John Foekens

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 P3-06-32: Topoisomerase 1 gene copy aberration is a frequent finding in clinical breast cancer samples

Background: The topoisomerase-1 (TOP1) targeting drug irinotecan may be a new effective drug in the treatment of metastatic breast cancer patients 1,2 . However, the use of irinotecan should be guided by predictive biomarkers to avoid drug-induced site effects in the non-responding subgroup of patients. A newly developed TOP1 fluorescence in situ hybridization (FISH) 3 probe mix (Dako, Denmark) may represent such a novel predictive biomarker assay. Materials and Methods: The TOP1 /CEN-20 FISH probe mix was applied to normal breast tissue (n = 100) and human breast cancer biopsy material (n = 42). TOP1 copy number variations (Affymetrix GeneChip 100k SNP array) and TOP1 mRNA levels (Affymetrix u133a GeneChip) were studied in 313 breast cancer samples 4 . Results: Employing FISH, the mean diploid TOP1 copy number in normal breast epithelium was 1.7 (range 1.45–1.90) and the diploid CEN-20 number was 1.6 (range 1.38–1.82). The normal diploid range was defined as the mean plus/minus 0.5 times the haploid gene copy number (Table 1 and 2). Using these values as cut-off, 13 (30.9%) of the 42 breast cancer samples had TOP1 gene copy number in the diploid range, 18 (42.9%) had 1 extra copy and 11 (26.2%) had ≥ 2 extra copies. CEN-20 counts showed that 19 (45.5%) had CEN-20 values within the diploid range, 21 (50%) had 1 extra copy and 2 (4.8%) had ≥ 2 extra copies. Calculating the TOP1 /CEN-20 ratio showed that 6 (14.3%) of the patients samples had a ratio above 1.5, while none had a ratio TOP1 and CEN-20 counts in the 42 breast cancer samples analyzed with FISH was 0.73. In the 313 breast cancer samples 34/313 (10.9%) had TOP1 copy gain and a significant association (p TOP1 copy number and TOP1 mRNA. Conclusions: This study shows that about 25% of breast cancers have ≥2 extra copies of TOP1 as determined by FISH analyses. The high correlation between TOP1 and CEN20 in the 42 breast cancer samples challenges the use of the ratio. Moreover, the significant association between TOP1 gene copy number (SNP array) and TOP1 mRNA expression in breast cancer biopsies suggests that TOP1 FISH results will correlate with TOP1 protein amounts and may therefore be used to predict sensitivity to the TOP1 targeting drug irinotecan in breast cancer. However, a prospective clinical study is needed to prove or disprove this hypothesis. References 1. Perez EA et al. J Clin Oncol. 2004, 22(14):2849–55. 2. Lee KS et al. Invest New Drugs. 2012 May 5. 3. Romer MU et al. Scand J Gastroenterol. 2012, 47(1):68–79. 4. Zhang Y et al. Cancer Res 2009, 69(9):3795–3801. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-06-32.

Which cyclin E prevails as prognostic marker for breast cancer? Results from a retrospective study involving 635 lymph node negative breast cancer patients

PURPOSEnTo evaluate the prognostic value of cyclin E with a quantitative method for lymph node-negative primary breast cancer patients.nnnPATIENTS AND METHODSnmRNA transcripts of full-length and splice variants of cyclin E1 (CCNE1) and cyclin E2 (CCNE2) were measured by real-time PCR in frozen tumor samples from 635 lymph node-negative breast cancer patients who had not received neoadjuvant or adjuvant systemic therapy.nnnRESULTSnNone of the PCR assays designed for the specific splice variants of the cyclins gave additional prognosis-related information compared with the common assays able to detect all variants. In Cox multivariate analysis, corrected for the traditional prognostic factors, high levels of cyclin E were independently associated with a short distant metastasis-free survival [hazard ratio (HR), 3.40; P < 0.001 for CCNE1 and HR, 1.76; P < 0.001 for CCNE2, respectively]. After dichotomizing the tumors at the median level of 70% tumor cells, the multivariate analysis showed particularly strong results for CCNE1 in the group of 433 patients with stroma-enriched primary tumors (HR, 5.12; P < 0.001). In these tumors, the worst prognosis was found for patients with estrogen receptor-negative tumors expressing high CCNE1 (HR, 9.89; P < 0.001) and for patients with small (T1) tumors expressing high CCNE1 (HR, 8.47; P < 0.001).nnnCONCLUSIONnOur study shows that both CCNE1 and CCNE2 qualify as independent prognostic markers for lymph node-negative breast cancer patients, and that CCNE1 may provide additional information for specific subgroups of patients.

