ER-positive Breast Cancer Patients Research Articles

Abstract P3-04-09: Genomic analysis to evaluate response to neoadjuvant anastrozole and fulvestrant in post-menopausal ER-positive HER2-negative breast cancer patients included in the UCBG CARMINA02 trial

Abstract Background: CARMINA02 is a non-comparative multicenter phase II randomized trial evaluating the clinical response rate after up to 6 months of neoadjuvant endocrine therapy (NET) in HR+/HER2- patients with 1 mg anastrozole (Arm A) or 500 mg fulvestrant (Arm B). Secondary objectives included predictive markers of response and outcome. Between 2007 and 2011, 116 women with operable infiltrating breast adenocarcinoma T2-T4 N0-N3 M0 were randomized. Clinical response rates at 6 months (RECIST criteria) were 52.6% [95%CI 41-64%] in Arm A and 36.8% [95%CI 25-49%] in Arm B (Cancer 2016, in press). We aimed to identify the molecular predictive markers of resistance or sensitivity common to both treatments. Methods: Ninety tumor RNA from clinical responder (n=34) and resistant patients (n=23) treated in arms A or B have been sequenced with Illumina Hiseq2500 technology leading to 2x100-nt paired-end RNA-seq reads. These samples are from pre-treatment (29 in arm A, 28 in arm B) and post-treatment tumors (6 months after, 17 in arm A and 16 in arm B). Alignment was performed with Tophat_2.0.6. Differential gene expression was analyzed with the Differential Expression analysis for Sequence count data package. Gene fusion was detected with ChimeraScan, TophatFusion and DeFuse tools. Variant calling including variations, insertions and deletions was processed following GATK recommendations for RNAseq datas. Quantitative RT-PCR experiments were done to confirm RNA-seq expression results in patient samples not selected for RNA-seq analysis (validation cohort). Results: We first analyzed differentially expressed genes (DEGs) between responders and non-responders in pre-treatment or post-treatment samples to select potential predictive markers of response. We identified 51 DEGs before treatment common to anastrozole and fulvestrant. Among these 51 genes, SGK2 was the only gene more intensely expressed in responders than in non-responders. Then we compared DEGs between pre and post-treatment samples for responders or non-reponders for both treatment arms. SGK2 expression remains stable after treatment. Furthermore we identified 7 DEGs specific to responders and 11 DEGs specific to non-responders. Concerning genes fusion detection predicted by at least 2 tools, none was specific to a response type. We noted a higher number of fusions in non-responders samples. Variants detected by RNA-Seq are being confirmed by DNA-Seq using a home-made next-generation sequencing panel including 95 genes frequently mutated in breast cancers (analysis ongoing). Conclusion:High expression of SGK2, encoding a kinase induced in response to signals that activate PI3kinase, may represent a predictive marker of sensitivity to NET. DEGs associated with NET response or resistance belong to cell cycle, DNA replication and repair, cell death and drug metabolism. Ongoing DNA-seq datas will complete this genomic analysis. This research was conducted with support from AstraZeneca and Institut Curie. Citation Format: Callens C, Bessoltane N, Ngo C, Chemlali W, Becette V, Bernard V, Delattre O, Lemonnier J, Mouret-Reynier M-A, Andre F, Bieche I, Lerebours F. Genomic analysis to evaluate response to neoadjuvant anastrozole and fulvestrant in post-menopausal ER-positive HER2-negative breast cancer patients included in the UCBG CARMINA02 trial [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 P3-04-09.

Abstract P2-05-10: UCBG 2-14: A prospective multicenter non-randomized trial evaluating the effect of EndoPredict® (EPclin®) clinico-genomic test on treatment decision making among patients with intermediate clinical risk

