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We propose a statistical boosting method, termed I-Boost, to integrate multiple types of high-dimensional genomics data with clinical data for predicting survival time. I-Boost provides substantially higher prediction accuracy than existing methods. By applying I-Boost to The Cancer Genome Atlas, we show that the integration of multiple genomics platforms with clinical variables improves the prediction of survival time over the use of clinical variables alone; gene expression values are typically more prognostic of survival time than other genomics data types; and gene modules/signatures are at least as prognostic as the collection of individual gene expression data.

Background: The seed and soil hypothesis was proposed over a century ago to describe why cancer cells (seeds) grow in certain organs (soil). Since then, the genetic properties that define the cancer cells have been heavily investigated; however, genomic mediators within the organ microenvironment that mediate successful metastatic growth are less understood. These studies sought to identify cancer- and organ-specific genomic programs that mediate metastasis., Methods: In these studies, a set of 14 human breast cancer patient-derived xenograft (PDX) metastasis models was developed and then tested for metastatic tropism with two approaches: spontaneous metastases from mammary tumors and intravenous injection of PDX cells. The transcriptomes of the cancer cells when growing as tumors or metastases were separated from the transcriptomes of the microenvironment via species-specific separation of the genomes. Drug treatment of PDX spheroids was performed to determine if genes activated in metastases may identify targetable mediators of viability., Results: The experimental approaches that generated metastases in PDX models were identified. RNA sequencing of 134 tumors, metastases, and normal non-metastatic organs identified cancer- and organ-specific genomic properties that mediated metastasis. A common genomic response of the liver microenvironment was found to occur in reaction to the invading PDX cells. Genes within the cancer cells were found to be either transiently regulated by the microenvironment or permanently altered due to clonal selection of metastatic sublines. Gene Set Enrichment Analyses identified more than 400 gene signatures that were commonly activated in metastases across basal-like PDXs. A Src signaling signature was found to be extensively upregulated in metastases, and Src inhibitors were found to be cytotoxic to PDX spheroids., Conclusions: These studies identified that during the growth of breast cancer metastases, there were genomic changes that occurred within both the cancer cells and the organ microenvironment. We hypothesize that pathways upregulated in metastases are mediators of viability and that simultaneously targeting changes within different cancer cell pathways and/or different tissue compartments may be needed for inhibition of disease progression.

Background: Use of cyclin D1 (CCND1) gene amplification as a breast cancer biomarker has been hampered by conflicting assessments of the relationship between cyclin D1 protein levels and patient survival. Here, we aimed to clarify its prognostic and treatment predictive potential through comprehensive long-term survival analyses., Methods: CCND1 amplification was assessed using SNP arrays from two cohorts of 1965 and 340 patients with matching gene expression array and clinical follow-up data of over 15 years. Kaplan-Meier and multivariable Cox regression analyses were used to determine survival differences between CCND1 amplified vs. non-amplified tumours in clinically relevant patient sets, within PAM50 subtypes and within treatment-specific subgroups. Boxplots and differential gene expression analyses were performed to assess differences between amplified vs. non-amplified tumours within PAM50 subtypes., Results: When combining both cohorts, worse survival was found for patients with CCND1-amplified tumours in luminal A (HR = 1.68; 95% CI, 1.15-2.46), luminal B (1.37; 1.01-1.86) and ER+/LN-/HER2- (1.66; 1.14-2.41) subgroups. In gene expression analysis, CCND1-amplified luminal A tumours showed increased proliferation (P < 0.001) and decreased progesterone (P = 0.002) levels along with a large overlap in differentially expressed genes when comparing luminal A and B-amplified vs. non-amplified tumours., Conclusions: Our results indicate that CCND1 amplification is associated with worse 15-year survival in ER+/LN-/HER2-, luminal A and luminal B patients. Moreover, luminal A CCND1-amplified tumours display gene expression changes consistent with a more aggressive phenotype. These novel findings highlight the potential of CCND1 to identify patients that could benefit from long-term treatment strategies.

Purpose and Methods: In human basal-like breast cancer, mutations and deletions in TP53 and BRCA1 are frequent oncogenic events. Thus, we interbred mice expressing the CRE-recombinase with mice harboring loxP sites at TP53 and BRCA1 (K14-Cre; p53 f/f Brca1 f/f ) to test the hypothesis that tissue-specific deletion of TP53 and BRCA1 would give rise to tumors reflective of human basal-like breast cancer., Results: In support of our hypothesis, these transgenic mice developed tumors that express basal-like cytokeratins and demonstrated intrinsic gene expression features similar to human basal-like tumors. Array comparative genomic hybridization revealed a striking conservation of copy number alterations between the K14-Cre; p53 f/f Brca1 f/f mouse model and human basal-like breast cancer. Conserved events included MYC amplification, KRAS amplification, and RB1 loss. Microarray analysis demonstrated that these DNA copy number events also led to corresponding changes in signatures of pathway activation including high proliferation due to RB1 loss. K14-Cre; p53 f/f Brca1 f/f also matched human basal-like breast cancer for a propensity to have immune cell infiltrates. Given the long latency of K14-Cre; p53 f/f Brca1 f/f tumors (~ 250 days), we created tumor syngeneic transplant lines, as well as in vitro cell lines, which were tested for sensitivity to carboplatin and paclitaxel. These therapies invoked acute regression, extended overall survival, and resulted in gene expression signatures of an anti-tumor immune response., Conclusion: These findings demonstrate that this model is a valuable preclinical resource for the study of human basal-like breast cancer.

Background: The dominant invasive breast cancer histologic subtype, ductal carcinoma, shows intrinsic subtype diversity. However, lobular breast cancers are predominantly Luminal A. Both histologic subtypes show distinct relationships with patient and tumor characteristics, but it is unclear if these associations remain after accounting for intrinsic subtype., Methods: Generalized linear models were used to estimate relative frequency differences (RFDs) and 95% confidence intervals (95% CIs) for the associations between age, race, tumor characteristics, immunohistochemistry (IHC) and RNA-based intrinsic subtype, TP53 status, and histologic subtype in the Carolina Breast Cancer Study (CBCS, n = 3,182) and The Cancer Genome Atlas (TCGA, n = 808)., Results: Relative to ductal tumors, lobular tumors were significantly more likely to be Luminal A [CBCS RNA RFD: 44.9%, 95% CI (39.6, 50.1); TCGA: RFD: 50.5%, 95% CI (43.9, 57.1)], were less frequent among young (≤ 50 years) and black women, were larger in size, low grade, less frequently had TP53 pathway defects, and were diagnosed at later stages. These associations persisted among Luminal A tumors (n = 242)., Conclusions: While histology is strongly associated with molecular characteristics, histologic associations with age, race, size, grade, and stage persisted after restricting to Luminal A subtype. Histology may continue to be clinically relevant among Luminal A breast cancers.

