Rachael Natrajan
email: rachael.natrajan@icr.ac.uk

Britta Weigelt

Alan Mackay

Felipe C Geyer

Anita Grigoriadis

David S P Tan

Chris Jones

Christopher J Lord

Radost Vatcheva

Socorro M Rodriguez-Pinilla

A E Britta

Weigelt Ae

A E Felipe

C Geyer Ae

A E David

S P Tan

A E Chris

Jones Ae

A E Radost

Vatcheva Ae

Jose Ae

A E Palacios

Ashworth Alan

Jorge S Ae

Reis-Filho

Á A Mackay

Á C J Lord

Á R Vatcheva

Á A Ashworth

J S Reis-Filho
email: jorge.reis-filho@icr.ac.uk

J S Reis

J Palacios

An integrative genomic and transcriptomic analysis reveals molecular pathways and networks regulated by copy number aberrations in basal

Keywords: Breast cancer, Molecular subtypes, Copy number, Gene expression, Data integration, Molecular pathways

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Introduction

Breast cancer is a heterogeneous disease comprising multiple entities, which have distinct biological features and clinical behaviour. In recent years, it has become apparent that breast cancer heterogeneity may be underpinned by distinct genomic changes [START_REF] Natrajan | Tiling path genomic profiling of grade 3 invasive ductal breast cancers[END_REF][START_REF] Bergamaschi | Distinct patterns of DNA copy number alteration are associated with different clinicopathological features and gene-expression subtypes of breast cancer[END_REF][START_REF] Chin | Genomic and transcriptional aberrations linked to breast cancer pathophysiologies[END_REF][START_REF] Chin | High-resolution aCGH and expression profiling identifies a novel genomic subtype of ER negative breast cancer[END_REF][START_REF] Hu | Genetic alterations and oncogenic pathways associated with breast cancer subtypes[END_REF][START_REF] Mackay | A high-resolution integrated analysis of genetic and expression profiles of breast cancer cell lines[END_REF][START_REF] Neve | A collection of breast cancer cell lines for the study of functionally distinct cancer subtypes[END_REF]. Microarray-based gene expression profiling of breast cancer has led to the identification of at least five distinct classes of breast cancers: luminal A, luminal B, HER2, normal breast-like and basallike breast cancer. It has been hypothesised that the differences between these molecular subgroups of breast cancer stem from differences in their cell of origin [START_REF] Perou | Molecular portraits of human breast tumours[END_REF][START_REF] Sorlie | Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications[END_REF]. However, luminal, HER2 and basal-like breast cancers have been shown to harbour discrete patterns of genetic aberrations, which may also contribute to their distinct transcriptomic profiles and clinical phenotypes [1-3, 5, 10].

Gene copy number aberrations have been observed in [95% of breast cancers and involve a substantial proportion of the genome of breast cancer cells [1-3, 5, 6, 10-12]. It is unclear, however, as to what extent copy number aberrations drive the expression of genes and molecular networks and pathways in breast cancer. Previous efforts in identifying these networks and pathways were either carried out in breast cancer cell lines [START_REF] Heiser | Integrated analysis of breast cancer cell lines reveals unique signaling pathways[END_REF] or made use of non-microdissected tissue [START_REF] Bergamaschi | Distinct patterns of DNA copy number alteration are associated with different clinicopathological features and gene-expression subtypes of breast cancer[END_REF][START_REF] Chin | Genomic and transcriptional aberrations linked to breast cancer pathophysiologies[END_REF][START_REF] Chin | High-resolution aCGH and expression profiling identifies a novel genomic subtype of ER negative breast cancer[END_REF][START_REF] Hu | Genetic alterations and oncogenic pathways associated with breast cancer subtypes[END_REF][START_REF] Adelaide | Integrated profiling of basal and luminal breast cancers[END_REF], which reduces the sensitivity of detection in copy number aberrations and confounds the transcriptomic profiles due to contamination with diploid stromal and inflammatory cells.

The aims of this study were twofold: (1) to determine the genes whose expression levels correlate with gene copy numbers and the genes which are overexpressed when amplified in grade III (GIII) breast cancers; and (2) to determine canonical pathways and networks significantly enriched for these genes. We microdissected and analysed a series of 48 GIII invasive ductal carcinomas of no special type (IDC-NSTs) with high resolution microarray-based comparative genomic hybridisation (aCGH) and mRNA expression arrays. Our study focused on GIII IDC-NSTs only to avoid bias introduced by distinct patterns of genetic aberrations between grade I and III cancers [START_REF] Natrajan | Loss of 16q in high grade breast cancer is associated with estrogen receptor status: Evidence for progression in tumors with a luminal phenotype?[END_REF] and between invasive ductal carcinomas and special types [START_REF] Marchio | Genomic and immunophenotypical characterization of pure micropapillary carcinomas of the breast[END_REF][START_REF] Simpson | Molecular profiling pleomorphic lobular carcinomas of the breast: evidence for a common molecular genetic pathway with classic lobular carcinomas[END_REF][START_REF] Weigelt | Refinement of breast cancer classification by molecular characterization of histological special types[END_REF].

Materials and methods

Sample selection

A series of 64 consecutive GIII IDC-NSTs cases were retrieved from University Hospital La Paz, Madrid, Spain. This project has been approved by the local Research Ethics Committees of the authors' institutions.

Four-lm thick sections of the tumours were subjected to immunohistochemistry with antibodies against oestrogen receptor, (ER, ID5, 1:30, Novocastra), progesterone receptor (PR, 1A6, 1:30, Novocastra), cytokeratin (CK) 5/6 (D5/16 B4, 1:25, DAKO) and epidermal growth factor receptor (EGFR) (EGFR, 31G7, 1:50, Zymed) essentially as previously described [START_REF] Natrajan | Tiling path genomic profiling of grade 3 invasive ductal breast cancers[END_REF]. Immunohistochemistry for HER2 was performed using the Herceptest kit (Dako, Glostrup, Denmark). HER2 expression was scored according to the ASCO/CAP guidelines [START_REF] Wolff | American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer[END_REF]; only cases displaying a 3? score were considered positive, cases displaying a 2? score were labelled as equivocal. In equivocal cases, HER2 gene status was determined using the FDA approved SpotLight chromogenic in situ hybridisation (CISH) HER2 probes. All markers were scored by two pathologists (SMP-R and JSR-F), blinded to the results of the aCGH, expression array and CISH analysis. Tumours were classified into HER2, luminal and basal-like subgroups utilising the criteria of Nielsen et al. [START_REF] Nielsen | Immunohistochemical and clinical characterization of the basal-like subtype of invasive breast carcinoma[END_REF].

