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An integrated genomics approach identifies drivers of proliferation in luminal subtype human breast cancer

Elucidating the molecular drivers of human breast cancers requires a strategy capable of integrating multiple forms of data and an ability to interpret the functional consequences of a given genetic aberration. Here we present an integrated genomic strategy based on the use of gene expression signatures of oncogenic pathway activity (n=52) as a framework to analyze DNA copy number alterations in combination with data from a genome-wide RNAi screen. We identify specific DNA amplifications, and importantly, essential genes within these amplicons representing key genetic drivers, including known and novel regulators of oncogenesis. The genes identified include eight that are essential for cell proliferation (FGD5, METTL6, CPT1A, DTX3, MRPS23, EIF2S2, EIF6 and SLC2A10) and are uniquely amplified in patients with highly proliferative luminal breast tumors, a clinical subset of patients for which few therapeutic options are effective. Our results demonstrate that this general strategy has the potential to identify putative therapeutic targets within amplicons through an integrated use of genetic, genomic, and genome-wide RNAi data sets.

critical to understand the relevance of these alterations for disease phenotypes but also to enable the development of rational therapeutic strategies that can match the characteristics of the individual patient's tumor. Many studies, including reports from The Cancer Genome Atlas (TCGA) project, have made use of the power of multi-platform genomic analyses to identify known and novel genetic drivers of tumor phenotypes [1][2][3] . This has led to the identification of disease subgroups with distinct characteristics and in some instances, distinct genetic mechanisms of disease 1,2,4 . The strength of this approach relies on the integration of large-scale genomic data to reveal biological co-variation that cannot be identified when using a single technology. A weakness of this approach is in the interpretation of the underlying biology, which generally represents an inference about pathway activity based upon prior knowledge concerning an individual gene mutation/ alteration. Altered signaling pathway activity is an important determinant of the biology of a tumor and may predict therapeutic response; therefore, identifying mechanisms driving key tumorigenic pathways is essential to understand the transformation process 2,5-8 . To take advantage of the vast amounts of existing genomic data, we utilized a series of experimentally derived gene expression signatures capable of measuring oncogene or tumor suppressor pathway activity, aspects of the tumor microenvironment, and other tumor characteristics including proliferation rate, as a framework by which to integrate multiple forms of genomic data. Our results identify patterns of oncogenic signaling within each of the molecular subtypes of breast cancer, many of which directly correlate with DNA copy number aberrations. By further analyzing functional data from a genome-wide RNAi screen 9 , we identified genes essential for cell viability in a pathway-dependent, and in some cases, a subtype-dependent manner. Our results identify a small number of DNA amplifications as potential drivers of proliferation in poor outcome luminal/ER+ breast cancers, and in general terms, we outline an approach that could be applied to many other tumor types where multi-platform genomic data exist.

Subtype-specific patterns of oncogenic signaling
To objectively identify genetic drivers of breast cancer, we examined genomic-based patterns of oncogenic pathway activity, the tumor microenvironment, and other important features in human breast tumors using a panel of 52 previously published gene expression signatures (Supplementary Tables 1 and 2)   . Each signature was applied to the breast cancer gene expression microarray data (n=476) from the TCGA project (Supplementary  Table 3) for which the molecular intrinsic subtype had been determined 2 . Consistent patterns of pathway activity emerged for each subtype, as illustrated in Figure 1A, and quantitatively assessed by an ANOVA test followed by a Tukey test for pair-wise comparison ( Figure 1B, Supplementary Table 4). Analyzing differences across subtypes based on these 52 features demonstrated that the strongest correlation between samples existed within each molecular subtype (Supplementary Figure 1). oncogenes, and/or tumor suppressor mutation status ( Figure 1). For example, basal-like tumors, which represent ~80% of triple negative breast cancers, are characterized by low HR signaling, mutant p53 signaling and high expression of proliferation pathway activity ( Figure 1). Likewise, HER2-enriched (HER2E) tumors show high expression of the Her2 11 and Her2-Amplicon (Amp) 12 signatures, while LumA tumors show high HR signaling and wild-type p53 signaling. Highly proliferative LumB tumors, which also show some HR signaling, are distinguished from less proliferative LumA samples by increased proliferation-associated pathways. Thus these data robustly recapitulate many previously published pathways versus subtype associations.
A Pearson correlation to assess the concordance between each of the 52 signatures (Supplementary Figure 2, Supplementary Table 5) identified strong relationships between independent signatures for a given pathway as well as between related pathways. For example, two Myc signatures 11,15,32 demonstrate an R value of 0.72, while a PIK3CA 18 and PTEN-deleted 27 signatures had a R value of 0.82. Signatures scoring different pathways were also concordant; for instance, Myc-mediated regulation of E2F signaling 33 was identified by the association between the RB-LOH 16 and Myc 15 signatures (R=0.79), while EGFR-mediated activation of Stat3 signaling 34 was recapitulated by the EGFR 11,32 and Stat3 11,32 (R=0.72) signatures. These results provide a measure of validity for each signature but, since differences do exist between signatures for a specific pathway, suggest that each provides an opportunity to investigate a particular pathway taking into account the genetic manipulation used to develop a given signature.