Abstract Background: Genomic tests can identify ER-positive Her2-negative localized breast cancer (BC) patients (pts) who may not drive any benefit from adjuvant chemotherapy (CT). Several genomic tests have reached a high level of analytical and clinical validity, as well as clinical utility in such situation. Recent results suggest that the safe de–escalation of adjuvant chemotherapy may be most beneficial in pts with clinical high or indetermediate risk, as assessed by classical variables or online tools, through the use of a genomic test. The clinical risk though remains quite uncertain with variable definition and grey zones. The present study aimed at determining if EPclin clinico-genomic test had a significant impact on treatment decision making among pts with predefined intermediate /borderline clinical risk. Patients and methods: Women were eligible for the present study if they had complete surgical removal of a localized ER+ Her2- pN0 or pN1mi BC, and were considered by the multidisciplinary team meeting (MTM) of the center as being in a "grey zone" of uncertain CT benefit based on classical clinic-pathological assessment. The MTM1 proposed a decision (chemo/no chemo). After informed consent, an EPclin signature classified the tumor as low risk (EPclin Score < 3.3, no chemo advised) or high risk (> 3.3, a theoretical indication for adjuvant chemo). Primary end point was the proportion of change between initial adjuvant CT decision at MTM1 and final administration of CT (yes/no). A 5 steps Fleming design was planned, considering that a change rate of 15% or less was not acceptable (low clinical utility). A one-step design was used (unilateral α = 2.5% and β = 1%). Results: 203 pts were included, of whom 198 are evaluable for the main end point. 74% of the tumors were grade 2, 72% T1 , 25% T2, 3% T3; 16% were pN1mi and 84% pN0. Median age was 57. EpClin® was low risk in 67% and high risk in 33% of the cases. The global rate of decision change between MTM1 and final administration of CT was 70/198 (35.4% IC-95% = [28.7-42.1]), with 55 (27.7%) decreases and 15 (7.6%) increases in CT indication. 27% instead of 47% of all pts finally received CT (43% decrease in CT prescription). In the multivariate analysis, the factors associated to decrease for less CT were EPclin score (OR 0.11 [0.03-0.35]), proliferation (OR 1.08 [1.03-1.13]) and higher grades (OR 5.22 [1.04-26.08]); while EPclin score was positively (OR 76.6 [7.11-824.8]), but higher grades inversely (OR 0.06 [0-0.86]) associated to an increase in CT administration. Of note, the change in final decision occurred after the MTM2 and after report of the results and discussion with the patient in 9 of the 70 cases (12% of changes; 4.5% of the whole population)(8 decreases and 1 increase in CT administration). Conclusion: Adendom met its primary objective, with 35% of intermediate clinico-pathological risk pts getting significant therapeutic changes upon the receipt of an EPclin gene expression profile. 43% of these "intermediate risk" pts planned for CT avoided it. 12% of the changes were discrepant with the test's results and occurred after discussion with the patient. Citation Format: Penault-Llorca F, Kwiatkovski F, Grenier J, Levy C, Leheurteur M, Uwer L, Derbel O, Le Rol A, Jacquin J-P, Jouannaud C, Quenel-Tueux N, Girre V, Foa C, Guardiola E, Lortholary A, Catala S, Lemonnier J, Delaloge S. UCBG 2-14: A prospective multicenter non-randomized trial evaluating the effect of EndoPredict® (EPclin®) clinico-genomic test on treatment decision making among patients with intermediate clinical risk [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 P2-05-10.

21-Gene Recurrence Score and Locoregional Recurrence in Node-Positive/ER-Positive Breast Cancer Treated With Chemo-Endocrine Therapy

The 21-gene recurrence score (RS) predicts risk of locoregional recurrence (LRR) in node-negative, estrogen receptor (ER)-positive breast cancer. We evaluated the association between RS and LRR in node-positive, ER-positive patients treated with adjuvant chemotherapy plus tamoxifen in National Surgical Adjuvant Breast and Bowel Project B-28. B-28 compared doxorubicin/cyclophosphamide (AC X 4) with AC X 4 followed by paclitaxel X 4. Tamoxifen was given to patients age 50 years or older and those younger than age 50 years with ER-positive and/or progesterone receptor-positive tumors. Lumpectomy patients received breast radiotherapy. Mastectomy patients received no radiotherapy. The present study includes 1065 ER-positive, tamoxifen-treated patients with RS assessment. Cumulative incidence functions and subdistribution hazard regression models were used for LRR to account for competing risks including distant recurrence, second primary cancers, and death from other causes. Median follow-up was 11.2 years. All statistical tests were one-sided. There were 80 LRRs (7.5%) as first events (68% local/32% regional). RS was low: 36.2%; intermediate: 34.2%; and high: 29.6%. RS was a statistically significant predictor of LRR in univariate analyses (10-year cumulative incidence of LRR = 3.3%, 7.2%, and 12.2% for low, intermediate, and high RS, respectively, P < .001). In multivariable regression analysis, RS remained an independent predictor of LRR (hazard ratio [HR] = 2.59, 95% confidence interval [CI] = 1.28 to 5.26, for a 50-point difference, P = .008) along with pathologic nodal status (HR = 1.91, 95% CI = 1.20 to 3.03, for four or more vs one to three positive nodes, P = .006) and tumor size (HR = 1.28, 95% CI = 1.05 to 1.55, for a 1 cm difference, P = .02). RS statistically significantly predicts risk of LRR in node-positive, ER-positive breast cancer patients after adjuvant chemotherapy plus tamoxifen. These findings can help in the selection of appropriate candidates for comprehensive radiotherapy.