Nucleic acid extraction

Ten consecutive 8-lm thick, frozen sections of 64 GIII tumours were microdissected as previously described [START_REF] Marchio | Genomic and immunophenotypical characterization of pure micropapillary carcinomas of the breast[END_REF][START_REF] Marchio | The genomic profile of HER2-amplified breast cancers: the influence of ER status[END_REF] to obtain a percentage of tumour cells in the remaining tissue greater than 90%. DNA was extracted as previously described [START_REF] Natrajan | Loss of 16q in high grade breast cancer is associated with estrogen receptor status: Evidence for progression in tumors with a luminal phenotype?[END_REF]. DNA concentration was measured with Picogreen Ò according to the manufacturer's instructions (Invitrogen, Paisley, UK). RNA was extracted using Trizol according to the manufacturers' instructions (Invitrogen, UK) and quantified using the Agilent 2100 Bioanalyzer with RNA Nano LabChip Kits (Agilent Biosystems). In 16 cases, DNA and/or RNA were of insufficient quantity for aCGH and/or expression array analysis and were excluded from the study. The final dataset comprises 48 cases, whose clinico-pathological characteristics are summarised in Supplementary Table 1.

Microarray-based comparative genomic hybridisation

The 32K bacterial artificial chromosome (BAC) re-array collection (CHORI) tiling path aCGH platform used for this study was constructed in the Breakthrough Breast Cancer Research Centre [START_REF] Marchio | Genomic and immunophenotypical characterization of pure micropapillary carcinomas of the breast[END_REF]. DNA labelling, array hybridisations and image acquisition were performed as described in Natrajan et al. [START_REF] Natrajan | Tiling path genomic profiling of grade 3 invasive ductal breast cancers[END_REF].

Cases with [10% of clones missing and clones where data were not available in C10% of cases were excluded. Log2 ratios were normalised for spatial and intensitydependent biases using a two-dimensional LOESS regression followed by a BAC-dependent bias correction as previously described [START_REF] Marchio | Genomic and immunophenotypical characterization of pure micropapillary carcinomas of the breast[END_REF][START_REF] Huang | Robust smooth segmentation approach for array CGH data analysis[END_REF]. Polymorphic BACs identified in an analysis of 50 male:female and female:female hybridisations were filtered out. This left a final dataset of 31,544 clones with unambiguous mapping information according to the March 2006 build (hg18) of the human genome (http://www.ensembl.org). Data were smoothed using the circular binary segmentation (cbs) algorithm [START_REF] Mackay | A high-resolution integrated analysis of genetic and expression profiles of breast cancer cell lines[END_REF]. A categorical analysis was applied to the BACs after classifying them as representing gain, loss or no-change according to their smoothed Log2 ratio values [START_REF] Arriola | Genomic analysis of the HER2/TOP2A amplicon in breast cancer and breast cancer cell lines[END_REF]. Threshold values were chosen to correspond to three standard deviations of the normal ratios obtained from the filtered clones mapping to chromosomes 1-22, assessed in multiple hybridisations between DNA extracted from a pool of male and female blood donors as previously described [START_REF] Arriola | Genomic analysis of the HER2/TOP2A amplicon in breast cancer and breast cancer cell lines[END_REF][START_REF] Reis-Filho | Pleomorphic lobular carcinoma of the breast: role of comprehensive molecular pathology in characterization of an entity[END_REF] (Log2 ratio of ±0.08). Low level gain was defined as a smoothed Log2 ratio of between 0.08 and 0.45, corresponding to approximately 3-5 copies of the locus, whilst gene amplification was defined as having a Log2 ratio[0.45, corresponding to more than 5 copies [START_REF] Marchio | The genomic profile of HER2-amplified breast cancers: the influence of ER status[END_REF].

Gene expression profiling

Gene expression profiling was performed using the Illumina human WG6 version 2 expression array according to the manufacturer's protocol. Briefly, 200 ng of each RNA sample was amplified using the Illumina Totalprep RNA amplification kit following the manufacturer's instructions (Ambion, UK). Labelled cRNA was subsequently hybridised to Illumina Sentrix-human 6 version 2 expression bead-chips. Full details on RNA amplification and hybridisation can be found at www.illumina.com. Raw gene expression values were robust-spline normalised using the Bioconductor lumi package (http://www.bioconductor.org/ packages/2.3/bioc/html/lumi.html) in R. Genes were mapped to their genomic location using the lumiHumanAllv2 annotation database available from Bioconductor. Only Illumina transcript probes with detection P values \0.01 in [25% of samples were included; this resulted in a dataset of 12,699 transcriptionally regulated probes with accurate and unequivocal mapping information. Gene expression data are publicly available at ArrayExpress http://www.ebi. ac.uk/microarray-as/ae/ (accession number: E-TABM-543).

Integrated aCGH and expression analysis

To identify genes whose expression correlated with genetic copy number changes, cbs-smoothed aCGH data were used to assign the median aCGH states for each of the 12,699 genes in the gene expression dataset using the median values for all BACs that overlap with the genomic positions of each gene. This resulted in a 1:1 matrix of expression data and aCGH values used in correlations. Pearson's correlations were performed between mRNA expression log intensity values and median cbs-smoothed ratios derived from aCGH analysis for each gene. P values for each test were adjusted with Benjamini and Hochberg multiple P value adjustment [START_REF] Mackay | A high-resolution integrated analysis of genetic and expression profiles of breast cancer cell lines[END_REF][START_REF] Benjamini | Controlling the false discovery rate: a practical and powerful approach to multiple testing[END_REF].

To identify genes consistently overexpressed when amplified in regions of recurrent amplification, Mann-Whitney U tests were performed and adjusted with Benjamini and Hochberg multiple P value adjustment. An adjusted P value of \0.05 was considered significant.

For unsupervised analysis, we performed hierarchical clustering of the 48 primary tumours using 848 unique genes identified from the 1,300 intrinsic genes, as described by Hu et al. [START_REF] Hu | The molecular portraits of breast tumors are conserved across microarray platforms[END_REF]. Genes were median centred, and average linkage clustering was performed using Pearson's correlation as the similarity metric on Cluster 3 [START_REF] Eisen | Cluster analysis and display of genome-wide expression patterns[END_REF]. Results were subsequently visualised using Java Treeview (http://jtreeview. sourceforge.net/). SAM software [START_REF] Tusher | Significance analysis of microarrays applied to the ionizing radiation response[END_REF] was employed to determine the genes significantly differentially expressed between basal-like, HER2 and luminal tumours, adopting a false discovery rate of 3% after 1,000 permutations.

Ingenuity pathway analysis

To determine pathways and networks that were significantly regulated in the gene expression data of basal-like, HER2 and luminal GIII tumours, we performed pathway analysis using the Ingenuity Pathway Analysis (IPA) program (http://www. ingenuity.com). HUGO gene identifiers were mapped to networks available in the Ingenuity database and ranked by score. The score indicates the likelihood of the genes in a network being found together due to random chance. Using a 99% confidence level, scores of C3 are considered significant. IPA was performed essentially as previously described [START_REF] Weigelt | Refinement of breast cancer classification by molecular characterization of histological special types[END_REF][START_REF] Weigelt | Metaplastic breast carcinomas are basal-like breast cancers: a genomic profiling analysis[END_REF] with the Gene-sets described in Fig. 1.

