Hormone-sensitive Breast Cancer Research Articles

Abstract P1-18-06: First-line treatment for endocrine sensitive bone-only metastatic breast cancer: Is more always better?

Abstract The standard first-line for endocrine sensitive metastatic breast cancer (BC) is represented by endocrine therapy. Several phase III clinical trials searched for more effective endocrine strategies. Nevertheless, the use of combinations for the first-line treatment of bone-only disease (BoD) is widely discussed, due to its indolent course. Our meta-analysis aims to explore the role of new endocrine strategies in BoD. A systematic review of electronic databases was conducted to identify the phase III clinical trials comparing the standard AI to novel experimental strategies. The hazard ratios (HR) for PFS were pooled in a meta-analysis. The heterogeneity of the data was evaluated by Chi-square Q test and I2 statistic. 8 studies were included in the analyses. 4 trials explored the role of CDK4/6 inhibitors (Monaleesa2 and 7, Monarch3 and Paloma2), 2 trials analyzed Fulvestrant + AI (SWOG and FACT), one trial studied Fulvestrant monotherapy (FALCON), while one trial evaluated the association between Bevacizumab and Letrozole (ALLIANCE). 6 trials reported data regarding the BoD, while 2 trials included the BoD in the non-visceral disease. Overall, the meta-analyses showed a PFS advantage for the experimental arms [HR 0.70 p 0.012], with a significant moderate/high heterogeneity [I2 66.48% p 0.004]. Only the FALCON and Paloma2 showed a significant improvement in PFS, respectively for Fulvestrant and Palbociclib + Letrozole. Considering only trials reporting data for BoD, the experimental arms significantly improved the PFS [HR 0.66 p 0.005], with a low/moderate non-significant heterogeneity [I2 44.95% p 0.106]. The novel strategies showed to be able to improve the PFS of BoD. Nonetheless, only Palbociclib + Letrozole provided statistically significant data of advantage in this setting. In clinical trials, BoD is often included in the non-visceral disease subgroup. Future clinical trials should take into account the differences in natural history and better prognosis of BoD, in order to define the best approach to these patients. Citation Format: Toss A, Venturelli M, Sperduti I, Isca C, Molinaro E, Barbieri E, Piacentini F, Omarini C, Cortesi L, Cascinu S, Moscetti L. First-line treatment for endocrine sensitive bone-only metastatic breast cancer: Is more always better? [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-18-06.

Abstract P5-04-20: Targeting CDK4/6 and IGF1R or insulin receptor synergistically inhibits growth of endocrine sensitive and endocrine resistant breast cancers

Abstract Estrogen receptor positive (ER+) breast cancers are treated with hormonal therapies such as tamoxifen or aromatase inhibitors. More than 30% of patients with early-stage ER+ breast cancer treated with endocrine therapy will relapse and all patients with metastatic breast cancer expressing ER eventually acquire resistance to hormonal therapy. Endocrine therapy resistant patients therefore require novel therapeutic options. Insulin-like growth factors (IGFs) and insulin signaling via the type I IGF receptor (IGF1R) and insulin receptor (IR) respectively regulate breast cancer biology. Unfortunately, IGF1R targeted therapies failed to show a benefit in prolonging either disease-free or overall survival in clinical trials. In pre-clinical models acquired resistance to tamoxifen results in loss of IGF1R and enhanced sensitivity to IR signaling. Inhibition of mTOR alone relieves the negative feedback loop regulating levels of the adaptor protein IRS-1, which mediates proliferative effects of IGFs and insulin and enhanced phosphorylation of Akt. The ribosomal protein S6 kinase (S6K) phosphorylates IRS-1 on serine residues targeting it for proteasomal degradation, and this negative feedback regulation is important in attenuating IGF and insulin signaling. Cyclin dependent kinases (CDKs) 4 and 6 are required for cell cycle progression. CDK4/6 inhibitors have recently been approved for treatment of ER+, Her2- advanced breast cancers and these such as palbociclib and abemaciclib block phosphorylation of retinoblastoma. IGFs and insulin stimulate cell cycle progression and increase cyclin D1 levels in breast cancers. Therefore, we hypothesized that CDK4/6 inhibition combined with IGF1R or IR targeting, to block mitogenic functions of IGF or insulin signaling, could be a viable therapeutic option in endocrine sensitive and endocrine resistant breast cancer, respectively. Parental MCF-7 and T47D were more sensitive to palbociclib compared to matched cells with acquired resistance to tamoxifen, MCF-7/TamR and T47D/TamR. Palbociclib also blocked IGF-I and insulin stimulated entry into cell cycle leading to G0/G1 arrest in ER+ breast cancer cells. Unlike mTOR inhibitors that upregulated IRS-1 levels leading to increased phosphorylation of Akt through IGF1R/IR, palbociclib did not affect IRS-1 levels and did not enhance phosphorylation of Akt in ER+ breast cancer cells. Combination of palbociclib with an IGF1R inhibitory antibody (huEM164), but not IR antibody (83-7), was better at inhibiting growth of endocrine sensitive MCF-7 and T-47D cells than either drug alone. Further, in a formal synergy study using the method of Chou-Talalay, the combination index of palbociclib and the IGF1R antibody was <1 for MCF-7 parent cells, indicating the two drugs synergistically inhibit growth. Combination of palbociclib with an IR antibody synergistically inhibited the growth of endocrine resistant MCF-7/TamR cells. Our data show that cotrageting CDK4/6 and IGF1R is more effective in endocrine sensitive but cotargeting CDK4/6 and IR is more effective in tamoxifen resistant breast cancer cells. These data indicate that targeting CDK4/6 and IR could be a therapeutic option for patients with endocrine resistant disease. Citation Format: Hoff K, Sachdev D. Targeting CDK4/6 and IGF1R or insulin receptor synergistically inhibits growth of endocrine sensitive and endocrine resistant breast cancers [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-04-20.

