches were used, i.e. differential gene locus mapping (DIGMAP) and the array CGH expression integration tool (ACE-it). Using these methods we found that increased gene expression was linked to increased gene copy numbers at 1q32.1, 3q13.32-22.3, 3q26.32-27.3, and 20q11.2113.33, whereas a loss at 11q22.3-25 correlated with recurrent decreased gene expression. Seven genes with significantly higher expression in carcinomas compared to normal epithelium were located within these regions. In conclusion, integrated genome-wide chromosomal and transcriptional analysis of cervical carcinomas highlighted 7 genes of which further investigations are warranted. O6 11.15 – 11.30 GAINS AND AMPLIFICATIONS OF THE ABLNUP214 REGION IN BLAST STAGE OF CML Diana Brazma; Anastasios Chanalaris; Junia Melo; Anna Virgilli; Jane Apperley; Elisabeth P. Nacheva; Colin Grace. Royal Free and UCL Medical School, UK E-mail: [email protected]; e.nacheva@ucl. ac.uk Introduction. Chronic myeloid leukaemia (CML) is a pluripotent haematopoietic stem cell disorder defined by expression of the BCR-ABL fusion gene, a constitutively activated tyrosine kinase. The fusion gene commonly results from formation of the Philadelphia chromosome (Ph) after a t(9;22)(q34;q11) or related variant rearrangementThe expression of the chimeric BCR/ABL gene is necessary but not sufficient to maintain the disease progression. The appearance of various chromosomal and molecular aberrations in the course of the disease is well documented but without any causal relationship. Methods and Materials. We undertook array CGH analysis of samples from 48 CML patients (22 chronic phase and 26 blast crisis) and 12 CML cell lines. Two array platforms were used – 1 Mbp BAC chip (Spectral Genomics 2600) and 60-mer oligonucleotide (44B Agilent). Experimental conditions followed manufacturers protocols. The same reference DNA (collections of 6 disease free individuals purchased from Invitrogene) was used throughout the study. Confirmation of the genome imbalances was sought using fluorescent in situ hybridisation and quantitative PCR. Results. A number of recurrent genome imbalances associated with disease progression were identified. Among these were copy number variations of the 9q34 region harboured by the Philadelphia chromosome. Presented as gains and/or high-level amplifications, these cryptic aberrations involve the 9q34.12 sequences distal of the ABL breakpoint. These imbalances were found only in blast crisis patient's samples. Furthermore, in cases with follow-up samples, the 9q34.1 imbalances were only present in the advanced stage of disease, thus confirming their secondary nature. FISH demonstrated that these cryptic aberrations affect the Philadelphia marker without affecting the G banding appearance. A common amplicon covering the ABL-NUP214 region was identified with estimated size of 1.2 Mbp and found to contain three known genes, namely FIBCD1 (fibrinogen family), LAMC3 (laminins family) and the NUP-214. The latter is a nucleoporin gene, recently associated with T-cell acute lymphoblastic leukaemia. A dual colour FISH probe targeting the ABL-NUP214 region was applied to samples from (1) an additional twenty-three CML patients either in an accelerated/blast stage of CML or known to carry a deletion of der(9) chromosome or known to be resistant to Imatinib treatment and (ii) 12 CML cell lines. Duplications and amplifications of the ABL-NUP214 region were found in five BC patients and 4 cell lines. Conclusions. The importance of these observations is two fold: firstly, the Philadelphia chromosome a product of a balanced translocation is shown to be unstable and prone to secondary changes during disease Abstracts of the 1 MC-GARD Meeting, 3–5 May 2007: Thursday 3 May 107s of the 1 MC-GARD Meeting, 3–5 May 2007: Thursday 3 May 107 progression and secondly, the gains/ amplifications of the ABL-NUP214 regions are non-random event in CML, the role of which in remains to be elucidated. O7 11.30 – 11.45 ESTROGEN RECEPTOR STATUS MODULATES THE GENOMIC PROFILE IN FAMILIAL AND SPORADIC BREAST CANCER: A STUDY BASED ON ARRAY CGH Lorenzo Melchor; Emiliano Honrado; Jia Huang; Sara Álvarez; Tara L. Naylor; María J. García; Ana Osorio; Michael R. Stratton; David Blesa; Juan C. Cigudosa; Barbara L. Weber; Nazneen Rahman; Katherine L. Nathanson; Javier Benítez. Human Genetics Group, Human Cancer Genetics Programme, Spanish National Cancer Centre (CNIO) E-mail: [email protected] Introduction. Familial breast cancer represents 5-10% of all breast tumors. Mutations in the two known major breast cancer susceptibility genes, BRCA1 and BRCA2, account for a minority of familial breast cancer, while families without mutations in these genes (BRCAX group) account for 70% of familial breast cancer cases. Our aim is the profiling of the genomic changes present in the three familial breast tumor groups and in sporadic breast cancer. Methods and Materials. We have analyzed 19 BRCA1, 24 BRCA2, and 31 BRCAX samples from familial breast cancer patients, and 19 sporadic breast tumors using a 1 Mb resolution BAC array-based comparative genomic hybridization. Results. We found that BRCA1/2 tumors showed a higher genomic instability than BRCAX and sporadic cancers. There were common genomic alterations present in all breast cancer groups, such as gains of 1q and 16p or losses of 8ptel-p12 and 16q. When we classified all tumors according to Estrogen Receptor (ER) expression status, we found that ER-negative tumors presented higher genomic instability and different altered regions than ER-positive ones, independently of the tumor type (familial or sporadic) and BRCA mutation status (BRCA1 or BRCA2). Conclusions. We describe a set of common genomic aberrations that would characterize the breast tumor development. We suggest that the presence/absence of ER may play a crucial role in driving tumor development through distinct genomic pathways, rather than the BRCA mutation status. According to our results, the BRCA genes mutation status (mainly BRCA1) would contribute to the genomic profile of abnormalities by increasing or modulating the genome instability. O8 11.45 – 12.00 LOW-GRADE B-CELL LYMPHOMA SUBTYPES SHOW DISTINCT PATTERNS OF GENOMIC INSTABILITY Bibiana I. Ferreira; Juan F. Garcia; Manuela Mollego; Patrocinio Algara; Francisca Camacho; Miguel A. Piris; Juan C. Cigudosa. Human Genetics Group, Human Cancer Genetics Programme, Spanish National Centre (CNIO), Spain E-mail: [email protected] Introduction. Low-grade lymphomas account for approximately 50% of all lymphomas. They are distinguished by a relatively low proliferative index, small cell size, large tumoural masses in hemopoietic organs, and a paradoxical combination of advanced clinical stages associated with low clinical aggressivity. Our aims are: 1) to characterize the genomic pattern (in terms of DNA copy number changes) of distinct groups of low-grade B cell lymphomas; and 2) to establish correlations between genomic aberrations and other histological, molecular or clinical features. We studied six different sub-types of low-grade B-cell lymphomas: follicular (FL), splenic (SMZL) and nodal (NMZL) marginal zone, lymphoplasmocytic (LPL), mantle cell (MCL) and B-cell chronic lymphocytic leukaemia (BCLL). Methods and Materials. We selected "gold-standard" samples from 10-15 different patients from each subtype. Two independent pathological confirmed the presence of at least 80% of tumour cells and the complete absence of proliferation markers. A Whole Human Genome CGH Microarray that contains 44000 60-mer oligonucleotides (Agilent Technologies) was used to delineating the genomic pattern. Results and Conclusions. 1. Varying degrees of genomic instability (assessed as gains and losses) affected all samples. The incidence of genomic aberrations ranged from 100% in MCL to 73% in SMZL. 2. Each subtype could be characterized by a distinct pattern of genomic aberrations. Among them, B-CLL and SMZL could be segregated in two classes that showed distinct numerical and structural genomic changes, probably reflecting different clinical behaviour. 3. On the other hand, we have identified some genomic aberrations that can be observed across most of the Abstracts of the 1 MC-GARD Meeting, 3–5 May 2007: Thursday 3 May 108s of the 1 MC-GARD Meeting, 3–5 May 2007: Thursday 3 May 108 subtypes. These changes (transversal aberrations) may represent progression markers. 