Exact hypergeometric probability analysis for gene list enrichment

Analysis for gene list enrichment was performed using hypergeometric probability analysis, whereby the number of genes in common between two groups was identified and a representation factor (the number of overlapping genes divided by the expected number of overlapping genes drawn from two independent groups) was calculated. A representation factor [1 indicates more overlap than expected of two independent groups, whereas a representation factor \1 indicates less overlap than expected. The probability of finding an overlap of that number of genes was then calculated using the hypergeometric probability formula: C (D, x) 9 C (N -D, nx)/C (N, n). http:// elegans.uky.edu/MA/progs/representation.stats.html.

Validation of aCGH and microarray-based expression profiling

All cases were reviewed by two pathologists (SMP-R and JSR-F), and representative areas of the 48 cases were included in a tissue microarray containing two 1-mm replicate cores from each case. These cases were used to validate selected amplifications found in aCGH.

Immunohistochemistry for cyclin D1 was performed with the SP4 rabbit monoclonal antibody (Neomarkers, Suffolk, UK) at 1:50 dilution as previously described [START_REF] Reis-Filho | Cyclin D1 protein overexpression and CCND1 amplification in breast carcinomas: an immunohistochemical and chromogenic in situ hybridisation analysis[END_REF] and scored using the Allred scoring system [START_REF] Reis-Filho | Cyclin D1 protein overexpression and CCND1 amplification in breast carcinomas: an immunohistochemical and chromogenic in situ hybridisation analysis[END_REF]. The immunohistochemical analysis was performed by two of the authors (SMP-R and JSR-F) with observers blinded to the results of the aCGH, gene expression and CISH analyses.

Chromogenic in situ hybridisation was performed using the Zymed/Invitrogen SpotLight CISH probes against HER2, CCND1 and EGFR [START_REF] Arriola | Genomic analysis of the HER2/TOP2A amplicon in breast cancer and breast cancer cell lines[END_REF][START_REF] Reis-Filho | Cyclin D1 protein overexpression and CCND1 amplification in breast carcinomas: an immunohistochemical and chromogenic in situ hybridisation analysis[END_REF][START_REF] Reis-Filho | EGFR amplification and lack of activating mutations in metaplastic breast carcinomas[END_REF], and an in-house generated PPM1D probe [START_REF] Tan | PPM1D is a potential therapeutic target in ovarian clear cell carcinomas[END_REF]. Hybridisations and washes were performed as previously described [START_REF] Arriola | Genomic analysis of the HER2/TOP2A amplicon in breast cancer and breast cancer cell lines[END_REF][START_REF] Reis-Filho | Cyclin D1 protein overexpression and CCND1 amplification in breast carcinomas: an immunohistochemical and chromogenic in situ hybridisation analysis[END_REF][START_REF] Reis-Filho | EGFR amplification and lack of activating mutations in metaplastic breast carcinomas[END_REF][START_REF] Tan | PPM1D is a potential therapeutic target in ovarian clear cell carcinomas[END_REF]. Gene amplification was defined as the presence of [5 signals, large gene clusters or a combination of signals and clusters in [50% of morphologically unequivocal neoplastic cells as previously described [START_REF] Arriola | Genomic analysis of the HER2/TOP2A amplicon in breast cancer and breast cancer cell lines[END_REF][START_REF] Reis-Filho | Cyclin D1 protein overexpression and CCND1 amplification in breast carcinomas: an immunohistochemical and chromogenic in situ hybridisation analysis[END_REF][START_REF] Reis-Filho | EGFR amplification and lack of activating mutations in metaplastic breast carcinomas[END_REF][START_REF] Tan | PPM1D is a potential therapeutic target in ovarian clear cell carcinomas[END_REF]. CISH analysis was performed by two of the authors (SMP-R and FCG) with observers blinded to the results of the aCGH, gene expression and immunohistochemical analyses. Nielsen et al. [START_REF] Nielsen | Immunohistochemical and clinical characterization of the basal-like subtype of invasive breast carcinoma[END_REF], MWU Mann-Whitney U test, adjp adjusted P value Quantitative real-time reverse transcriptase PCR RNA from 48 microdissected cases was available for quantitative real-time reverse transcriptase PCR (qRT-PCR) analysis. First strand synthesis was performed as previously described [START_REF] Natrajan | Tiling path genomic profiling of grade 3 invasive ductal breast cancers[END_REF], and qRT-PCR was performed with TaqMan chemistry on the ABI Prism 7900HT (Applied Biosystems), using the standard curve method [START_REF] Natrajan | Tiling path genomic profiling of grade 3 invasive ductal breast cancers[END_REF][START_REF] Arriola | Genomic analysis of the HER2/TOP2A amplicon in breast cancer and breast cancer cell lines[END_REF][START_REF] Tan | PPM1D is a potential therapeutic target in ovarian clear cell carcinomas[END_REF]. Assays for PPM1D and TFRC were purchased from Applied Biosystems. PPM1D expression levels were normalised to gene TFRC (Assay on demand ID: Hs00174609_m1-TFRC and Hs00186230_m1-PPM1D) [START_REF] Tan | PPM1D is a potential therapeutic target in ovarian clear cell carcinomas[END_REF].

Results

Breast cancer molecular subtype classification

To determine the basal-like, luminal and HER2 molecular subtype classes, we subjected 48 GIII IDC-NSTs to a validated immunohistochemical surrogate of the molecular subtypes of breast cancer described by Nielsen et al. [START_REF] Nielsen | Immunohistochemical and clinical characterization of the basal-like subtype of invasive breast carcinoma[END_REF]. This taxonomy was employed given that it can be used to classify archival tumour samples and more accurately identifies the clinically relevant subgroup of HER2-positive/amplified tumours (i.e. amenable to therapy with anti-HER2 agents). Using Hu et al.'s [START_REF] Hu | The molecular portraits of breast tumors are conserved across microarray platforms[END_REF] or Parker et al.'s [START_REF] Parker | Supervised risk predictor of breast cancer based on intrinsic subtypes[END_REF] microarray-based classification, a proportion of ER-positive HER2 amplified cancers are classified as of luminal B phenotype. Based on the expression of HER2, ER, CK 5/6 and EGFR, 13, 22 and 13 tumours were classified as HER2, luminal and basal-like phenotype, respectively (Supplementary Table 1). As expected, a good agreement between the classification of tumours into basal-like, HER2 and luminal using Nielsen et al.'s [START_REF] Nielsen | Immunohistochemical and clinical characterization of the basal-like subtype of invasive breast carcinoma[END_REF] immunohistochemical surrogate and by expression profiling using the Hu et al.'s centroids [START_REF] Hu | The molecular portraits of breast tumors are conserved across microarray platforms[END_REF] was observed (Kappa score = 0.753, P \ 1 910 -11 , Supplementary Table 1). As expected [START_REF] Parker | Supervised risk predictor of breast cancer based on intrinsic subtypes[END_REF], cases harbouring HER2 amplification were consistently classified as of HER2 phenotype by Nielsen et al. [START_REF] Nielsen | Immunohistochemical and clinical characterization of the basal-like subtype of invasive breast carcinoma[END_REF] and fell into either HER2 or luminal subgroup using the Hu et al.'s centroids [START_REF] Hu | The molecular portraits of breast tumors are conserved across microarray platforms[END_REF].