A Clinical Proteomic Tumor Analysis Consortium (CPTAC) proteogenomic analysis prioritized dihydropyrimidinase-like-3 (DPYSL3) as a multilevel (RNA/protein/phosphoprotein) expression outlier specific to the claudin-low (CLOW) subset of triple-negative breast cancers. A PubMed informatics tool indicated a paucity of data in the context of breast cancer, which further prioritized DPYSL3 for study. DPYSL3 knockdown in DPYSL3-positive ([Formula: see text]) CLOW cell lines demonstrated reduced proliferation, yet enhanced motility and increased expression of epithelial-to-mesenchymal transition (EMT) markers, suggesting that DPYSL3 is a multifunctional signaling modulator. Slower proliferation in DPYSL3-negative ([Formula: see text]) CLOW cells was associated with accumulation of multinucleated cells, indicating a mitotic defect that was associated with a collapse of the vimentin microfilament network and increased vimentin phosphorylation. DPYSL3 also suppressed the expression of EMT regulators SNAIL and TWIST and opposed p21 activated kinase 2 (PAK2)-dependent migration. However, these EMT regulators in turn induce DPYSL3 expression, suggesting that DPYSL3 participates in negative feedback on EMT. In conclusion, DPYSL3 expression identifies CLOW tumors that will be sensitive to approaches that promote vimentin phosphorylation during mitosis and inhibitors of PAK signaling during migration and EMT., Competing Interests: Conflict of interest statement: M.J.E. received ad hoc consulting fees from Pfizer, AstraZeneca, Sermonix, Celgene, Nanostring, Puma, and Novartis, as well as patents and royalty income for PAM50-based diagnostics to Nanostring/Prosigna. C.M.P. and M.J.E. are equity stockholders, consultants, and board of director members of BioClassifier LLC, are listed as inventors on patent applications on the Breast PAM50 assay, and receive income from the “Prosigna” breast cancer assay. All other authors have no potential conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Purpose: Prognostic value of pathologic complete response (pCR) and extent of pathologic response attained with anthracycline-free platinum plus taxane neoadjuvant chemotherapy (NAC) in triple-negative breast cancer (TNBC) is unknown. We report recurrence-free survival (RFS) and overall survival (OS) according to degree of pathologic response in patients treated with carboplatin plus docetaxel NAC., Patients and Methods: One-hundred and ninety patients with stage I-III TNBC were treated with neoadjuvant carboplatin (AUC6) plus docetaxel (75 mg/m 2 ) every 21 days × 6 cycles. pCR (no evidence of invasive tumor in breast and axilla) and Residual cancer burden (RCB) were evaluated. Patients were followed for recurrence and survival. Extent of pathologic response was associated with RFS and OS using the Kaplan-Meier method., Results: Median age was 51 years, and 52% were node-positive. pCR and RCB I rates were 55% and 13%, respectively. Five percent of pCR patients, 0% of RCB I patients, and 58% of RCB II/III patients received adjuvant anthracyclines. Three-year RFS and OS were 79% and 87%, respectively. Three-year RFS was 90% in patients with pCR and 66% in those without pCR [HR = 0.30; 95% confidence interval (CI), 0.14-0.62; P = 0.0001]. Three-year OS was 94% in patients with pCR and 79% in those without pCR (HR = 0.25; 95% CI, 0.10-0.63; P = 0.001). Patients with RCB I demonstrated 3-year RFS (93%) and OS (100%) similar to those with pCR. On multivariable analysis, higher tumor stage, node positivity, and RCB II/III were associated with worse RFS., Conclusions: Neoadjuvant carboplatin plus docetaxel yields encouraging efficacy in TNBC. Patients achieving pCR or RCB I with this regimen demonstrate excellent 3-year RFS and OS without adjuvant anthracycline., (©2018 American Association for Cancer Research.)

Background: Based on promising phase II data, the histone deacetylase inhibitor entinostat is in phase III trials for patients with metastatic estrogen receptor-positive breast cancer. Predictors of sensitivity and resistance, however, remain unknown., Methods: A total of eight cell lines and nine mouse models of breast cancer were treated with entinostat. Luminal cell lines were treated with or without entinostat at their IC 50 doses, and MMTV/Neu luminal mouse tumors were untreated or treated with entinostat until progression. We investigated these models using their gene expression profiling by microarray and copy number by arrayCGH. We also utilized the network-based DawnRank algorithm that integrates DNA and RNA data to identify driver genes of resistance. The impact of candidate drivers was investigated in The Cancer Genome Atlas and METABRIC breast cancer datasets., Results: Luminal models displayed enhanced sensitivity to entinostat as compared to basal-like or claudin-low models. Both in vitro and in vivo luminal models showed significant downregulation of Myc gene signatures following entinostat treatment. Myc gene signatures became upregulated on tumor progression in vivo and overexpression of Myc conferred resistance to entinostat in vitro. Further examination of resistance mechanisms in MMTV/Neu tumors identified a portion of mouse chromosome 4 that had DNA copy number loss and low gene expression. Within this region, Jun was computationally identified to be a driver gene of resistance. Jun knockdown in cell lines resulted in upregulation of Myc signatures and made these lines more resistant to entinostat. Jun-deleted samples, found in 17-23% of luminal patients, had significantly higher Myc signature scores that predicted worse survival., Conclusions: Entinostat inhibited luminal breast cancer through Myc signaling, which was upregulated by Jun DNA loss to promote resistance to entinostat in our models. Jun DNA copy number loss, and/or high MYC signatures, might represent biomarkers for entinostat responsiveness in luminal breast cancer.

Purpose: Response to a complex trastuzumab-based regimen is affected by multiple features of the tumor and its microenvironment. Developing a predictive algorithm is key to optimizing HER2-targeting therapy. Experimental Design: We analyzed 137 pretreatment tumors with mRNA-seq and DNA exome sequencing from CALGB 40601, a neoadjuvant phase III trial of paclitaxel plus trastuzumab with or without lapatinib in stage II to III HER2-positive breast cancer. We adopted an Elastic Net regularized regression approach that controls for covarying features within high-dimensional data. First, we applied 517 known gene expression signatures to develop an Elastic Net model to predict pCR, which we validated on 143 samples from four independent trials. Next, we performed integrative analyses incorporating clinicopathologic information with somatic mutation status, DNA copy number alterations (CNA), and gene signatures. Results: The Elastic Net model using only gene signatures predicted pCR in the validation sets (AUC = 0.76). Integrative analyses showed that models containing gene signatures, clinical features, and DNA information were better pCR predictors than models containing a single data type. Frequently selected variables from the multiplatform models included amplifications of chromosome 6p, TP53 mutation, HER2-enriched subtype, and immune signatures. Variables predicting resistance included Luminal/ER + features. Conclusions: Models using RNA only, as well as integrated RNA and DNA models, can predict pCR with improved accuracy over clinical variables. Somatic DNA alterations (mutation, CNAs), tumor molecular subtype (HER2E, Luminal), and the microenvironment (immune cells) were independent predictors of response to trastuzumab and paclitaxel-based regimens. This highlights the complexity of predicting response in HER2-positive breast cancer. Clin Cancer Res; 24(21); 5292-304. ©2018 AACR ., (©2018 American Association for Cancer Research.)

CDH1 and PIK3CA are the two most frequently mutated genes in invasive lobular carcinoma (ILC) of the breast. Transcription profiling has identified molecular subtypes for ILC, one of which, immune-related (IR), is associated with gene expression linked to lymphocyte and macrophage infiltration. Here, we report that deletion of Cdh1, together with activation of Pik3ca in mammary epithelium of genetically modified mice, leads to formation of IR-ILC-like tumors with immune cell infiltration, as well as gene expression linked to T-regulatory (Treg) cell signaling and activation of targetable immune checkpoint pathways. Interestingly, these tumors show enhanced Rac1- and Yap-dependent transcription and signaling, as well as sensitivity to PI3K, Rac1, and Yap inhibitors in culture. Finally, high-dimensional immunophenotyping in control mouse mammary gland and IR-ILC tumors by mass cytometry shows dramatic alterations in myeloid and lymphoid populations associated with immune suppression and exhaustion, highlighting the potential for therapeutic intervention via immune checkpoint regulators., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

RNA-based, multi-gene molecular assays are available and widely used for patients with ER-positive/HER2-negative breast cancers. However, RNA-based genomic tests can be costly and are not available in many countries. Methods for inferring molecular subtype from histologic images may identify patients most likely to benefit from further genomic testing. To identify patients who could benefit from molecular testing based on H&E stained histologic images, we developed an image analysis approach using deep learning. A training set of 571 breast tumors was used to create image-based classifiers for tumor grade, ER status, PAM50 intrinsic subtype, histologic subtype, and risk of recurrence score (ROR-PT). The resulting classifiers were applied to an independent test set ( n  = 288), and accuracy, sensitivity, and specificity of each was assessed on the test set. Histologic image analysis with deep learning distinguished low-intermediate vs. high tumor grade (82% accuracy), ER status (84% accuracy), Basal-like vs. non-Basal-like (77% accuracy), Ductal vs. Lobular (94% accuracy), and high vs. low-medium ROR-PT score (75% accuracy). Sampling considerations in the training set minimized bias in the test set. Incorrect classification of ER status was significantly more common for Luminal B tumors. These data provide proof of principle that molecular marker status, including a critical clinical biomarker (i.e., ER status), can be predicted with accuracy >75% based on H&E features. Image-based methods could be promising for identifying patients with a greater need for further genomic testing, or in place of classically scored variables typically accomplished using human-based scoring., Competing Interests: C.M.P. is an equity stock holder and Board of Directors member of BioClassifier, L.L.C. and C.M.P. has filed patents on the PAM50 subtyping assay. The other authors declare no competing interests.