ISY-3-4 - Multi-gene assay “Curebest 95GC” for recurrence prediction for ER+/HER2-/n0 breast cancer patients

In recent years, one of the priority questions in breast cancer research has been the prediction of recurrence for ER-positive, HER2-negative and node-negative (ER+/HER2-/n0) breast cancer patients. This is because the indication for adjuvant chemotherapy for such patients is often very difficult to determine on the basis of the results of conventional histological examinations including immunohistochemistry for ER, PR, HER2 and Ki67. Then by the comprehensive gene expression analysis of DNA microarray for 549 primary sites of ER+ breast cancer, we developed a multi-gene classifier “Curebest 95GC” to predict recurrence for ER+/HER2-/n0 breast cancer treated by adjuvant hormonal therapy alone. The merit of 95GC is to divide the Intermediate-risk patients of 21GC(Oncotype DX) furtherly into the two risk groups (High/Low-risk). 95GC: http://www.ncbi.nlm.nih.gov/pubmed/23884597 The other priority question has been the prediction of sensitivity to neo-adjuvant chemotherapy (NAC: P-FAC) and prognosis after NAC. However, it is very difficult to predict it with high NPV (negative predictive value). Then we also developed multi-gene classifiers to predict it precisely. IRSN23 is the sensitivity prediction model for NAC for ER positive and negative breast cancer, and MPCP155 is the prediction model for sensitivity to NAC and prognosis after NAC only for ER positive breast cancer. IRSN23: http://www.ncbi.nlm.nih.gov/pubmed/24356621 MPCP155: http://www.ncbi.nlm.nih.gov/pubmed/26052094 Such classifiers have been shown to be superior to conventional histological examination findings and “Curebest 95GC” has already commercially available and in practical use. Here we review these multi-gene classifiers with a special emphasis on “Curebest 95GC”.

Ridaforolimus (MK-8669) synergizes with Dalotuzumab (MK-0646) in hormone-sensitive breast cancer.

BackgroundMammalian target of rapamycin (mTOR) represents a key downstream intermediate for a myriad of oncogenic receptor tyrosine kinases. In the case of the insulin-like growth factor (IGF) pathway, the mTOR complex (mTORC1) mediates IGF-1 receptor (IGF-1R)-induced estrogen receptor alpha (ERα) phosphorylation/activation and leads to increased proliferation and growth in breast cancer cells. As a result, the prevalence of mTOR inhibitors combined with hormonal therapy has increased in recent years. Conversely, activated mTORC1 provides negative feedback regulation of IGF signaling via insulin receptor substrate (IRS)-1/2 serine phosphorylation and subsequent proteasomal degradation. Thus, the IGF pathway may provide escape (e.g. de novo or acquired resistance) from mTORC1 inhibitors. It is therefore plausible that combined inhibition of mTORC1 and IGF-1R for select subsets of ER-positive breast cancer patients presents as a viable therapeutic option.MethodsUsing hormone-sensitive breast cancer cells stably transfected with the aromatase gene (MCF-7/AC-1), works presented herein describe the in vitro and in vivo antitumor efficacy of the following compounds: dalotuzumab (DALO; “MK-0646”; anti-IGF-1R antibody), ridaforolimus (RIDA; “MK-8669”; mTORC1 small molecule inhibitor) and letrozole (“LET”, aromatase inhibitor).ResultsWith the exception of MK-0646, all single agent and combination treatment arms effectively inhibited xenograft tumor growth, albeit to varying degrees. Correlative tissue analyses revealed MK-0646 alone and in combination with LET induced insulin receptor alpha A (InsR-A) isoform upregulation (both mRNA and protein expression), thereby further supporting a triple therapy approach.ConclusionThese data provide preclinical rationalization towards the combined triple therapy of LET plus MK-0646 plus MK-8669 as an efficacious anti-tumor strategy for ER-positive breast tumors.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2847-3) contains supplementary material, which is available to authorized users.

