Mphm Jansen

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.

Additional value of the 70-gene signature and levels of ER and PR for the prediction of outcome in tamoxifen-treated ER-positive breast cancer

BACKGROUND Breast cancer patients with node positive disease can have an excellent outcome with tamoxifen only. It is unclear whether analysing both the 70-gene signature and hormone receptors provides superior prediction of outcome in tamoxifen-treated patients than either alone. METHODS Three series were evaluated: 121 patients (81% node positive) received adjuvant tamoxifen, 151 patients did not receive tamoxifen (10% node positive) and 92 patients received tamoxifen for metastatic disease. The 70-gene signature was analysed using MammaPrint. Oestrogen receptor (ER) and progesterone receptor (PR) immunohistochemistry was evaluated following St. Gallen Consensus (Highly Endocrine Responsive: ER and PR ≥ 50%, Incompletely Endocrine Responsive: ER and/or PR low or either one absent). RESULTS In patients treated with adjuvant tamoxifen, both the 70-gene signature (adjusted for Endocrine Response Categories HR 2.17, 95%CI 1.01-4.66) as well as the Endocrine Response Categories (adjusted for 70-gene signature HR 6.35, 95%CI 1.90-21.3) were associated with breast-cancer-specific-survival (BCSS). Also in patients treated with tamoxifen for metastatic disease, combined analysis of the 70-gene signature and ER/PR revealed additional value (multivariate Cox regression, p = 0.013). In patients who did not receive tamoxifen, only the 70-gene signature was associated with outcome. CONCLUSION In the series analysed, the 70-gene signature was mainly a prognostic factor, while ER and PR levels were mainly associated with outcome after tamoxifen. Combination of these three factors may improve outcome prediction in tamoxifen-treated patients.

Gene expression profiles and molecular classification to predict distant metastasis and tamoxifen-resistant breast cancer.

Genome-wide measures of gene expression can identify patterns of gene activity that subclassify tumours and might provide better means than are currently available for individual risk assessment in patients with primary breast cancer and for prediction of tamoxifen resistance.

Abstract P1-02-02:ESR1mutations in circulating tumor cell versus circulating cell-free DNA of metastatic breast cancer patients before first-line endocrine therapy and at progression

Background Mutations in ESR1 , the gene encoding the estrogen receptor, have been linked to endocrine resistance in metastatic breast cancer (MBC). It is thought that these mutations are selected during endocrine treatment (ET), but direct evidence that these ESR1 mutations (m ESR1 ) emerge during treatment with endocrine agents is scant. We set out to evaluate m ESR1 in circulating tumor cells (CTCs) and matched plasma cell-free DNA (cfDNA) of MBC patients before start of 1 st line ET and at progression. Materials & Methods CellSearch-enriched CTCs (≥ 5 CTC/7.5 mL) of 37 MBC patients before start of 1 st line ET (baseline cohort; BL) and 38 MBC patients who had progressed on any line of ET for metastatic disease (progressive disease cohort; PD) were evaluated. 52% of the PD patients received one line of ET and 48% more lines, of which 92% contained an aromatase inhibitor. In addition, 10 CellSearch-enriched fractions from healthy blood donors (HBDs) and 46 matched plasma samples (7xHBD, 15xBL, 24xPD) were included. DNA was isolated using the AllPrep kit and cfDNA with the QIAamp CNA kit (Qiagen). Hotspot mutations for ESR1 (D538G, Y537S, Y537C and Y537N) were evaluated with mutation-specific Taqman assays by chip-based digital PCR (QuantStudio 3D). m ESR1 status was assessed after target-specific ESR1 amplification capturing all 4 mutations, with thresholds for positivity based on the highest variant allele frequencies in HBDs. Results Of all the CTC samples in the BL cohort, 1 patient had mutated Y537N copies, while this mutation was not detected in the matched cfDNA. This patient had received adjuvant treatment with tamoxifen. Also none of the other 14 BL cfDNA samples analyzed harbored m ESR1 . Three PD patients (8%) were positive for m ESR1 in their CTCs (2x D538G and 1x Y537S). These D538G variants identified in CTCs were also detected in the corresponding cfDNA of these patients; for the Y537S mutation no matched cfDNA was available. Seven additional m ESR1 carriers were identified in the other 22 matched cfDNA PD samples, resulting in 38% m ESR1 positivity of the PD plasma samples (7x D538G, 1x Y537C and 1x Y537C). Conclusion Sensitivity for detecting m ESR1 in CTC fractions (identified in 8% of the PD patients) was lower than for cfDNA samples. Using cfDNA for m ESR1 detection, we found an higher prevalence of m ESR1 variants in samples obtained at progression to ET (38%) compared to baseline (0%). These findings further substantiate the role of m ESR1 in endocrine resistance. Citation Format: Sieuwerts AM, Beije N, Kraan J, Van M, Onstenk W, Vitale SR, van der Vlugt – Daane M, Hamberg P, Dirix LY, Brouwers A, de Jongh FE, Jager A, Seynaeve CM, Jansen MPHM, Foekens JA, Martens JWM, Sleijfer S. ESR1 mutations in circulating tumor cell versus circulating cell-free DNA of metastatic breast cancer patients before first-line endocrine therapy and at progression [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-02-02.