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 P6-08-10: Mutational signatures impact the breast cancer transcriptome and distinguish mitotic from immune response pathways

A comprehensive whole genome analysis of a large breast cancer cohort of 560 cases (Nik-Zainal et al, submitted 2015) reports novel and existing DNA substitution and rearrangement signatures next a comprehensive list of events driving the breast cancer cell to its malignant potency. In the current study, we linked the observed genetic diversity to the breast cancer transcriptome for 260 cases for which whole genome and whole transcriptome data were both available. Cluster analysis of the global gene expression showed the familiar view of a coherent basal-like and a heterogeneous luminal subgroup. New and previously reported 1 subtype-specific aberrations with concordant expression changes were found in TP53, PIK3CA, PTEN, CCND1, CDH1 and GATA3, and mutations in PIK3CA, PTEN, AKT1 and AKT2 were mutually exclusive confirming they are active in the same pathway in breast cancer. Integrating the identified DNA substitutions signatures with the transcriptome, we observed that the total number of substitutions in a cancer, irrespective of substitution type, was positively associated with cell cycle regulated gene expression and with adverse outcome. In addition and more remarkably, we observed that the number substitution of two substitution signatures 2 particularly associated with immune-response specific gene expression, with increased amount of tumor infiltrating lymphocytes and with a better outcome. These two signatures comprised 1) mutations of the APOBEC-type (predominant C>G in a TCN context), and 2) mutations which lacks specific features but which are strongly associated with genetic and epigenetic inactivating aberrations in BRCA1 and BRCA2. Thus, while earlier reports 3-5 imply that the sheer number of driver events triggers an immune-response, we refine this statement by observing that substitutions of a particular type are much very effective in doing so explaining the superior outcome of cancer having these particular types of substitutions. This result also implies that purposefully augmenting T-cell reactivity against amino-acid substitutions resulting from either of these two DNA substitution types could potentially improve immunotherapies in breast cancer. 1. Comprehensive molecular portraits of human breast tumours. Nature 490, 61-70 (2012). 2. Alexandrov, L.B., et al. Signatures of mutational processes in human cancer. Nature 500, 415-421 (2013). 3. Rizvi, N.A., et al. Cancer immunology. Mutational landscape determines sensitivity to PD-1 blockade in non-small cell lung cancer. Science 348, 124-128 (2015). 4. Schumacher, T.N. & Schreiber, R.D. Neoantigens in cancer immunotherapy. Science 348, 69-74 (2015). 5. Snyder, A., et al. Genetic basis for clinical response to CTLA-4 blockade in melanoma. N Engl J Med 371, 2189-2199 (2014). Citation Format: Martens JWM, Smid M, Rodriguez-Gonzalez G, Sieuwerts AM, Prager-Van der Smissen WJC, Van Der Vlugt - Daane M, Van Galen A, Nik-Zainal S, Staaf J, Brinkman AB, Van de Vijver MJ, Richardson AL, Berentsen K, Caldas C, Butler A, Martin S, Davies HD, Debets R, Meijer-Van Gelder ME, Van Deurzen CHM, Ramakrishna MR, Ringner M, Viari A, Birney E, Borresen-Dale A-L, Stunnenberg HG, Stratton M, Foekens JA. Mutational signatures impact the breast cancer transcriptome and distinguish mitotic from immune response pathways. [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 P6-08-10.

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.