Esther A. Reijm

Hallmarks of Aromatase Inhibitor Drug Resistance Revealed by Epigenetic Profiling in Breast Cancer

Aromatase inhibitors are the major first-line treatment of estrogen receptor-positive breast cancer, but resistance to treatment is common. To date, no biomarkers have been validated clinically to guide subsequent therapy in these patients. In this study, we mapped the genome-wide chromatin-binding profiles of estrogen receptor α (ERα), along with the epigenetic modifications H3K4me3 and H3K27me3, that are responsible for determining gene transcription (n = 12). Differential binding patterns of ERα, H3K4me3, and H3K27me3 were enriched between patients with good or poor outcomes after aromatase inhibition. ERα and H3K27me3 patterns were validated in an additional independent set of breast cancer cases (n = 10). We coupled these patterns to array-based proximal gene expression and progression-free survival data derived from a further independent cohort of 72 aromatase inhibitor-treated patients. Through this approach, we determined that the ERα and H3K27me3 profiles predicted the treatment outcomes for first-line aromatase inhibitors. In contrast, the H3K4me3 pattern identified was not similarly informative. The classification potential of these genes was only partially preserved in a cohort of 101 patients who received first-line tamoxifen treatment, suggesting some treatment selectivity in patient classification.

High protein expression of EZH2 is related to unfavorable outcome to tamoxifen in metastatic breast cancer.

BACKGROUND Metastatic breast cancer (MBC) is a highly heterogeneous disease with great differences in outcome to both chemo- and endocrine therapy. Better insight into the mechanisms underlying resistance is essential to better predict outcome to therapy and to obtain a more tailored treatment approach. We have previously described that increased mRNA expression levels of Enhancer of Zeste homolog (EZH2) are associated with worse outcome to tamoxifen therapy in MBC. Here, we explored whether this is also the case for EZH2 protein expression. PATIENTS AND METHODS A tissue microarray (TMA) was created using formalin-fixed, paraffin-embedded estrogen receptor (ER)-positive primary breast tumor tissues of 250 MBC patients treated with first-line tamoxifen. Quantity and intensity of EZH2 expression were determined by immunohistochemistry (IHC) and both were used to generate and group scores according to a previously described method for scoring EZH2. RESULTS In total, 116 tumors (46%) were considered to be EZH2 positive. The presence of EZH2 protein expression was significantly associated with progression-free survival (PFS) in both univariate [hazard ratio (HR) 1.51, 95% confidence interval (CI) 1.17-1.97, P = 0.002] and multivariate analysis including traditional factors associated with tamoxifen outcome (HR 1.41, 95% CI 1.06-1.88, P = 0.017). Considering quantity irrespective of intensity, tumors with >50% EZH2-positive cells had the worst PFS (HR 2.15, 95% CI 1.42-3.27, P < 0.001), whereas intensity alone did not show a significant association with PFS. Application of other methods of scoring EZH2 positivity resulted in a similar significant association between the amount of EZH2 positive cells and PFS. CONCLUSION In addition to EZH2 mRNA levels, these results suggest that protein expression of EZH2 can be used as a marker to predict outcome to tamoxifen therapy. This provides new rationale to explore EZH2 inhibition in the clinical setting and increases the possibilities for a more personalized treatment approach in MBC patients.

Gene expression profiles of circulating tumor cells versus primary tumors in metastatic breast cancer.

Before using circulating tumor cells (CTCs) as liquid biopsy, insight into molecular discrepancies between CTCs and primary tumors is essential. We characterized CellSearch-enriched CTCs from 62 metastatic breast cancer (MBC) patients with ≥5 CTCs starting first-line systemic treatment. Expression levels of 35 tumor-associated, CTC-specific genes, including ESR1, coding for the estrogen receptor (ER), were measured by reverse transcription quantitative polymerase chain reaction and correlated to corresponding primary tumors. In 30 patients (48%), gene expression profiles of 35 genes were discrepant between CTCs and the primary tumor, but this had no prognostic consequences. In 15 patients (24%), the expression of ER was discrepant. Patients with ER-negative primary tumors and ER-positive CTCs had a longer median TTS compared to those with concordantly ER-negative CTCs (8.5 versus 2.1 months, P = 0.05). From seven patients, an axillary lymph node metastasis was available. In two patients, the CTC profiles better resembled the lymph node metastasis than the primary tumor. Our findings suggest that molecular discordances between CTCs and primary tumors frequently occur, but that this bears no prognostic consequences. Alterations in ER-status between primary tumors and CTCs might have prognostic implications.

