Comprehensive molecular portraits of human breast tumors

Summary We analyzed primary breast cancers by genomic DNA copy number arrays, DNA methylation, exome sequencing, mRNA arrays, microRNA sequencing and reverse phase protein arrays. Our ability to integrate information across platforms provided key insights into previously-defined gene expression subtypes and demonstrated the existence of four main breast cancer classes when combining data from five platforms, each of which shows significant molecular heterogeneity. Somatic mutations in only three genes (TP53, PIK3CA and GATA3) occurred at > 10% incidence across all breast cancers; however, there were numerous subtype-associated and novel gene mutations including the enrichment of specific mutations in GATA3, PIK3CA and MAP3K1 with the Luminal A subtype. We identified two novel protein expression-defined subgroups, possibly contributed by stromal/microenvironmental elements, and integrated analyses identified specific signaling pathways dominant in each molecular subtype including a HER2/p-HER2/HER1/p-HER1 signature within the HER2-Enriched expression subtype. Comparison of Basal-like breast tumors with high-grade Serous Ovarian tumors showed many molecular commonalities, suggesting a related etiology and similar therapeutic opportunities. The biologic finding of the four main breast cancer subtypes caused by different subsets of genetic and epigenetic abnormalities raises the hypothesis that much of the clinically observable plasticity and heterogeneity occurs within, and not across, these major biologic subtypes of breast cancer.


Introduction
Breast cancer is one of the most common cancers with greater than 1,300,000 cases and 450,000 deaths each year world-wide. Clinically, this heterogeneous disease is categorized into three basic therapeutic groups. The Estrogen Receptor (ER) positive group are the most numerous and diverse, with several genomic tests to assist in predicting outcomes for ER+ patients receiving endocrine therapy 1,2 . The HER2/ERBB2 amplified group 3 is a great clinical success because of effective therapeutic targeting of HER2/ERBB2, which has led to intense efforts to characterize other DNA copy number aberrations 4,5 . Triple Negative Breast Cancers (TNBC), also known as Basal-like breast cancers 6 , are a group with only chemotherapy options, and an increased incidence in patients with germline BRCA1 mutations 7,8 or of African ancestry 9 .
Most molecular studies of breast cancer have focused on just one or two high information content platforms, most frequently mRNA expression profiling or DNA copy number analysis, and more recently massively parallel sequencing [10][11][12] . Supervised clustering of mRNA expression data has reproducibly established that breast cancers encompass several distinct disease entities, often referred to as the intrinsic subtypes of breast cancer 13,14 . The recent development of additional high information content assays focused on abnormalities in DNA methylation, microRNA expression and protein expression, provide further opportunities to more completely characterize the molecular architecture of breast cancer. In this study, a diverse set of breast tumors were assayed using six different technology platforms. Individual platform and integrated pathway analyses identified many subtypespecific mutations and copy number changes that identify therapeutically tractable genomic aberrations and other events driving tumor biology.

Samples and clinical data
Tumor and germline DNA samples were obtained from 825 patients. Different subsets of patients were assayed on each platform: 466 tumors from 463 patients had data available on five platforms including Agilent mRNA expression microarrays (n=547), Illumina Infinium DNA methylation chips (n=802), Affymetrix 6.0 SNP arrays (n=773), microRNA sequencing (n=697), and whole exome sequencing (n=507); in addition, 348 of the 466 samples also had Reverse Phase Protein Array (RPPA) data (n=403). Due to the short median overall follow up (17 months) and the small number of overall survival events (93/818), survival analyses will be presented in a later publication. Demographic and clinical characteristics are presented in Supplemental Table 1.
involved in Holt-Oram Syndrome 17 . Two other transcription factors, CTCF and FOXA1, were at or near significance harboring 13 and 8 mutations, respectively. RUNX1 and CBFB, both rearranged in Acute Myeloid Leukemia and interfering with hematopoietic differentiation, harbored 19 and 9 mutations, respectively. PIK3R1 contained 14 mutations, most of which clustered in the PIK3CA interaction domain similar to previously identified mutations in glioma 18 and endometrial cancer 19 . We also observed a statistically significant exclusion pattern among PIK3R1, PIK3CA, PTEN, and AKT1 mutations (P = 0.025). Mutation of splicing factor SF3B1, previously described in Myelodysplastic Syndromes 20 and Chronic Lymphocytic Leukemia 21 , was significant with 15 non-silent mutations, of which 4 were a recurrent K700E substitution. Two protein tyrosine phosphatases (PTPN22 and PTPRD) were also significantly mutated; frequent deletion/mutation of PTPRD is observed in lung adenocarcinoma 22 .