Characterization of pathway-specific copy number alterations
We next utilized DNA copy number data from the TCGA project (n=476) to identify Copy Number Alterations (CNA) associated with pathway activity (Figure 2A). We first identified genes for which CNAs were positively (or inversely) correlated with pathway activity using a Spearman Rank correlation (Bonferroni corrected to control the family-wise error rate) to assess the relationship between pathway score and gene-level DNA segment score (Supplementary Figure 3, Supplementary Tables 6-57). Secondly, we used a Fisher's exact test (Bonferroni corrected) to calculate the frequency of CNA gains (including high-level amplification and gains) or losses (including loss of heterozygosity and deletions) in samples with high (top quartile) pathway activity compared to all other samples (low activity) (Supplementary Figure 4, Supplementary Tables 6-57). To reduce potential falsepositive results associated with either strategy alone, for each signature we focused on those genes that were significant in both analyses ( Figure 2A); potential drivers of pathway activity had a positive correlation and a higher amplification frequency in samples with high pathway activity, while potential repressors had a negative correlation and increased frequency of copy number losses. Mapping genes that met these criteria to chromosomal loci identified pathway-specific patterns of CNA ( Figure 2B). Consistent with previous studies reporting that basal-like tumors have a higher incidence and larger spectrum of CNAs 2,35 , pathways associated with basal-like tumors have more complex patterns of CNA when compared to luminal-associated pathways.
To further assess the validity of this strategy, we investigated the relationship between pathway activity and a chromosomal alteration of known causative activity. We first focused on the Her2-Amp signature 12 since this signature is comprised of genes located at the 17q loci and since ERBB2/17q amplification is the dominant driver of this pathway. As illustrated in Figure 2C (Supplementary Table 27) ERBB2 is amplified in 84.9% of samples with high (top quartile) pathway activity compared to 7.3% of low scoring samples (q= 1.1 × 10 −55 ); likewise this relationship has a positive Spearman rank correlation (q= 2.4× 10 −108 ). While several other alterations, including MYC amplification (q=1.1× 10 −02 , q=6.3× 10 −03 ) were also associated with this signature, thus identifying a previously known relationship 36 , ERBB2 /17q amplification was the dominant alteration identified thus providing a robust positive control for this strategy. As expected, similar results were observed when analyzing the Her2 pathway using the independently developed Her2 11,32 signature (Supplementary Table 26).
This strategy was further validated by assessing the relationship between CNA and pathways associated with a more complex genomic landscape. Previous studies from our group suggest that the Her1-C2 13 signature predominantly measures the RAS/RAF/MEK arm of the EGFR pathway 13 . Consistent with this observation, we detected a correlation between the Her1-C2 signature (q<0.01) and GRB2, SOS1, KRAS, BRAF, PIK3CA, PIK3CB, and MYC genomic DNA amplifications as well as a negative correlation (q<0.01) with loss of NF1 and PI3K repressors INPP4B and PTEN ( Figure 2D, Supplementary Table 24). Finally, we analyzed CNA associated with the RB-LOH 16 signature ( Figure 2E, Supplementary Table 47) and identified associations between it and CNA of known RB/E2F components including loss of RB1 and gains of E2F1 and/or E2F3. Consistent with the role of the RB/E2F pathway in mediating cell cycle progression and proliferation 37 , CCND2, CCND3, and MYC amplification also correlated with this signature. Collectively these results demonstrate that this strategy is able to link CNA with pathway activity and does so by focusing on all aspects of the pathway, often beyond the dominant regulator, potentially allowing for the identification of novel regulatory components.

