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XLSX file - 87K, Gene Expression Changes Comparing Control Rat Tumors and Rat Tumors Treated with Vorozole

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PDF file - 322K, Gene Expression Changes Comparing Control Breast Cancer and Breast Cancers Treated with an Aromatase Inhibitor in Humans (GSE 16391)

XLSX file - 244K, Gene Expression Changes Comparing Control Breast Cancers and Letrozole Treated Breast Cancer in Humans (GSE 5462)

XLSX file - 138K, Gene Expression Changes Comparing Control Breast Cancers and Letrozole Treated Breast Cancer in Humans (GSE 10281)

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PDF file - 556KB, Supplementary Figure 1a: Overview of pre-surgical studies: Fulvestrant (NEWEST), detailing study design (i) and samples (ii). S1b: Anastrozole (FAIMoS), detailing study design (i) and samples (ii). S1c: Summary of patient demographics and tumour characteristics for NEWEST and FAIMoS; Supplementary Figure 2ai: Spearman correlations between fulvestrant (F) and E-deprivation (ED) related and specific genes, selected proliferation-associated and E-regulated index genes in pre-treatment samples from NEWEST (n=22). Direct correlations are indicated in red and inverse correlations in green, with their strength reflected by colour intensity. S2aii: Spearman correlations between fulvestrant (F) and E-deprivation (ED) related and specific genes, selected proliferation-associated and E-regulated index genes in pre-treatment samples from FAIMoS (n=81). Direct correlations are indicated in red and inverse correlations in green, with their strength reflected by colour intensity. Supplementary Figure 2b: Summary of Spearman correlations between up-regulated (n=13) and down-regulated (n=28) genes which were fulvestrant-related (n=41), AURKA and ESR1, in pre-treatment in samples from (i) NEWEST and (ii) FAIMoS. Supplementary Figure 3a/b: mpact of ESR1 knockdown with an siRNA SMARTpool and four constituent oligonucleotides (a), on the mRNA expression of genes differentially down-regulated by fulvestrant (b). S3c: Impact of ESR1 knockdown with an siRNA SMARTpool and four constituent oligonucleotides (a), on the mRNA expression of genes differentially up-regulated by fulvestrant (c).

Purpose: To investigate potential associations between gene modules representing key biologic processes and response to aromatase inhibitors (AI) in estrogen receptor–positive (ER+) breast cancer.Patients and Methods: Paired gene expression and Ki67 protein expression were available from 69 postmenopausal women with ER+ early breast cancer, at baseline and 2 weeks post-anastrozole treatment, in the presurgical setting. Functional gene modules (n = 26) were retrieved from published studies and their module scores were computed before and after elimination of proliferation-associated genes (PAG). Ki67 and module scores were assessed at baseline and 2 weeks post-anastrozole. Unsupervised clustering was used to assess associations between modules and Ki67.Results: Proliferation-based modules were highly correlated with Ki67 expression both pretreatment and on-treatment. At baseline with and without PAGs, Ki67 expression was significantly inversely correlated with ERG, ESR1.2, SET, and PIK3CA modules. Modules measuring estrogen signaling strongly predicted antiproliferative response to therapy with and without PAGs. Baseline expression of insulin-like growth factor-1 (IGF-I) module predicted a poor change in Ki67-implicating genes within the module as involved in de novo resistance to AIs. High expression of Immune.2.STAT1 module pretreatment predicted poor antiproliferative response to therapy. A significant association between estrogen-regulated genes modules (ESR1, ESR1-2, SET, and ERG) was evident post AI.Conclusions: Multiple processes and pathways are affected by AI treatment in ER+ breast cancer. Modules closely associated with ESR1 expression were predictive of good antiproliferative response to AIs, but modules representing immune activity and IGF-I/MAPK were predictive of poor Ki67 response, supporting their therapeutic targeting in combination with AIs. Clin Cancer Res; 20(9); 2485–94. ©2014 AACR.

Spearman Rank Correlation Matrix (rho and p-value) of expression change in Gene Module Scores and Ki67, ∆log2Ki67 (log2(Ki67post) - log2(Ki67pre)). A: Including Proliferation-Associated Genes. B: Excluding Proliferation-Associated Genes.