Association between mutations and mRNA-expression subtypes
We analyzed the somatic mutation spectrum within the context of the four mRNAexpression subtypes, excluding the Normal-like group due to small numbers (n=8) ( Figure  1). Several SMGs showed mRNA-subtype (Supplemental Figures 1-3) and clinical-subtype specific patterns of mutation (Supplemental Table 2). SMGs were considerably more diverse and recurrent within Luminal A and Luminal B tumors than within Basal-like and HER2E subtypes; however, the overall mutation rate was lowest in Luminal A subtype and highest in the Basal-like and HER2E subtypes. The Luminal A subtype harbored the most SMGs, with the most frequent being PIK3CA (45%), followed by MAP3K1, GATA3, TP53, CDH1, and MAP2K4. 12% of Luminal A tumors contained likely inactivating mutations in MAP3K1 and MAP2K4, which represent two contiguous steps in the p38/JNK1 stress kinase pathway 23 . Luminal B cancers exhibited a diversity of SMGs with TP53 and PIK3CA (29% each) being the most frequent. The luminal tumor subtypes markedly contrasted with Basallike cancers where TP53 mutations occurred in 80% of cases and the majority of the Luminal SMG repertoire, except PIK3CA (9%), were absent or near absent. The HER2-Enriched subtype (HER2E), which has frequent HER2/ERBB2 amplification (80%), had a hybrid pattern with a high frequency of TP53 (72%) and PIK3CA(39%) mutations and a much lower frequency of other SMGs including PIK3R1 (4%).
Intrinsic mRNA-subtypes differed not only by mutation frequencies, but also by mutation type. Most notably, TP53 mutations in Basal-like tumors were mostly nonsense and frameshift, while missense mutations predominated in Luminal A and B tumors (Supplemental Figure 1). 58 somatic GATA3 mutations, some of which were previously described 24 , were detected including a hot spot 2bp deletion within intron 4 only in the Luminal A subtype (13/13 mutants) (Supplemental Figure 2). In contrast, 7/9 frame-shift mutations in exon 5 (DNA binding domain) occurred in Luminal B cancers. PIK3CA mutation frequency and spectrum also varied by mRNA-subtype (Supplemental Figure 3); the recurrent PIK3CA E545K mutation was present almost exclusively within Luminal A (25/27) tumors. CDH1 mutations were common (30/36) within the lobular histologic subtype and corresponded with lower CDH1 mRNA (Supplemental Figure 4) and protein expression. Finally, we identified 4/8 somatic variants in HER2/ERBB2 within lobular cancers, 3 of which were within the tyrosine kinase domain.
We performed analyses on a selected set of genes 25 using the normal tissue DNA data and detected a number of germline predisposing variants. These analyses identified 47/507 patients with deleterious germline variants, representing nine different genes (ATM, BRCA1, BRCA2, BRIP1, CHEK2, NBN, PTEN, RAD51C, and TP53; Supplementary Table 3), supporting the hypothesis that ~10% of sporadic breast cancers may have a strong germline contribution. These data confirmed the association between the presence of germline BRCA1 mutations and Basal-like breast cancers 7,8 .