The mammary gland consists of cells with gene expression patterns reflecting their cellular origins, function, and spatiotemporal context. However, knowledge of developmental kinetics and mechanisms of lineage specification is lacking. We address this significant knowledge gap by generating a single-cell transcriptome atlas encompassing embryonic, postnatal, and adult mouse mammary development. From these data, we map the chronology of transcriptionally and epigenetically distinct cell states and distinguish fetal mammary stem cells (fMaSCs) from their precursors and progeny. fMaSCs show balanced co-expression of factors associated with discrete adult lineages and a metabolic gene signature that subsides during maturation but reemerges in some human breast cancers and metastases. These data provide a useful resource for illuminating mammary cell heterogeneity, the kinetics of differentiation, and developmental correlates of tumorigenesis., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

RNA sequencing (RNA-seq) detects estrogen receptor alpha gene (ESR1) fusion transcripts in estrogen receptor-positive (ER + ) breast cancer, but their role in disease pathogenesis remains unclear. We examined multiple ESR1 fusions and found that two, both identified in advanced endocrine treatment-resistant disease, encoded stable and functional fusion proteins. In both examples, ESR1-e6>YAP1 and ESR1-e6>PCDH11X, ESR1 exons 1-6 were fused in frame to C-terminal sequences from the partner gene. Functional properties include estrogen-independent growth, constitutive expression of ER target genes, and anti-estrogen resistance. Both fusions activate a metastasis-associated transcriptional program, induce cellular motility, and promote the development of lung metastasis. ESR1-e6>YAP1- and ESR1-e6>PCDH11X-induced growth remained sensitive to a CDK4/6 inhibitor, and a patient-derived xenograft (PDX) naturally expressing the ESR1-e6>YAP1 fusion was also responsive. Transcriptionally active ESR1 fusions therefore trigger both endocrine therapy resistance and metastatic progression, explaining the association with fatal disease progression, although CDK4/6 inhibitor treatment is predicted to be effective., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Buparlisib is an oral pan-class I phosphotidyinositol-3-kinase (PI3K) inhibitor. The present phase I study evaluated the safety, pharmacokinetics, and efficacy of buparlisib with capecitabine in patients with metastatic breast cancer., Patients and Methods: Patients received buparlisib once daily (range, 50 to 100 mg) for 3 weeks with capecitabine twice daily (range, 1000 to 1250 mg/m 2 ) for 2 weeks with a 1-week break. Dose escalation used a traditional "3 + 3" design with standard definitions of dose-limiting toxicity (DLT) and maximum tolerated dose., Results: Of the 25 patients enrolled, 23 were evaluable for DLT and 17 were evaluable for response. The maximum tolerated dose of the combination was buparlisib 100 mg daily and capecitabine 1000 mg/m 2 twice daily. DLTs included grade 3 hyperglycemia and grade 3 confusion. The most common grade 3 toxicities were diarrhea and elevation of aspartate aminotransferase and alanine transaminase. One patient exhibited a complete response to treatment and four had a confirmed partial response. In cohorts 3 and 4, in which the buparlisib dose remained constant but the capecitabine dose was increased, significant increases in the buparlisib plasma concentration were noted., Conclusion: The combination of buparlisib with capecitabine in patients with metastatic breast cancer was generally well-tolerated, with several patients demonstrating prolonged responses. Unexpectedly low rates of PIK3CA mutations (3 of 17) were seen, and only 2 of 7 tumors with subtyping were luminal, making exploration of these putative predictive markers impossible. Further study of the combination is not unreasonable, with expanded pharmacokinetics and sequencing analysis to better elucidate potential drug-drug interactions and more accurate predictive biomarkers of response., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Cooperativity between WNT and FGF signaling is well documented in embryonic development and cancer progression, but the molecular mechanisms underlying this cross-talk remain elusive. In this study, we interrogated the dynamics of RNA levels, ribosome occupancy, and protein expression as a function of inducible FGF signaling in mouse mammary glands with constitutive WNT hyperactivation. Multiomics correlation analysis revealed a substantial discrepancy between RNA and ribosome occupancy levels versus protein levels. However, this discrepancy decreased as cells became premalignant and dynamically responded to FGF signaling, implicating the importance of stringent gene regulation in nontransformed cells. Analysis of individual genes demonstrated that acute FGF hyperactivation increased translation of many stem cell self-renewal regulators, including WNT signaling components, and decreased translation of genes regulating cellular senescence. WNT pathway components translationally upregulated by FGF signaling had long and structured 5' UTRs with a high frequency of polypurine sequences, several of which harbored (CGG) 4 motifs that can fold into either stable G-quadruplexes or other stable secondary structures. The FGF-mediated increase in translation of WNT pathway components was compromised by silvestrol, an inhibitor of EIF4A that clamps EIF4A to polypurine sequences to block 43S scanning and inhibits its RNA-unwinding activity important for translation initiation. Moreover, silvestrol treatment significantly delayed FGF-WNT-driven tumorigenesis. Taken together, these results suggest that FGF signaling selectively enhances translation of structured mRNAs, particularly WNT signaling components, and highlight their vulnerability to inhibitors that target the RNA helicase EIF4A. Significance: The RNA helicase EIF4A may serve as a therapeutic target for breast cancers that require FGF and WNT signaling. Cancer Res; 78(15); 4229-40. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Mounting evidence supports a role for the immune system in breast cancer outcomes. The ability to distinguish highly immunogenic tumors susceptible to anti-tumor immunity from weakly immunogenic or inherently immune-resistant tumors would guide development of therapeutic strategies in breast cancer. Genomic, transcriptomic and clinical data from The Cancer Genome Atlas (TCGA) and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) breast cancer cohorts were used to examine statistical associations between tumor mutational burden (TMB) and the survival of patients whose tumors were assigned to previously-described prognostic immune subclasses reflecting favorable, weak or poor immune-infiltrate dispositions (FID, WID or PID, respectively). Tumor immune subclasses were associated with survival in patients with high TMB (TMB-Hi, P  < 0.001) but not in those with low TMB (TMB-Lo, P  = 0.44). This statistical relationship was confirmed in the METABRIC cohort (TMB-Hi, P  = 0.047; TMB-Lo, P  = 0.39), and also found to hold true in the more-indolent Luminal A tumor subtype (TMB-Hi, P  = 0.011; TMB-Lo, P  = 0.91). In TMB-Hi tumors, the FID subclass was associated with prolonged survival independent of tumor stage, molecular subtype, age and treatment. Copy number analysis revealed the reproducible, preferential amplification of chromosome 1q immune-regulatory genes in the PID immune subclass. These findings demonstrate a previously unappreciated role for TMB as a determinant of immune-mediated survival of breast cancer patients and identify candidate immune-regulatory mechanisms associated with immunologically cold tumors. Immune subtyping of breast cancers may offer opportunities for therapeutic stratification.

Inactivation of the von Hippel-Lindau (VHL) E3 ubiquitin ligase protein is a hallmark of clear cell renal cell carcinoma (ccRCC). Identifying how pathways affected by VHL loss contribute to ccRCC remains challenging. We used a genome-wide in vitro expression strategy to identify proteins that bind VHL when hydroxylated. Zinc fingers and homeoboxes 2 (ZHX2) was found as a VHL target, and its hydroxylation allowed VHL to regulate its protein stability. Tumor cells from ccRCC patients with VHL loss-of-function mutations usually had increased abundance and nuclear localization of ZHX2. Functionally, depletion of ZHX2 inhibited VHL-deficient ccRCC cell growth in vitro and in vivo. Mechanistically, integrated chromatin immunoprecipitation sequencing and microarray analysis showed that ZHX2 promoted nuclear factor κB activation. These studies reveal ZHX2 as a potential therapeutic target for ccRCC., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The 2013 update of the American Society of Clinical Oncology-College of American Pathologists (ASCO-CAP) human epidermal growth factor receptor 2 (HER2) testing guidelines recommend using an alternative chromosome 17 probe assay to resolve HER2 results determined to be equivocal by immunohistochemistry (IHC) or fluorescence in-situ hybridization (FISH). However, it is unclear if cases considered HER2-positive (HER2 + ) by the alternative probe method are similar to those classified as HER2 + by traditional IHC and FISH criteria and benefit the same from HER2-targeted therapies. We studied the clinical and pathologic features of all 31 breast cancers classified as HER2 + by the alternative probe method at our institution since 2013 and determined their PAM50 intrinsic molecular subtypes. For comparison, we analyzed 19 consecutive cases that were classified as HER2 + by traditional FISH criteria during the same time period. Thirty (97%) cancers in the alternative probe cohort were estrogen receptor (ER)-positive (ER + ), while only 9/19 (47%) of traditional HER2 controls were ER + (p = 0.0002). Sufficient tissue for intrinsic subtype analysis was available for 20/31 cancers in the alternative probe cohort and 9/19 in the traditional HER2 + group. None (0%) of the 20 alternative probe-positive cases were of the HER2-enriched intrinsic subtype, while 8/9 (89%) of those HER2 + by traditional FISH criteria were HER2-enriched (p = 0.0001). These findings suggest that breast cancers classified as HER2 + only by the alternative probe method are biologically distinct from those classified as HER2 + by traditional criteria, and raises questions as to whether or not they derive the same benefit from HER2-targeted therapies.