Purpose: Although most patients with estrogen receptor α (ER)-positive breast cancer initially respond to endocrine therapy, many ultimately develop resistance to antiestrogens. However, mechanisms of antiestrogen resistance and biomarkers predictive of such resistance are underdeveloped.Experimental Design: We adapted four ER+ human breast cancer cell lines to grow in an estrogen-depleted medium. A gene signature of estrogen independence was developed by comparing expression profiles of long-term estrogen-deprived (LTED) cells to their parental counterparts. We evaluated the ability of the LTED signature to predict tumor response to neoadjuvant therapy with an aromatase inhibitor and disease outcome following adjuvant tamoxifen. We utilized Gene Set Analysis (GSA) of LTED cell gene expression profiles and a loss-of-function approach to identify pathways causally associated with resistance to endocrine therapy.Results: The LTED gene expression signature was predictive of high tumor cell proliferation following neoadjuvant therapy with anastrozole and letrozole, each in different patient cohorts. This signature was also predictive of poor recurrence-free survival in two studies of patients treated with adjuvant tamoxifen. Bioinformatic interrogation of expression profiles in LTED cells revealed a signature of MYC activation. The MYC activation signature and high MYC protein levels were both predictive of poor outcome following tamoxifen therapy. Finally, knockdown of MYC inhibited LTED cell growth.Conclusions: A gene expression signature derived from ER+ breast cancer cells with acquired hormone independence predicted tumor response to aromatase inhibitors and associated with clinical markers of resistance to tamoxifen. Activation of the MYC pathway was associated with this resistance. Clin Cancer Res; 17(7); 2024–34. ©2011 AACR.

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Purpose: The majority of breast cancer patients who have estrogen receptor positive (ER+) tumors whose proliferation is reduced after estrogen deprivation by aromatase inhibitors (AI). This study investigates any link between proliferation and hypoxia, a major determinant of tumor biology, and defines the effect of estrogen deprivation on hypoxia-associated genes.Methods: Genome-wide expression profiles were obtained from tumor biopsies from 81 ER+ postmenopausal patients, before and after 2 weeks' anastrozole treatment. A hypoxia metagene was developed by identifying genes clustered with classical hypoxia-regulated genes, excluding those associated with proliferation. Proliferation was measured by Ki67 and a proliferation metagene derived from two published breast cancer data sets.Results: Hypoxia and proliferation metagenes were associated at baseline (Pearson correlation coefficient, r = 0.67, P < 10−4) and after 2 weeks (r = 0.71, P < 10−4). Hypoxia metagene at baseline was associated with 2-week Ki67 (r = 0.43, P = 0.0002) and more weakly with poor 2-week Ki67 change consistent with a weak association with AI resistance. Hypoxia metagene was significantly downregulated with AI. This downregulation was significantly associated with change in the proliferation metagene and with Ki67 but, importantly, not with the substantial change in expression of classical estrogen-dependent genes.Conclusions: Hypoxia metagene is closely associated with proliferation before and after AI treatment. The downregulation of hypoxia metagene after AI therapy is most likely the result of changes in proliferation. There may be a weak effect of hypoxia metagene on de novo resistance to AIs. These findings are important to consider in coapplication of antiproliferative agents with antiangiogenic or antihypoxia agents. Clin Cancer Res; 17(9); 3005–12. ©2011 AACR.

PDF file - 109K, Derivation of samples for microarray analysis.

PDF file - 33KB, Impact of fulvestrant and E-deprivation on selected index proliferation-associated or E-regulated genes.

Purpose: Estrogen withdrawal by treatment with aromatase inhibitors is the most effective form of endocrine therapy for postmenopausal estrogen receptor–positive (ER+) breast cancer. However, response to therapy varies markedly and understanding of the precise molecular effects of aromatase inhibitors and causes of resistance is limited. We aimed to identify in clinical breast cancer those genes and pathways most associated with resistance to aromatase inhibitors by examining the global transcriptional effects of AI treatment.Experimental Design: Baseline and 2-week posttreatment biopsies were obtained from 112 postmenopausal women with ER+ breast cancer receiving neoadjuvant anastrozole. Gene expression data were obtained from 81 baseline and 2-week paired samples. Pathway analysis identified (i) the most prevalent changes in expression and (ii) the pretreatment genes/pathways most related to poor antiproliferative response.Results: A total of 1,327 genes were differentially expressed after 2-week treatment (false discovery rate < 0.01). Proliferation-associated genes and classical estrogen-dependent genes were strongly downregulated whereas collagens and chemokines were upregulated. Pretreatment expression of an inflammatory signature correlated with antiproliferative response to anastrozole and this observation was validated in an independent study. Higher expression of immune-related genes such as SLAMF8 and TNF as well as lymphocytic infiltration were associated with poorer response (P < 0.001) and validated in an independent cohort.Conclusions: The molecular response to aromatase inhibitor treatment varies greatly between patients consistent with the variable clinical benefit from aromatase inhibitor treatment. Higher baseline expression of an inflammatory signature is associated with poor antiproliferative response and should be assessed further as a novel biomarker and potential target for aromatase inhibitor-treated patients. Clin Cancer Res; 19(10); 2775–86. ©2013 AACR.