Gene expression analyses (mRNA and microRNA)
Several approaches were used to look for structure in the mRNA expression data. We performed an unsupervised hierarchical clustering analysis of 525 tumors and 22 tumoradjacent normal tissues using the top 3,662 variably expressed genes (Supplemental Figure  5); SigClust analysis identified 12 classes (5 classes with >9 samples/class). We performed a semi-supervised hierarchical cluster analysis using a previously published "intrinsic gene list" 14 , which identified 13 classes (9 classes with >9 samples/class) (Supplemental Figure  6). We also classified each sample using the 50-gene PAM50 model 14 (Supplemental Figure  5). High concordance was observed between all three, therefore, we used the PAM50defined subtype predictor as a common classification metric. There were only 8 Normal-like and 8 Claudin-low tumors 26 , thus no analyses focused on these two subtypes were performed.
MicroRNA expression levels were assayed via Illumina sequencing, using 1222 miRBase 27 v16 mature and star strands as the reference database of microRNA transcripts/genes. Seven subtypes were identified by consensus NMF clustering using an abundance matrix containing the 25% most variable microRNAs (306 transcripts/genes or MIMATs). These subtypes correlated with mRNA-subtypes, ER, PR and HER2 clinical status (Supplemental Figure 7). Of note, microRNA groups 4 and 5 showed high overlap with the Basal-like mRNA subtype and contained many TP53 mutations. The remaining microRNA groups (1-3, 6 and 7) were composed of a mixture of Luminal A, Luminal B and HER2-positive tumors with little correlation with the PAM50 defined subtypes. With the exception of TP53, which showed a strong positive correlation, and PIK3CA and GATA3 that showed negative associations with groups 4 and 5 respectively, there was little correlation with mutation status and microRNA subtype.

DNA methylation
llumina Infinium DNA methylation arrays were used to assay 802 breast tumors. Data from HumanMethylation27 (HM27) and HumanMethylation450 (HM450) arrays were combined and filtered to yield a common set of 574 probes used in an unsupervised clustering analysis, which identified five distinct DNA methylation groups (Supplemental Figure 8). Group 3 showed a hyper-methylated phenotype and was significantly enriched for Luminal B mRNA-subtype and under-represented for PIK3CA and MAP3K1/MAP2K4 mutations. Group 5 showed the lowest levels of DNA methylation, overlapped with the Basal-like mRNA-subtype, and showed a high frequency of TP53 mutations. HER2-positive clinical status, or the HER2E mRNA subtype, had only a modest association with the methylation subtypes.
A supervised analysis of the DNA methylation and mRNA expression data was performed to compare the DNA methylation Group 3 (N=49) versus all tumors in groups 1, 2, and 4 (excluding Group 5 that consisted predominantly of Basal-like tumors). This analysis identified 4,283 genes differentially methylated (3,735 higher in Group 3 tumors) and 1,899 genes differentially expressed (1,232 downregulated); 490 genes were both methylated and showed lower expression in Group 3 tumors (Supplemental Table 4). A DAVID functional annotation analysis identified 'Extracellular region part' and 'Wnt signaling pathway' to be associated with this 490 gene-set; the Group 3 hyper-methylated samples showed fewer PIK3CA and MAP3K1 mutations, and lower expression of Wnt-pathway genes.

DNA copy number
773 breast tumors were assayed using Affymetrix 6.0 SNP arrays. Segmentation analysis and GISTIC were used to identify focal amplifications/deletions and arm-level gains and losses (Supplemental Table 5). These analyses confirmed all previously reported copy number variations and highlighted a number of SMGs including focal amplification of regions containing PIK3CA, HER1/EGFR, FOXA1, and HER2/ERBB2, as well as focal deletions of regions containing MLL3, PTEN, RB1 and MAP2K4 (Supplemental Figure 9); in all cases, multiple genes were included within each altered region. Importantly, many of these copy number changes correlated with mRNA-subtype including characteristic loss of 5q and gain of 10p in Basal-like cancers 5,28 and gain of 1q and/or 16q loss in Luminal tumors 4 . NMF clustering of GISTIC segments identified five copy number clusters/groups that correlated with mRNA-subtypes, ER, PR and HER2 clinical status, and TP53 mutation status (Supplemental Figure 10). In addition, this aCGH subtype classification was highly correlated with the aCGH subtypes recently defined by Curtis et al. 29 (Supplemental Figure  11).