Abstract P5-08-51: SIAH2 protein expression is inversely correlated with the ER status and outcome to tamoxifen therapy in metastatic breast cancer patients

Introduction: In a previous study we observed a positive correlation between Seven in Absentia Homolog 2 (SIAH2) and Estrogen Receptor (ER) mRNA levels. Additionally, high SIAH2 mRNA levels were related to a favorable progression-free survival (PFS) after first-line tamoxifen. In contrast, others showed high SIAH2 protein levels in ER-negative breast cancer associated with an unfavorable relapse-free survival. In this study, we investigated the above discrepancy between SIAH2 protein and mRNA findings and evaluated the prognostic and predictive value of SIAH2 protein in breast cancer patients. Patients and methods: Tissue microarrays (TMAs) of formalin-fixed, paraffin-embedded primary breast tumors were immunohistochemically stained for SIAH2 protein. The TMAs contained core specimens of 759 patients with early disease and of 245 ER-positive patients with advanced disease treated with first-line tamoxifen. SIAH2 protein staining was scored for its intensity and proportion positive cells and subsequently evaluated for its relationship with metastasis-free survival (MFS) and PFS in uni- and multivariate analyses including traditional prognostic or predictive factors, respectively. Results: The proportion SIAH2-positive cells had a relationship with MFS and PFS, whereas staining intensity and a previous described score for SIAH2 combining intensity and proportion were not related with clinical outcome. Based on these results, tumors with more than 20% positive cells were considered as SIAH2-positive. In early disease, 267 patients (35%) had SIAH2-positive tumors, which were further characterized by decreased expression of ER at protein and mRNA levels (P Conclusions: SIAH2 protein expression is especially observed in ER-negative tumors and has no additional prognostic value in breast cancer. The proportion SIAH2-positive cells in ER-positive tumors can be used as biomarker to predict tamoxifen treatment failure in breast cancer patients with advanced disease. Future studies should establish if expression of certain microRNAs explain the observed discrepancy in SIAH2 mRNA and protein levels. Citation Format: van der Willik KD, Timmermans MM, Look MP, Reijm EA, van Deurzen CHM, den Bakker MA, Westenend PJ, Martens JWM, Berns EMJJ, Jansen MPHM. SIAH2 protein expression is inversely correlated with the ER status and outcome to tamoxifen therapy in metastatic breast cancer patients. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-08-51.

Abstract A15: High miRNA-26a and low EZH2 expression levels are associated with favorable outcome to tamoxifen in advanced breast cancer

Background: In breast cancer we have recently discovered that decreased EZH2 expression levels are associated with a favorable outcome to tamoxifen in advanced disease. Furthermore, EZH2 knockdown in the breast cancer cell line MCF7 resulted in estrogen receptor (ER) upregulation and increased sensitivity to anti-estrogens. Interestingly, EZH2 has been identified as target of miRNA-26a and miRNA-101. Objective: The main objective of this study is to investigate miRNA-26a and miRNA-101 for: A) their association with EZH2 and B) their predictive value in breast cancer patients treated with first-line tamoxifen monotherapy. Materials & Methods: Expression levels of miRNA-26a, miRNA-101, EZH2 and references were measured using quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) in 235 ER-positive primary breast cancer specimens from patients with advanced disease. The levels of expression were related to clinicopathologic factors and disease outcome. Computations were performed with STATA and P-values Results: Expression levels of miRNA-26a and miRNA-101 were not correlated with age, tumor grade, tumor size, and nodal status. The miRNA-26a levels were significantly associated with progesterone (PgR) levels, HER2-status and EGFR levels, whereas miRNA-101 levels showed significant relations with PgR expression and menopausal status. The miRNA-26a and miRNA-101 levels showed an inverse relation with EZH2 mRNA levels (Spearman Rank Correlation of −0.21 and −0.15, respectively, P Conclusion & Discussion: The miRNA-26a and miRNA-101 levels have an inverse relation with EZH2, but only miRNA-26a has predictive value in advanced breast cancer. High levels of miRNA-26a and low EZH2 levels are associated with a favorable outcome to tamoxifen therapy. Interestingly, estrogens repress miRNA-26a expression suggesting that ER-positive tumors have less miRNA-26a than ER-negative tumors. This contradicts, however, with the lower EZH2 levels observed in ER-positive compared to ER-negative breast cancers. To clarify this paradox, future (functional) studies will focus on miRNA-26a and EZH2 in different breast cancer subtypes and determine their activated pathways in relation with response to anti-estrogens. Citation Information: Clin Cancer Res 2010;16(7 Suppl):A15