Identification of genes whose expression is associated with gene copy number

To define genes whose expression is significantly correlated with gene copy number, we extracted DNA and RNA from microdissected samples and performed aCGH analysis, using a BAC array platform (actual resolution 50 kb) and genome-wide gene expression profiling using the Illumina WG6 v2 platform. We next overlaid the aCGH and gene expression data of our 48 GIII IDC-NSTs. Out of the 12,699 significantly regulated genes, 5,931 (46.70%) displayed mRNA expression levels that significantly correlated with copy number (Pearson's correlation adjusted P \ 0.05, Supplementary Table 2, Gene-set A, Fig. 1a). This list included genes that have previously been found to have increased expression levels determined by gains in copy number, such as ORAOV1, ASH2L, FADD, RPS6 KB1, NME1, EIF4EBP2, GRB7, INTS2, PERLD1, MYST3, PIGS, INTS4, TLK2, PPM1D, PROSC, STARD3, ERBB2 and CTTN [START_REF] Chin | Genomic and transcriptional aberrations linked to breast cancer pathophysiologies[END_REF][START_REF] Chin | High-resolution aCGH and expression profiling identifies a novel genomic subtype of ER negative breast cancer[END_REF][START_REF] Hu | Genetic alterations and oncogenic pathways associated with breast cancer subtypes[END_REF][START_REF] Mackay | A high-resolution integrated analysis of genetic and expression profiles of breast cancer cell lines[END_REF][START_REF] Adelaide | Integrated profiling of basal and luminal breast cancers[END_REF].

To identify potential amplicon drivers, we determined genes whose expression correlates with copy number and are recurrently amplified (n = 811, Pearson's correlation adjusted P \ 0.05; amplified in 2 or more tumours, Supplementary Table 3; Gene-set B) and genes significantly overexpressed when amplified (n = 469, Mann-Whitney U test adjusted P \ 0.05, Supplementary Table 3; Gene-set C). Not surprisingly, these lists of genes included known drivers of amplicons on 1q, 8p, 8q, 11q13, 17q12-q21, 17q22-q25 and 20q13 [1, 3-5, 7, 11, 32-36]. This unbiased approach accurately identified known amplicon drivers, including HER2, STARD3, GRB7 [START_REF] Kao | RNA interference-based functional dissection of the 17q12 amplicon in breast cancer reveals contribution of coamplified genes[END_REF] on 17q12, ORAOV1 [START_REF] Jiang | Oral cancer overexpressed 1 (ORAOV1): a regulator for the cell growth and tumor angiogenesis in oral squamous cell carcinoma[END_REF] on 11q13 and PPM1D [1, 5, 30] on 17q23.2, genes whose expression and activity have been shown to be required for the proliferation and/or survival of cancer cells harbouring their amplification. In addition, this analysis has also revealed additional genes on chromosome 17q that do not pertain to the smallest region of amplification on 17q12-q21 or on 17q22-q25, but are significantly overexpressed when amplified (e.g. MRPL45, FBXL20, MED1 and ORM DL3, Fig. 2a), suggesting that the reported complex patterns of genetic aberrations on chromosome 17 [START_REF] Orsetti | Genomic and expression profiling of chromosome 17 in breast cancer reveals complex patterns of alterations and novel candidate genes[END_REF] may have a biological impact (i.e. overexpression of specific genes) and are not mere genomic 'noise'. Similar findings were observed in the genomic regions adjacent to the amplicons on 11q13.2-q13.3 and 17q21-q23 (Fig. 2b,c).

On the other hand, the 8q23.2-24.3 amplicon displayed a significantly more complex pattern of genetic aberrations without a well-defined core. Our unbiased analysis demonstrates that UTP23, RAD21, MED30, MRPL13, MTBP, C8orf76, ZHX1, C8orf32, FBXO32, NDUFB9, KIAA0196, NSMCE2, DDEF1, EFR3A, PHF20L1, ST3GAL1 and TRAPPC9 (Fig. 2d) are overexpressed when amplified and constitute potential drivers of this amplicon. It should be noted that MYC, which maps to this region, has expression levels that correlate with copy number, but is not consistently overexpressed when amplified, as previously described [START_REF] Guan | Amplification of PVT1 contributes to the pathophysiology of ovarian and breast cancer[END_REF]. Taken together with the results of previous functional and correlative studies [START_REF] Chin | Genomic and transcriptional aberrations linked to breast cancer pathophysiologies[END_REF][START_REF] Chin | High-resolution aCGH and expression profiling identifies a novel genomic subtype of ER negative breast cancer[END_REF][START_REF] Neve | A collection of breast cancer cell lines for the study of functionally distinct cancer subtypes[END_REF][START_REF] Adelaide | Integrated profiling of basal and luminal breast cancers[END_REF][START_REF] Heiser | Integrated analysis of breast cancer cell lines reveals unique signaling pathways[END_REF][START_REF] Kao | RNA interference-based functional dissection of the 17q12 amplicon in breast cancer reveals contribution of coamplified genes[END_REF][START_REF] Jiang | Oral cancer overexpressed 1 (ORAOV1): a regulator for the cell growth and tumor angiogenesis in oral squamous cell carcinoma[END_REF], our analysis seems to identify accurately genes whose expression correlates with copy number and potential amplicon drivers.

Molecular pathways regulated by gene copy number differ in basal-like, HER2 and luminal cancers

To determine which of the 5,931 genes whose expression levels are significantly correlated with gene copy number are associated with each molecular subtype of GIII breast cancers (i.e. basal-like, HER2 and luminal), multiclass significance analysis of microarray (SAM) was performed [START_REF] Tusher | Significance analysis of microarrays applied to the ionizing radiation response[END_REF] using 1,000 permutations and accepting a false discovery rate of 3%. SAM analysis revealed that 1,897 genes (Geneset D; Fig. 1a) were differentially expressed between GIII IDC-NSTs of basal-like, HER2 and luminal molecular subtypes (Supplementary Table 4). As expected, genes pertaining to the HER2 amplicon, PERLD1, C17orf37, GRB7, STARD3 and HER2 (ERBB2) were significantly overexpressed in cancers of HER2 phenotype and were the top ranking genes in the SAM analysis.

Ingenuity Pathway Analysis (IPA) of these 1,897 transcripts revealed networks and pathways enriched for genes whose expression correlates with copy number and are differentially expressed between molecular subtypes. These included networks related to DNA replication and repair, and cell cycle (Fig. 3; Supplementary Fig. 1; Supplementary Table 5). For instance, the network 'Gene Expression, DNA Replication, Recombination, and Repair, Cell Cycle, Cancer' (score 40, Fig. 3) encompasses MYC and other key genes such as MDC1, a mediator of DNA damage checkpoint; MSH3, MLH1 and MLH3, which play a role in mismatch repair; and RAD50, which is involved in DNA double-strand break repair. Basal-like cancers displayed recurrent copy number gains/amplifications and upregulation of MYC and MDC1 and losses and downregulation of RAD50 and genes related to mismatch repair (i.e. MSH3, MLH1 and MLH3) compared to HER2 and luminal cancers (Fig. 3). The network 'Gene Expression, DNA Replication, Recombination, and Repair, Cell Cycle' (score 40, Supplementary Fig. 1) was centred around key genes involved in cell cycle such as CCND1, E2F3, CCNA2 and CCNDBP1, with basal-like tumours showing recurrent deletions and downregulation of CCND1 and 1).