Reverse Phase Protein Arrays (RPPA)
Quantified expression of 171 cancer-related proteins and phospho-proteins by RPPA was performed on 403 breast tumors 30 . Unsupervised hierarchical clustering analyses identified seven subtypes; one class contained too few cases for further analysis (Supplemental Figure  12). These protein subtypes were highly concordant with the mRNA-subtypes, particularly with Basal-like and HER2E mRNA subtypes. Closer examination of the HER2-containing RPPA-defined subgroup showed coordinated overexpression of HER2 and HER1 with a strong concordance with phosphorylated HER2 (pY1248) and HER1 (pY992), likely from heterodimerization and cross-phosphorylation. While there is a potential for modest cross reactivity of antibodies against these related total and phosphoproteins, the concordance of phosphorylation of HER2 and HER1 was confirmed using multiple independent antibodies.
In RPPA-defined Luminal tumors, there was high protein expression of ER, PR, AR, BCL2, GATA3 and INPP4B, defining mostly Luminal A cancers and a second more heterogeneous protein subgroup composed of both Luminal A and Luminal B cancers. Two potentially novel protein-defined subgroups were identified: Reactive I consisted primarily of a subset of Luminal A tumors, whereas Reactive II consisted of a mixture of mRNA-subtypes. These groups are termed 'Reactive' because many of the characteristic proteins are likely produced by the microenvironment and/or cancer-activated fibroblasts including Fibronectin, Caveolin 1 and Collagen VI. These two RPPA groups did not have a marked difference in the % tumor cell content versus each other, or the other protein subtypes, as assessed by SNP array analysis or pathological examination. In addition, supervised analyses of Reactive I vs. II groups using microRNA expression, DNA methylation, mutation, or DNA copy number data identified no significant differences between these groups, while similar supervised analyses using protein and mRNA expression identified many differences.

Multi-platform subtype discovery
To reveal higher order structure in breast tumors based on multiple data types, significant clusters/subtypes from each of five platforms were analyzed using a multi-platform data matrix subjected to unsupervised Consensus Clustering ( Figure 2). This "Cluster of Clusters" (C of C) approach illustrated that Basal-like cancers had the most distinct multiplatform signature since all the different platforms for the Basal-like groups clustered together. To a great extent, the four major C of C subdivisions correlated well with the previously-published mRNA-subtypes (driven in part by the fact that the four intrinsic subtypes were one of the inputs). Therefore, we also performed C of C analysis with no mRNA data present (Supplemental Figure 13) or with the 12 unsupervised mRNA subtypes (Supplemental Figure 14), and in each case 4-6 groups were identified. Recent work by Curtis et al. identified 10 copy number based subgroups in a 997 breast cancer set 29 . We evaluated this classification in a C of C analysis instead of our 5-class copy number subtypes, with either the PAM50 (Supplemental Figure 15) or 12 unsupervised mRNA subtypes (Supplemental Figure 16); each of these C of C classifications were highly correlated with PAM50 mRNA-subtypes and with the other C of C analyses ( Figure 2). The transcriptional profiling and RPPA platforms demonstrated a high correlation with the consensus structure suggesting that the information content from copy number aberrations, microRNAs, and methylation is captured at the level of gene expression and protein function.