Identification of amplified genes linked to pathway activity
Given the ability of this strategy to identify known CNA of pathway activity, we next used this approach to identify novel drivers of pathway activity. Because highly proliferative luminal/ER+ tumors have a poor prognosis and poor responses to existing therapies 38,39 , we sought to identify amplified genes/CNA associated with our previously published 11-gene PAM50 Proliferation signature with the hope that these might represent targetable drivers of oncogenesis.
To identify those genes that are specifically altered in highly proliferative luminal tumors, while excluding those genes that are associated with proliferation irrespective of subtype, analyses were performed on two subsets of samples: all tumors and all non-basal-like tumors (henceforth called luminal tumors). Some rationale for this binary distinction comes from recent TCGA studies where 12 tumor types were studied simultaneously and showed that breast tumors formed two groups, namely Basal-like and all other breast tumors (called Luminal and including HER2+ tumors), suggesting that breast cancer might broadly be considered two main disease types 40 .
Examining the TCGA breast cancer dataset using the PAM50 Proliferation signature 31 , Basal-like, LumB, and HER2E tumors were found to have the highest proliferation levels ( Figures 3A and 3B) with the top quartile ( Figure 3C) comprised of Basal-like (49.6%), LumB (33.6%) and HER2E (16.8%) patients, whereas the top quartile of proliferative luminal tumors ( Figure 3D) contained LumB (68.0%) and HER2E (32.0%) patients. Using the PAM50 Proliferation signature, we examined the frequency of CNA gains and losses in highly proliferative (top quartile) tumors relative to less proliferative samples irrespective of subtype using the previously discussed statistical strategies ( Figure 3E-F, Supplementary Table 43). To identify those genes that are specifically amplified in highly proliferative luminal breast cancer, these analyses were repeated using the luminal tumor subset (Figures 3G-H, Supplementary Table 58). Analyzing both populations of patients identified three classes of proliferation-associated regions (q<0.05): (1) CNAs associated irrespective of subtype, (2) those altered in basal-like tumors, and (3) those altered in highly proliferative luminal tumors. These results allowed us to focus our analyses on those genes within regions that were uniquely altered in highly proliferative luminal tumors by censoring proliferationassociated genes altered in basal-like breast cancer (e.g.TP53 or INPP4B loss), or that were altered irrespective of molecular subtype (e.g. RB1 loss or MYC amplification). These analyses identified a number of regions including 3p25, 5p15, 11q13, 17q22, and 20q11-13 that were uniquely amplified in highly proliferative luminal tumors.

Identification of pathway-specific essential genes
To distinguish essential from non-essential genes in amplified regions associated with proliferation in luminal tumors, we next examined data from a genome-wide RNAi screen of multiple breast tumor-derived cell lines 9 . The 52 gene expression signatures were applied to a panel (GSE12777) 41 Supplementary  Table 59), of which 27 had both mRNA expression data and were part of a RNAi proliferation screen in which a genome-wide shRNA library (~16,000 genes) was used to identify essential genes ( Figure 4A) 9 . For each signature, a negative Spearman rank correlation was used to identify pathway-specific essential genes ( Figure 4B, Supplementary  Table 60) by comparing the pathway score against the normalized shRNA score across the panel of 27 cell lines. These analyses identified inverse relationships between the abundance of shRNAs targeting key regulatory genes and pathway scores. For instance, examining the ER 11,32 , Her2 11,32 , or Stat1 42 signatures as controls ( Figures 4C-4E) showed a negative correlation between pathway score and shRNA against ESR1 (p=0.0143), ERBB2 (p=0.0227), and STAT1 (p=0.0049) or JAK3 (p=0.00013), respectively. These associations were expected for the ER and Her2 pathways given the relationship between HER2 or ERα mRNA and/or protein expression and the response of cell lines or tumors to trastuzumab or anti-estrogen therapies, respectively. These results confirm that this approach is able to identify essential genes known to be functionally associated with pathway activity, thereby suggesting that these data can serve as a biological filter to distinguish pathway-specific essential from non-essential genes.