In agreement with the above, IPA also identified the canonical pathways 'Cell Cycle: G1/S Checkpoint Regulation' and 'Role of BRCA1 in DNA Damage Response' as significantly enriched for genes whose expression correlates with copy number and are significantly differentially expressed in breast cancer molecular subtypes (P \ 0.001 and P = 0.0069, respectively) (Supplementary Table 6). This analysis revealed that the expression levels of distinct components of the G1/S checkpoint regulation pathway are differentially expressed in basal-like, luminal and HER2 cancers due to specific patterns of copy number aberrations (Fig. 4). Recurrent gains/amplifications and upregulation of CDK6, p16INK4A, E2F, CCNE1 and MYC were observed in basal-like breast cancers, whereas CCNE1 and CCND1 were recurrently gained/amplified and upregulated in HER2 tumours, and CCND1 in luminal tumours. SKP1 and SKP2 were recurrently deleted and downregulated in basal-like breast cancers, whereas recurrent gains and overexpression of SKP2 were found in HER2 cancers, and recurrent gains and overexpression of both genes were observed in luminal cancers. Interestingly, p16 (CDKN2A), a gene that has been reported to be overexpressed in the majority of basal-like breast cancers [START_REF] Subhawong | Most basallike breast carcinomas demonstrate the same Rb-/p16? immunophenotype as the HPV-related poorly differentiated squamous cell carcinomas which they resemble morphologically[END_REF], was recurrently gained and overexpressed in these tumours; whereas HER2 and luminal cancers harboured recurrent deletions and downregulation of this gene. Genes involved in BRCA1 DNA damage control and DNA repair mechanisms, including RB, MLH1 and RAD50, were recurrently deleted and downregulated and E2F3, MRE11A and FANCE were recurrently copy number gained and upregulated in basal-like cancers compared with HER2 and luminal tumours (Supplementary Fig. 2). Although BRCA1 did not reach significance in our multiclass SAM analysis, its expression is associated with copy number (Pearson's correlation adjusted P = 0.0433), and lower expression is associated with basal-like tumours (P = 0.0283, t-test, homoscedastic; adjusted P = 0.01499, Mann-Whitney U test). In addition, 'Protein Ubiquitination' (P \ 0.001) and 'IGF-1' (P = 0.0018) were amongst the top canonical pathways significantly enriched for genes whose expression correlates with copy number (Supplementary Table 6).

These results suggest that the transcriptional regulation of molecular pathways may be determined by the pattern of genetic aberrations in breast cancers and that distinct pathways and networks are driven by specific patterns of copy number aberrations in basal-like, HER2 and luminal tumours. To corroborate the above hypotheses, we overlaid the aCGH and expression data for each subtype separately (Pearson's correlation adjusted P \ 0.05, Supplementary Table 7). This resulted in a list of 730 (Gene-set E1), 933 (Gene-set F1) and 2,137 (Gene-set G1; see Fig. 1b) genes whose expression is associated with copy number in 13 basal-like, 13 HER2 and 22 luminal cancers, respectively. A significant overlap as defined by exact hypergeometric probability analysis was found for the genes identified as copy number regulated with both approaches in basal-like (Gene-set D vs. Gene-set E1, Representation factor: 2.0, P \ 10 -25 ), HER2 (Gene-set D vs. Gene-set F1, Representation factor: 2.1, P \ 10 -37 ) and luminal (Gene-set D vs. Gene-set G1, Representation factor: 1.9, P \ 10 -71 ) cancers.

Ingenuity Pathway Analysis of Gene-sets E1, F1 and G1 (Pearson's correlation adjusted P \ 0.05) revealed 6,8). This analysis revealed a heterogeneous pattern of losses, gains and amplifications of genes whose expression levels are correlated with copy number between the tumour subtypes leading to the activation of distinct networks and pathways. In basal-like cancers (Gene-set E1), the 'Oestrogen Receptor Signalling' (P = 0.00178, Fig. 5) and 'Protein Ubiquitination' (P = 0.00181) canonical pathways were significantly enriched for genes whose expression correlates with copy number (Supplementary Table 6). Of these genes pertaining to the 'Oestrogen Receptor Signalling' pathway, the vast majority were lost and downregulated, with the exception of PgR, which was gained in 3 and amplified in one basal-like case (B11, which expressed high mRNA and protein levels of progesterone receptor (PR), Supplementary Table 1). A strong correlation was observed between ER and PR status as defined by immunohistochemistry and ESR1 and PgR mRNA expression levels as defined by microarray-based expression analysis (Mann-Whitney U test, P = 5.5 9 10 -9 and P = 1.2 9 10 -7 , respectively; Supplementary Table 1). The 'Oestrogen Receptor Signalling' canonical pathway was also significantly enriched for genes whose expression correlates with copy number in HER2 cancers (Gene-set F1, P = 0.01288, Fig. 5); however, the genes that were copy number regulated in each subtype were largely distinct. In HER2 tumours, RAF1 and ERK1/2 were recurrently lost, whereas gains of multiple components of the RNA polymerase II complex, including POLR2K and TATA-binding proteinassociated factor genes (i.e. TAF4 and TAF15), were observed. In HER2 cancers, the 'Nucleotide Excision Repair Pathway' was significantly enriched for genes whose expression correlates with copy number (P = 0.0214), with recurrent gains and amplifications of POLR2K and losses of RPA2 (Fig. 5; Supplementary Table 6).

These results suggest that several of the molecular pathways and networks specifically associated with basallike, HER2 and luminal cancers are driven, at least in part, by specific genetic aberrations. Importantly, DNA copy number changes appear to be involved in the downregulation of oestrogen receptor signalling, one of the hallmark features of basal-like cancers [START_REF] Reis-Filho | Triple negative tumours: a critical review[END_REF][START_REF] Rakha | Basal-like breast cancer: a critical review[END_REF], and in the dysfunctional status of gene pathways and networks related to various types of DNA repair mechanisms.