152PD - Serum metabolomic profiles identify ER-positive early breast cancer patients at increased risk of disease recurrence in a multicentre population

152PD - Serum metabolomic profiles identify ER-positive early breast cancer patients at increased risk of disease recurrence in a multicentre population

190P - Measurement of molecular biomarkers to predict tumor response in estrogen receptor positive breast cancer after dose-dense (biweekly) paclitaxel/carboplatin neoadjuvant chemotherapy

Dose-dense (biweekly) paclitaxel/carboplatin(PC) as neoadjuvant Chemotherapy (NCT) for operable breast cancer is feasible and efficient. This study was to analysis the relationship between the molecular biomarkers and tumor response in estrogen receptor(ER) positive breast cancer. 84 ER-positive breast cancer patients treated with Dose-dense (biweekly) paclitaxel/carboplatin NCT were analyzed for expression of progesrone receptor (PgR), Tau, ki67, HER2, Bcl-2 by immunohistochemistry (IHC), these data were used to test whether these biomarkers can predict tumor response. The primary endpoint was a pathologically complete response (pCR). The second endpoint was the change in tumor size between pre and post NCT. Univariate analysis showed that HER2 positive (53.85% vs 8.62%, p < 0.01,Tau negative (40.91% vs 16.13%,p = 0.017, BCL-2 negative (48.15% vs 10.53%, p < 0.01) expression were associated with higher pCR rate. Multivariate analysis revealed that HER2 (OR: 8.116; 95%CI: 1.931-34.115; p = 0.04), BCL-2(OR: 0.176; 95%CI: 0.041-0.746; p = 0.018) were independent pCR predictive biomarkers. Tumor size was significant reduced in HER2 positive (p = 0.001, high-level ki67 (p = 0.007, Tau negative (p = 0.002, BCL-2 negative (p = 0.008) breast cancer. This study investigates the value of traditional biological markers, Bcl-2 and Tau in ER-positive patients treated with dose-dense (biweekly) paclitaxel/carboplatin NCT.

Lack of progesterone receptor expression predicts poor prognosis in patients with operable ER-positive invasive breast cancer

To investigate the impact of lack of progesterone receptor (PR) expression on the prognosis of patients with operable ER (estrogen receptor)-positive invasive breast cancer. We retrospectively analyzed the clinicopathological features, treatment and survival data of 318 women with ER+ /PR+ and ER+ /PR- invasive breast cancer. Among the 318 patients, there were 219 PR-positive and 99 PR-negative cases. The 5-year overall survival (OS) rate was 92.5%, and the 5-year disease-free survival (DFS) rate was 87.2% in the 318 ER-positive patients. Among them, the 5-year OS rates were significantly different between the PR-positive group (94.6%) and PR-negative group (87.8%, P=0.020), and the 5-year DFS rates were also significantly different from each other (89.8% and 81.6%, respectively, P=0.019). Univariate analysis showed that PR status, tumor size, T stage, axillary lymph node metastasis, and clinical stage were prognostic factors for OS (P<0.05 for all). Multivariate analysis showed that lack of PR expression, T stage ≥2, and positive axillary lymph node metastasis were independent risk factors for poor DFS and OS in ER-positive breast cancer patients (P<0.05 for all). Subgroup analysis showed that lack of PR expression was not significant in predicting poor DFS or OS when patients were in stage Ⅰ or with a small tumor (≤2 cm) (P>0.05 for all), and also showed that premenopausal women with PR-negative disease had poorer DFS and OS than PR-positive patients (P<0.05 for both). Lack of PR expression is an independent risk factor for poor prognosis in patients with operable ER-positive invasive breast cancer, especially in patients with a large tumor (>2 cm), advanced clinical stage (Stage Ⅱ or Ⅲ) or in premenopausal status.

Association of polymorphisms in intron 2 of FGFR2 and breast cancer risk in Chinese women

Recent genome-wide association studies (GWAS) demonstrated that genetic variation in intron 2 of fibroblast growth factor receptor 2 (FGFR2) was a novel risk for breast cancer. We investigated whether two SNPs rs1219648 and rs2981582 in intron 2 of FGFR2 were associated with the risk of breast cancer in Chinese women. A total of 340 female breast cancer patients and 400 normal age-matched controls were recruited. Two SNPs were genotyped using matrix-assisted laser desorption/ionization mass spectrometry. The two SNPs rs1219648 and rs2981582 showed no association with the risk of breast cancer. A subgroup analysis by menopausal status demonstrated that the distribution of rs2981582 T alleles, including CT and TT genotypes, was significantly higher in premenopausal patients compared with postmenopausal patients. The TT genotype in rs2981582 was more strongly associated with ER-positive than with ER-negative tumors by ER status analysis. Analysis by haplotypes showed that no haplotypes associated with breast cancer. The results showed no association between two SNPs, rs1219648 and rs2981582 and breast cancer risk, although in a stratified analysis rs2981582 strongly associated with premenopausal and ER-positive breast cancer patients in Chinese women.