Abstract P4-02-16: High miRNA26A1 and Low EZH2 Expression Levels Are Associated with Favorable Outcome to Tamoxifen in Advanced Breast Cancer through Similar Molecular Pathways

Background: We showed that decreased expression levels of EZH2 are associated with a favorable outcome to tamoxifen in advanced breast cancer. Furthermore, EZH2 knockdown in MCF7 cells resulted in estrogen receptor (ER) upregulation and increased sensitivity to anti-estrogens. Recently, EZH2 has been identified as a target of miRNA26A1 and miRNA101. Objective: To associate miRNA26A1 and miRNA101 expression levels with: A) EZH2 and B) molecular pathways and C) outcome to first-line tamoxifen monotherapy for advanced disease. Materials & Methods: Expression levels of miRNA26A1, miRNA101, EZH2 and references (miRNA-132 and miRNA-374) were measured using quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) in 235 ER-positive primary breast cancer specimens from patients with advanced disease. The levels of expression were related to clinicopathologic factors and disease outcome. Pathway analysis was performed in a subset of 65 ER-positive tumors with available gene expression microarray data available. Computations were performed with STATA and P-values Results: The miRNA26A1 levels were significantly associated with levels of ER, progesterone (PgR), HER2 and EGFR, whereas miRNA101 levels showed significant relations with PgR expression and menopausal status. The miRNA26A1 and miRNA101 levels showed an inverse relation with EZH2 mRNA levels (Spearman Rank Correlation of -0.21 and -0.15, respectively, P Conclusions: The miRNA26A1 and miRNA101 levels have an inverse relation with levels of EZH2, however, only miRNA26A1 has predictive value in advanced breast cancer. Pathways comparison between miRNA26A1 and EZH2 identified 2 overlapping cell cycle related pathways and the genes CCNE1 and CDC2. Low levels of EZH2, CCNE1 and CDC2 and high levels of miRNA26A1 are associated with a favorable outcome to tamoxifen therapy. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-02-16.

Integrated Genomic Profiling of Endocrine Therapy Response in Advanced Breast Cancer.

Purpose In hormone receptor positive breast cancer the response rates for endocrine treatment, i.e. tamoxifen (TAM) or aromatase inhibitors (AIs), are only 50 to 70% in the advanced disease setting. The overall aim of this retrospective study is to identify a molecular signature using integrated genomic profiling to improve prediction of endocrine treatment outcome in the advanced disease setting. Objectives A) To compare mRNA expression profiles of TAM- and AI-treated patients and to identify genes and pathways associated with treatment outcome.B) To discover miRNA and mRNA signatures predictive for AI response. Patients and Methods Fresh frozen Estrogen Receptor (ER)-positive primary breast cancer specimens from patients with advanced disease treated with first-line AIs (N=55) or TAM (N=109) were analyzed. Expression profiles of 670 miRNAs and 44K mRNAs were generated using multiplex qRT-PCR and microarrays. Profiles were related to clinical response and time to progression (TTP). Statistical and bio-informatic tools were applied to discover and combine markers into an integrated genomic predictive signature. The nearest centroid prediction method of BRB-ArrayTools (Version3.7.0) was used to assess the predictive value. Results The quality controlled and informative expression profiles of 277 miRNAs and 14112 mRNAs in 50 AI-treated tumors and 10433 mRNAs in 101 TAM-treated tumors were included for further analysis in the discovery phase.Global testing of mRNAs linked to Biocarta pathways demonstrated the involvement of the interferon pathway in endocrine therapy response in both AI- and TAM-treated patients. Using BRB-ArrayTools survival analysis to find genes associated with TTP (P Conclusion This is the first study that combines miRNA and mRNA profiling in an attempt to define an integrated genomic signature for endocrine treatment outcome. Additional prospective multicenter studies are needed to confirm the predictive value of this signature. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3029.