Mutations in tumor suppressor TP53 have been inconsistently linked to breast cancer risk factors and survival. Immunohistochemistry (IHC) staining, a primary clinical means of TP53 mutation determination, only detects mutations that facilitate protein accumulation (e.g., missense mutations). RNA-based pathway methods capture functional status and may aid in understanding the role of TP53 function in racial disparities of breast cancer. TP53 status was assessed among invasive breast cancer cases from the Carolina Breast Cancer Study (CBCS) (2008-2013) using IHC and an established RNA-based TP53 signature (CBCS and The Cancer Genome Atlas (TCGA)). Frequency of TP53 status (IHC, RNA-based) was estimated in association with tumor characteristics, PAM50 intrinsic subtype, age, and race using relative frequency differences (RFDs) and 95% confidence intervals (95% CI) as the measure of association. Approximately 60% of basal-like tumors were TP53 protein positive (IHC), while nearly 100% were TP53 mutant-like (RNA). Luminal A tumors had low frequency of TP53 positivity (IHC: 7.9%) and mutant-like status (RNA: 1.7%). Mutant-like TP53 (RNA) was strongly associated with age ≤50 years, high tumor grade, advanced stage of disease, large tumor size, and basal-like and HER2 intrinsic subtypes. Black race was strongly associated with TP53 mutant-like status (RNA ) (RFD: 24.8%, 95% CI: 20.5, 29.0) even after adjusting for age, grade, stage (RFD: 11.3%; 95% CI: 7.6, 15.0). Associations were attenuated and non-significant when measured by IHC. IHC-based TP53 status is an insensitive measurement of TP53 functional status. RNA-based methods suggest a role for TP53 in tumor prognostic features and racial disparities., Competing Interests: Charles M. Perou is an equity stock holder of BioClassifier LLC and University Genomics and filed a patent on the PAM50 intrinsic breast cancer subtyping assay.

Breast cancers detected after a negative breast screening examination and prior to the next screening are referred to as interval cancers. These cancers generally have poor clinical characteristics compared with screen-detected cancers, but associations between interval cancer and genomic cancer characteristics are not well understood. Mammographically screened women diagnosed with primary invasive breast cancer from 1993 to 2013 ( n = 370) were identified by linking the Carolina Breast Cancer Study and the Carolina Mammography Registry. Among women with a registry-identified screening mammogram 0 to 24 months before diagnosis, cancers were classified as screen-detected ( N = 165) or interval-detected ( N = 205). Using logistic regression, we examined the association of mode of detection with cancer characteristics (clinical, IHC, and genomic), overall, and in analyses stratified on mammographic density and race. Interval cancer was associated with large tumors [>2 cm; OR, 2.3; 95% confidence interval (CI), 1.5-3.7], positive nodal status (OR, 1.8; 95% CI, 1.1-2.8), and triple-negative subtype (OR, 2.5; 95% CI, 1.1-5.5). Interval cancers were more likely to have non-Luminal A subtype (OR, 2.9; 95% CI, 1.5-5.7), whereas screen-detected cancers tended to be more indolent (96% had low risk of recurrence genomic scores; 71% were PAM50 Luminal A). When stratifying by mammographic density and race, associations between interval detection and poor prognostic features were similar by race and density status. Strong associations between interval cancers and poor-prognosis genomic features (non-Luminal A subtype and high risk of recurrence score) suggest that aggressive tumor biology is an important contributor to interval cancer rates. Cancer Prev Res; 11(6); 327-36. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Gene expression profiling has had a considerable impact on our understanding ofbreastcancer biology. Duringthelast decade, 4 intrinsic molecular subtypes of breast cancer (Luminal A, Luminal B, HER2-enriched [HER2-E] and Basal-like) have been identified and intensively studied. In this article, we review and discuss the clinical implications of the 2 non-luminal subtypes (i.e. HER2-E and Basal-like) identified within hormone receptor (HR)-positive disease. After reviewing 32 studies for a total of 13,091 samples, ∼8% and ∼ 15% of early and metastatic HR+/HER2-negative breast cancer, respectively, were found to be non-luminal. Clinically, HR+/HER2-negative/non-luminal subtypes have been associated with estrogen independence, chemo-sensitivity, resistance to CDK4/6 inhibition and poor outcome. Interestingly, EGFR/HER2 tyrosine kinase inhibition might be of value in the HR+/HER2-negative/HER2-E subtype. Finally, the HER2-E subtype within HR+/HER2 + disease represents ∼ 30% and has been associated with anti-HER2 sensitivity, chemo-sensitivity and resistance to CDK4/6 inhibition. In the upcoming years, retrospective and prospective clinical trials evaluating both biomarkers should lead to improvements in patient outcomes., (Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Germline mutations in BRCA1/2 predispose individuals to breast cancer (termed germline-mutated BRCA1/2 breast cancer, gBRCA-BC) by impairing homologous recombination (HR) and causing genomic instability. HR also repairs DNA lesions caused by platinum agents and PARP inhibitors. Triple-negative breast cancers (TNBCs) harbor subpopulations with BRCA1/2 mutations, hypothesized to be especially platinum-sensitive. Cancers in putative 'BRCAness' subgroups-tumors with BRCA1 methylation; low levels of BRCA1 mRNA (BRCA1 mRNA-low); or mutational signatures for HR deficiency and those with basal phenotypes-may also be sensitive to platinum. We assessed the efficacy of carboplatin and another mechanistically distinct therapy, docetaxel, in a phase 3 trial in subjects with unselected advanced TNBC. A prespecified protocol enabled biomarker-treatment interaction analyses in gBRCA-BC and BRCAness subgroups. The primary endpoint was objective response rate (ORR). In the unselected population (376 subjects; 188 carboplatin, 188 docetaxel), carboplatin was not more active than docetaxel (ORR, 31.4% versus 34.0%, respectively; P = 0.66). In contrast, in subjects with gBRCA-BC, carboplatin had double the ORR of docetaxel (68% versus 33%, respectively; biomarker, treatment interaction P = 0.01). Such benefit was not observed for subjects with BRCA1 methylation, BRCA1 mRNA-low tumors or a high score in a Myriad HRD assay. Significant interaction between treatment and the basal-like subtype was driven by high docetaxel response in the nonbasal subgroup. We conclude that patients with advanced TNBC benefit from characterization of BRCA1/2 mutations, but not BRCA1 methylation or Myriad HRD analyses, to inform choices on platinum-based chemotherapy. Additionally, gene expression analysis of basal-like cancers may also influence treatment selection.

Purpose: Triple-negative breast cancer (TNBC) requires the iden- tification of reliable predictors of response to neoadjuvant chemotherapy (NACT). For this purpose, we aimed to evaluate the performance of the TNBCtype-4 classifier in a cohort of patients with TNBC treated with neoadjuvant carboplatin and docetaxel (TCb). Methods: Patients with TNBC were accrued in a nonrandomized trial of neoadjuvant carboplatin AUC 6 and docetaxel 75 mg/m 2 for six cycles. Response was evaluated in terms of pathologic complete response (pCR, ypT0/is ypN0) and residual cancer burden by Symmans and colleagues. Lehmann's subtyping was performed using the TNBCtype online tool from RNAseq data, and germline sequencing of a panel of seven DNA damage repair genes was conducted. Results: Ninety-four out of the 121 patients enrolled in the trial had RNAseq available. The overall pCR rate was 44.7%. Lehmann subtype distribution was 34.0% BL1, 20.2% BL2, 23.4% M, 14.9% LAR, and 7.4% were classified as ER+. Response to NACT with TCb was significantly associated with Lehmann subtype ( P = 0.027), even in multivariate analysis including tumor size and nodal involvement, with BL1 patients achieving the highest pCR rate (65.6%), followed by BL2 (47.4%), M (36.4%), and LAR (21.4%). BL1 was associated with a significant younger age at diagnosis and higher ki67 values. Among our 10 germline mutation carriers, 30% were BL1, 40% were BL2, and 30% were M. Conclusions: TNBCtype-4 is associated with significantly different pCR rates for the different subtypes, with BL1 and LAR displaying the best and worse responses to NACT, respectively. Clin Cancer Res; 24(8); 1845-52. ©2018 AACR ., (©2018 American Association for Cancer Research.)

We systematically studied the association between somatic copy number aberration (SCNA), DNA methylation and gene expression using -omic data from The Cancer Genome Atlas (TCGA) on six cancer types: breast cancer, colon cancer, glioblastoma, leukemia, lower-grade glioma and prostate cancer. A major challenge for such integrated study is that the association between DNA methylation and gene expression is severely confounded by tumor purity and cell type composition, which are often unobserved and difficult to estimate. To overcome this challenge, we developed a method to remove confounding effects by calculating the principal components that span the space of the latent factors. Another intriguing findings of our study is that there could be both positive and negative associations between SCNA and DNA methylation, while the CpGs with negative/positive associations with SCNA are often located around CpG islands/ocean, respectively. A joint study of SCNA, DNA methylation, and gene expression suggest that SCNA often affect DNA methylation and gene expression independently.