The 29.5 kb APOBEC3B Deletion Polymorphism Is Not Associated with Clinical Outcome of Breast Cancer.

Increased APOBEC3B mRNA levels are associated with a hypermutator phenotype and poor prognosis in ER-positive breast cancer patients. In addition, a 29.5 kb deletion polymorphism of APOBEC3B, resulting in an APOBEC3A-B hybrid transcript, has been associated with an increased breast cancer risk and the hypermutator phenotype. Here we evaluated whether the APOBEC3B deletion polymorphism also associates with clinical outcome of breast cancer. Copy number analysis was performed by quantitative PCR (qPCR) in primary tumors of 1,756 Dutch breast cancer patients. The APOBEC3B deletion was found in 187 patients of whom 16 carried a two-copy deletion and 171 carried a one-copy deletion. The prognostic value of the APOBEC3B deletion for the natural course of the disease was evaluated among 1,076 lymph-node negative (LNN) patients who did not receive adjuvant systemic treatment. No association was found between APOBEC3B copy number values and the length of metastasis-free survival (MFS; hazard ratio (HR) = 1.00, 95% confidence interval (CI) = 0.90–1.11, P = 0.96). Subgroup analysis by ER status also did not reveal an association between APOBEC3B copy number values and the length of MFS. The predictive value of the APOBEC3B deletion was assessed among 329 ER-positive breast cancer patients who received tamoxifen as the first-line therapy for recurrent disease and 226 breast cancer patients who received first-line chemotherapy for recurrent disease. No association between APOBEC3B copy number values and the overall response rate (ORR) to either tamoxifen (odds ratio (OR) = 0.88, 95% CI = 0.69–1.13, P = 0.31) or chemotherapy (OR = 0.97, 95% CI = 0.71–1.33, P = 0.87) was found. Thus, in contrast to APOBEC3B mRNA levels, the APOBEC3B deletion polymorphism has neither a prognostic nor a predictive value for breast cancer patients. Although a correlation exists between APOBEC3B copy number and mRNA expression, it is relatively weak. This suggests that other mechanisms exist that may affect and therefore determine the prognostic value of APOBEC3B mRNA levels.

Anti-miR-203 suppresses ER-positive breast cancer growth and stemness by targeting SOCS3.

Breast cancer is a major public health problem worldwide in women and existing treatments are not adequately effective for this deadly disease. microRNAs (miRNAs) regulate the expression of many target genes and play pivotal roles in the development, as well as in the suppression of many cancers including breast cancer. We previously observed that miR-203 was highly upregulated in breast cancer tissues and in ER-positive breast cancer cell lines. In our present study, we observed that anti-miR-203 suppresses breast cancer cell proliferation in vitro. Orthotopic implantation of miR-203 depleted MCF-7 cells into nude mice displays smaller tumor growth as compared to control MCF-7 cells. Furthermore, miR-203 expression is significantly higher in ER-positive breast cancer patients as compared to ER-negative patients. We identified suppressor of cytokine signaling 3 (SOCS3) as a direct target of miR-203. Here we observed that miR-203 expression is inversely correlated with SOCS3 expression in ER-positive breast cancer samples. Additionally, we found that anti-miR-203 suppressed the expression of pStat3, pERK and c-Myc in MCF-7 and ZR-75-1 cells. We also demonstrated that anti-miR-203 decreased mammospheres formation and expression of stem cell markers in MCF-7 and ZR-75-1 cells. Taken together, our data suggest that anti-miR-203 has potential as a novel therapeutic strategy in ER-positive breast cancer treatment.

Lower Beclin 1 downregulates HER2 expression to enhance tamoxifen sensitivity and predicts a favorable outcome for ER positive breast cancer.

Tamoxifen(TAM) is one of the most effective endocrine treatment for estrogen receptor(ER)-positive breast cancer, however drug resistance greatly limits benefit of it. Our purpose is to uncover the role of Beclin 1 in tamoxifen resistance and prognosis of ER positive breast cancer. We established a tamoxifen resistant ER-positive breast cancer cell subline MCF-7R presenting with higher Beclin 1 and human epidermal growth factor receptor 2(HER2) levels than MCF-7. Silencing Beclin 1 decreased levels of HER2 and significantly promoted TAM sensitivity of MCF-7 and MCF-7R in vitro. Overexpression of HER2 could reverse TAM sensitivity, which was formerly increased in Beclin 1 downregulated cell. Beclin 1 level was not only positively correlated with level of HER2 but also negatively correlated with overall survival of ER-positive breast cancer patients. Using bioinformatic methods, Beclin 1 mRNA was found to be negatively correlated with overall survival in breast cancer patients receiving TAM treatment. This study indicated for the first time that lower HER2 expression by Beclin 1 downregulation contributes to alteration of tamoxifen sensitivity and low Beclin 1 predicts favorable outcome in ER-positive breast cancer.