Amplified essential genes linked to luminal tumor proliferation
We next sought to distinguish between essential and non-essential genes within regions specifically amplified in highly proliferative luminal tumors. For each subset of tumors we identified genes in amplified regions that were positively correlated with proliferation and showed an increased amplification frequency (q<0.05). We next examined the RNAi data in all breast cancer cell lines (Supplementary Figure 6A) and in luminal/HER2+ cell lines (Supplementary Figure 6B) in the context of the PAM50 Proliferation signature (Supplementary Table 61). Comparing the results of these four analyses ( Figure 5A) identified 19 genes that were uniquely essential for cell viability in luminal cell lines and that were amplified in highly proliferative luminal tumors ( Figure 5B). Two additional genes, DNAJC5 and SNX21, were identified by RNAi analysis but were initially overlooked in the CNA analyses since they were located at the cusp of two segmented regions; however, since genes overlapping both 5' and 3' of these genes were amplified, these were included for further investigation. Of these 21 candidate genes, twelve showed a significant relationship between DNA copy number levels and mRNA expression in luminal tumors (Supplementary Figure 7). Interestingly, half of these genes were located at 20q11-13 (EIF2S2, EIF6, SLC2A10, SNX21, ZBTB46 and DNAJC5), with two located at 3p25.1 (FGD5 and METTL6), and the remaining genes located at 5p15 (TRIO), 11q13 (CPT1A), 12q13 (DTX3), and 17q22-23 (MRPS23). In contrast, permuting the data labels 1000 times for each analysis, in all samples and in luminal samples alone, identified no gene that met this statistical threshold, suggesting that the 21 candidate genes could not have been identified by chance alone.

Validation of identified candidate genes
We next confirmed that the majority of the identified genes were significantly amplified in highly proliferative luminal breast tumors by analyzing an independent breast tumor dataset (METABRIC, n=1,992) for which both mRNA expression and genomic DNA CNA data were available 3 . Of the twelve identified genes, nine (FGD5, METTL6, TRIO, CPT1A, DTX3, MRPS23, EIFS2S, EIF6, and SLC2A10) were present on both platforms used in the METABRIC study. Each of these genes (Supplementary Figure 8) showed a significant relationship between CNA status and mRNA expression in luminal breast tumors (n=1,333). Importantly, eight of the nine, the exception being TRIO, also showed an increased amplification frequency in highly proliferative (top quartile) luminal tumors (Supplementary Figure 9), thus recapitulating one of our main findings.
To confirm that DNA mutations of genes associated with proliferation in luminal tumors did not confound these results, we examined the relationship between the 11-gene Proliferation score and the mutation frequency of the previously identified 35 significantly mutated genes in human breast cancers reported by TCGA 2 . Using a Fisher's exact test (Bonferroni corrected), we determined that only TP53 (q=7.0×10 −10 ) and MAP3K1 (q=5.0×10 −03 ) mutations occurred at significantly different frequencies in highly proliferative (top quartile) luminal tumors compared to all other samples; TP53 mutations occurred more frequently (51.6% vs. 18.6%) while MAP3K1 (2.1% vs.12.4%) mutations occurred less frequently in highly proliferative luminal tumors (Supplementary Table 62). Moreover, we found no significant relationship between MAP3K1 or TP53 mutation status (Bonferroni corrected Fisher's exact test, q>0.05) and the amplification status of each candidate gene (Supplementary Table 63) in highly proliferative luminal tumors.
Lastly, we investigated whether expression of the candidate genes, independent of CNA status, was associated with proliferation in luminal breast tumors. By comparing the mRNA expression patterns of each candidate gene in highly proliferative luminal tumor samples (top quartile) against all other samples, we found that tumors lacking CNA of each candidate gene fell into three categories: those that exhibited a positive relationship between mRNA expression and the PAM50 proliferation signature (EIF2S2, EIF6, CPT1A, and MRPS23), those that were anti-correlated with the signature (DTX3), and those that showed no correlation (FGD5, METTL6, and SLC2A10) between datasets (Supplementary Figure 10). These data suggest that amplification is a key mechanism driving the expression of these genes. However, our data also suggest, not surprisingly, that for some genes overall high expression may be the driver, which can be accomplished by amplification or through other unknown means.