Supplementary Tables S1-S10.

PDF file - 147KB, S7: Ingenuity based analysis of genes affected by E-deprivation: (a) In vitro and (b) FAIMoS (two weeks) detailing (i) canonical pathways (p

PDF file - 127KB, S9: Fold-changes and statistical significance of genes significantly and differentially affected by fulvestrant and E-deprivation in pre-surgical studies and in vitro models: (a) Up-regulated and (b) Down-regulated. Probes are annotated with gene symbols where available; S10: GO functional gene sets significantly and differentially affected by fulvestrant and E-deprivation in pre-surgical studies and in vitro models: (a) Up-regulated and (b) Down-regulated.

XLSX file - 250K, Excel file of differentially expressed and correlated genes.

PDF file - 87K, Lymphocytic infiltration in tumours.

PDF file - 58KB, S11: Summary of Spearman correlations between up-regulated (n=13) and down-regulated (n=28) genes which were fulvestrant-related (n=41), AURKA and ESR1, in pre-treatment in samples from (a) NEWEST and (b) FAIMoS; S12: Summary of treatment-related genes for which pre-treatment expression correlates significantly with treatment-induced changes in expression of the proliferation-associated gene AURKA: (a) High pre-treatment expression correlates with good response, (b) Low pre-treatment expression correlates with good response, and (c) high pre-treatment expression correlates with good response to low-dose fulvestrant. E-deprivation (ED), fulvestrant (F). Probes are annotated with gene symbols where available; S13: Impact of sixteen weeks of anastrozole on transcripts found to be specifically affected by fulvestrant: (a) Up-regulated and (b) Down-regulated.

Supplemental Figure Legend from Effect of Aromatase Inhibition on Functional Gene Modules in Estrogen Receptor–Positive Breast Cancer and Their Relationship with Antiproliferative Response

PDF file - 826K, Clustering of the tumours based on immunohistochemical parameters.

Analysis results of assessing the effect of the impact of the gene expression of PAGs on FAIMoS dataset and their association with Ki67 protein expression.

PDF file - 46K, Prediction analysis of correlated genes.

PDF file - 68K, Multivariable analysis of inflammatory metagene, grade, ER H-score, baseline Ki67 and proportional change in Ki67.

PDF file - 44K, Patient demographics.

Supplementary Figure S1; Supplementary Tables S1-S6.

Supplementary Figure S1; Supplementary Methods.

PDF file - 81K, IPA analysis and genes in the inflammatory immune response signature.

XLSX file - 1531KB, Details of transcripts and Gene Ontology (GO) functional sets resulting from comparative analyses. Probes are annotated with gene symbols where available.

PDF file - 134KB, S3: Top 30 genes significantly affected by E-deprivation: (a) In vitro and (b) FAIMoS (two weeks), (i) Up-regulated and (ii) Down-regulated. Probes are annotated with gene symbols where available; S4: Top 30 genes significantly affected by fulvestrant: (a) In vitro and (b) NEWEST (high-dose), (i) Up-regulated and (ii) Down-regulated. Probes are annotated with gene symbols where available; S5: Top 30 GO functional gene sets significantly affected by E-deprivation: (a) In vitro and (b) FAIMoS (two weeks), (i) Up-regulated and (ii) Down-regulated; S6: Top 30 GO functional gene sets significantly affected by fulvestrant: (a) In vitro and (b) NEWEST (high-dose), (i) Up-regulated and (ii) Down-regulated.

Purpose: Endocrine therapies include aromatase inhibitors and the selective estrogen receptor (ER) downregulator fulvestrant. This study aimed to determine whether the reported efficacy of fulvestrant over anastrozole, and high- over low-dose fulvestrant, reflect distinct transcriptional responses.Experimental Design: Global gene expression profiles from ERα-positive breast carcinomas before and during presurgical treatment with fulvestrant (n = 22) or anastrozole (n = 81), and corresponding in vitro models, were compared. Transcripts responding differently to fulvestrant and estrogen deprivation were identified and integrated using Gene Ontology, pathway and network analyses to evaluate their potential significance.Results: The overall transcriptional response to fulvestrant and estrogen deprivation was correlated (r = 0.61 in presurgical studies, r = 0.87 in vitro), involving downregulation of estrogen-regulated and proliferation-associated genes. The transcriptional response to fulvestrant was of greater magnitude than estrogen deprivation (slope = 0.62 in presurgical studies, slope = 0.63 in vitro). Comparative analyses identified 28 genes and 40 Gene Ontology categories affected differentially by fulvestrant. Seventeen fulvestrant-specific genes, including CAV1/2, SNAI2, and NRP1, associated with ERα, androgen receptor (AR), and TP53, in a network regulating cell cycle, death, survival, and tumor morphology. Eighteen genes responding differently to fulvestrant specifically predicted antiproliferative response to fulvestrant, but not anastrozole. Transcriptional effects of low-dose fulvestrant correlated with high-dose treatment, but were of lower magnitude (ratio = 0.29).Conclusions: The transcriptional response to fulvestrant has much in common with estrogen deprivation, but is stronger with distinctions potentially attributable to arrest of estrogen-independent ERα activity and involvement of AR signaling. Genes responding differently to fulvestrant may have predictive utility. These data are consistent with the clinical efficacy of fulvestrant versus anastrozole and higher dosing regimens. Clin Cancer Res; 20(15); 3962–73. ©2014 AACR.