This corrects the article DOI: 10.1038/nature25465.

Breast cancer metastasis remains a clinical challenge, even within a single patient across multiple sites of the disease. Genome-wide comparisons of both the DNA and gene expression of primary tumors and metastases in multiple patients could help elucidate the underlying mechanisms that cause breast cancer metastasis. To address this issue, we performed DNA exome and RNA sequencing of matched primary tumors and multiple metastases from 16 patients, totaling 83 distinct specimens. We identified tumor-specific drivers by integrating known protein-protein network information with RNA expression and somatic DNA alterations and found that genetic drivers were predominantly established in the primary tumor and maintained through metastatic spreading. In addition, our analyses revealed that most genetic drivers were DNA copy number changes, the TP53 mutation was a recurrent founding mutation regardless of subtype, and that multiclonal seeding of metastases was frequent and occurred in multiple subtypes. Genetic drivers unique to metastasis were identified as somatic mutations in the estrogen and androgen receptor genes. These results highlight the complexity of metastatic spreading, be it monoclonal or multiclonal, and suggest that most metastatic drivers are established in the primary tumor, despite the substantial heterogeneity seen in the metastases.

GATA3 is frequently mutated in breast cancer; these mutations are widely presumed to be loss-of function despite a dearth of information regarding their effect on disease course or their mechanistic impact on the breast cancer transcriptional network. Here, we address molecular and clinical features associated with GATA3 mutations. A novel classification scheme defines distinct clinical features for patients bearing breast tumors with mutations in the second GATA3 zinc-finger (ZnFn2). An engineered ZnFn2 mutant cell line by CRISPR-Cas9 reveals that mutation of one allele of the GATA3 second zinc finger (ZnFn2) leads to loss of binding and decreased expression at a subset of genes, including Progesterone Receptor. At other loci, associated with epithelial to mesenchymal transition, gain of binding correlates with increased gene expression. These results demonstrate that not all GATA3 mutations are equivalent and that ZnFn2 mutations impact breast cancer through gain and loss-of function.

Using a functional model of breast cancer heterogeneity, we previously showed that clonal sub-populations proficient at generating circulating tumour cells were not all equally capable of forming metastases at secondary sites. A combination of differential expression and focused in vitro and in vivo RNA interference screens revealed candidate drivers of metastasis that discriminated metastatic clones. Among these, asparagine synthetase expression in a patient's primary tumour was most strongly correlated with later metastatic relapse. Here we show that asparagine bioavailability strongly influences metastatic potential. Limiting asparagine by knockdown of asparagine synthetase, treatment with l-asparaginase, or dietary asparagine restriction reduces metastasis without affecting growth of the primary tumour, whereas increased dietary asparagine or enforced asparagine synthetase expression promotes metastatic progression. Altering asparagine availability in vitro strongly influences invasive potential, which is correlated with an effect on proteins that promote the epithelial-to-mesenchymal transition. This provides at least one potential mechanism for how the bioavailability of a single amino acid could regulate metastatic progression.

Background: Breast cancer subtype can be classified using standard clinical markers (estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2)), supplemented with additional markers. However, automated biomarker scoring and classification schemes have not been standardized. The aim of this study was to optimize tumor classification using automated methods in order to describe subtype frequency in the African American Breast Cancer Epidemiology and Risk (AMBER) consortium., Methods: Using immunohistochemistry (IHC), we quantified the expression of ER, PR, HER2, the proliferation marker Ki67, and two basal-like biomarkers, epidermal growth factor receptor (EGFR) and cytokeratin (CK)5/6, in 1381 invasive breast tumors from African American women. RNA-based (prediction analysis of microarray 50 (PAM50)) subtype, available for 574 (42%) cases, was used to optimize classification. Subtype frequency was calculated, and associations between subtype and tumor characteristics were estimated using logistic regression., Results: Relative to ER, PR and HER2 from medical records, central IHC staining and the addition of Ki67 or combined tumor grade improved accuracy for classifying PAM50-based luminal subtypes. Few triple negative cases (< 2%) lacked EGFR and CK5/6 expression, thereby providing little improvement in accuracy for identifying basal-like tumors. Relative to luminal A subtype, all other subtypes had higher combined grade and were larger, and ER-/HER2+ tumors were more often lymph node positive and late stage tumors. The frequency of basal-like tumors was 31%, exceeded only slightly by luminal A tumors (37%)., Conclusions: Our findings indicate that automated IHC-based classification produces tumor subtype frequencies approximating those from PAM50-based classification and highlight high frequency of basal-like and low frequency of luminal A breast cancer in a large study of African American women.

Background: African American breast cancer patients have lower frequency of hormone receptor-positive (HR+)/human epidermal growth factor receptor 2 (HER2)-negative disease and higher subtype-specific mortality. Racial differences in molecular subtype within clinically defined subgroups are not well understood., Methods: Using data and biospecimens from the population-based Carolina Breast Cancer Study (CBCS) Phase 3 (2008-2013), we classified 980 invasive breast cancers using RNA expression-based PAM50 subtype and recurrence (ROR) score that reflects proliferation and tumor size. Molecular subtypes (Luminal A, Luminal B, HER2-enriched, and Basal-like) and ROR scores (high vs low/medium) were compared by race (blacks vs whites) and age (≤50 years vs > 50 years) using chi-square tests and analysis of variance tests., Results: Black women of all ages had a statistically significantly lower frequency of Luminal A breast cancer (25.4% and 33.6% in blacks vs 42.8% and 52.1% in whites; younger and older, respectively). All other subtype frequencies were higher in black women (case-only odds ratio [OR] = 3.11, 95% confidence interval [CI] = 2.22 to 4.37, for Basal-like; OR = 1.45, 95% CI = 1.02 to 2.06, for Luminal B; OR = 2.04, 95% CI = 1.33 to 3.13, for HER2-enriched). Among clinically HR+/HER2- cases, Luminal A subtype was less common and ROR scores were statistically significantly higher among black women., Conclusions: Multigene assays highlight racial disparities in tumor subtype distribution that persist even in clinically defined subgroups. Differences in tumor biology (eg, HER2-enriched status) may be targetable to reduce disparities among clinically ER+/HER2- cases., (© The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com)

Background: Invasive lobular breast tumors display unique reproductive risk factor profiles. Lobular tumors are predominantly Luminal A subtype, and it is unclear whether reported risk factor associations are independent of molecular subtype., Methods: Polytomous logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (95% CIs) for the associations between risk factors and histologic subtype [ductal (n = 2,856), lobular (n = 326), and mixed ductal-lobular (n = 473)] in the Carolina Breast Cancer Study (1993-2013). Three-marker immunohistochemical clinical subtypes were defined as Luminal A (ER+ or PR+/HER2-), Luminal B (ER+ or PR+/HER2+), Triple Negative (ER-/PR-/HER2-), and HER2+ (ER-/PR-/HER2+)., Results: In case-case analyses compared to ductal, lobular tumors were significantly associated with lactation duration > 12 months [OR 1.86, 95% CI (1.33-2.60)], age at first birth ≥ 26 years [OR: 1.35, 95% CI: (1.03-1.78)], and current oral contraceptive use [OR: 1.86, 95% CI: (1.08-3.20)]. Differences in risk factor associations between ductal and lobular tumors persisted after restricting to Luminal A subtype., Conclusions: Lobular tumors were associated with older age at first birth, increased lactation duration, and current oral contraceptive use. Etiologic heterogeneity by histology persisted after restricting to Luminal A subtype, suggesting both tumor histology and intrinsic subtype play integral parts in breast cancer risk.