Candidate gene amplification correlates with poor prognosis
Previous studies have shown that highly proliferative luminal tumors have a poor prognosis 38,39 , therefore we investigated what impact amplification of each candidate gene had on overall survival. From the TCGA (n=388) 2 and METABRIC (n=1,333) 3 datasets we extracted the subset of LumA, LumB, or HER2E patients for which survival data was available (Supplementary Tables 64-65). We first analyzed data from the TCGA ( Figure  6A-E), and despite the relatively short follow-up (median: 1.7 years), determined that amplification of FGD5 (P<0.0001, HR: 8.0), METTL6 (P=0.0003, HR: 5.9), DTX3 (P=0.0387, HR: 2.6), and MRPS23 (P=0.0078, HR: 2.9) predicted a significantly worse outcome in luminal breast cancer patients whereas CPT1A amplification had no effect on patient survival (P=0.3738). Extending these analyses to the METABRIC dataset ( Figures  6F-J), which has a longer median survival time (7.2 years), confirmed that FGD5 (P=0.0170, HR: 2.0), METTL6 (P=0.0081, HR: 2.1), DTX3 (P=0.0098, HR: 1.8), and MRPS23 (P=0.0020, HR: 1.5) amplification correlated with a poor prognosis, while gain of CPT1A had no effect (P=0.099) on luminal breast cancer survival. The remaining three genes, showed no consistent effect on prognosis (Supplementary Figure 11). While it is possibly that other genes within these chromosomal loci are also prognostic, these amplified genes were associated with proliferation in vivo, were prognostic in multiple patient cohorts, and essential for cell viability in vitro.
Likewise, we determined that for most of the identified candidate genes that failed to meet all our predetermined criteria, amplification alone, without a coordinate increase in mRNA expression, was not sufficient to affect prognosis since only one (TMEM117) of these genes showed a consistently poor prognosis in the TCGA and METABRIC datasets (Supplementary Table 66). Lastly, we investigated whether the 12 initial candidate genes were predictive of a poor prognosis when compared with standard prognostic markers including molecular subtype, tumor stage, node status, ER status, HER2 status, age at diagnosis, and the 11-gene proliferation score when tested using a multivariate analysis (Cox model). We determined that amplification of a single candidate gene did not consistently outperformed or improve the prognostic capacity of these clinical and genomic variables (Supplementary Table 67). However, these candidate genes were not identified to be prognostic markers, especially given that they correlate with proliferation, but instead were selected to be likely drivers of proliferation, a highly important prognostic feature.