Genomic profiling of tumours in patients in clinical trials enables rapid testing of multiple hypotheses to confirm which genomic events determine likely responder groups for targeted agents. A key challenge of this new capability is defining which specific genomic events should be classified as 'actionable' (that is, potentially responsive to a targeted therapy), especially when looking for early indications of patient subgroups likely to be responsive to new drugs. This Opinion article discusses some of the different approaches being taken in early clinical development to define actionable mutations, and describes our strategy to address this challenge in early-stage exploratory clinical trials.

6548 Background: Baseline tumor and germline biomarkers in R/M HNSCC were analyzed for predictive potential in pts benefitting from D or D+T. Methods: In HAWK (NCT02207530), 112 pts (PD-L1 tumor cells [TC]≥25%) received D (10 mg/kg Q2W for ≤12 m); in CONDOR (NCT02319044), 67 pts (PD-L1 TC < 25%) received D (10 mg/kg Q2W for ≤12 m), 133 pts received D+T (D 20 mg/kg Q4W, T 1 mg/kg Q4W for ≤12 m), and 67 pts received T (10 mg/kg Q4W [7 doses] then Q12W [2 doses] for ≤12 m) VENTANA PD-L1 (SP263) Assay determined PD-L1 status. Paired FFPE archival tumor and PBMC samples (as germline control) in the HAWK and CONDOR trials were evaluated by whole exome sequencing (WES). Tumor mutation burden (TMB) was number of somatic mutations/megabase. HLA class I types were obtained via WES of PBMCs (CONDOR only). HPV and neutrophil-to-lymphocyte ratio (NLR) were tested locally in CONDOR. Wilcoxon, log-rank tests, and COX-PH models were used. Pooled D & D+T data were analyzed unless noted. Results: 153 pts had paired evaluable FFPE tumor and PBMC samples (HAWK, n = 48; CONDOR, n = 105). TMB distributions were similar between studies ( P= 0.43). TMB correlated with smoking ( P= 0.02) but not HPV ( P= 0.24), NLR ( P= 0.66), or PD-L1 status ( P= 0.43). Overall, high TMB (≥upper tertile) trended with longer OS vs low TMB in all evaluable pts (N = 153; 9.0 vs 5.6 m; HR = 0.70; 95% CI = 0.48-1.01); P= 0.06). In HAWK, there was no association of TMB with OS. In CONDOR, pts (D and D+T arms) with high TMB vs low had significantly longer OS (N = 76; 16.3 vs 5.3 m; HR = 0.53; 95% CI = 0.31-0.92). TMB and OS association was further assessed by increasing TMB cutoffs. Improved HRs trended with higher cutoffs; cutoffs ≥upper quartile significantly linked to OS.TMB was not associated with PFS or ORR. Pts with low PD-L1 and low TMB had worse OS compared to pts with high PD-L1 or high TMB. Pts with high NLR (≥median) and low TMB had significantly worse OS than pts with low NLR and high TMB (HR = 2.63, P< 0.001). Analysis of germline HLA alleles revealed significantly poorer survival for carriers of the HLA-B*15:01 allele (9.4%) (HLA-B variant status did not affect TMB and OS association in CONDOR). Germline HLA heterozygosity did not impact OS. Pts with mutations in ATM (5%), a DNA damage repair gene, also trended with prolonged OS. Conclusions: TMB is a possible predictive biomarker of IO HNSCC therapy. Combined analysis of NLR and TMB may provide additional PD-L1 data in assessing pts most likely to have long-term benefit. Clinical trial information: NCT002207530, NCT02319044 .