Pathway analysis identifies novel potential therapeutic targets in GIII IDC-NSTs of basal-like, HER2 and luminal molecular subtype

Previous studies have demonstrated that genes whose expression correlates with copy number and are recurrently amplified and genes consistently overexpressed when amplified are likely to be amplicon drivers and may constitute therapeutic targets [START_REF] Natrajan | Tiling path genomic profiling of grade 3 invasive ductal breast cancers[END_REF][START_REF] Chin | Genomic and transcriptional aberrations linked to breast cancer pathophysiologies[END_REF][START_REF] Hu | Genetic alterations and oncogenic pathways associated with breast cancer subtypes[END_REF][START_REF] Tan | PPM1D is a potential therapeutic target in ovarian clear cell carcinomas[END_REF][START_REF] Reis-Filho | FGFR1 emerges as a potential therapeutic target for lobular breast carcinomas[END_REF][START_REF] Bernard-Pierrot | Characterization of the recurrent 8p11-12 amplicon identifies PPAPDC1B, a phosphatase protein, as a new therapeutic target in breast cancer[END_REF]. To identify potential therapeutic targets specific for subgroups of basal-like, HER2 and luminal cancers, we defined (1) genes whose expression correlates with copy number in each molecular subtype individually (Pearson's correlation adjusted P \ 0.05) and are recurrently amplified in each group and (2) genes overexpressed when amplified in each group individually (Mann-Whitney U adjusted P \ 0.05) (Fig. 1b). The first analysis resulted in a list of 32, 157 and 265 genes in basal-like, HER2 and luminal tumours, respectively (Gene-sets E2, F2 and G2, respectively), whereas the second resulted in 16, 33 and 159 genes in basal-like, HER2 and luminal tumours, respectively (Genesets E3, F3 and G3, respectively), (Supplementary Tables 9,10). A significant overlap as defined by exact hypergeometric probability analysis was found between the genes identified as recurrently amplified and genes overexpressed when amplified in basal-like (Gene-set E2 vs. Gene-set E3, Representation factor: 372.0, P \ 10 -40 ), HER2 (Gene-set F2 vs. Gene-set F3, Representation factor: 68.8, P \ 10 -50 ) and luminal (Gene-set G2 vs. Gene-set G3, Representation factor: 44.3, P \ 10 -252 ) tumours.

Genes whose expression correlates with copy number and are recurrently amplified in basal-like cancers (Gene-set E2) mapped to 1q22-q24, 1q44, 8q24.1, 10p15 and 19q12-q13. IPA revealed that the networks 'Gene Expression, Cancer, Gastrointestinal Disease' (score 46) and 'Gene Expression, Cancer, Cell Death' (score 17) were significantly enriched for these genes (Supplementary Table 11; Supplementary Fig. 3). Interestingly, different components of these networks were amplified in individual cases. Given the limited sample size, only genes mapping to 19q12-q13 were significantly overexpressed when amplified in basal-like cancers after multiple comparison adjustment (Gene-set E3; Fig. 1b). These genes, including C19orf2, MED30, SUPT 5H, SERTAD1 and SERTAD3, were part of the network 'Cancer, Cell Death, Endocrine System Disorders' (score 40, Supplementary Fig. 3). Notably, the p21-activated kinase 4 (PAK4) was present in both 'Gene expression, Cancer, Cell Death' and 'Cancer, Cell Death, Endocrine System Disorders' networks and has recently emerged as a potential therapeutic target [START_REF] Eswaran | Targeting group II PAKs in cancer and metastasis[END_REF].

In HER2 cancers, the network 'Lipid Metabolism, Molecular Transport, Nucleic Acid Metabolism' (score 37) was significantly enriched for recurrently amplified genes whose expression correlates with copy number (Gene-set F2) and the network 'Amino Acid Metabolism, Post-Translational Modification, Small Molecule Biochemistry' (score 29, Fig. 6; Supplementary Tables 11,12) was significantly enriched for genes overexpressed when amplified (Gene-set F3; Fig. 1b). These networks were composed of genes mapping not only to the HER2 amplicon (17q12-q21), but also to distinct amplicons, including 8p12, 8q22-q24, 11q13-q14, 17p11 and 17q23, which were amplified in multiple combinations in each tumour (Fig. 6; Supplementary Tables 9,10). Interestingly, PPM1D, a negative regulator of the p53 pathway and potential therapeutic target for a subgroup of patients with HER2 amplified breast cancers [START_REF] Natrajan | Tiling path genomic profiling of grade 3 invasive ductal breast cancers[END_REF], was present in both networks.

In luminal tumours, the 'ERK/MAPK Signalling' (P = 0.00631 and P = 0.00978, respectively) and 'PI3K/AKT Signalling' (P = 0.01445 and P = 0.01047, respectively) canonical pathways were significantly enriched for recurrently amplified genes whose expression correlates with copy number (Gene-set G2) and for genes overexpressed when amplified (Gene-set G3) (e.g., PAK1, PRKAR1A, RPS6KB1, PPP2R5A) (Supplementary Table 6; Fig. 7; Supplementary Fig. 4). Protein kinases of these two canonical pathways may constitute targets for therapeutic intervention by PI3K and/or MEK inhibitors [START_REF] Heiser | Integrated analysis of breast cancer cell lines reveals unique signaling pathways[END_REF][START_REF] Ihle | Take your PIK: phosphatidylinositol 3kinase inhibitors race through the clinic and toward cancer therapy[END_REF][START_REF] Crowder | PIK3CA and PIK3CB inhibition produce synthetic lethality when combined with estrogen deprivation in estrogen receptor-positive breast cancer[END_REF]. Luminal breast cancers were also significantly enriched for genes in the networks 'Cell Cycle, Reproductive System Development and Function, Haematological System Development and Function' (score 40) and 'Reproductive System Disease, Cellular Assembly and Organisation, Cellular Movement' (score 33) (Supplementary Tables 11,12; Fig. 7; Supplementary Fig. 4). Of note, PPM1D was part of the cell cycle network, providing additional evidence to our previous findings that PPM1D may also be a potential therapeutic target in a subgroup luminal breast cancers harbouring amplification of this gene [START_REF] Natrajan | Tiling path genomic profiling of grade 3 invasive ductal breast cancers[END_REF].

Validation of aCGH and microarray-based expression profiling analysis

Two tissue microarrays containing the 48 tumours in our study were subjected to CISH with probes for CCND1, EGFR, PPM1D and HER2. A perfect agreement between aCGH and CISH results for EGFR, PPM1D and HER2 was observed (Supplementary Table 1; Supplementary Fig. 5), whereas for CCND1, all but two cases showed concordant results (unweighted Kappa score = 0.86297; 95% CI 0.7099-1.0000).

A good correlation was observed between the microarray and qRT-PCR-defined expression levels of PPM1D (Pearson's r value = 0.81421, P = 1.8 9 10 -11 ). In agreement with the results of the aCGH and expression array analysis, CISH and qRT-PCR-based assessment of PPM1D copy numbers and mRNA expression also Immunohistochemical analysis of cyclin D1 using a semiquantitative scoring system showed a good correlation with CCND1 mRNA levels as defined by microarray analysis (Spearman's correlation coefficient = 0.68943, P \ 1 9 10 -6 ). Despite the impact of ER expression on cyclin D1 expression levels [START_REF] Reis-Filho | Cyclin D1 protein overexpression and CCND1 amplification in breast carcinomas: an immunohistochemical and chromogenic in situ hybridisation analysis[END_REF], CISH and immunohistochemistry based assessment of CCND1 copy numbers and cyclin D1 protein expression, respectively, confirmed that CCND1 is overexpressed when amplified (Mann-Whitney U test = 62.0, P = 0.00694), in agreement with aCGH and expression array analysis. Our results provide direct evidence for the accuracy of the aCGH and expression profiling methods employed in this study.

