Research Article Distinct Genomic Profiles in Hereditary Breast Tumors Identified by Array-Based Comparative Genomic Hybridization Go¨ran Jo¨nsson,1 Tara L. Naylor,5 Johan Vallon-Christersson,1 Johan Staaf,1 Jia Huang,5 M. Renee Ward,5 Joel D. Greshock,5 Lena Luts,4 Ha˚kan Olsson,1 Nazneen Rahman,6 Michael Stratton,6 Markus Ringne´r,3 A˚ke Borg,1,2 and Barbara L. Weber5 1Department of Oncology, University Hospital; 2Lund Strategic Research Center for Stem Cell Biology and Cell Therapy and 3Department of Theoretical Physics, Lund University; and 4Department of Pathology, University Hospital, Lund, Sweden; 5Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, Pennsylvania; and 6Section of Cancer Genetics, Institute of Cancer Research, Sutton, Surrey, United Kingdom Abstract of additional predisposing genes, although technical limitations Mutations in BRCA1 and BRCA2 account for a significant and the complexity of BRCA gene regulation and mutation proportion of hereditary breast cancers. Earlier studies have spectrum can probably explain why some disease-causing muta- shown that inherited and sporadic tumors progress along tions are missed (2). BRCA1 and BRCA2 function as classic tumor different somatic genetic pathways and that global gene suppressor genes with frequent loss of the wild-type allele in expression profiles distinguish between these groups. To tumors of mutation carriers. The BRCA1 protein has been determine whether genomic profiles similarly discriminate implicated in a broad range of cellular functions, including repair among BRCA1, BRCA2, and sporadic tumors, we established of double-strand breaks by homologous recombination, cell cycle DNA copy number profiles using comparative genomic checkpoint control, chromatin remodeling, and transcriptional hybridization to BAC-clone microarrays providing <1 Mb regulation. The role of BRCA2 is more restricted to DNA resolution. Tumor DNA was obtained from BRCA1 (n = 14) and recombination and repair processes, being particular important BRCA2 (n = 12) mutation carriers, as well as sporadic cases in RAD51 regulation. It is thought that part of the tumor (n = 26). Overall, BRCA1 tumors had a higher frequency of suppressor function of BRCA1 and BRCA2 is attributed to this copy number alterations than sporadic breast cancers (P = genome caretaker activity (3). 0.00078). In particular, frequent losses on 4p, 4q, and 5q in Earlier studies have suggested different somatic genetic path- BRCA1 tumors and frequent gains on 7p and 17q24 in BRCA2 ways in progression of inherited and sporadic tumors, and several tumors distinguish these from sporadic tumors. Distinct histopathologic and clinical features differ among BRCA1, BRCA2, amplicons at 3q27.1-q27.3 were identified in BRCA1 tumors and sporadic breast cancers. For instance, BRCA1 tumors are often and at 17q23.3-q24.2 in BRCA2 tumors. A homozygous estrogen and progesterone receptor (ER/PR) negative, whereas deletion on 5q12.1 was found in a BRCA1 tumor. Using a set BRCA2 tumors predominantly are ER and PR positive (4–7). BRCA1 of 169 BAC clones that detect significantly (P < 0.001) different tumors are primarily of high histologic grade and manifest high frequencies of copy number changes in inherited and sporadic lymphocyte infiltration and continuous ‘‘pushing’’ tumor margins. tumors, these could be discriminated into separate groups BRCA2 tumors are more heterogeneous but have significantly less using hierarchical clustering. By comparing DNA copy number tubule formation compared with sporadic tumors, which account and RNA expression for genes in these regions, several for an overall higher histologic grade (7). Using global gene candidate genes affected by up- or down-regulation were expression profiling, we have previously identified a set of genes to identified. Moreover, using support vector machines, we distinguish among BRCA1, BRCA2, and sporadic tumors (8). In a correctly classified BRCA1 and BRCA2 tumors (P < 0.0000004 parallel fashion, we used conventional metaphase comparative ge- and 0.00005, respectively). Further validation may prove this nomic hybridization (CGH) to characterize chromosomal aberrations tumor classifier to be useful for selecting familial breast in hereditary breast tumors (9–11). We found a higher frequency of cancer cases for further mutation screening, particularly, as copy number alterations among BRCA1 and BRCA2 tumors when these data can be obtained using archival tissue. (Cancer Res compared with sporadic cases. BRCA1 tumors harbor frequent loss 2005; 65(17): 7612-21) at 2q, 4p, 4q, 5q, and 12q, whereas BRCA2 tumors are characterized by a higher frequency of 6q and 13q losses as well as gains on 17q22- Introduction 24 and 20q13. Others have also