Purpose: To investigate potential associations between gene modules representing key biologic processes and response to aromatase inhibitors (AI) in estrogen receptor–positive (ER+) breast cancer. Patients and Methods: Paired gene expression and Ki67 protein expression were available from 69 postmenopausal women with ER+ early breast cancer, at baseline and 2 weeks post-anastrozole treatment, in the presurgical setting. Functional gene modules (n = 26) were retrieved from published studies and their module scores were computed before and after elimination of proliferation-associated genes (PAG). Ki67 and module scores were assessed at baseline and 2 weeks post-anastrozole. Unsupervised clustering was used to assess associations between modules and Ki67. Results: Proliferation-based modules were highly correlated with Ki67 expression both pretreatment and on-treatment. At baseline with and without PAGs, Ki67 expression was significantly inversely correlated with ERG, ESR1.2, SET, and PIK3CA modules. Modules measuring estrogen signaling strongly predicted antiproliferative response to therapy with and without PAGs. Baseline expression of insulin-like growth factor-1 (IGF-I) module predicted a poor change in Ki67-implicating genes within the module as involved in de novo resistance to AIs. High expression of Immune.2.STAT1 module pretreatment predicted poor antiproliferative response to therapy. A significant association between estrogen-regulated genes modules (ESR1, ESR1-2, SET, and ERG) was evident post AI. Conclusions: Multiple processes and pathways are affected by AI treatment in ER+ breast cancer. Modules closely associated with ESR1 expression were predictive of good antiproliferative response to AIs, but modules representing immune activity and IGF-I/MAPK were predictive of poor Ki67 response, supporting their therapeutic targeting in combination with AIs. Clin Cancer Res; 20(9); 2485–94. ©2014 AACR.

Purpose: Estrogen withdrawal by treatment with aromatase inhibitors is the most effective form of endocrine therapy for postmenopausal estrogen receptor–positive (ER+) breast cancer. However, response to therapy varies markedly and understanding of the precise molecular effects of aromatase inhibitors and causes of resistance is limited. We aimed to identify in clinical breast cancer those genes and pathways most associated with resistance to aromatase inhibitors by examining the global transcriptional effects of AI treatment. Experimental Design: Baseline and 2-week posttreatment biopsies were obtained from 112 postmenopausal women with ER+ breast cancer receiving neoadjuvant anastrozole. Gene expression data were obtained from 81 baseline and 2-week paired samples. Pathway analysis identified (i) the most prevalent changes in expression and (ii) the pretreatment genes/pathways most related to poor antiproliferative response. Results: A total of 1,327 genes were differentially expressed after 2-week treatment (false discovery rate < 0.01). Proliferation-associated genes and classical estrogen-dependent genes were strongly downregulated whereas collagens and chemokines were upregulated. Pretreatment expression of an inflammatory signature correlated with antiproliferative response to anastrozole and this observation was validated in an independent study. Higher expression of immune-related genes such as SLAMF8 and TNF as well as lymphocytic infiltration were associated with poorer response (P < 0.001) and validated in an independent cohort. Conclusions: The molecular response to aromatase inhibitor treatment varies greatly between patients consistent with the variable clinical benefit from aromatase inhibitor treatment. Higher baseline expression of an inflammatory signature is associated with poor antiproliferative response and should be assessed further as a novel biomarker and potential target for aromatase inhibitor-treated patients. Clin Cancer Res; 19(10); 2775–86. ©2013 AACR.

Background: Most breast cancers (BC) at primary diagnosis are estrogen receptor positive (ER+). Estrogen (E) mediates its effects by binding to the ER. Therapies targeting the estrogenic stimulation of tumor growth reduce mortality from ER+ BC. However, resistance remains a clinical problem. To identify molecular mechanisms associated with early resistance to E-deprivation, we overlaid global gene transcription data from patients treated with neoadjuvant anastrazole with those from MCF7 cells adapted to long-term E-deprivation (LTED). Methods: Global gene expression and Ki67 data were available from 81 postmenopausal women with ER+ early BC, at baseline and 2-weeks post anastrazole treatment. Genes, that changed expression in response to anastrozole, were identified using multiple testing corrected multi-group analyses. In vitro global gene expression was available from MCF7 cells adapted to LTED. Functional consequence of target genes on proliferation, ER-mediated transcription and downstream cell signaling was assessed using siRNA. Results: Overlay of genes that predicted for a poor change in Ki67 in patients treated with anastrazole (p < 0.01) with those from LTED cells (p < 0.001) identified 32 genes of interest. Genes associated with inflammation and immunity such as chemokine receptors (CXCR) were highlighted as potential biomarkers of poor response. LTED cells showed a 17-fold higher level of CXCR7 and 1.5-fold higher level of CXCR4 versus wt-MCF7. High CXCR7 but not CXCR4 was associated with reduced relapse free survival in ER+ BC patients in 4 clinical public data sets. SiRNA knockdown of CXCR4 had no specific effect on the proliferation of wt-MCF7 or LTED cells. In contrast, siCXCR7 significantly inhibited the proliferation of the LTED cells versus wt-MCF7. Furthermore, LTED cells were inhibited in a dose-dependent manner by CXC733, a CXCR7 antagonist, whilst wt-MCF7 cells were not. We hypothesized CXCR7 via BCL2 may lead to pro-survival signals. However, siCXCR7 had a minimal effect on BCL2 expression in LTED cells and did not increase apoptosis. Of note, siCXCR7 significantly decreased expression of cyclinD1, CDK4 and phosphorylated-Rb in LTED cells but not wt MCF7 ± estradiol, indicating a likely role in cell cycle arrest. As LTED cells require ER for proliferation, we hypothesized CXCR7 was required for ER-transactivation. SiCXCR7 significantly inhibited ER-mediated transcription of an estrogen response element linked reporter construct in LTED but not wt MCF7. Previously CDK4 mediated phosphorylation of PELP1, a major coactivator of ER, was shown to be critical for its interaction with ER. Of note, siCXCR7 decreased association between ER and PELP1 in LTED cells and was associated with a decrease in expression of the endogenous E-regulated gene TFF1. Conclusion: We have demonstrated for the first time that CXCR7 expression associates with poor relapse free survival in ER+ BC patients. Our in vitro model of endocrine resistance, provided evidence of novel cross talk between ER and CXCR7 leading to ligand-independent ER-mediated transactivation via PELP1. These data highlight the role of CXCR7 as a potential therapeutic target in endocrine resistant BC. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD01-05.