Abstract P1-09-20: An optimized workflow to analyzeESR1mutations in both circulating cell-free and circulating tumor cell DNA by digital PCR

Background In metastatic breast cancer (MBC) patients ESR1 mutations (mESR1) in cell-free DNA (cfDNA) have been related to endocrine therapy (ET) resistance. Such mutations might also be detectable in circulating tumor cells (CTCs). Mutation detection in small amounts of cfDNA and in CTCs in a background of leukocytes is highly challenging. The current study evaluated how to reliably investigate mESR1 status in such minute amounts of cfDNA and in DNA from CellSearch-enriched CTCs. Materials & Methods Plasma (200 µL) and matched CellSearch-enriched CTC fractions of 7 healthy blood donors (HBD) and 29 MBC patients at baseline and after ET (≥ 5 CTC/7.5 mL) were evaluated. cfDNA was isolated from plasma with the QIAamp CNA kit and CTC-enriched DNA with the AllPrep kit (Qiagen). mESR1 status in both cfDNA and CTC-enriched DNA fractions was compared with or without whole genome amplification (repli-g WGA, Qiagen) or ESR1 target specific amplification. Quantitative PCR (qPCR) for wild type (WT) ESR1 was used to control the number of WT copies loaded into the chips for digital PCR (dPCR) analysis. The variant allele frequencies (VAF) of hotspot mutations for ESR1 (D538G, Y537S, Y537C and Y537N) were evaluated with mutation-specific Taqman assays by chip-based dPCR (QuantStudio 3D, Thermo Fischer Scientific). Results To allow inclusion of as many samples as possible, we successfully downscaled the volume of required plasma from 1 mL to 200 µL as this resulted in the same VAF. Sample-type specific thresholds for mESR1 presence were established (2% for the cell-free plasma samples, at which percentage all HBDs were negative, and 0.5% for the CTCs to allow identification of one mutated CTC-specific copy in a background of ~1,000 leukocytes). WGA was unable to adequately amplify fragmented cfDNA, resulting in a too low DNA yield. However, locus-specific target pre-amplification of a 136 bp fragment covering all 4 different mutations followed by mutant specific dPCR performed well for both cfDNA and CTC DNA, but only if the loading of the pre-amplified product into the dPCR chips was optimized by qPCR for the number of WT ESR1 copies. The most optimal results for dPCR data interpretation were obtained after: 1. including at least one positive sample in each dPCR session; 2. using a “safe loading window”, 3. loading and reading chips at least twice in QuantStudio 3D ; 4. critically evaluating the contribution by a non-specific “comet effect”; and 5. after loading the data in the software, performing at least two independent data analyses to exclude intra-observer variations. Summary Here we describe our workflow to assess mESR1 in a limited amount of plasma cfDNA or CellSearch enriched CTC DNA. This workflow has been successfully used to investigate the mESR1 VAF status in DNA from matched CTC DNA and cfDNA of MBC patients before start of 1st line endocrine therapy and at progression (see also abstract number 851017). Citation Format: Vitale SR, Sieuwerts AM, Helmijr J, Beije N, van der Vlugt – Daane M, Foekens JA, Sleijfer S, Jansen MPHM, Martens JWM. An optimized workflow to analyze ESR1 mutations in both circulating cell-free and circulating tumor cell DNA by digital PCR [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-09-20.

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 P6-04-08: FOXA1 expression: regulated by EZH2 and associated with favorable outcome to tamoxifen in advanced breast cancer

Background: Enhancer of Zeste Homolog 2 (EZH2) is a member of the polycomb complex 2 and serves as a histone methyltransferase. Through trimethylation of histone 3 of lysine 27, EZH2 regulates ‘genome wide’ gene transcription silencing. Downregulation of EZH2 is associated with increased expression of the estrogen receptor (ER) and favorable outcome to tamoxifen in metastatic breast cancer. The binding of ER to its target genes is enhanced by Forkhead Box A1 (FOXA1), a pioneer factor that binds to and opens chromatized DNA. The aim of this study is to investigate the potential association between EZH2 and FOXA1 and their relation with treatment outcome in patients with advanced breast cancer. Experimental design: EZH2 and FOXA1 mRNA levels were analysed using available gene expression profile-data of 344 primary breast cancer specimens of lymph-node-negative (LNN) patients and in 109 ER-positive (ER+) tumors of patients with metastatic disease treated with first-line tamoxifen. To functionally analyze EZH2, cell lines were generated with different knockdown-constructs for EZH2, followed by evaluation of mRNA and protein expression levels and chromatin immunoprecipitation (ChIP) experiments combined with qPCR and/or next generation sequencing (NGS; ChIPseq). Results: In the 344 LNN breast tumors, EZH2 was highly expressed in breast tumors of the basal subtype. In contrast, FOXA1 was hardly expressed in this subtype, but highly in the luminal A and B subtypes. FOXA1 was related to a prolonged time to progression (TTP) after tamoxifen (HR = 0.51 [0.35–0.74], P We obtained 50–70% EZH2 knockdown in the breast cancer cell lines MCF7 (ER+/PR+/FOXA1+), SUM52PE (ER+/PR−/FOXA1+), and MM231 (ER−/PR−/FOXA1−). ChIP followed by qPCR demonstrated higher levels of H3K27 trimethylation at the ER-locus in the parental cell lines compared to the EZH2-knockdowns for MM231 and at the PR-locus for SUM52PE and MM231. ChIPseq evaluation in the EZH2 knockdown of MM231 identified 200 loci with a significant decrease in read numbers for H3K27me3 compared to the parental. Interestingly, amongst these 200 loci the FOXA1-locus was identified. These results suggest FOXA1 as an EZH2-target since its expression was not seen in the parental MM231. Conclusion: High EZH2 and low FOXA1 mRNA levels are associated with poor clinical outcome for tamoxifen therapy of advanced breast cancer. Functional studies indicate that EZH2 might regulate the expression of FOXA1. These results suggest that EZH2 and/or FOXA1 could be used as a predictive marker to identify patients at risk for tamoxifen therapy failure and possibly as a target for therapy. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-04-08.

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.