Maxime P. Look

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

BACKGROUNDnMetastatic 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.nnnPATIENTS AND METHODSnA 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.nnnRESULTSnIn 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.nnnCONCLUSIONnIn 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.

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 S6-05: High levels of APOBEC3B, a DNA deaminase and an enzymatic source of C-to-T transitions, are a validated marker of poor outcome in estrogen receptor-positive breast cancer

Two recent observations have connected the innate immune DNA cytosine deaminase APOBEC3B to the genetic evolution of breast cancer. First, APOBEC3B was shown to be up-regulated in the majority of breast cancers, and, in breast cancer cell lines, its activity was causally linked to a doubling of the number of C-to-T transitions over time and to a delay in cell cycle progression (1). Second, sequencing of the complete genome of 21 breast cancers independently suggested that APOBEC deaminase activity could be responsible for 2 of 5 mutational imprints identified, which involved clustered (also called kataegis) and dispersed C-to-T transition mutations in the context of 5’TC dinucleotide motifs (2). In the current study, we addressed a possible association of APOBEC3B expression with outcome in clinical breast cancer. For this we measured using real-time RT-PCR APOBEC3B mRNA levels in 1,491 primary invasive breast cancers and correlated these levels with disease-free survival (DFS), metastasis-free survival (MFS) and overall survival (OS) using univariate and multivariable Cox regression analysis. In addition, we independently validated our findings in available gene expression datasets with appropriate follow-up. In univariate analyses including all patients, increasing levels of APOBEC3B mRNA analyzed as a continuous variable were significantly associated with shorter DFS, MFS and OS (Hazard Ratio [HR] = 1.29, 1.31 and 1.36, respectively, all P To substantiate and validate our findings, we analysed 4 independent available datasets containing in total 5,760 breast cancer cases in which APOBEC3B mRNA expression was measured by probes on microarrays and found that higher APOBEC3B mRNA expression (dichotomised by mean) was significantly associated with poor outcome in all 4 cohorts ([Metabric, 1,491 ER+ cases, HR = 1.82; P 5 years], and [Affymetrix dataset-2, 643 ER+ cases, HR = 2.04; P = 0.001]). Altogether, our analyses show that APOBEC3B mRNA - and as a result likely DNA deamination – is a validated predictor of poor outcome in breast cancer, supporting the notion that APOBEC3B is a potentially interesting clinical target for therapeutic intervention to prevent breast cancer progression and metastasis, particularly in ER+ disease. 1. Burns, M.B. et al. Nature 494, 366-70 (2013); 2. Nik-Zainal, S. et al. Cell 149, 979-93 (2012). Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr S6-05.