The preoperative setting is increasingly popular for the clinical investigation of hormonal agents and new biological drugs. The effectiveness of endocrine agents is well established for estrogen receptor–positive disease, and the emphasis in preoperative studies is on their combination with agents targeted at resistance mechanisms over 3 or more months. New agents are also being assessed for early evidence of clinical efficacy in shorter-term window-of-opportunity studies. The establishment of Ki67 as an intermediate marker of treatment benefit and of long-term outcome, with endocrine drugs, provides the opportunity for new trial designs with Ki67 as the primary endpoint. The PeriOperative Endocrine Therapy for Individualizing Care (POETIC) trial is randomizing (2:1) 4000 estrogen receptor–positive patients to 2 weeks presurgical treatment with a nonsteroidal aromatase inhibitor or no presurgical treatment. It provides a unique opportunity for detailed study of the determinants of response and resistance to estrogen deprivation as well as testing the role of presurgical therapy for improved biomarker-based estimates of prognosis. J Natl Cancer Inst Monogr 2011;43:120–123

Most estrogen receptor α (ER)-positive breast cancers initially respond to antiestrogens, but many eventually become estrogen-independent and recur. We identified an estrogen-independent role for ER and the CDK4/Rb/E2F transcriptional axis in the hormone-independent growth of breast cancer cells. ER downregulation with fulvestrant or small interfering RNA (siRNA) inhibited estrogen-independent growth. Chromatin immunoprecipitation identified ER genomic binding activity in estrogen-deprived cells and primary breast tumors treated with aromatase inhibitors. Gene expression profiling revealed an estrogen-independent, ER/E2F-directed transcriptional program. An E2F activation gene signature correlated with a lesser response to aromatase inhibitors in patients' tumors. siRNA screening showed that CDK4, an activator of E2F, is required for estrogen-independent cell growth. Long-term estrogen-deprived cells hyperactivate phosphatidylinositol 3-kinase (PI3K) independently of ER/E2F. Fulvestrant combined with the pan-PI3K inhibitor BKM120 induced regression of ER+ xenografts. These data support further development of ER downregulators and CDK4 inhibitors, and their combination with PI3K inhibitors for treatment of antiestrogen-resistant breast cancers. Significance: ERα retains genomic activity and drives a CDK4/E2F-dependent transcriptional program despite estrogen deprivation therapy. Combined inhibition of ER and PI3K induced regression of ER+ xenografts, supporting further development of strong ER downregulators and CDK4 inhibitors, and their combination with PI3K inhibitors for the treatment of antiestrogen-resistant breast cancers. Cancer Discovery; 1(4); 338–51. ©2011 AACR. Read the Commentary on this article by Van Tine et al., p. 287 This article is highlighted in the In This Issue feature, p. 275

Aromatase inhibitors (AI) have improved the treatment of oestrogen receptor positive (ER+) breast cancer. Despite the efficacy of these agents over 40% of patients relapse with endocrine resistant disease. Here we describe an in vitro model of acquired resistance to long-term oestrogen deprivation (LTED). The LTED cells retain expression of the ER and appear hypersensitive to oestrogen as a result of altered kinase activity. Furthermore analysis of temporal changes in gene expression during the acquisition of resistance highlight growth factor receptor pathways as key mediators of this adaptive process.