used metaphase CGH analysis to classify BRCA1 tumors based on patterns of genomic alterations and Germ line mutations in the two major breast cancer suscepti- suggested gain of 3q and loss of 3p and 5q to distinguish BRCA1 from bility genes, BRCA1 and BRCA2, confer a highly elevated risk of sporadic tumors (12). breast and ovarian cancer and account for a significant proportion Taken together, these findings imply that tumor profiling can be of inherited breast cancer (1). However, many familial breast cancer useful as diagnostics tools and encouraged us to extend our cases cannot be attributed to BRCA1 and BRCA2, suggesting a role previous studies with an array-based CGH technique, providing higher consistency and resolution, to build classifiers based on genomic aberration patterns in tumor tissue. In the familial cancer Requests for reprints: A˚ke Borg, Department of Oncology, Lund University, SE-221 clinic, candidates for BRCA1 and BRCA2 mutation screening are 85 Lund, Sweden, SE 22185. Phone: 46-46-177502; Fax: 46-46-147327; E-mail: ake.borg@ selected based on family history, age at onset, bilateral disease, and onk.lu.se. I2005 American Association for Cancer Research. cases of ovarian cancers in the family. Because screening of these doi:10.1158/0008-5472.CAN-05-0570 genes is laborious and costly, more precise criteria for selection and Cancer Res 2005; 65: (17). September 1, 2005 7612 www.aacrjournals.org Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2005 American Association for Cancer Research. Genomic Profiling of Hereditary Breast Cancer diagnosis is needed (e.g., a rationale corresponding to the tests for treatment before proteinase K digestion and phenol chloroform purifica- microsatellite instability and mismatch repair protein expression tion. For all samples, 1 Ag of genomic DNA was labeled according to in diagnosis of hereditary nonpolyposis colon cancer; ref. 13). In published protocols (14) using a random labeling kit (Invitrogen Life the present study, we established DNA copy number profiles for Technologies, Carlsbad, CA). Tumor DNA and male reference DNA was differentially labeled, pooled, mixed with human COT-1 DNA, dried down, BRCA1, BRCA2, and sporadic breast tumors using a microarray f and resuspended in a formamide-based buffer (14). The hybridization containing 5,000 individual BAC clones providing an average reactions were applied to arrays and the arrays were incubated under resolution of 1 Mbp. Furthermore, by comparing DNA copy number coverslips for 48 to 72 hours at 37jC. Slides were washed according to and gene expression in 11 BRCA1/2 tumors, we found novel published protocols (15) and scanned using an Axon 4000A scanner (Axon genes affected by genomic aberrations in hereditary breast cancer. Instruments, Wheatherford, TX). All experiments were done with ‘‘dye- Our results form the basis for a new classification system of swap’’ to account for variations in dye labeling efficiency and fluorescence inherited breast cancer and reveal new molecular targets in tumor emission and all BACs are spotted on the array in duplicate; thus, each data progression of inherited tumors. The approach of using array CGH point is represented by four replicates. in diagnostics may be more advantageous than gene expression RNA extraction and gene expression analysis. RNA was extracted profiling because paraffin-embedded material can be used and thus from freshly frozen tumor tissue using Trizol reagent (Invitrogen Life Technologies) followed by RNeasy Midi kit (Qiagen, Chatsworth, CA). RNA more feasible in the clinical setting. quality was assessed using the RNA 6000 Nano LabChip Kit for Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, CA) and concentration was Materials and Methods determined using a NanoDrop Spectrophotometer (NanoDrop Technolo- Patients and tumors. Freshly frozen breast tumor tissue was obtained gies, Wilmington, DE). Arrays were produced by the SWEGENE DNA from the Southern Sweden Breast Cancer Group’s tissue bank at the Microarray Resource Centre, Department of Oncology at Lund University. Department of Oncology, Lund University Hospital and from the HWP and Human Genome Oligo Set Version 2.1 (containing 21,329 70-mer probes) Delaware Memorial Hospital. Breast cancer patients from which BRCA1 and and Human Genome Oligo Set Version 2.1 Upgrade (containing 5,462 70-mer BRCA2 tumors were derived had been screened for germ line mutations in probes) were obtained from Operon Biotechnologies, Inc. (Huntsville, AL). the BRCA1 and BRCA2 genes according to standard techniques (6). From cDNA synthesis, labeling, and cleanup were overall done according to Lund University Hospital, eight frozen BRCA1 tumors were obtained from manufacturers’ instructions