Luminal/ER-positive summary analysis
Luminal/ER-positive breast cancers are the most heterogeneous in terms of gene expression (Supplemental Figure 5), mutation spectrum (Figure 1), copy number changes (Supplemental Figure 9) and patient outcomes 1,14 . One of the most dominant features is high mRNA and protein expression of the luminal expression signature (Supplemental Figure 5), which contains ESR1, GATA3, FOXA1, XBP1 and cMYB; the Luminal/ERpositive cluster also contained the largest number of SMGs. Most notably, GATA3 and FOXA1 were mutated in a mutually exclusive fashion, while ESR1 and XBP1 were typically highly expressed but infrequently mutated. Mutations in RUNX1 and its dimerization partner CBFB may also play a role in aberrant ER-signaling in Luminals as RUNX1 functions as an ER "DNA tethering factor" 31 . PARADIGM 32 analysis comparing Luminal vs. Basal-like cancers further emphasized the presence of a hyperactivated FOXA1/ER complex as a critical network hub differentiating these two tumor subtypes (Supplemental Figure 17).
A confirmatory finding here was the high mutation frequency of PIK3CA in Luminal/ERpositive breast cancers 33,34 . Through multiple technology platforms, we examined possible relationships between PIK3CA mutation, PTEN loss, INPP4B loss and multiple gene and protein expression signatures of pathway activity. RPPA data demonstrated that pAKT, pS6 and p4EBP1, typical markers of PI3K pathway activation, were not elevated in PIK3CAmutated Luminal A cancers; instead, they were highly expressed in Basal-like and HER2E mRNA-subtypes (the latter having frequent PIK3CA mutations) and correlated strongly with INPP4B and PTEN loss, and to a degree with PIK3CA amplification. Similarly, protein 35 and three mRNA signatures [36][37][38] of PI3K pathway activation were enriched in Basal-like over Luminal A cancers ( Figure 3A). This apparent disconnect between the presence of PIK3CA mutations and biomarkers of pathway activation has been previously noted 35 .
Another striking Luminal/ER-positive subtype finding was the frequent mutation of MAP3K1 and MAP2K4, which represent two contiguous steps within the p38/JNK1 pathway 23,39 . These mutations are predicted to be inactivating, with MAP2K4 also a target of focal DNA loss in Luminal tumors (Supplemental Figure 9). To explore the possible interplay between PIK3CA and MAP3K1/MAP2K4 signaling, MEMo analysis 40 was performed to identify mutually exclusive alterations targeting frequently altered genes likely to belong to the same pathway ( Figure 4). Across all breast cancers, MEMo identified a set of modules that highlight the differential activation events within the Receptor Tyrosine Kinase (RTK)/PI3K pathway ( Figure 4A); mutations of PIK3CA were very common in Luminal/ER-positive cancers while PTEN loss was more common in Basal-like tumors. Almost all MAP3K1/MAP2K4 mutations were in Luminal tumors, yet MAP3K1 and MAP2K4 appeared almost mutually exclusive relative to one another.
The TP53 pathway was differentially inactivated in Luminal/ER-positive breast cancers, with a low TP53 mutation frequency in Luminal A (12%) and a higher frequency in Luminal B (29%) (Figure 1). In addition to TP53 itself, a number of other pathway-inactivating events occurred including ATM loss and MDM2 amplification ( Figure 3B and 4B), both of which occurred more frequently within Luminal B cancers. Gene expression analysis demonstrated that individual markers of functional TP53 (GADD45A and CDKN1A), and TP53 activity 41,42 signatures, were highest in Luminal A ( Figure 3B). These data suggest that the TP53 pathway remains largely intact in Luminal A but is often inactivated in the more aggressive Luminal B 43 . Other PARADIGM-based pathway differences driving Luminal B versus Luminal A included hyperactivation of transcriptional activity associated with cMYC and FOXM1/proliferation.
The critical retinoblastoma/RB1 pathway also showed mRNA-subtype specific alterations ( Figure 3C). RB1 itself, by mRNA and protein expression, was detectable in most Luminal cancers with highest levels within Luminal A. A common oncogenic event was Cyclin D1 amplification and high expression, which preferentially occurred within Luminal tumors, and more specifically within Luminal B. In contrast, the presumed tumor suppressor p18/ CDKN2C was at its lowest levels in Luminal A, consistent with observations from mouse models 44 . Lastly, RB1 activity signatures were also high in Luminal cancers [45][46][47] . Luminal A tumors, which have the best prognosis, are the most likely to retain activity of the major tumor suppressors RB1 and TP53. These genomic characterizations also provided clues for druggable targets. We compiled a drug target table in which we defined a target as a gene/protein for which there is an approved or investigational drug in human clinical trials targeting the molecule or canonical pathway (Supplemental Table 6). In Luminal/ER-positive cancers, the high frequency of PIK3CA mutations suggests that inhibitors of this activated kinase or its signaling pathway may be beneficial. Other potential SMG drug candidates include AKT1 inhibitors (11/12 AKT1 variants were Luminal) and PARP inhibitors for BRCA1/2 mutations. Though still unapproved as biomarkers, many potential copy number-based drug targets were identified including amplifications of FGFRs and IGFR1, as well as Cyclin D1/CDK4/CDK6. A summary of the general findings in Luminal tumors and the other subtypes is presented in Table 1.