Purpose: Although most patients with estrogen receptor α (ER)-positive breast cancer initially respond to endocrine therapy, many ultimately develop resistance to antiestrogens. However, mechanisms of antiestrogen resistance and biomarkers predictive of such resistance are underdeveloped. Experimental Design: We adapted four ER+ human breast cancer cell lines to grow in an estrogen-depleted medium. A gene signature of estrogen independence was developed by comparing expression profiles of long-term estrogen-deprived (LTED) cells to their parental counterparts. We evaluated the ability of the LTED signature to predict tumor response to neoadjuvant therapy with an aromatase inhibitor and disease outcome following adjuvant tamoxifen. We utilized Gene Set Analysis (GSA) of LTED cell gene expression profiles and a loss-of-function approach to identify pathways causally associated with resistance to endocrine therapy. Results: The LTED gene expression signature was predictive of high tumor cell proliferation following neoadjuvant therapy with anastrozole and letrozole, each in different patient cohorts. This signature was also predictive of poor recurrence-free survival in two studies of patients treated with adjuvant tamoxifen. Bioinformatic interrogation of expression profiles in LTED cells revealed a signature of MYC activation. The MYC activation signature and high MYC protein levels were both predictive of poor outcome following tamoxifen therapy. Finally, knockdown of MYC inhibited LTED cell growth. Conclusions: A gene expression signature derived from ER+ breast cancer cells with acquired hormone independence predicted tumor response to aromatase inhibitors and associated with clinical markers of resistance to tamoxifen. Activation of the MYC pathway was associated with this resistance. Clin Cancer Res; 17(7); 2024–34. ©2011 AACR.

Purpose: The majority of breast cancer patients who have estrogen receptor positive (ER+) tumors whose proliferation is reduced after estrogen deprivation by aromatase inhibitors (AI). This study investigates any link between proliferation and hypoxia, a major determinant of tumor biology, and defines the effect of estrogen deprivation on hypoxia-associated genes. Methods: Genome-wide expression profiles were obtained from tumor biopsies from 81 ER+ postmenopausal patients, before and after 2 weeks' anastrozole treatment. A hypoxia metagene was developed by identifying genes clustered with classical hypoxia-regulated genes, excluding those associated with proliferation. Proliferation was measured by Ki67 and a proliferation metagene derived from two published breast cancer data sets. Results: Hypoxia and proliferation metagenes were associated at baseline (Pearson correlation coefficient, r = 0.67, P < 10−4) and after 2 weeks (r = 0.71, P < 10−4). Hypoxia metagene at baseline was associated with 2-week Ki67 (r = 0.43, P = 0.0002) and more weakly with poor 2-week Ki67 change consistent with a weak association with AI resistance. Hypoxia metagene was significantly downregulated with AI. This downregulation was significantly associated with change in the proliferation metagene and with Ki67 but, importantly, not with the substantial change in expression of classical estrogen-dependent genes. Conclusions: Hypoxia metagene is closely associated with proliferation before and after AI treatment. The downregulation of hypoxia metagene after AI therapy is most likely the result of changes in proliferation. There may be a weak effect of hypoxia metagene on de novo resistance to AIs. These findings are important to consider in coapplication of antiproliferative agents with antiangiogenic or antihypoxia agents. Clin Cancer Res; 17(9); 3005–12. ©2011 AACR.

Aim: To identify molecular mechanisms and predictors of response to aromatase inhibitors (AIs) in primary ER+ breast cancer. Background: Although AIs are highly effective suppressants of estrogen synthesis in postmenopausal women, up to 50% of patients derive little or no clinical benefit from AI treatment. The proliferation marker Ki67 provides a valuable pharmacodynamic marker for response in the presurgical setting and 2 week measurements of this marker have been shown to predict recurrence-free survival1. However, understanding of the precise molecular effects of AIs and causes of resistance is limited. We present, to our knowledge, the largest study of the transcriptional effects of AI treatment in the neoadjuvant setting. Methods: Baseline and 2wk post-treatment core-cut tumor biopsies were obtained from 112 postmenopausal women with stage I to IIIB ER+ early breast cancer who received single agent neoadjuvant anastrozole2. RNA extracted from biopsies was analysed on Illumina 48K microarrays. Genes which changed expression upon anastrozole treatment were identified using paired class comparison of the 81 matched baseline and 2wk pairs from which gene expression data was available. Correlates of response were determined using multiple testing corrected Spearman correlation analysis. Pathway analysis was performed on the ranked list of correlated genes. IHC for Ki67, ER and PgR was performed centrally on FFPE sections from duplicate core biopsies. Results: At a false discovery rate (FDR) of 0.01, 1154 genes were differentially expressed (792 down-regulated and 362 up-regulated). Proliferation-associated genes and classical estrogen-dependent genes such as TOP2A, CCNB2, TFF1, and PDZK1 were strongly downregulated (FDR 1. Dowsett et al., JNCI, 2007, 99, 167-70. 2. Smith et al., JCO, 2007, 3816-22. Supported by the Mary-Jean Mitchell Green Foundation and Breakthrough Breast Cancer. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr S2-5.