Discussion

The actual impact of gene copy number changes on the transcriptomic profiles of breast cancer molecular subtypes has been a matter of contention. Whilst HER2 tumours by definition are biologically driven by amplification and overexpression of ERBB2, the impact of genomic alterations on the transcriptome and phenotype of basal-like and luminal tumours is less clear [START_REF] Chin | Genomic and transcriptional aberrations linked to breast cancer pathophysiologies[END_REF]. Here, we show that Green copy number loss; black no copy number change; dark red copy number gain; red gene amplification distinct gene networks and canonical pathways are enriched for genes whose expression levels correlate with copy number in basal-like, HER2 and luminal tumours, providing evidence that differences between these molecular subtypes may be determined, at least in part, by their patterns of genetic aberrations.

Meta-analyses of microarray expression data have demonstrated that the main separation between molecular subtypes is driven by ER, HER2 and proliferation and that proliferation is a major determinant of breast cancer clinical behaviour [START_REF] Desmedt | Biological processes associated with breast cancer clinical outcome depend on the molecular subtypes[END_REF][START_REF] Wirapati | Meta-analysis of gene expression profiles in breast cancer: toward a unified understanding of breast cancer subtyping and prognosis signatures[END_REF]. Our analysis demonstrates that networks and canonical pathways directly related to ER signalling (Fig. 5), HER2 signalling (Fig. 6) and proliferation (Fig. 4) are enriched for genes whose expression correlates with copy number. It should be noted that although ER pathway was enriched for genes whose expression correlates with copy number, ESR1 mRNA levels and DNA copy number failed to show any statistically significant correlation (Pearson's correlation adjusted P = 0.5667) in our study, and ESR1 was only amplified in a single ER-positive case (1.8%). This concurs with previous reports suggesting that only a small minority of ER-positive cancers harbour ESR1 gene amplification and that ER expression is not significantly associated with ESR1 copy numbers [START_REF] Reis-Filho | ESR1 gene amplification in breast cancer: a common phenomenon?[END_REF][START_REF] Brown | ESR1 gene amplification in breast cancer: a common phenomenon?[END_REF][START_REF] Horlings | ESR1 gene amplification in breast cancer: a common phenomenon?[END_REF][START_REF] Vincent-Salomon | Identification of typical medullary breast carcinoma as a genomic subgroup of basal-like carcinomas, a heterogeneous new molecular entity[END_REF].

Our group and others have demonstrated that tumours arising in patients harbouring BRCA1 germline mutations predominantly display a basal-like phenotype and that a subgroup of sporadic basal-like breast cancers harbours a dysfunctional BRCA1 pathway [START_REF] Lakhani | Prediction of BRCA1 status in patients with breast cancer using estrogen receptor and basal phenotype[END_REF][START_REF] Foulkes | Germline BRCA1 mutations and a basal epithelial phenotype in breast cancer[END_REF][START_REF] Turner | Basal-like breast cancer and the BRCA1 phenotype[END_REF][START_REF] Turner | BRCA1 dysfunction in sporadic basal-like breast cancer[END_REF]. Given the rarity of BRCA1 somatic mutations, several mechanisms of BRCA1 pathway dysfunction in sporadic breast cancers have been proposed [START_REF] Rakha | Basal-like breast cancer: a critical review[END_REF][START_REF] Turner | Basal-like breast cancer and the BRCA1 phenotype[END_REF][START_REF] Turner | BRCA1 dysfunction in sporadic basal-like breast cancer[END_REF], including BRCA1 gene promoter methylation and overexpression of ID4, a negative regulator of BRCA1 [START_REF] Turner | BRCA1 dysfunction in sporadic basal-like breast cancer[END_REF]. In this study, we provide evidence that the BRCA1 pathway may be dysfunctional in sporadic basal-like cancers due to gene copy number aberrations affecting genes pertaining to this pathway. The 'BRCA1 DNA Damage Response' pathway was enriched for genes whose expression correlates with copy number and are differentially expressed between the molecular subtypes (Supplementary Fig. 2). BRCA1 expression itself was found to be associated with copy number and downregulated in basal-like tumours, suggesting a role for homologous recombination DNA repair defects in this subgroup of sporadic breast cancer. In addition, we provide evidence to suggest that overexpression of the BRCA1 negative regulator ID4 in basal-like cancers is driven by copy number gains, as (1) the expression levels of this gene correlated with copy number in the whole cohort (Pearson's correlation adjusted P \ 0.01669, Supplementary Table 2), (2) ID4 was expressed at significantly higher levels in basallike breast cancer (Supplementary Table 4), and (3) ID4 copy number gains were significantly more frequent in basal-like than in non-basal-like tumours (9/13 basal-like cancers and 10/35 non-basal-like cancers; Fisher's exact test, P = 0.0187). Taken together, our results support the contention that BRCA1 pathway dysfunction in sporadic basal-like breast cancers may be driven by genetic aberrations. Given the reported sensitivity of tumours with BRCA1 loss of function to PARP inhibitors, our findings provide further credence to the molecular basis for its potential efficacy in sporadic basal-like breast cancers [START_REF] Ashworth | A synthetic lethal therapeutic approach: poly(ADP) ribose polymerase inhibitors for the treatment of cancers deficient in DNA double-strand break repair[END_REF].

By comparing our list of copy number regulated genes with that of published 'intrinsic gene' lists, we observed that 61/207 (29%) intrinsic genes described by Perou et al. [START_REF] Perou | Molecular portraits of human breast tumours[END_REF] were present in our dataset, 166/365 (45.5%) intrinsic genes by Sorlie et al. [START_REF] Sorlie | Repeated observation of breast tumor subtypes in independent gene expression data sets[END_REF], 435/929 (46.8%) unique identifiers of the 'Intrinsic/UNC' gene list by Hu et al. [START_REF] Hu | The molecular portraits of breast tumors are conserved across microarray platforms[END_REF] and 684/1,488 (46%) intrinsic genes by Parker et al. [START_REF] Parker | Supervised risk predictor of breast cancer based on intrinsic subtypes[END_REF] were significantly associated with gene copy number. The substantial enrichment of the published 'intrinsic gene' lists with genes whose expression correlates with copy number further emphasises the potential importance of genetic alterations in the molecular classification of breast cancers. Furthermore, it suggests that the differential expression of the 'intrinsic genes' in the distinct molecular subtypes may not only stem from their cell of origin [START_REF] Perou | Molecular portraits of human breast tumours[END_REF][START_REF] Sorlie | Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications[END_REF], but also as a result of genetic instability and subsequent acquisition of stochastic genetic aberrations.