HER2-based classifications and summary analysis
DNA amplification of HER2/ERBB2 was readily evident in this study (Supplemental Figure  9), together with overexpression of multiple HER2-amplicon associated genes that in part, define the HER2-Enriched (HER2E) mRNA-subtype (Supplemental Figure 5). However, not all clinically HER2+ tumors are of the HER2E mRNA-subtype, and not all tumors in the HER2E mRNA-subtype are clinically HER2+. Integrated analysis of the RPPA and mRNA data clearly identified a HER2+ group (Supplemental Figure 12). When the HER2-positive protein and HER2E mRNA-subtypes overlapped, a strong signal of HER1, p-HER1, HER2, and p-HER2 was observed. However, only ~50% of clinically HER2-positive tumors fall into this HER2E-mRNA-subtype/HER2-protein group, the rest of the clinically HER2positive tumors were observed predominantly in the Luminal mRNA subtypes.
These data suggest that there exist at least two types of clinically defined HER2-positive tumors. To identify differences between these groups, a supervised gene expression analysis comparing 36 HER2E-mRNA-subtype/HER2-positive versus 31 Luminal-mRNA-subtype/ HER2-positive tumors was performed and identified 302 differentially expressed genes (qvalue = 0%) (Supplemental Figure 18, Supplemental Table 7). These genes largely track with ER status but also indicated that HER2E-mRNA-subtype/HER2-positive tumors showed significantly higher expression of a number of RTKs including FGFR4, HER1/ EGFR, HER2 itself, as well as genes within the HER2-amplicon (i.e. GRB7). Conversely, the Luminal-mRNA-subtype/HER2-positive tumors showed higher expression of the Luminal cluster of genes including GATA3, BCL2, and ESR1. Further support for two types of clinically-defined HER2+ disease was evident in the somatic mutation data supervised by either mRNA-subtype or ER status; TP53 mutations were significantly enriched in HER2E or ER-negative tumors while GATA3 mutations were only observed in Luminal subtypes or ER-positive tumors.
Analysis of the RPPA data according to mRNA-subtype identified 36 differentially expressed proteins (q-value <5%) (Supplemental Figure 18G, Supplemental Table 8). The HER1/p-HER1/HER2/p-HER2 signal was again observed and present within the HER2E-mRNA-subtype/HER2-positive tumors, as was high p-SRC and p-S6; conversely, many protein markers of Luminal cancers again distinguished the Luminal-mRNA-subtype/HER2positive tumors. Given the importance of clinical HER2 status, a more focused analysis was Page 8 Nature. Author manuscript; available in PMC 2013 April 04. performed based upon the RPPA-defined protein expression of HER2 (Supplemental Figure  19); the results strongly recapitulated findings from the RPPA and mRNA-subtypes including a high correlation between HER2 clinical status, HER2 protein by RPPA, p-HER2, HER1, and p-HER1. These multiple signatures, namely HER2E-mRNA-subtype, HER2-amplicon genes by mRNA expression, and RPPA HER1/p-HER1/HER2/p-HER2 signature, ultimately identify at least two groups/subtypes within clinically HER2+ tumors (Table 1). These signatures represent breast cancer biomarker(s) that could potentially predict response to anti-HER2 targeted therapies.
Many therapeutic advances have been made for clinically HER2-positive disease. This study has identified additional somatic mutations that represent potential therapeutic targets within this group, including a high frequency of PIK3CA mutations (39%), a lower frequency of PTEN and PIK3R1 mutations (Supplemental Table 6), and genomic losses of PTEN and INPP4B. Other possible druggable mutations included variants within HER-family members including two somatic mutations in HER2, two within HER1/EGFR, and five within HER3. Pertuzumab, in combination with trastuzumab, targets the HER2-HER3 heterodimer 48 , however, these data suggest that targeting HER1 with HER2 could also be beneficial. Lastly, the HER2E-mRNA-subtype typically showed high aneuploidy, the highest somatic mutation rate (Table 1), and DNA amplification of other potential therapeutic targets including FGFRs, HER1/EGFR, CDK4 and Cyclin D1.