Down Regulation of EZH2 Is Associated with ESR1 Upregulation and Response to Endocrine Therapy in Breast Cancer.

Background: We have previously identified a gene signature for resistance to first-line tamoxifen therapy in advanced breast cancer. One of these genes is Enhancer of Zeste Homolog 1 (EZH1), a member of the EZH family of which EZH2 has been identified as being prognostic. Both genes are involved in transcriptional control and epigenetic memory maintenance and act as polycomb repressors. The aim of this study is to investigate these genes for their predictive value and, if associated with clinical outcome, to evaluate the functional role in treatment response in vitro . Experimental design: Expression levels of EZH1 and EZH2 expression were measured using quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) in primary breast cancer specimens and related to clinicopathologic factors and disease outcome. Functional studies were done in MCF7, a human estrogen sensitive breast cancer cell line. Expression levels of EZH2 were downregulated with siRNAs, and changes in cell numbers were determined treating with (the anti-estrogen) ICI164.384. In addition, alterations in expression levels of putative downstream genes, including the estrogen receptor (ESR1), were measured. Results: When analysed as continuous variable in univariate analysis, only EZH2 was significantly associated with therapy resistance and a shorter Progression Free Survival (PFS) in 278 patients with advanced disease treated with first-line tamoxifen monotherapy. In univariate analysis the tertile with highest EZH2 levels was associated with clinical benefit (OR=0.48, 95%CI: 0.26-0.89; P=0.02) and with PFS (HR=1.80, 95%CI: 1.32-2.46;P Conclusion: High levels of EZH2 are associated with poor clinical outcome for tamoxifen therapy of advanced breast cancer. EZH2 silencing in MCF7 cells inhibits cell proliferation, it upregulates ESR1 levels and increases sensitivity to ICI164.384. Further validation is needed to confirm that silencing of EZH2 leads to an upregulation of the ESR1 and as a consequence to a better response to anti-estrogens. This finding sheds new light on the involvement of EZH2 in anti-estrogen treatment offering possibilities for novel management strategies. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5131.

Decreased expression of EZH2 is associated with ESR1 upregulation and response to anti-estrogens.

Abstract #6127 Background: In prostate and breast cancer high levels of Enhancer of Zeste Homolog 2 (EZH2) are associated with tumor progression. EZH2, a histone H3 methyl transferase, is part of the polycomb complex. Together with histone deacetylases (HDACs) EZH2 regulates “genome wide” gene transcription silencing. The aims of this study are a) to correlate EZH2 expression with endocrine therapy response in patients with recurrent disease treated with first-line tamoxifen monotherapy and b) to determine the role of EZH2 in endocrine therapy response using in vitro cell line models.
 Material and Methods: EZH2 mRNA levels were measured with quantitative real-time PCR (qRT-PCR) in 297 retrospectively collected hormone receptor positive (HR+) primary breast tumor specimens of patients with recurrent disease who did respond (N=110) or were resistant (N=187) to first-line tamoxifen monotherapy. In vitro, EZH2 and estrogen receptor (ESR1) expression was downregulated with siRNAs in the human breast cancer cell line MCF7 and assessed for their sensitivity to the selective estrogen receptor degrader ICI164.384 after 96hrs treatment (N=3). To establish therapy response in vitro, cell number counts were determined. All p-values are two-sided and significant if P Results: In 297 HR+ breast tumors, EZH2 as continuous variable, associated significantly with poor response (OR= 0.67 [0.49-0.91]; P=0.001) and a shorter progression-free survival (PFS) (HR=1.28 [1.11-1.47], P Conclusion: In primary breast tumors, high EZH2 mRNA levels are associated with poor outcomes after tamoxifen therapy. These results suggest that EZH2 may identify patients at risk for tamoxifen therapy failure. In vitro studies show that downregulation of EZH2 inhibits cell proliferation and on the other hand results in upregulation of ESR1 levels. The latter may explain the increased sensitivity to ICI164.384 of EZH2 silenced cells. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6127.