To identify possible therapeutic targets, we used our integrative approach to distinguish recurrently amplified genes whose expression is associated with copy number and also genes that are consistently overexpressed when amplified. Our genome-wide, unbiased approach identified genes that have been functionally validated as potential therapeutic targets in specific breast cancer molecular subtypes (e.g. HER2, PPM1D, PAK1 and PPAPDC1B-Supplementary Tables 9,10) [START_REF] Natrajan | Tiling path genomic profiling of grade 3 invasive ductal breast cancers[END_REF][START_REF] Heiser | Integrated analysis of breast cancer cell lines reveals unique signaling pathways[END_REF][START_REF] Bernard-Pierrot | Characterization of the recurrent 8p11-12 amplicon identifies PPAPDC1B, a phosphatase protein, as a new therapeutic target in breast cancer[END_REF] and canonical pathways that have been exploited in preclinical therapeutic models for breast cancer (e.g. PI3K/AKT signalling in luminal cancers-Fig. 7 and Supplementary Fig. 4) [START_REF] Crowder | PIK3CA and PIK3CB inhibition produce synthetic lethality when combined with estrogen deprivation in estrogen receptor-positive breast cancer[END_REF], as well as a comprehensive list of genes that may constitute amplicon drivers and novel therapeutic targets.

Molecular subtype-specific analyses of genes consistently significantly overexpressed when amplified highlighted networks and canonical pathways enriched for potential therapeutic targets. The networks identified in basal-like tumours were found to be enriched for multiple genes involved in transcriptional regulation, suggesting that preclinical studies testing these transcriptional regulators as potential targets may be warranted. The p21activated kinase 4 PAK4, which plays a role in integrinmediated breast cancer cell motility [START_REF] Zhang | P21-activated kinase 4 interacts with integrin alpha v beta 5 and regulates alpha v beta 5mediated cell migration[END_REF], was significantly overexpressed when amplified in basal-like cancers. Novel strategies for targeting PAK4 and its family members in cancer are under investigation [START_REF] Eswaran | Targeting group II PAKs in cancer and metastasis[END_REF]. Ingenuity Pathway Analysis of HER2 cancers substantiates previous findings for the use of PPM1D-targeted therapies in this subgroup [START_REF] Natrajan | Tiling path genomic profiling of grade 3 invasive ductal breast cancers[END_REF][START_REF] Rayter | A chemical inhibitor of PPM1D that selectively kills cells overexpressing PPM1D[END_REF] and revealed that PI3K and ERK/MAPK canonical pathways are significantly enriched for genes that are amplified and overexpressed in luminal tumours (e.g. PAK1, PPP2R5A and PRKAR1A). Interestingly, PAK1 overexpressing luminal breast cancer cell lines have recently been shown to be significantly more sensitive to MEK inhibition [START_REF] Heiser | Integrated analysis of breast cancer cell lines reveals unique signaling pathways[END_REF]. Moreover, the PI3K catalytic subunit inhibitor BEZ235 has been reported to cause selective cell growth inhibition in ER-positive breast cancer cell lines [START_REF] Crowder | PIK3CA and PIK3CB inhibition produce synthetic lethality when combined with estrogen deprivation in estrogen receptor-positive breast cancer[END_REF] and may constitute an interesting therapeutic approach for subgroups of patients with luminal breast cancer harbouring amplification of these genes.

In conclusion, high resolution integrative genomic analysis of basal-like, HER2 and luminal GIII breast cancers using microdissected tissues identified pathways and networks specific to each tumour type that are regulated by changes at the DNA level, and has identified key genes that govern distinct pathways. These results provide strong circumstantial evidence to suggest that the diversity of breast cancer and the molecular subtypes may stem, at least in part, from the distinct pattern of genetic aberrations found in these cancers. Furthermore, we have identified canonical pathways enriched for genes recurrently amplified or overexpressed when amplified in basal-like, HER2 and luminal cancers. Functional genomic studies to test the biological importance of these pathways and the therapeutic implication of inhibition of these pathways in appropriate preclinical models are warranted.

Fig. 1

 1 Fig. 1 Work flow diagram of integration of aCGH and gene expression data. a Analysis of cohort of 48 grade III invasive ductal carcinomas of no special type. b Analysis of 48 grade III invasive ductal carcinomas of no special type assigned to the molecular subtypes basal-like (n = 13), HER2 (n = 13) and luminal (n = 22) separately.

1

 1

Fig. 2

 2 Fig. 2 Matched heatmaps of gene expression and aCGH within regions of recurrent amplification. a 17q12-q21.1, b 11q13.2-q13.3, c 17q21-q23 and d 8q23.3-q24.3. For each amplicon, genes within the amplified region are recovered and median aCGH values and states are assigned. Samples are separated into those harbouring amplification within the region and those that do not. Expression and aCGH values are depicted in two matching heatmaps (aCGH states on the left and expression values on the right) in which the genes are ordered

Fig. 3

 3 Fig. 3 Network enriched for genes whose expression correlates with copy number and are significantly differentially expressed between basal-like, HER2 and luminal GIII invasive ductal carcinomas of no special type, defined by Ingenuity Pathway Analysis software. a 'Gene Expression, DNA Replication, Recombination, and Repair, Cell Cycle, Cancer' network (score 40) in (I) basal-like, (II) HER2

Fig. 4

 4 Fig. 4 Canonical pathway 'Cell Cycle: G1/S Checkpoint Regulation' significantly enriched for genes whose expression correlates with copy number and differentially expressed between basal-like, HER2 and luminal GIII tumours (P \ 0.001), defined by Ingenuity Pathway Analysis software. a Canonical pathway in (I) basal-like, (II) HER2

Fig. 5

 5 Fig. 5 Canonical pathways enriched for genes whose expression correlates with copy number in 13 basal-like and 13 HER2 GIII tumours, defined by Ingenuity Pathway Analysis software. a Canonical pathways (I) 'Oestrogen Receptor Signalling' (P = 0.0018) in basal-like, (II) 'Oestrogen Receptor Signalling' (P = 0.013) in HER2 and (III) 'Nucleotide Excision Repair Pathway' (P = 0.0214) in

Fig. 6

 6 Fig. 6 Top ranked networks in 13 HER2 tumours enriched for genes whose expression correlates with copy number and are recurrently amplified (Pearson's correlation) and genes that are overexpressed when amplified (Mann-Whitney U test), defined by Ingenuity Pathway Analysis. a Top ranked networks in HER2 tumours (I) 'Lipid Metabolism, Molecular Transport, Nucleic Acid Metabolism'

Fig. 7

 7 Fig. 7 Top ranking networks and canonical pathways identified by Ingenuity Pathway Analysis in 22 luminal tumours enriched for genes whose expression correlates with copy number and are recurrently amplified (Pearson's correlation). a Top ranked network and canonical pathways in luminal tumours (I) 'Cell Cycle, Reproductive System Development and Function, Haematological System Development and

Breast Cancer Res Treat (2010) 121:575-589

Acknowledgments This study was funded in part by Breakthrough Breast Cancer. B. Weigelt is supported by a Cancer Research UK Fellowship, J Palacios by RETICS: RD06/0020/0013 and PI 08/