PTP1B promotes aggressiveness of breast cancer cells by regulating PTEN but not EMT.

Metastasis is a complicated, multistep process and remains the major cause of cancer-related mortality. Exploring the molecular mechanisms underlying tumor metastasis is crucial for development of new strategies for cancer prevention and treatment. In this study, we found that protein tyrosine phosphatase 1B (PTP1B) promoted breast cancer metastasis by regulating phosphatase and tensin homolog (PTEN) but not epithelial-mesenchymal transition (EMT). By detecting PTP1B expression of the specimens from 128 breast cancer cases, we found that the level of PTP1B was higher in breast cancer tissues than the corresponding adjacent normal tissues. Notably, PTP1B was positively associated with lymph node metastasis (LNM) and estrogen receptor (ER) status. In vitro, disturbing PTP1B expression obviously attenuated cell migration and invasion. On the contrary, PTP1B overexpression significantly increased migration and invasion of breast cancer cells. Mechanistically, PTP1B knockdown upregulated PTEN, accompanied with an abatement of AKT phosphorylation and the expression of matrix metalloproteinase 2 (MMP2) and MMP7. Conversely, forced expression of PTP1B reduced PTEN and increased AKT phosphorylation as well as the expression of MMP2 and MMP7. Notably, neither EMT nor stemness of breast cancer cells was regulated by PTP1B. We also found that PTP1B acted as an independent prognostic factor and predicted poor prognosis in ER-positive breast cancer patients. Taken together, our findings provide advantageous evidence for the development of PTP1B as a potential therapeutic target for breast cancer, especially for ER-positive breast cancer patients.

Abstract 885: Epigenetic regulation of estrogen receptor transcription by the PI3K pathway in breast cancer

Abstract Mutations in the PIK3CA gene are the most frequent genomic alterations in estrogen receptor (ER)-positive breast cancers. Direct pharmacological inhibition of PI3K signaling is therefore an attractive clinical strategy and a number of PI3K pathway inhibitors are currently under clinical development. Unfortunately, although the majority of ER-positive PIK3CA-mutant patients respond, mechanisms of resistance to these inhibitors inevitably emerge. By studying both mouse models and human samples, our laboratory has previously uncovered that inhibition of PI3K pathway increases ER transcriptional activity, which in turn renders cells more susceptible to endocrine therapy. The mechanisms by which ER and PI3K signaling pathway regulate each other in breast cancer cells, however, remain elusive. To better understand the cross-talk between the PI3K pathway and the ER transcriptional program, we developed an unbiased transposon activation mutagenesis screen with the goal of identifying modulators of resistance to PI3K inhibitors in ER-positive tumors. Among the genes identified, we found a number of key regulators of ER function including the pioneer transcription factors FOXA1 and PBX1. We further confirmed that FOXA1 and PBX1 expression and transcriptional activity was enhanced upon PI3K inhibition and validated these observations in both xenograft models and samples from patients undergoing treatment with the PI3Ká inhibitor BYL719. Moreover, chromatin imunoprecipitation (ChIP)-sequencing against ER and FOXA1 demonstrated that these factors occupy the same genomic regions, and their binding is increased upon PI3K inhibition. Silencing FOXA1 or PBX1 impaired the activation of the ER-dependent transcriptional program following PI3K blockade and sensitized cells to PI3K inhibition. To better understand the role of FOXA1 and PBX1 in the ER-PI3K crosstalk, we have then studied in detail the chromatin changes upon BYL719 treatment using transposase-accessible chromatin using high-throughput sequencing (ATAC-seq) in breast cancer cells. Epigenomic profiling using ATAC-seq is also being done on patient samples collected before BYL719 administration (pre-treatment) and during therapy (on-treatment). ATAC-seq and RNA-seq data from the same patients will be integrated using novel computational approaches. These analyses will help to dissect the ER-dependent epigenetic changes occurring upon PI3K inhibition and how the cells use these chromatin modifications to adapt to the pharmacological stress. Elucidating the interconnection between the PI3K pathway and ER activity may uncover novel mechanisms of resistance to either PI3K inhibitors or endocrine therapy in ER-positive breast cancer patients. Citation Format: Eneda Toska, Hatice Osmanbeyoglu, Moshe Elkabets, Carmen Chan, Pau Castel, Maura Dickler, Scott Armstrong, Christina Leslie, Maurizio Scaltriti, Baselga José. Epigenetic regulation of estrogen receptor transcription by the PI3K pathway in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 885.