Basal-like summary analysis
The Basal-like subtype was discovered more than a decade ago by first-generation cDNA microarrays 13 . These tumors are often referred to as Triple-Negative Breast Cancers (TNBC) because most Basal-like tumors are typically negative for ER, PR and HER2. However, ~75% of TNBC are Basal-like with the other 25% comprised of all other mRNAsubtypes 6 . In this data set, there was a high degree of overlap between these two distinctions with 76 TNBC, 81 Basal-like, and 65 that were both TNBC and Basal-like. Given the known heterogeneity of TNBC, and that the Basal-like subtype proved to be distinct on every platform, we choose to use the Basal-like distinction for comparative analyses.
Basal-like tumors showed a high frequency of TP53 mutations (80%) 9 , which when combined with inferred TP53 pathway activity suggests that loss of TP53 function occurs within most, if not all, Basal-like cancers ( Figure 3B). In addition to loss of TP53, a MEMo analysis reconfirmed that loss of RB1 and BRCA1 are Basal-like features ( Figure 4C) 46,49 . PIK3CA was the next most commonly mutated gene (~9%); however, inferred PI3K pathway activity, whether from gene 36-38 , protein 35 , or high PI3K/AKT pathway activities, was highest in Basal-like cancers ( Figure 3A). Alternative means of activating the PI3Kpathway in Basal-like cancers likely includes loss of PTEN and INPP4B and/or amplification of PIK3CA. A recent paper from Shah et al. 12 performed exome sequencing of 102 TNBC. Five of the top six most frequent TNBCs mutations in Shah et al. were also observed at a similar frequency in our TNBC subset (Myo3A not present here); of those five, three passed our test as a significantly mutated gene in TNBCs (Supplemental Table 2).

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Expression features of Basal-like tumors include a characteristic signature containing Keratins 5, 6 and 17 and high expression of genes associated with cell proliferation (Supplemental Figure 5). A PARADIGM 32 analysis of Basal-like versus Luminal tumors emphasized the importance of hyperactivated FOXM1 as a transcriptional driver of this enhanced proliferation signature (Supplemental Figure 17). PARADIGM also identified hyperactivated cMYC and HIF1α/ARNT network hubs as key regulatory features of Basallike cancers. Even though chromosome 8q24 is amplified across all subtypes (Supplemental Figure 9), high cMYC activation appears to be a Basal-like characteristic 50 .
Given the striking contrasts between Basal-like and Luminal/HER2E subtypes, we performed a MEMo analysis on Basal-like tumors alone. The top-scoring module included ATM mutations, BRCA1 and BRCA2 inactivation, RB1 loss and Cyclin E1 amplification ( Figure 4C). Interestingly, these same modules were identified previously for Serous Ovarian cancers 40 . Furthermore, the Basal-like (and TNBC) mutation spectrum was reminiscent of the spectrum seen in Serous Ovarian cancers 51 with only one gene (i.e. TP53) at >10% mutation frequency. To explore possible similarities between Serous Ovarian and the breast Basal-like cancers, we performed a number of analyses comparing Ovarian vs. breast Luminal, Ovarian vs. breast Basal-like, and breast Basal-like vs. breast Luminal ( Figure 5). Comparing copy number landscapes, we observed several common features between Ovarian and Basal-like tumors including widespread genomic instability and common gains of 1q, 3q, 8q and 12p, and loss of 4q, 5q and 8p (Supplemental Figure 20A). Using a more global copy number comparison, we examined the overall fraction of the genome altered and the overall copy number correlation of Ovarian cancers versus each breast cancer mRNA-subtype (Supplemental Figure 20A, B); in both cases, Basal-like tumors were the most similar to the Serous Ovarian carcinomas.
We systematically looked for other common features between Serous Ovarian and Basallike when each was compared to Luminal. We identified: 1) BRCA1 inactivation, 2) RB1 loss and Cyclin E1 amplification, 3) high expression of AKT3, 4) cMYC amplification and high expression, and 5) a high frequency of TP53 mutations ( Figure 5A). An additional supervised analysis of a large, external multi-tumor type transcriptomic dataset (GSE2109) was performed where each TCGA breast tumor expression profile was compared via a correlation analysis to that of each tumor in the multi-tumor set. Basal-like breast cancers clearly showed high mRNA expression correlations with Serous Ovarian cancers, as well as with Lung Squamous carcinomas ( Figure 5B). A PARADIGM analysis that calculates whether a gene or pathway feature is both differentially activated in Basal-like versus Luminal cancers and has higher overall activity across the TCGA Ovarian samples was performed; this identified comparably high pathway activity of the HIF1α/ARNT, cMYC, and FOXM1 regulatory hubs in both Ovarian and Basal-like cancers (Supplemental Figure  20C). The common findings of TP53, RB1 and BRCA1 loss, with cMYC amplification strongly suggest that these are shared driving events for Basal-like and Serous Ovarian carcinogenesis. This suggests that common therapeutic approaches should be considered, which is supported by the activity of platinum analogs, and taxanes, in breast Basal-like and Serous Ovarian cancers. Given that most Basal-like cancers are TNBC, finding new drug targets for this group is critical. Unfortunately, the somatic mutation repertoire for Basal-like breast cancers has not provided a common target aside from BRCA1 and BRCA2. Here we note that ~20% of Basal-like tumors had a germline (n=12) and/or somatic (n=8) BRCA1 or BRCA2 variant, which suggests 1 in 5 Basal-like patients might benefit from PARP inhibitors and/or platinum compounds 52,53 . The copy number landscape of Basal-like cancers showed multiple amplifications and deletions, some of which may provide therapeutic targets (Supplemental Table 6). Potential targets include losses of PTEN and INPP4B, both of which have been shown to sensitize cell lines to PI3K pathway inhibitors 54,55 . Interestingly, many of the components of the PI3K and RAS-RAF-MEK pathway were amplified (but not typically mutated) in Basal-like cancers including PIK3CA (49%), KRAS (32%), BRAF (30%), and HER1/EGFR (23%). Other RTKs that are plausible drug targets and amplified in some Basal-like cancers include FGFR1, FGFR2, IGFR1, c-KIT, MET and PDGFRA. Lastly, the PARADIGM identification of high HIF1α/ARNT pathway activity suggest that these malignancies might be susceptible to angiogenesis inhibitors and/or bioreductive drugs that become activated under hypoxic conditions.