Abstract P4-01-15: Theprevalence of estrogen receptor-1 mutation in advanced breast cancer: The estrogen receptor one study (EROS)

Abstract Background Estrogen receptor alpha (ESRα), encoded by the estrogen receptor-1 (ESR1) gene, is expressed in approximately 70% of all breast cancers, and hormonal therapy represents a major treatment modality in ESRα-positive cancers. The most commonly used endocrine therapies inhibit ESR activity by either targeting the ESR protein itself or depriving the receptor of its ligand. Endocrine therapy has become the mainstay of prevention and treatment of ESR+ breast cancers in all stages of the disease. Furthermore, ESR status might be a strong predictor of response to endocrine therapy. About 20% of patients who present with early disease will develop resistance manifested as recurrences either during or after adjuvant endocrine treatments. While in metastatic breast cancer (MBC) patients, the resistance rate could be as high as 30%. Recent studies unveiled that these ESR1 mutations lead to constitutive activity of the ESR, meaning that the receptor is active in absence of its ligand estrogen conferring resistance against endocrine therapy. Purpose: The goal of the present study is to determine the frequency rate of ESR1 mutations in hormone-sensitive advanced breast cancer by using digital droplet PCR (ddPCR) technique. Materials and Methods This retrospective study was conducted in the Multidisciplinary Breast Clinic of the Antwerp University Hospital. The seven most common ESR1mutations (c.1138G>C (p. (E380Q)), c.1610A>G (p.(Y537C)), c.1613A>G (p.(p.D538G)), c.1607T>G (p.(L536R)), c.1387T>C (p.S463R)), c.16410A>C (p.(Y537S)), c.609T>A (p.(Y537N)) were assessed in available baseline plasma samples of women with hormone sensitive progressive breast cancer. Inclusion criteria for study participation were: female, age above 18 years, breast cancer, positive ESR expression, 5 years endocrine therapy of the primary disease, disease progression under endocrine therapy. ESR mutations were analyzed in cell-free DNA (cfDNA) by using ddPCR. Results In EROS study, ESR1 mutations were successfully examined in cfDNA from 21 patients with advanced breast cancer. In the current study, we reported positive ESR1 mutation in 19% of patients (4/21; 95% CI, 5%-42%). The test sensitivity was lower than the targeted value <0.1% in 29% of patients (6/21). No significant statistical difference in baseline clinical characteristics was observed in patients with wild-type and mutant ESR (p>0.05). All the patients had received AI with a variable period of good response. Adjuvant endocrine therapy for primary disease was Tamoxifen (TAM) for 57% of patients (12 of 21) of whom 8 patients had received aromatase inhibitor (AI) after two years, while 43% of patients (9 of 21) had received AI as first line adjuvant hormonal therapy. However, there was no sufficient number of samples to formally analyze the clinical impact of ESR mutation on the type of endocrine therapy. Conclusion ESR1 mutation analysis should be considered in hormone-sensitive MBC patients to improve the therapeutic strategies in controlling ESR signaling before the occurrence of wide spread disease metastasis. Key words: Estrogen receptor, Mutation, breast cancer, hormonal therapy, and metastasis. Citation Format: Najim O, Huizing M, Tjalma W. Theprevalence of estrogen receptor-1 mutation in advanced breast cancer: The estrogen receptor one study (EROS) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-01-15.