Prognostic value of the ubiquitin ligase carboxyl terminus of the Hsc70-interacting protein in postmenopausal breast cancer.

The carboxyl terminus of the Hsc70‐interacting protein (CHIP) is considered to induce the ubiquitination and degradation of several oncogenic proteins, and play a role in the inhibition of tumor progression and invasion under experimental conditions. However, the impact of CHIP expression on the prognosis of breast cancer patients has not yet been established. In this study, using an immunohistochemical method, 272 patients with invasive breast cancer were assessed for the expression of CHIP (graded scores 0‐3) and the statuses of biomarkers, such as estrogen receptor (ER), progesterone receptor (PgR), and HER2. The relationships between the statuses of CHIP and biomarkers as well as clinical features were also evaluated, and that between the expression of CHIP and patient prognosis was analyzed. We revealed that the strong expression of CHIP correlated with positive ER (P < 0.001), positive PgR (P < 0.001), and negative HER2 (P = 0.02). In postmenopausal patients, relapse‐free survival (RFS) was significantly better in the high CHIP group than in the low CHIP group (P = 0.042). In addition, RFS and cancer‐specific survival (CSS) were significantly better in patients with ER‐positive/CHIP score 3 tumors than in those with ER‐negative/CHIP score 0 tumors (RFS: P = 0.038, CSS: P = 0.0098). The methylation status of CHIP gene promoter did not always account for the down‐regulation of its expression. In conclusion, the overexpression of CHIP is a potent prognostic factor of a good prognosis in ER‐positive breast cancer patients in the postmenopausal phase.

Long non-coding RNA metastasis associated in lung adenocarcinoma transcript 1 (MALAT1) interacts with estrogen receptor and predicted poor survival in breast cancer.

Metastasis associated in lung adenocarcinoma transcript 1 (MALAT1), a lncRNA that was first recognized as a prognostic parameter for patient survival of stage I lung cancer, is up-regulated in multiple human malignancies, including breast cancer. However, the mechanism of its function remained elusive. In the current study, by examining MALAT1 expression on mRNA level, we demonstrated that compared with MCF10A, MALAT1 expression was up-regulated in the majority of breast cancer cell lines (9/12). In 26 pairs of estrogen receptor (ER)-positive breast cancer samples, MALAT1 expression was significantly up-regulated compared with adjacent normal tissues (P = 0.012). Furthermore, of 204 breast cancer patients, high MALAT1 expression was associated with positive ER (P = 0.023) and progesterone receptor (PR) (P = 0.024) status. Further analysis using TCGA database revealed that ER and its target genes PGR and CCND1, were overexpressed in MALAT1 altered group compared with unaltered group, both on the mRNA and protein level. Lastly, we verified MALAT1's prognostic value in breast cancer. At the cut-off value of 75%, MALAT1 was the only independent prognostic factor of recurrence-free survival (RFS) in ER-negative patients in a multivariate Cox regression model (hazard ratio [HR] = 2.83, 95% confidence interval [CI] 1.02–7.83). MALAT1 overexpression was also associated with poor RFS in tamoxifen treated ER-positive breast cancer patients, which might serve as a potential biomarker to predict endocrine treatment sensitivity.

Single-nucleotide polymorphism in microRNA-binding site of SULF1 target gene as a protective factor against the susceptibility to breast cancer: a case-control study.