Purpose: Gene signatures and Ki67 stratify the same breast tumor into opposing good/poor prognosis groups in approximately 20% of patients. Given this discrepancy, we hypothesized that the combination of a clinically relevant signature and IHC markers may provide more prognostic information than either classifier alone. Experimental Design: We assessed Ki67 alone or combined with ER, PR and HER2 (forming IHC subtypes), and the research versions of the Genomic Grade Index, 70-gene, cell-cycle score, recurrence score (RS), and PAM50 signatures on matching TMA/whole tumor sections and microarray data in two Swedish breast cancer cohorts of 379 and 209 patients, with median follow-up of 12.4 and 12.5 years, respectively. First, we fit Cox proportional hazards models and used the change in likelihood ratio (Δ LR) to determine the additional prognostic information provided by signatures beyond that of (i) Ki67 and (ii) IHC subtypes. Second and uniquely, we then assessed whether signatures could compete well with pathology-based IHC classifiers by calculating the additional prognostic information of Ki67/IHC subtypes beyond signatures. Results: In cohort 1, only RS and PAM50 provided additional prognostic information beyond Ki67 and IHC subtypes (Δ LR-χ 2 Ki67: RS = 12.8, PAM50 = 20.7, IHC subtypes: RS = 12.9, PAM50 = 11.7). Conversely, IHC subtypes added prognostic information beyond all signatures except PAM50. Similar results were observed in cohort 2. Conclusions: RS and PAM50 provided more prognostic information than the IHC subtypes in all breast cancer patients; however, the IHC subtypes did not add any prognostic information to PAM50. Clin Cancer Res; 23(24); 7512-20. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Background: We examined racial differences in the expression of eight genes and their associations with risk of recurrence among 478 white and 495 black women who participated in the Carolina Breast Cancer Study Phase 3., Methods: Breast tumor samples were analyzed for PAM50 subtype and for eight genes previously found to be differentially expressed by race and associated with breast cancer survival: ACOX2, MUC1, FAM177A1, GSTT2, PSPH, PSPHL, SQLE, and TYMS. The expression of these genes according to race was assessed using linear regression and each gene was evaluated in association with recurrence using Cox regression., Results: Compared to white women, black women had lower expression of MUC1, a suspected good prognosis gene, and higher expression of GSTT2, PSPHL, SQLE, and TYMS, suspected poor prognosis genes, after adjustment for age and PAM50 subtype. High expression (greater than median versus less than or equal to median) of FAM177A1 and PSPH was associated with a 63% increase (hazard ratio (HR) = 1.63, 95% confidence interval (CI) = 1.09-2.46) and 76% increase (HR = 1.76, 95% CI = 1.15-2.68), respectively, in risk of recurrence after adjustment for age, race, PAM50 subtype, and ROR-PT score. Log 2 -transformed SQLE expression was associated with a 20% increase (HR = 1.20, 95% CI = 1.03-1.41) in recurrence risk after adjustment. A continuous multi-gene score comprised of eight genes was also associated with increased risk of recurrence among all women (HR = 1.11, 95% CI = 1.04-1.19) and among white (HR = 1.14, 95% CI = 1.03-1.27) and black (HR = 1.11, 95% CI = 1.02-1.20) women., Conclusions: Racial differences in gene expression may contribute to the survival disparity observed between black and white women diagnosed with breast cancer.

Importance: African Americans have the highest breast cancer mortality rate. Although racial difference in the distribution of intrinsic subtypes of breast cancer is known, it is unclear if there are other inherent genomic differences that contribute to the survival disparities., Objectives: To investigate racial differences in breast cancer molecular features and survival and to estimate the heritability of breast cancer subtypes., Design, Setting, and Participants: Among a convenience cohort of patients with invasive breast cancer, breast tumor and matched normal tissue sample data (as of September 18, 2015) were obtained from The Cancer Genome Atlas., Main Outcomes and Measures: Breast cancer–free interval, tumor molecular features, and genetic variants., Results: Participants were 930 patients with breast cancer, including 154 black patients of African ancestry (mean [SD] age at diagnosis, 55.66 [13.01] years; 98.1% [n = 151] female) and 776 white patients of European ancestry (mean [SD] age at diagnosis, 59.51 [13.11] years; 99.0% [n = 768] female). Compared with white patients, black patients had a worse breast cancer-free interval (hazard ratio, HR=1.67; 95% CI, 1.02-2.74; P = .043). They had a higher likelihood of basal-like (odds ratio, 3.80; 95% CI, 2.46-5.87; P < .001) and human epidermal growth factor receptor 2 (ERBB2 [formerly HER2])–enriched (odds ratio, 2.22; 95% CI, 1.10-4.47; P = .027) breast cancer subtypes, with the Luminal A subtype as the reference. Blacks had more TP53 mutations and fewer PIK3CA mutations than whites. While most molecular differences were eliminated after adjusting for intrinsic subtype, the study found 16 DNA methylation probes, 4 DNA copy number segments, 1 protein, and 142 genes that were differentially expressed, with the gene-based signature having an excellent capacity for distinguishing breast tumors from black vs white patients (cross-validation C index, 0.878). Using germline genotypes, the heritability of breast cancer subtypes (basal vs nonbasal) was estimated to be 0.436 (P = 1.5 × 10−14). The estrogen receptor–positive polygenic risk score built from 89 known susceptibility variants was higher in blacks than in whites (difference, 0.24; P = 2.3 × 10−5), while the estrogen receptor–negative polygenic risk score was much higher in blacks than in whites (difference, 0.48; P = 2.8 × 10−11)., Conclusions and Relevance: On the molecular level, after adjusting for intrinsic subtype frequency differences, this study found a modest number of genomic differences but a significant clinical survival outcome difference between blacks and whites in The Cancer Genome Atlas data set. Moreover, more than 40% of breast cancer subtype frequency differences could be explained by genetic variants. These data could form the basis for the development of molecular targeted therapies to improve clinical outcomes for the specific subtypes of breast cancers that disproportionately affect black women. Findings also indicate that personalized risk assessment and optimal treatment could reduce deaths from aggressive breast cancers for black women.

Purpose: Transcriptional pathway activity and the molecular subtypes of breast cancer metastases have been shown to significantly influence patient postrelapse survival. Here, we further determine the relevance of clinically employed gene signatures in the advanced breast cancer (ABC) setting. Experimental Design: Sufficient RNA for expression profiling was obtained from distant metastatic or inoperable loco-regional relapse tissue by fine-needle aspiration from 109 patients of the Swedish TEX clinical trial. Gene signatures (GGI, 70 gene, recurrence score, cell-cycle score, risk of recurrence score, and PAM50) were applied to all metastases, and their relationship to long- (5-year) and short-term (1.5-year) postrelapse survival at all and locoregional lymph nodes ( n = 40) versus other metastatic sites ( n = 69) combined was assessed using Kaplan-Meier and/or multivariate Cox regression analyses. Results: The majority of metastases were classified into intermediate or high-risk groups by all signatures, and a significant association was found between metastatic signature subgroups and primary tumor estrogen receptor status and histologic grade ( P < 0.05). When considering all sites of metastasis, only PAM50 was statistically significant in Kaplan-Meier analysis (Log-rank P = 0.008 and 0.008 for long- and short-term postrelapse breast cancer-specific survival, respectively). This significance remained in both uni- and multivariate models when restricting analyses to lymph node metastases only, and a similar trend was observed in other metastatic sites combined, but did not reach formal significance. Conclusions: Our findings are the first to demonstrate that the PAM50 signature can provide prognostic information from the lymph node metastases of ABC patients. Clin Cancer Res; 23(23); 7225-31. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Cellular heterogeneity is a common feature in breast cancer, yet an understanding of the coexistence and regulation of various tumor cell subpopulations remains a significant challenge in cancer biology. In the current study, we approached tumor cell heterogeneity from the perspective of Wnt pathway biology to address how different modes of Wnt signaling shape the behaviors of diverse cell populations within a heterogeneous tumor landscape. Using a syngeneic TP53-null mouse model of breast cancer, we identified distinctions in the topology of canonical Wnt β-catenin-dependent signaling activity and non-canonical β-catenin-independent Ror2-mediated Wnt signaling across subtypes and within tumor cell subpopulations in vivo. We further discovered an antagonistic role for Ror2 in regulating canonical Wnt/β-catenin activity in vivo, where lentiviral shRNA depletion of Ror2 expression augmented canonical Wnt/β-catenin signaling activity across multiple basal-like models. Depletion of Ror2 expression yielded distinct phenotypic outcomes and divergent alterations in gene expression programs among different tumors, despite all sharing basal-like features. Notably, we uncovered cell state plasticity and adhesion dynamics regulated by Ror2, which influenced Ras Homology Family Member A (RhoA) and Rho-Associated Coiled-Coil Kinase 1 (ROCK1) activity downstream of Dishevelled-2 (Dvl2). Collectively, these studies illustrate the integration and collaboration of Wnt pathways in basal-like breast cancer, where Ror2 provides a spatiotemporal function to regulate the balance of Wnt signaling and cellular heterogeneity during tumor progression.