Summary
The integrated molecular analyses of breast carcinomas we report significantly extends our knowledge base to produce a comprehensive catalog of likely genomic drivers of the most common breast cancer subtypes (Table 1). Our novel observation that diverse genetic and epigenetic alterations converge phenotypically into four main breast cancer classes is not only consistent with convergent evolution of gene circuits as seen across multiple organisms, but also with models of breast cancer clonal expansion and in vivo cell selection proposed to explain the phenotypic heterogeneity observed within defined breast cancer subtypes.

Methods summary
Specimens were obtained from patients with appropriate consent from institutional review boards. Using a co-isolation protocol, DNA and RNA were purified. In total, 800 patients were assayed on at least one platform. Different numbers of patients were used for each platform using the largest number of patients available at time of data freeze; 466 samples (463 patients) were in common across 5/6 platforms (excluding RPPA) and 348 patients were in common on 6/6 platforms. Technology platforms used include: 1) gene expression DNA microarrays 51 , 2) DNA methylation arrays, 3) microRNA sequencing, 4) Affymetrix SNP arrays, 5) exome sequencing, and 6) Reverse Phase Protein Arrays. Each platform, except for the exome sequencing, was used in a de novo subtype discovery analysis (Supplemental Methods) which were included in a single analysis to define an overall subtype architecture. Additional integrated across platform computational analyses were preformed including PARADIGM 32 and MEMo 40 .
All of the primary sequence files are deposited in CGHub (https://cghub.ucsc.edu/); all other data including mutation annotation file are deposited at the Data Coordinating Center (DCC) (http://cancergenome.nih.gov/). Sample lists, data matrices and supporting data can be found Page 11 Nature. Author manuscript; available in PMC 2013 April 04.    Mutual exclusivity modules are represented by their gene components and connected to reflect their activity in distinct pathways. For each gene, the frequency of alteration in Basallike (right box) and non-Basal (left box) is reported. Next to each module is a fingerprint indicating what specific alteration is observed for each gene (row) in each sample (column). a) MEMo identified several overlapping modules that recapitulate the RTK/PI3K and p38/ JNK1 signaling pathways and whose core was the top-scoring module. b) MEMo identified alterations to TP53 signaling as occurring within a statistically significant mutually exclusive trend. c) A Basal-like only MEMo analysis identified one module that included ATM mutations, defects at BRCA1 and BRCA2, and deregulation of the RB1-pathway. A gene expression heatmap is below the fingerprint to show expression levels.