4. We are elaborating a list of genes and/or genomic regions that may be involved in the understanding of low-grade lymphoma pathogenesis. O9 12.00 – 12.15 COMPLEX GENOMIC ARCHITECTURE AT XQ28 RESULTS IN DUPLICATION OF MECP2 COMMONLY FOUND IN A SPECIFIC SUBSET OF MR PATIENTS Marijke Bauters; Hilde Van Esch; Mike Friez; Odile Boespflug-Tanguy; Martin Zenker; Angela M. ViannaMorgante; Jaakko Ignatius; Martine Raynaud; Karen Hollanders; Kris Vandenreijt; Pierre Blanc; Claude Moraine; Roger Stevenson; Peter Marynen; Jean-Pierre Fryns; Charles Schwartz; Guy Froyen. Human Genome Laboratory, VIB11, Department of Human Genetics, University of Leuven, Belgium E-mail: [email protected] Recently, we identified a 2-fold increased dosage of MECP2 as the cause of severe mental retardation (MR) with progressive spasticity in 4 unrelated patients, thereby demonstrating a new disease mechanism in mental retardation. In order to assess the prevalence of submicroscopic duplications at Xq28, including the MECP2 gene, we screened well-defined groups of MR patients (12 positives) as well as a heterogeneous group of male MR patients (1 positive) with MLPA, arrayCGH or quantitative PCR. This brings the total MECP2 duplication carriers to 17 unrelated cases/families, which enables a comprehensive genotype/phenotype analysis and the search for a common recombination mechanism. The duplication size varied from 0.3 Mb till 2.2 Mb with a common overlap of the smallest duplication. This 0.3 Mb interval contains 8 genes of which MECP2 is the only one that is highly expressed in brain. FISH in 3 patients showed a tandem orientation, and SNP and marker analysis suggests an intrachromosomal rearrangement inherited from the maternal line. Extensive in silico analysis of the breakpoint regions revealed the presence of numerous repeats, such as Low Copy Repeats (LCRs) and Alu repeats, thus rendering this region unstable and prone to rearrangements. More precise mapping of the breakpoints clearly showed clustering of at least one of the breakpoints in these repetitive regions in 14 out of 17 patients. Interestingly, we were able to clone the breakpoint in only one patient, strongly suggesting that complex rearrangements have occurred. The single cloned breakpoint revealed a mechanism of non-homologous end joining (NHEJ). Therefore, MECP2 duplications most likely originate from combined homologous and non-homologous recombination, and are stimulated but not necessarily mediated by the complex genomic architecture surrounding the MECP2 gene. IL3 12.15 – 13.00 TOWARDS CLINICAL GENOMICS: AN ARRAY OF POSSIBILITIES Norma Jean Nowak; Jeffrey Conroy; Susan Christian; Henry Sadowski; Jeffrey Miecznikowski; Daniel Gaile; Ping Liang. Roswell Park Cancer Institute and University at Buffalo, USA E-mail:{Norma.Nowak, Jeffrey.Conroy}@roswellpark. org The development and evolution of array Comparative Genomic Hybridization (aCGH) has revolutionized the field of molecular cytogenetics. Chromosome abnormalities reflected as discrete copy number gains and losses can now be delineated on the sequence of the human genome at the molecular level. We have identified at high resolution, copy number changes in several thousand samples including 30 types of cancer, developmental disorders including autism, and various mental health diseases. DNA prepared from blood, solid tumors and hematologic malignancies, and FFPE tissues, including a subset with matched frozen tissue, were included. aCGH was performed on ~2000 cancer samples, ~500 developmental disorders and ~400 normal individuals utilizing a 19k BAC array developed at the Roswell Park Cancer Institute. Similarly, 244k oligonucleotide arrays (Agilent Technologies) were utilized for selected samples. The FFPE DNA was screened using the BioScoreTM Screening and Amplification kit (Enzo Life Sciences) and those meeting the criterion for aCGH were subsequently assayed. All samples were fluorescently labeled and hybridized to BAC and Agilent oligonucleotide arrays. The copy number aberration profiles that have emerged from our large scale analysis will be discussed including: 1) tumor specific changes, 2) common changes within tumor types, 3) copy number changes in autism, 4) copy number variation in normal individuals, 5) FFPE derived DNA samples, and 6) BAC and Agilent oligonucleotide platform comparisons. In conclusion, array CGH has emerged as a tool not only for disease gene discovery but Abstracts of the 1 MC-GARD Meeting, 3–5 May 2007: Thursday 3 May 109s of the 1 MC-GARD Meeting, 3–5 May 2007: Thursday 3 May 109 as a robust technology that is amenable to automation for detailing genomic aberrations. As a result molecular karyotyping by aCGH will likely become a routine test in clinical labs for tumor and pre/postnatal diagnostics. 13.00 – 14.00 LUNCH AND COMMERCIAL EXHIBITION STRUCTURAL VARIATION OF THE