Jwm Martens

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.

Survival and contralateral breast cancer in CHEK2 1100delC breast cancer patients: impact of adjuvant chemotherapy

Background:We assessed the sensitivity to adjuvant chemotherapy in cell cycle checkpoint kinase 2 (CHEK2) vs non-CHEK2 breast cancer patients by comparing the contralateral breast cancer incidence and distant disease-free and breast cancer-specific survival between both groups, stratified for adjuvant chemotherapy.Methods:One Dutch hereditary non-BRCA1/2 breast cancer patient cohort (n=1220) and two Dutch cohorts unselected for family history (n=1014 and n=2488, respectively) were genotyped for CHEK2 1100delC. Hazard ratios for contralateral breast cancer, distant disease-free and breast cancer-specific death for mutation carriers vs noncarriers were calculated using the Cox proportional hazard method, stratified for adjuvant chemotherapy.Results:The CHEK2 mutation carriers (n=193) had an increased incidence of contralateral breast cancer (multivariate hazard ratio 3.97, 95% confidence interval 2.59–6.07). Distant disease-free and breast cancer-specific survival were similar in the first 6 years in mutation carriers compared with noncarriers, but diverted as of 6 years after breast cancer diagnosis (multivariate hazard ratios and 95% confidence intervals 2.65 (1.79–3.93) and 2.05 (1.41–2.99), respectively). No significant interaction between CHEK2 and adjuvant chemotherapy was observed.Conclusions:The CHEK2 1100delC-associated breast cancer is associated with a higher contralateral breast cancer rate as well as worse survival measures beyond 6 years after diagnosis. No differential sensitivity to adjuvant chemotherapy was observed in CHEK2 patients.

Performance of BRCA1/2 mutation prediction models in male breast cancer patients

To establish whether existing mutation prediction models can identify which male breast cancer (MBC) patients should be offered BRCA1 and BRCA2 diagnostic DNA screening, we compared the performance of BOADICEA (Breast and Ovarian Analysis of Disease Incidence and Carrier Estimation Algorithm), BRCAPRO (BRCA probability) and the Myriad prevalence table (“Myriad”). These models were evaluated using the family data of 307 Dutch MBC probands tested for BRCA1/2, 58 (19%) of whom were carriers. We compared the numbers of observed vs predicted carriers and assessed the Area Under the Receiver Operating Characteristic (ROC) Curve (AUC) for each model. BOADICEA predicted the total number of BRCA1/2 mutation carriers quite accurately (observed/predicted ratio: 0.94). When a cut‐off of 10% and 20% prior probability was used, BRCAPRO showed a non‐significant better performance (observed/predicted ratio BOADICEA: 0.81, 95% confidence interval [CI]: [0.60‐1.09] and 0.79, 95% CI: [0.57‐1.09], vs. BRCAPRO: 1.02, 95% CI: [0.75‐1.38] and 0.94, 95% CI: [0.68‐1.31], respectively). Myriad underestimated the number of carriers in up to 69% of the cases. BRCAPRO showed a non‐significant, higher AUC than BOADICEA (0.798 vs 0.776). Myriad showed a significantly lower AUC (0.671). BRCAPRO and BOADICEA can efficiently identify MBC patients as BRCA1/2 mutation carriers. Besides their general applicability, these tools will be of particular value in countries with limited healthcare resources.

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.

Association of microRNA-7 and its binding partner CDR1-AS with the prognosis and prediction of 1st-line tamoxifen therapy in breast cancer

The large number of non-coding RNAs (ncRNAs) and their breadth of functionalities has fuelled many studies on their roles in cancer. We previously linked four microRNAs to breast cancer prognosis. One of these microRNAs, hsa-miR-7, was found to be regulated by another type of ncRNA, the circular non-coding RNA (circRNA) CDR1-AS, which contains multiple hsa-miR-7 binding sites. Based on this finding, we studied the potential clinical value of this circRNA on breast cancer prognosis in a cohort based on a cohort that was previously analysed for hsa-miR-7 and in an adjuvant hormone-naïve cohort for 1st-line tamoxifen treatment outcomes, in which we also analysed hsa-miR-7. A negative correlation was observed between hsa-miR-7 and CDR1-AS in both cohorts. Despite associations with various clinical metrics (e.g., tumour grade, tumour size, and relapse location), CDR1-AS was neither prognostic nor predictive of relevant outcomes in our cohorts. However, we did observe stromal CDR1-AS expression, suggesting a possible cell-type specific interaction. Next to the known association of hsa-miR-7 expression with poor prognosis in primary breast cancer, we found that high hsa-miR-7 expression was predictive of an adverse response to tamoxifen therapy and poor progression-free and post-relapse overall survival in patients with recurrent disease.

PO-462 Functional ex vivo assay reveals homologous recombination deficiency in breast cancer beyond brca gene defects

Introduction Tumours of germline BRCA1/2 mutated carriers show homologous recombination (HR) deficiency (HRD), resulting in impaired DNA double strand break (DSB) repair and high sensitivity to Poly-(ADP-Ribose)-Polymerase (PARP) inhibitors. Although this therapy is expected to be effective beyond germline BRCA1/2 mutated carriers, a robust validated test to detect HRD tumours is lacking. In the present study we therefore evaluated a functional HR assay exploiting the formation of RAD51 foci in proliferating cells after ex vivo irradiation of fresh breast cancers (BrC) tissue: the RECAP test. Material and methods Fresh samples of 170 primary BrC were analysed using the RECAP test. The molecular explanation for the HRD phenotype was investigated by exploring BRCA deficiencies, mutational signatures, amount of tumour infiltrating lymphocytes (TILs) and microsatellite instability (MSI). Results and discussions RECAP was completed successfully in 148 out of 170 samples (87%). 24 tumours showed HRD (16%), while 6 tumours were HR intermediate (HRi) (4%). HRD was explained by BRCA deficiencies (mutations, promoter hypermethylation, deletions) in 16 cases. Several non-BRCA deficient HRD tumours showed BRCAness related mutational signatures, suggesting that they are likely also bona fide HRD cases. HRD tumours showed an increased incidence of high TIL counts (p=0.023) compared to HR proficient (HRP) tumours and MSI was more frequently observed in the HRD group (2/20, 10%) than expected in unselected BrC (1%) (p=0.017). Conclusion The RECAP test is a robust assay detecting both BRCA1/2 deficient and BRCA1/2 proficient HRD tumours. This test identifies approximately 50% more patients that may benefit from PARPi treatment than BRCA gene testing only.

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 P6-08-10: 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.