DISCUSSION
Numerous studies, including many focused on human breast cancer, have made use of largescale analyses to investigate the genomic landscape of human cancers and to identify molecular heterogeneity within tumor types not previously recognized 2,3,6,11 . The challenge presented by these studies, and by the enormous amount of genomic data available from resources such as the TCGA and METABRIC projects, is how to integrate multiple forms of genomic data to investigate the biology of the disease, and how to interpret the significance of identified genomic alterations without relying on inferences of "known" biology to determine the role that these alterations play in tumorigenesis.
In this study, we utilize gene expression signatures of signaling pathways to identify patterns that can distinguish the known subtypes of breast cancer. These signatures are largely developed from controlled manipulations of the relevant pathways in vitro, and are thus based on experimental evidence for pathway activation as opposed to extrapolations of pathway activity achieved from analyses of annotated gene lists. Therefore, use of an experimentally derived pathway signature, as opposed to the analysis of a single genomic alteration, provides a measure of pathway activity irrespective of how the pathway may have been activated. For instance, a given pathway can be active in a subset of tumors, either as a result of an activating alteration (i.e. E2F1 or E2F3 amplification) or an independent event that inactivates a negative regulator of the pathway (i.e. RB1 loss and/or mutation), which nevertheless achieves the same end result (i.e. DNA replication and cell proliferation); importantly, we identified these four genetic events as being statistically associated with the RB-LOH signature 16 , which is dominated by E2F-regulated genes and is a strong indicator of cell proliferation and prognosis.
Proliferation is one of the most powerful prognostic features in breast cancers, especially for ER+ cancers 38,39 . Since proliferation is so important, we utilized a gene expression signature of proliferation as a means to integrate the DNA copy number data, along with data from a genome-wide RNAi screen of luminal breast cancer cell lines, to identify luminal-specific genetic drivers of proliferation. We identified 12 genes that were uniquely amplified in highly proliferative luminal tumors in the TCGA dataset, have a correlation between mRNA expression and DNA copy number, and were shown to be essential for luminal breast cancer cell line viability; eight were validated using the independent METABRIC dataset. While FGD5, METTL6, DTX3, and MRPS23 amplification was prognostic in luminal patients, these and many of the other identified genes have been previously reported to regulate tumorigenic characteristics, albeit not necessarily in human breast cancer. For example, FGD5 has been shown to regulate the pro-angiogenic function of VEGF 43 , potentially leading to increased proliferation. DTX3 purportedly promotes Notch signaling 44,45 , while EIF6 is a Notch-dependent regulator of cell invasion and migration 46 and its inhibition restricts lymphomagenesis and tumor progression 47 . MRPS23 expression is associated with proliferation, oxidative phosphorylation, invasiveness, and tumor size in uterine cervical cancer 48 . METTL6 has been reported to contribute to cytotoxic chemotherapy sensitivity in lung cancers 49 .
Several previous studies have identified chromosomal regions specifically altered in subsets of breast cancer, including 3p25 (encompassing METTL6, FGD5) 2 and 11q13 (CPT1A) 3 in luminal breast tumors; however these studies neither discriminated between essential and non-essential genes within a specific amplicon, nor did they identified the functional consequence of these alterations. In contrast, we have shown that these regions are uniquely amplified in highly proliferative luminal tumors and importantly, we distinguish between amplified genes that are essential for cell proliferation and thus likely contribute to tumorigenesis, versus those that are amplified but not essential. For instance SRC (20q12-13), which is co-amplified with EIF6, is similarly amplified in a significant percentage of highly proliferative luminal tumors ( Supplementary Tables 43 and 58), but was not identified as essential in highly proliferative luminal breast cancer cell lines in the RNAi screen (Supplementary Table 60). Interestingly, in addition to its role in regulating translation 50 and Notch signaling 46 , EIF6 has been reported to link Integrin-β4 to the intermediate filament cytoskeleton 51 , potentially leading to down-stream activation of SRC signaling. These results may explain some of the paradoxical findings of SRC in that it may contribute to proliferation status, but may not be essential, whereas a gene very near it, also linked to proliferation, is essential for cell viability in vitro. Clearly additional experiments are needed to address this issue, but these results highlight the complex nature, and importance, of this specific amplicon.
A significant challenge to translating these findings into the clinic is the identification of genes within amplicons that are therapeutically targetable. One such event may be amplification of 11q13-14/CPT1A, which was recently reported 3 to be a defining feature of a high-risk ER+ subgroup (Integrative Cluster 2) and correlates with a poor prognosis in esophageal squamous cell carcinoma 52 . We identified CPT1A as the only gene within the amplified 11q13 locus required for cell viability within the confines of the proliferation signature and luminal cell lines, suggesting that repression of CPT1A could affect the proliferative phenotype of these tumors. Consistent with this hypothesis, it was recently reported that RNAi-mediated down-regulation, or drug-mediated inhibition of CPT1A, inhibited cancer cell line proliferation, migration and metastasis 53-55 , albeit not in breast cancer cell lines. Lastly, a specific inhibitor of CPT1A (ST-1326) repressed tumor formation and proliferation in an Eμ-myc mouse model of Burkett's lymphoma 55 .
Collectively these data demonstrate the ability of this across-platforms genomics approach to identify novel oncogenes amplified in a subset of highly proliferative luminal breast cancer patients which are essential for cell viability. These data suggest that not only are these identified genes potential drivers of oncogenesis and that an emphasis should be placed on elucidating their role in breast tumorigenesis, but also that they, or their associated pathways, may serve as novel therapeutic targets in a subset of human breast cancers for which limited therapeutic opportunities currently exist.