Claudin-low breast cancer is an aggressive subtype that confers poor prognosis and is found largely within the clinical triple-negative group of breast cancer patients. Here, we have shown that intrinsic and immune cell gene signatures distinguish the claudin-low subtype clinically as well as in mouse models of other breast cancer subtypes. Despite adaptive immune cell infiltration in claudin-low tumors, treatment with immune checkpoint inhibitory antibodies against cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and programmed death receptor 1 (PD-1) were ineffective in controlling tumor growth. CD4+FoxP3+ Tregs represented a large proportion of the tumor-infiltrating lymphocytes (TILs) in claudin-low tumors, and Tregs isolated from tumor-bearing mice were able to suppress effector T cell responses. Tregs in the tumor microenvironment highly expressed PD-1 and were recruited partly through tumor generation of the chemokine CXCL12. Antitumor efficacy required stringent Treg depletion combined with checkpoint inhibition; delays in tumor growth were not observed using therapies that modestly diminished the number of Tregs in the tumor microenvironment. This study provides evidence that the recruitment of Tregs to the tumor microenvironment inhibits an effective antitumor immune response and highlights early Treg recruitment as a possible mechanism for the lack of response to immune checkpoint blockade antibodies in specific subtypes of cancer that are heavily infiltrated with adaptive immune cells.

The vast majority of cancer-related deaths are due to metastatic disease, whereby primary tumor cells disseminate and colonize distal sites within the body. Triple negative breast cancer typically displays aberrant Wnt signaling, lacks effective targeted therapies, and compared with other breast cancer subtypes, is more likely to recur and metastasize. We developed a Wnt-driven lung metastasis model of triple negative breast cancer (metM-Wnt lung ) through serial passaging of our previously described, nonmetastatic, claudin-low M-Wnt cell line. metM-Wnt lung cells displayed characteristics of epithelial-to-mesenchymal transition (e.g., increased invasiveness) with some re-epithealization (e.g., increased adhesion, tight colony formation, increased E-cadherin expression, and decreased Vimentin and Fibronectin expression). When orthotopically transplanted into syngeneic mice, metM-Wnt lung cells readily formed tumors and metastasized in vivo, and tumor growth and metastasis were enhanced in obese mice compared with non-obese mice. Gene expression analysis revealed several genes and pathways altered in metM-Wnt lung cells compared with M-Wnt cells, including multiple genes associated with epithelial-to-mesenchymal transition, energy metabolism and inflammation. Moreover, obesity caused significant transcriptomic changes, especially in metabolic pathways. Metabolic flux analyses showed greater metabolic plasticity, with heightened mitochondrial and glycolytic energetics in metM-Wnt lung cells relative to M-Wnt cells. Similar metabolic profiles were found in a second triple negative breast cancer progression series, M6 and M6C cells. These findings suggest that metabolic reprogramming is a feature of metastatic potential in triple negative breast cancer. Thus, targeting metastases-associated metabolic perturbations may represent a novel strategy for reducing the burden of metastatic triple negative breast cancer, particularly in obese women.

Background: Compelling evidence shows that progestins regulate breast cancer growth. Using preclinical models, we demonstrated that antiprogestins are inhibitory when the level of progesterone receptor isoform A (PR-A) is higher than that of isoform B (PR-B) and that they might stimulate growth when PR-B is predominant. The aims of this study were to investigate ex vivo responses to mifepristone (MFP) in breast carcinomas with different PR isoform ratios and to examine their clinical and molecular characteristics., Methods: We performed human breast cancer tissue culture assays (n = 36) to evaluate the effect of MFP on cell proliferation. PR isoform expression was determined by immunoblotting (n = 282). Tumors were categorized as PRA-H (PR-A/PR-B ≥ 1.2) or PRB-H (PR-A/PR-B ≤ 0.83). RNA was extracted for Ribo-Zero-Seq sequencing to evaluate differentially expressed genes. Subtypes and risk scores were predicted using the PAM50 gene set, the data analyzed using The Cancer Genome Atlas RNA-seq gene analysis and other publicly available gene expression data. Tissue microarrays were performed using paraffin-embedded tissues (PRA-H n = 53, PRB-H n = 24), and protein expression analyzed by immunohistochemistry. All statistical tests were two-sided., Results: One hundred sixteen out of 222 (52.3%) PR+ tumors were PRA-H, and 64 (28.8%) PRB-H. Cell proliferation was inhibited by MFP in 19 of 19 tissue cultures from PRA-H tumors. A total of 139 transcripts related to proliferative pathways were differentially expressed in nine PRA-H and seven PRB-H tumors. PRB-H and PRA-H tumors were either luminal B or A phenotypes, respectively ( P = .03). PRB-H cases were associated with shorter relapse-free survival (hazard ratio [HR] = 2.70, 95% confidence interval [CI] = 1.71 to 6.20, P = .02) and distant metastasis-free survival (HR = 4.17, 95% CI = 2.18 to 7.97, P <  .001). PRB-H tumors showed increased tumor size ( P <  .001), Ki-67 levels ( P <  .001), human epidermal growth factor receptor 2 expression ( P =  .04), high grades ( P =  .03), and decreased total PR ( P =  .004) compared with PRA-H tumors. MUC-2 ( P <  .001) and KRT6A ( P =  .02) were also overexpressed in PRB-H tumors., Conclusion: The PRA/PRB ratio is a prognostic and predictive factor for antiprogestin responsiveness in breast cancer., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Purpose: Hormone receptor-positive (HR + ) breast cancer is clinically and biologically heterogeneous, and subgroups with different prognostic and treatment sensitivities need to be identified. Experimental Design: Research-based PAM50 subtyping and expression of additional genes was performed on 63 patients with HR + /HER2 - disease randomly assigned to neoadjuvant multiagent chemotherapy versus endocrine therapy in a phase II trial. The biology associated with treatment response was used to derive a PAM50-based chemoendocrine score (CES). CES's predictive ability was evaluated in 4 independent neoadjuvant data sets ( n = 675) and 4 adjuvant data sets ( n = 1,505). The association of CES, intrinsic biology, and PAM50 risk of relapse (ROR) was explored across 6,007 tumors. Results: Most genes associated with endocrine sensitivity were also found associated with chemotherapy resistance. In the chemotherapy test/validation data sets, CES was independently associated with pathologic complete response (pCR), even after adjusting for intrinsic subtype. pCR rates of the CES endocrine-sensitive (CES-E), uncertain (CES-U), and chemotherapy-sensitive (CES-C) groups in both data sets combined were 25%, 11%, and 2%, respectively. In the endocrine test/validation data sets, CES was independently associated with response. Compared with ROR, >90% of ROR-low and ROR-high tumors were identified as CES-E and CES-C, respectively; however, each CES group represented >25% of ROR-intermediate disease. In terms of survival outcome, CES-C was associated with poor relapse-free survival in patients with ROR-intermediate disease treated with either adjuvant endocrine therapy only or no adjuvant systemic therapy, but not in patients treated with (neo)adjuvant chemotherapy. Conclusions: CES is a genomic signature capable of estimating chemoendocrine sensitivity in HR + breast cancer beyond intrinsic subtype and risk of relapse. Clin Cancer Res; 23(12); 3035-44. ©2016 AACR ., (©2016 American Association for Cancer Research.)

Immune checkpoint inhibitors have emerged as a potent new class of anticancer therapy. They have changed the treatment landscape for a range of tumors, particularly those with a high mutational load. To date, however, modest results have been observed in breast cancer, where tumors are rarely hypermutated. Because BRCA1 -associated tumors frequently exhibit a triple-negative phenotype with extensive lymphocyte infiltration, we explored their mutational load, immune profile, and response to checkpoint inhibition in a Brca1 -deficient tumor model. BRCA1 -mutated triple-negative breast cancers (TNBCs) exhibited an increased somatic mutational load and greater numbers of tumor-infiltrating lymphocytes, with increased expression of immunomodulatory genes including PDCD1 ( PD-1 ) and CTLA4 , when compared to TNBCs from BRCA1 -wild-type patients. Cisplatin treatment combined with dual anti-programmed death-1 and anti-cytotoxic T lymphocyte-associated antigen 4 therapy substantially augmented antitumor immunity in Brca1 -deficient mice, resulting in an avid systemic and intratumoral immune response. This response involved enhanced dendritic cell activation, reduced suppressive FOXP3 + regulatory T cells, and concomitant increase in the activation of tumor-infiltrating cytotoxic CD8 + and CD4 + T cells, characterized by the induction of polyfunctional cytokine-producing T cells. Dual (but not single) checkpoint blockade together with cisplatin profoundly attenuated the growth of Brca1 -deficient tumors in vivo and improved survival. These findings provide a rationale for clinical studies of combined immune checkpoint blockade in BRCA1 -associated TNBC., (Copyright © 2017, American Association for the Advancement of Science.)

