Genomic Profiling Reveals Association of Chromosomal Aberrations on 1 q and 16 pwith Histologic and Genetic Subgroups of Invasive Breast Cancer

Purpose: Invasive ductal carcinoma and invasive lobular carcinoma (ILC) represent the major histologic subtypes of invasive breast cancer. They differ with regard to presentation, metastatic spread, and epidemiologic features. To elucidate the genetic basis of these differences, we analyzed copy number imbalances that differentiate the histologic subtypes. Experimental Design: High-resolution genomic profiling of 40 invasive breast cancers using matrix-comparative genomic hybridization with an average resolution of 0.5 Mb was conducted on bacterial artificial chromosome microarrays. The data were subjected to classification and unsupervised hierarchical cluster analyses. Expression of candidate genes was analyzed in tumor samples. Results:Thehighest discriminatingpowerwas achievedwhencombining the aberrationpatterns of chromosome arms1q and16p,whichwere significantlymore oftengained in ILC.These regions were further narrowed down to subregions 1q24.2-25.1, 1q25.3-q31.3, and 16p11.2. Located within the candidate gains on1q are two genes, FMO2 and PTGS2, known to be overexpressed in ILC relative to invasive ductal carcinoma. Assessment of four candidate genes on 16p11.2 by real-time quantitative PCR revealed significant overexpression of FUS and ITGAX in ILC with 16p copy number gain. Unsupervised hierarchical cluster analysis identified three molecular subgroups that are characterized by different aberration patterns, in particular concerning gain of MYC (8q24) and the identified candidate regions on 1q24.2-25.1, 1q25.3-q31.3, and 16p11.2. These genetic subgroups differed with regard to histology, tumor grading, frequency of alterations, and estrogen receptor expression. Conclusions: Molecular profiling using bacterial artificial chromosome arrays identified DNA copy number imbalances on 1q and 16p as significant classifiers of histologic and molecular subgroups. Invasive ductal carcinoma (IDC) represents the predominant histologic subtype of breast cancer, constituting 40% to 75% of mammary carcinomas, whereas invasive lobular carcinoma (ILC) ranges second in frequency and accounts for 5% to 15% of cases. Besides differences in the histopathologic morphology, ILC and IDC differ with regard to clinical and epidemiologic features. There has been a steady and disproportionate increase in the incidence of ILC compared with IDC in women over 50 years during the last 20 years, which has been attributed to the increased use of hormone replacement therapy (1, 2). The molecular basis for this development as well as for differences in phenotype and clinical behavior between ILC and IDC is not yet understood. The molecular portrait of breast cancer was previously explored by a number of groups (3, 4), with two recent studies specifically comparing the gene expression profiles of IDC and ILC (5, 6). Using unsupervised cluster analysis, Zhoa et al. (5) were able to subdivide ILC into a ‘‘typical’’ and a ‘‘ductal-like’’ subgroup. The typical ILC showed expression patterns similar to those of tumors that previously were grouped into a normallike subgroup (4) because their expression profiles are more similar to normal than to cancerous breast tissue. Korkola et al. (6) were unable to distinguish ILC and IDC by unsupervised hierarchical clustering of gene expression data; however, they also identified a group of tumors, which was similar to the normal-like subgroup (4) and was characterized by low expression of proliferation associated genes. Using supervised statistical methods, sets of genes distinguishing ILC and IDC Human Cancer Biology Authors’ Affiliations: Divisions of Molecular Genetics and Theoretical Bioinformatics, German Cancer Research Center, Heidelberg, Germany; and Institute of Pathology, Medizinische Hochschule Hannover, Hannover, Germany Received 7/27/05; revised10/24/05; accepted11/7/05. Grant support: Deutsche Forschungsgemeinschaft, Graduiertenkolleg 886 (D.E. Stange), and Bundesministerium fu« r Bildung und Forschung grants 01GR0417, 01GS0460 (B. Radlwimmer and P. Lichter), and 01GR0417 (P. Lichter). The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section1734 solely to indicate this fact. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Requests for reprints: Peter Lichter, Division of Molecular Genetics (B060), German Cancer Research Center, Im Neuenheimer Feld 580, 69120 Heidelberg, Germany. Phone: 49-6221-42-4619; Fax: 49-6221-42-4639; E-mail: m.macleod@ dkfz.de. F2006 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-05-1633 www.aacrjournals.org Clin Cancer Res 2006;12(2) January 15, 2006 345 Research. on July 16, 2017. © 2006 American Association for Cancer clincancerres.aacrjournals.org Downloaded from were defined in both studies. Only three genes (CDH1, FHL1, and ADH1C) overlap between both gene signatures (33 and 378 genes in size). Although gene expression profiling has not allowed reliable classification of IDC and ILC, independent evidence for genetic differences between these tumor types comes from analyses of DNA copy number using chromosomal comparative genomic hybridization (CGH). These studies revealed correlation of gain of 8q and 20q with IDC and loss of 16q and loss of 22q with ILC (7–9). Additionally, preferential loss of 17q and 5p, both of which occur at low frequency in breast cancer, were reported for ILC by one study each (7, 9). In recent years, CGH was increasingly replaced by CGH to bacterial artificial chromosome (BAC) microarrays (matrixCGH; refs. 10, 11), which uses large genomic DNA fragments that are arrayed onto glass slides instead of metaphase chromosomes. This method offers superior resolution, which is only limited by the length and the spacing of the genomic fragments used (12, 13). Recently, matrix-CGH was applied for the first time to compare IDC and ILC (14). Differences in the frequency of copy number imbalances were detected on 1q and 11q; however, neither of these imbalances reached statistical significance. Here, we report the high-resolution genomic profiling of 40 invasive breast cancers using matrix-CGH with an average resolution of 0.5 Mb. The data were subjected to classification and unsupervised hierarchical clustering analyses to improve our understanding of the molecular differences between IDC and ILC. Materials and Methods Tumor material. Fresh frozen material from 46 breast tumors was obtained after informed consent and stored in an anonymous fashion according to an approval of the local ethics committee of the Medizinische Hochschule Hannover. The tumors were classified histologically as IDC (18 cases), ILC (21 cases), and one case with overlapping features of ILC and IDC. Six further cases were excluded from the analysis after repeated hybridizations because the tumor materials were consistently not analyzable. Sampling period encompassed the years 1998 to 2003. Histopathologic variables are summarized in Supplementary Table S1. Preparation of DNA microarrays and labeling. A set of f3,200 sequence-verified BAC genomic fragments (15, 16) covering the genome at f1 Mb resolution was kindly provided to us by the Mapping Core and Map Finishing groups of the Wellcome Trust Sanger Institute (Nigel Carter and Heike Fiegler). Additionally, 3,000 gene– and region-specific genomic fragments were ordered from the RZPD (Berlin, Germany) and CalTech BAC libraries (Invitrogen, Karlsruhe, Germany). A complete list of genomic fragments spotted on the array will be available upon publication on Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) under the platform accession number GPL1432. The chromosomal mapping information was based on the Ensembl (version 17) or the University of California at Santa Cruz genome database (Freeze, July 2003) and for regions of interest a more recent version of Ensembl was used (v31.35d). Detailed description of target DNA preparation and labeling procedures is described in detail in Zielinski et al. (17). Briefly, genomic DNA from tumor tissue and blood of healthy donors was isolated using the Blood and Cell Culture kit (Qiagen, Hilden, Germany) following the instructions of the suppliers. Tumor DNA and sex-matched reference DNA (pooled DNA from four healthy individuals) were labeled differentially using the Bioprime Labeling kit (Invitrogen). Hybridization to microarrays. Ten to 15 Ag of labeled test and sexmatched reference DNA each, plus 100 to 150 Ag of human Cot1-DNA (Roche, Mannheim, Germany), were precipitated and resuspended in 130 AL of hybridization buffer (ULTRAhyb, Ambion, TX). Samples were denatured for 10 minutes at 75jC and reannealed 1 hour at 42jC. Rubber cement was applied around the array to enclose an area of 2 3 cm. Arrays were prehybridized as follows: 750 Ag of salmon sperm (Invitrogen) was precipitated, resuspended in 180 AL of hybridization buffer, denatured for 10 minutes at 75jC, and then added to the array. The array was placed in a humidity chamber on a rocking table at 5 rpm at 37jC for 60 minutes. The prehybridization solution was then removed and replaced by the solution containing reannealed genomic DNA and the array was transferred in a humidity chamber on a rocking table at 5 rpm and incubated at 37jC for 48 hours. The hybridization solution was washed away with 2 SSC/0.05% Tween 20; slides were then incubated for 2 15 minutes with 50% formamide, 2 SSC, 0.1% Tween 20 at 43jC, followed by 15 minutes wash in 2 SSC, 0.05% Tween 20 at 43jC, and 10 minutes in 1 PBS, 0.05% Tween 20 at room temperature. All washing buffers were adjusted to pH 7.0. The slides were dried by centrifugation. Image and data analysis. Arrays were scanned with an Axon 4000B scanner (Axon Instruments, Burlingame, CA) and images were analyzed using GenePix Pro 4.0 software (Axon Instrument