Gene expression data
Agilent custom 244K whole genome gene expression microarray data for human breast cancer samples was acquired from The Cancer Genome Atlas (TCGA) project 2 data portal. Samples were filtered to include only those 476 samples for which Affymetrix SNP 6.0 data was present. As previously described, (TCGA) data were median centered for each gene. Illumina HT-29 v3 expression data for the METABRIC (Molecular Taxonomy of Breast Cancer International Consortium) project (n=1,992 samples) was acquired from the European Genome-phenome Archive at the European Bioinformatics Institute and data were median centered for each gene 3 . Expression data for a panel of 51 breast cancer cell lines was acquired from GEO (GSE12777) 41 . Affymetrix U133+2 data were MAS5.0 normalized using Affymetrix Expression Console (ver1.2.1.20), and log 2 transformed. Expression probes were collapsed using the median gene value with the GenePattern 56 module CollapseProbes.
Affymetrix SNP 6.0 data DNA copy number values were determined in 490 TCGA primary breast tumors (of which 476 had matched mRNA expression data) and 1,992 METABRIC primary breast tumors using Affymetrix 6.0 SNP arrays as previous described 2, 3 . Copy number segmentation and segment calls (i.e. NEUT, AMP, GAIN, HOMD or HETD) was performed using the Circular Binary Segmentation (CBS) algorithm as previously described 2,3 . Using the hg19 build annotation from the UCSC genome browser, genes were selected if they fell completely within a CBS identified copy number segment. Genes that were not found completely within a copy number segment across any sample were filtered out. In the METABRIC dataset, the copy number call gene-matrix was determined from genes that fell completely within a CBS identified copy number segment. Out of the 12 genes of interest, SNX21, ZBTB46, and DNAJC5 were not found completely within a CBS identified segment among the METABRIC samples and were excluded from further analyses.

Gene expression signatures
A panel of 52 previous published gene expression signatures was used to examine patterns of pathway activity and/or microenvironmental states (Supplementary Table 1). In order to implement each signature, the methods detailed in the original studies were followed as closely as possible. The 22 signatures from Gatza et al. 10,11,32 were originally developed based on a Bayesian binary regression strategy and identified a signature comprised of Affymetrix probe sets with positive and negative regression weights; to translate these signatures to a form that could be applied to non-Affymetrix expression data, the original reported signatures were altered. For each signature, we excluded those probe sets with a negative correlation coefficient. The remaining probe sets with a positive coefficient were then translated to the gene level, and replicate genes were merged. To apply a given signature to a new dataset, the expression data was filtered to contain only those genes that met the previous criteria and the mean expression value was calculated using all genes within a given signature that were present in more than 80% of samples. The list of genes in each modified signature is reported in Supplementary Table 2 and the scores for the TCGA  dataset (Supplementary Table 3) and cell line dataset are provided (Supplementary Table  59).

Statistical analyses of signature scores
To quantify differences in patterns of signature scores across subtypes, an ANOVA followed by a Tukey's post-test for pairwise comparisons was used (as shown in Figure 1B). To investigate the level of concordance between each of the 52 signatures, the pathway scores calculated for each sample in the TCGA dataset (Supplementary Table 3) were analyzed. The R values calculated by a Pearson correlation are reported in Supplementary Figure 2 and Supplementary Table 5.