PurposeNumerous clinical studies have suggested that chemopreventive drugs for breast cancer such as tamoxifen and exemestane can effectively reduce the incidence of estrogen receptor (ER)-positive breast cancer. However, it remains unclear how to identify those who are susceptible to ER-positive breast cancer. Accordingly, there is a great demand for a probe into the predisposing factors so as to provide precise chemoprevention. Recent evidence has indicated that ERα expression can be regulated by microRNAs (miRNAs), such as miR-206, in breast cancer. We assumed that single-nucleotide polymorphisms (SNPs) in the miR-206-binding sites of the target genes may be associated with breast cancer susceptibility with different ER statuses.MethodsWe genotyped the SNPs that reside in and around the miR-206-binding sites of two target genes – heparan sulfatase 1 (SULF1) and RPTOR-independent companion of mammalian target of rapamycin Complex 2 (RICTOR) – which were related to the progression or metastasis of breast cancer cells in 710 breast cancer patients and 294 controls by the matrix-assisted laser desorption ionization-time of flight mass spectrometry method. Modified odds ratios (ORs) with their 95% confidence intervals (CIs) were calculated by a multivariate logistic regression analysis to evaluate the potential association between the SNPs and breast cancer susceptibility.ResultsFor rs3802278, which is located in the 3′-untranslated region (3′-UTR) of SULF1, the frequency of the AA genotype was less in breast cancer patients than that in the controls as compared to that of the GG + GA genotype not only for ER-positive breast cancer patients (adjusted OR =0.663, P=0.032) but also for hormone receptor-positive breast cancer patients (adjusted OR =0.610, P=0.018). Besides, the frequency of the AA genotype was less than that of the GG genotype between the ER-positive breast cancer patients and the controls (adjusted OR =0.791, P=0.038). For rs66916453, which is located in the 3′-UTR of RICTOR, no significant difference was observed between the case and the control group for the genotypes or alleles (P>0.05).ConclusionThe SNPs in the miRNA-binding sites within the 3′-UTR of SULF1 may serve as protective factors against the susceptibility to breast cancer, especially to ER-positive breast cancer in the Chinese population. These SNPs are promising candidate biomarkers to predict the susceptibility of breast cancer and guide the administration of targeted preventive endocrine therapy.

GATA3 mRNA expression, but not mutation, associates with longer progression-free survival in ER-positive breast cancer patients treated with first-line tamoxifen for recurrent disease

In breast cancer, GATA3 mutations have been associated with a favorable prognosis and the response to neoadjuvant aromatase inhibitor treatment. Therefore, we investigated whether GATA3 mutations predict the outcome of tamoxifen treatment in the advanced setting. In a retrospective study consisting of 235 hormone-naive patients with ER-positive breast cancer who received tamoxifen as first-line treatment for recurrent disease, GATA3 mutations (in 14.0% of patients) did not significantly associate with either the overall response rate (ORR) or with the length of progression-free survival (PFS) after the start of tamoxifen therapy. Interestingly, among 148 patients for whom both mutation and mRNA expression data were available, GATA3 mutations associated with an increased expression of GATA3. However, only 23.7% of GATA3 high tumors had a mutation. Evaluation of the clinical significance of GATA3 mRNA revealed that it was associated with prolonged PFS, but not with the ORR, also in multivariate analysis. Thus, GATA3 mRNA expression, but not GATA3 mutation, is an independent predictor of prolonged PFS in ER-positive breast cancer patients who received first-line tamoxifen for recurrent disease. Besides GATA3 mutation, other mechanisms must exist that underlie increased GATA3 levels.

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

Abstract 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 &amp;lt;0.001 and P = 0.003, respectively). These SIAH2-positive tumors correlated with significant unfavorable MFS in lymph node negative, ER-positive breast cancer patients, but only in univariate analysis. In advanced disease, 86 patients (35%) had SIAH2-positive tumors which was associated with an unfavorable PFS after first-line tamoxifen in both uni- and multivariate analyses (HR = 1.45; 95% CI, 1.07 to 1.96; P = 0.015). 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 IA20: Chromatin looping factors and breast cancer

Abstract Estrogen signaling in breast cancer cells relies on chromatin interactions connecting distal regulatory elements bound by the estrogen receptor alpha (ER) to target gene promoters. This ensures stimulus and subtype-specific transcriptional responses. Chromatin looping factors, including CTCF, ZNF143 and RAD21, are genetically altered in breast cancer. However, the impact of these alterations on breast cancer development is ill defined. Here we demonstrate that ZNF143 directly regulates the formation of chromatin interactions by marking promoters connecting with distal regulatory elements. ZNF143 occupies the promoter of most early-response estrogen target genes in ER-positive breast cancer cells. Its chromatin occupancy is unaffected by estrogen stimulation suggesting that chromatin interactions are stable as opposed to modulated by stimulus. ZNF143 overexpression within ER-positive breast cancer patients associates with a worse outcome. Its loss abrogates the estrogen response in breast cancer cells. Overall, these results suggest that ZNF143 is a critical effector of the estrogen response and highlights the contribution of the chromatin looping machinery to ER-positive breast cancer development. Citation Format: Aislinn Treloar, Xue Wu, Nadia Penrod, Swneke D. Bailey, Xiaoyang Zhang, Kinjal Desai, Balazs Gyorffy, Mathieu Lupien. Chromatin looping factors and breast cancer. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr IA20.