Introduction: Molecular subtyping of lung adenocarcinoma (AD) and lung squamous cell carcinoma (SCC) reveal biologically diverse tumors that vary in their genomic and clinical attributes., Methods: Published immune cell signatures and several lung AD and SCC gene expression data sets, including The Cancer Genome Atlas, were used to examine immune response in relation to AD and SCC expression subtypes. Expression of immune cell populations and other immune related genes, including CD274 molecule gene (CD274) (programmed death ligand 1), was investigated in the tumor microenvironment relative to the expression subtypes of the AD (terminal respiratory unit, proximal proliferative, and proximal inflammatory) and SCC (primitive, classical, secretory, and basal) subtypes., Results: Lung AD and SCC expression subtypes demonstrated significant differences in tumor immune landscape. The proximal proliferative subtype of AD demonstrated low immune cell expression among ADs whereas the secretory subtype showed elevated immune cell expression among SCCs. Tumor expression subtype was a better predictor of immune cell expression than CD274 (programmed death ligand 1) in SCC tumors but was a comparable predictor in AD tumors. Nonsilent mutation burden was not correlated with immune cell expression across subtypes; however, major histocompatibility complex class II gene expression was highly correlated with immune cell expression. Increased immune and major histocompatibility complex II gene expression was associated with improved survival in the terminal respiratory unit and proximal inflammatory subtypes of AD and in the primitive subtype of SCC., Conclusions: Molecular expression subtypes of lung AD and SCC demonstrate key and reproducible differences in immune host response. Evaluation of tumor expression subtypes as potential biomarkers for immunotherapy should be investigated., (Copyright © 2017 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.)

Biological changes that occur during metastatic progression of breast cancer are still incompletely characterized. In this study, we compared intrinsic molecular subtypes and gene expression in 123 paired primary and metastatic tissues from breast cancer patients. Intrinsic subtype was identified using a PAM50 classifier and χ 2 tests determined the differences in variable distribution. The rate of subtype conversion was 0% in basal-like tumors, 23.1% in HER2-enriched (HER2-E) tumors, 30.0% in luminal B tumors, and 55.3% in luminal A tumors. In 40.2% of cases, luminal A tumors converted to luminal B tumors, whereas in 14.3% of cases luminal A and B tumors converted to HER2-E tumors. We identified 47 genes that were expressed differentially in metastatic versus primary disease. Metastatic tumors were enriched for proliferation-related and migration-related genes and diminished for luminal-related genes. Expression of proliferation-related genes were better at predicting overall survival in metastatic disease (OSmet) when analyzed in metastatic tissue rather than primary tissue. In contrast, a basal-like gene expression signature was better at predicting OSmet in primary disease compared with metastatic tissue. We observed correlations between time to tumor relapse and the magnitude of changes of proliferation, luminal B, or HER2-E signatures in metastatic versus primary disease. Although the intrinsic subtype was largely maintained during metastatic progression, luminal/HER2-negative tumors acquired a luminal B or HER2-E profile during metastatic progression, likely reflecting tumor evolution or acquisition of estrogen independence. Overall, our analysis revealed the value of stratifying gene expression by both cancer subtype and tissue type, providing clinicians more refined tools to evaluate prognosis and treatment. Cancer Res; 77(9); 2213-21. ©2017 AACR ., (©2017 American Association for Cancer Research.)

We previously demonstrated that altered activity of lysophosphatidic acid in murine mammary glands promotes tumorigenesis. We have now established and characterized a heterogeneous collection of mouse-derived syngeneic transplants (MDSTs) as preclinical platforms for the assessment of personalized pharmacological therapies. Detailed molecular and phenotypic analyses revealed that MDSTs are the most heterogeneous group of genetically engineered mouse models (GEMMs) of breast cancer yet observed. Response of MDSTs to trametinib, a mitogen-activated protein kinase (MAPK) kinase inhibitor, correlated with RAS/MAPK signaling activity, as expected from studies in xenografts and clinical trials providing validation of the utility of the model. Sensitivity of MDSTs to talazoparib, a poly(adenosine 5'-diphosphate-ribose) polymerase (PARP) inhibitor, was predicted by PARP1 protein levels and by a new PARP sensitivity predictor (PSP) score developed from integrated analysis of drug sensitivity data of human cell lines. PSP score-based classification of The Cancer Genome Atlas breast cancer suggested that a subset of patients with limited therapeutic options would be expected to benefit from PARP-targeted drugs. These results indicate that MDSTs are useful models for studies of targeted therapies, and propose novel potential biomarkers for identification of breast cancer patients likely to benefit from personalized pharmacological treatments.

Changes in the quantity of genetic material, known as somatic copy number alterations (CNAs), can drive tumorigenesis. Many methods exist for assessing CNAs using microarrays, but considerable technical issues limit current CNA calling based upon DNA sequencing. We present SynthEx, a novel tool for detecting CNAs from whole exome and genome sequencing. SynthEx utilizes a "synthetic-normal" strategy to overcome technical and financial issues. In terms of accuracy and precision, SynthEx is highly comparable to array-based methods and outperforms sequencing-based CNA detection tools. SynthEx robustly identifies CNAs using sequencing data without the additional costs associated with matched normal specimens.

Purpose: The PI3K/Akt signaling axis contributes to the dysregulation of many dominant features in breast cancer including cell proliferation, survival, metabolism, motility, and genomic instability. While multiple studies have demonstrated that basal-like or triple-negative breast tumors have uniformly high PI3K/Akt activity, genomic alterations that mediate dysregulation of this pathway in this subset of highly aggressive breast tumors remain to be determined., Methods: In this study, we present an integrated genomic analysis based on the use of a PI3K gene expression signature as a framework to analyze orthogonal genomic data from human breast tumors, including RNA expression, DNA copy number alterations, and protein expression. In combination with data from a genome-wide RNA-mediated interference screen in human breast cancer cell lines, we identified essential genetic drivers of PI3K/Akt signaling., Results: Our in silico analyses identified SOX4 amplification as a novel modulator of PI3K/Akt signaling in breast cancers and in vitro studies confirmed its role in regulating Akt phosphorylation., Conclusions: Taken together, these data establish a role for SOX4-mediated PI3K/Akt signaling in breast cancer and suggest that SOX4 may represent a novel therapeutic target and/or biomarker for current PI3K family therapies.

Genomic instability is a hallmark of breast cancer, contributes to tumor heterogeneity, and influences chemotherapy resistance. Although Gap 2 and mitotic checkpoints are thought to prevent genomic instability, the role of these checkpoints in breast cancer is poorly understood. Here, we assess the Gap 2 and mitotic checkpoint functions of 24 breast cancer and immortalized mammary epithelial cell lines representing four of the six intrinsic molecular subtypes of breast cancer. We found that patterns of cell cycle checkpoint deregulation were associated with the intrinsic molecular subtype of breast cancer cell lines. Specifically, the luminal B and basal-like cell lines harbored two molecularly distinct Gap 2/mitosis checkpoint defects (impairment of the decatenation Gap 2 checkpoint and the spindle assembly checkpoint, respectively). All subtypes of breast cancer cell lines examined displayed aberrant DNA synthesis/Gap 2/mitosis progression and the basal-like and claudin-low cell lines exhibited increased percentages of chromatid cohesion defects. Furthermore, a decatenation Gap 2 checkpoint gene expression signature identified in the cell line panel correlated with clinical outcomes in breast cancer patients, suggesting that breast tumors may also harbor defects in decatenation Gap 2 checkpoint function. Taken together, these data imply that pharmacological targeting of signaling pathways driving these phenotypes may lead to the development of novel personalized treatment strategies for the latter two subtypes which currently lack targeted therapeutic options because of their triple negative breast cancer status.

Recent advances in mass spectrometry (MS) have enabled extensive analysis of cancer proteomes. Here, we employed quantitative proteomics to profile protein expression across 24 breast cancer patient-derived xenograft (PDX) models. Integrated proteogenomic analysis shows positive correlation between expression measurements from transcriptomic and proteomic analyses; further, gene expression-based intrinsic subtypes are largely re-capitulated using non-stromal protein markers. Proteogenomic analysis also validates a number of predicted genomic targets in multiple receptor tyrosine kinases. However, several protein/phosphoprotein events such as overexpression of AKT proteins and ARAF, BRAF, HSP90AB1 phosphosites are not readily explainable by genomic analysis, suggesting that druggable translational and/or post-translational regulatory events may be uniquely diagnosed by MS. Drug treatment experiments targeting HER2 and components of the PI3K pathway supported proteogenomic response predictions in seven xenograft models. Our study demonstrates that MS-based proteomics can identify therapeutic targets and highlights the potential of PDX drug response evaluation to annotate MS-based pathway activities.