Identification of point mutations as a function of pathway activity
To compare the frequency of mutations, the 35 genes identified as being significantly mutated in human breast cancer 2 were assessed in the context of the 11-gene PAM50 Proliferation signature 31 . A Fisher's exact test (Bonferroni corrected) was used to compare the frequency of mutations in samples with high (top quartile) and low (all other samples) in LumA, LumB and HER2E (n=388) samples. The frequency of mutations associated with each group for each signature is summarized in Supplementary Table 60-61.

Identification of copy number alterations as a function of pathway activity
To identify copy number alterations two analysis methods were independently used. A Spearman rank correlation, both positive and negative, was used to compare gene-level segment scores with predicted pathway activity. To compare the frequency of amplification and losses, a Fisher's exact test was used to compare the frequency of either gene-specific copy number gains and amplification or deletions (both loss of heterozygosity of deletion) against non-amplified or non-deleted samples. Samples in the top quartile of calculated pathway activity were compared to those in the bottom three quartiles. For each analysis, the negative log 10

Analysis of genome-wide RNAi proliferation data
In order to identify genes required for cell viability in a signature dependent manner, data from a previously published genome-wide RNAi screen carried out on a panel breast cancer cell lines were analyzed 9 . The Gene Active Ranking Profile (GARP)-normalized data were obtained from the COLT database and filtered to include only those 27 cell lines for which gene expression data (GSE12777) were also available (acquired February 2013). To identify gene essential for pathway-dependent cell proliferation, a negative Spearman correlation was performed comparing predicted pathway activity and GARP score for each sample. A threshold of p<0.05 was considered significant for all analyses.

Analysis of mRNA expression in copy number neutral samples
To assess mRNA expression in luminal tumors lacking CNA of each candidate genes, luminal and HER2E samples from the TCGA (n=388) and METABRIC (n=1,333) studies were grouping to high (top quartile) and low (all other samples). Samples with copy number gains (including high-level amplification or gain) or losses (both loss of heterozygosity and homozygous deletion) were excluded and a t-test was used to examine statistical differences between the expression levels of genes in each cohort.

Survival analyses
To investigate the effect that candidate gene amplification has on disease-specific survival, clinical data for the 1,992 patients in the METABRIC study were obtained 3 . The 11-gene PAM50 Proliferation signature 31 was applied to all 1,992 samples by calculating the median value of the signature, for each sample. For survival analyses, patients that died of causes unrelated to breast cancer and patients without a date of death were censored. We extracted patients classified as Luminal A, Luminal B or HER2E for which survival data was reported (n=1,333). For survival analysis of the TCGA dataset 2 , we extracted patients classified as LumA, LumB or HER2E for which clinical data were available (September 2012). Diseasespecific survival was calculated comparing samples with an amplification of a candidate gene against those without. In each dataset, patients without a CNA call for a specific gene were excluded from the survival analysis. For each analysis, significance was calculated by a log rank test and the hazard ratio (HR) is reported. To compare the effect of candidate gene copy number status on common prognostic markers including proliferation (PAM50 proliferation signature), molecular subtype (PAM50), tumor stage, node status, ER status, HER2 status, or age at diagnosis, a multivariate Cox model was used.  activity and a Fisher's exact test was used to compare the frequency of copy number gains (red) or losses (light blue) for the (C) Her2 Amp (D) Her1-C2 signature, and (E) RB-LOH signature. Yellow arrows indicate known pathway drivers with q<0.01 for each analysis; black arrow indicates q<0.01 for a single analysis. In each figure, chromosomal boundaries are indicated by vertical black lines.
blue) Spearman rank correlation and Fisher's exact test of amplification (red) or deletion (light blue) frequency. (G) Frequency of CNA in highly proliferative luminal tumors; color key same as (E). (H) Statistical analyses of CNA in proliferative luminal tumors; color key same as (F). Chromosomal boundaries in (E-H) are defined by vertical black lines.
were originally excluded, but mentioned here. Genes identified by (#) were not included on mRNA expression microarrays, and the correlation between DNA and mRNA expression was not assessed.