Aim: To compare changes in whole genome transcription elicited by anastrozole (ANA) and fulvestrant (FUL) in estrogen receptor positive (ER+) breast cancer. Background: Aromatase inhibitors (AIs), such as ANA, and the selective estrogen receptor down-regulator, FUL, are both effective in the treatment of ER+ breast cancer. AIs reduce stimulation of ER by suppressing estrogen synthesis while FUL acts as an antagonist and receptor down-regulator. Better understanding of their mechanism of action may lead to a more rational clinical application of these two drugs. Methods: Pre-and post-treatment core-cut tumor biopsies were obtained from 105 postmenopausal women with ER+ early breast cancer who received neoadjuvant FUL[1]. Genome-wide expression data were available from 22 matched baseline and post-treatment pairs from the patients who received 500mg of FUL every 28 days and 500mg on day 14 of the first month (FUL500), also, from 16 patients who received 250mg of FUL every 28 days (FUL250). In addition, microarray data were obtained from 81 matched pre-and post-treatment pairs of tumor biopsies from postmenopausal women with ER+ breast cancer who received single agent neoadjuvant ANA[2]. Results: At a false discovery rate (FDR) of 5%, expression of 1875 genes changed with FUL500 but no genes changed with FUL250. Each of 4 index proliferation genes (TOP2A,AURKA, CCNB2, CDC20) and 4 index estrogen responsive genes (ERGs) (TFF1, PGR, PDZK1, GREB1) showed greater suppression with FUL500 than FUL250. Further study of FUL was conducted only in the FUL500 group. At 5% FDR, 1457 genes decreased in expression and 1064 increased with ANA while 1083 decreased and 792 increased with FUL500. Most genes that showed significant changes with ANA also showed changes of a similar magnitude with FUL500 but there were some individual striking differences. There was more commonality in the genes that decreased in expression than those that increased. The reduction in a proliferation metagene was similar with ANA and FUL500 (geometric mean suppression, 23.1% and 23.2% respectively). Each of the 4 proliferation index genes showed a very similar degree of change using the 2 drugs as did the 4 ERGs. However, while IQ motif containing GTPase activating protein 3 was among the 5 most significantly changed genes with ANA (mean 52% suppression, FDR Conclusions: Gene expression changes with FUL250 were modest but with FUL500 were similar in extent to those observed with ANA. Evaluation of the substantial differences in individual gene responses between the two drugs may allow their more rational clinical use. Kuter I, et al; Breast Cancer Res Treat 2008; 109: 589 Smith I.E, et al ; J Clin Oncol 2007; 25:3816 Supported by AstraZeneca, the Mary-Jean Mitchell Green Foundation and Breakthrough Breast Cancer. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P1-12-02.

Purpose To determine whether plasma estradiol (E2) levels are related to gene expression in estrogen receptor (ER)–positive breast cancers in postmenopausal women. Materials and Methods Genome-wide RNA profiles were obtained from pretreatment core-cut tumor biopsies from 104 postmenopausal patients with primary ER-positive breast cancer treated with neoadjuvant anastrozole. Pretreatment plasma E2 levels were determined by highly sensitive radioimmunoassay. Genes were identified for which expression was correlated with pretreatment plasma E2 levels. Validation was performed in an independent set of 73 ER-positive breast cancers. Results The expression of many known estrogen-responsive genes and gene sets was highly significantly associated with plasma E2 levels (eg, TFF1/pS2, GREB1, PDZK1 and PGR; P < .005). Plasma E2 explained 27% of the average expression of these four average estrogen-responsive genes (ie, AvERG; r = 0.51; P < .0001), and a standardized mean of plasma E2 levels and ER transcript levels explained 37% (r, 0.61). These observations were validated in an independent set of 73 ER-positive tumors. Exploratory analysis suggested that addition of the nuclear coregulators in a multivariable analysis with ER and E2 levels might additionally improve the relationship with the AvERG. Plasma E2 and the standardized mean of E2 and ER were both significantly correlated with 2-week Ki67, a surrogate marker of clinical outcome (r = −0.179; P = .05; and r = −0.389; P = .0005, respectively). Conclusion Plasma E2 levels are significantly associated with gene expression of ER-positive breast cancers and should be considered in future genomic studies of ER-positive breast cancer. The AvERG is a new experimental tool for the study of putative estrogenic stimuli of breast cancer.