Abstract P4-14-03: Influence of competing risks of death on the interpretation of adjuvant endocrine therapy trials for breast cancer

Abstract Background: Early stage, hormone sensitive breast cancer is associated generally with a good prognosis, with only a minority of patients expected to die of breast cancer. Death from causes other than breast cancer can dilute the patients at risk of breast cancer events and result in over-estimation of risk of recurrence and consequently the benefit from breast cancer therapy, a so-called immortal time bias. The MA.17R trial (Goss et al 2016) evaluated the role of extending adjuvant treatment with letrozole from 5 to 10 years. Here we determine the effect of analyzing the MA.17R trial using methods accounting for competing risks. Methods: We compared conventional and competing risk methods for disease-free survival (DFS) and for distant recurrence-free survival (DRFS). In Kaplan-Meier analyses death from any cause was considered an event while cumulative incidence functions (CIFs) assumed death without recurrence to be a competing risk. The complement of the survival function (one minus the survival function) was used to estimate incidence of the primary event of interest. This was compared to estimates obtained using CIFs accounting for the occurrence of competing events. Results: Non-breast cancer death was the most common event defining DFS and DRFS. Over the course of follow-up, there was increasing discrepancy between the risk of disease recurrence measured using Kaplan-Meier and CIF. Among letrozole treated patients the estimated distant recurrence at 5 years of follow-up was 5.4% using CIF and 9.6% using Kaplan-Meier. At 10 years of follow-up, the estimated distant recurrence was 8.4% using CIF and 20.0% using Kaplan-Meier. Similar results were observed for the placebo group (8.5% vs 12.1% at 5 years and 14.8% vs 27.3% at 10 years), and in patients with baseline cardiovascular disease (see Table). Benefit from letrozole on DFS and DRFS was greater when accounting for competing risk (hazard ratio [HR] for DFS 0.66, 95%CI 0.48-0.90; DFRS HR 0.75, 0.50-1.14) compared to the conventional method (DFS HR 0.79, 0.62-0.99; DRFS HR 0.91, 0.70-1.18). In women with baseline cardiovascular risk, the benefits of extended adjuvant letrozole when considering competing risk (DFS HR 0.38, 0.16-0.89; DRFS HR 0.46, 0.16-1.35) were also greater than those observed in the conventional analysis (DFS HR 0.55, 0.32-0.93; DRFS HR 0.59, 0.33-1.04). Treatment with extended letrozole did not influence non-breast cancer death in women who died with disease recurrence (HR 1.06, 0.74 -1.50) or in those with competing risk or censored from the analysis (HR 1.05, 0.73 -1.49). Conclusion: Over the course of follow-up, estimates of DFS and DRFS differ increasingly if measured using Kaplan-Meier or CIF, with CIF estimates of risk being substantially lower. Using a competing risk model, the reduction in distant recurrence at 8 years with extended letrozole is less than 1%. Additional competing risk analyses of the MA.17 (Goss 2006) and MA.27 (Goss 2013) trials are ongoing. Cumulative incidence of disease recurrence in patients with baseline cardiovascular riskTime (years)CIF (%)1-KM (%)Letrozole11.51.534.46.555.813.8Placebo13.53.538.311.8512.520.3 Citation Format: Ethier J-L, Parulekar W, Shepherd L, Summers L, Strasser-Weippl K, Tu D, Amir E. Influence of competing risks of death on the interpretation of adjuvant endocrine therapy trials for breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-14-03.

Abstract P5-04-16: Total estrogenic activity during neoadjuvant therapy with letrozole and exemestane – An intra-patient cross-over comparison using the AroER tri-screen

Abstract Background. Aromatase inhibitors (AIs), letrozole (Femarâ / Femaraâ) and exemestane (Aromasinâ), are widely used anti-hormonal drugs for breast cancer. Both compounds strongly reduce circulating estradiol levels in postmenopausal women. In the setting of metastatic breast cancer, these drugs may be used after another, causing new responses in selected patients by switching to the alternative drug after progressing on the first choice. This well-known ”lack of cross resistance” has been recognized for some time and is documented by several clinical trials. However, the precise explanation for this clinical observation is still unknown. The availability of mechanistic information may lead to an improved strategy against hormone-sensitive breast cancer. Patients and methods. NEO-LET-EXE was a neoadjuvant, randomized, open-label, intra-patient cross-over trial. Postmenopausal patients with estrogen receptor (ER) positive (>50%), HER-2 negative, locally advanced breast cancer were enrolled. Sequential blood samples (obtained at baseline, after 2 months and 4 months of treatment) were available from 29 patients. All patients were randomized to sequential treatment starting with either letrozole (2.5 mg o.d.) or exemestane (25 mg o.d) for 2 months followed by another 2 months on the alternative compound. The total estrogenic activities in the collected blood samples were determined using AroER tri-screen assay developed by the Chen laboratory. The assay utilizes MCF-7aro ERE cells which contain both aromatase and ER. The samples were assayed in the presence as well as the absence of letrozole, to estimate relative contributions of estrogen and estrogen+androgen to the activities, respectively. Results. Using the highly sensitive AroER tri-screen assay, estrogenic activity were detected at three time points in all blood samples. Importantly, a significantly higher total estrogenic activity was found during therapy with exemestane compared to letrozole in 23 out of 26 patients. Only in three patients, the activity was higher during therapy with letrozole compared to exemestane. When letrozole was included in the assay, the estrogenic activities in most samples collected during exemestane treatment were further reduced, suggesting that low levels of androgen were present in samples from exemestane treatment. Four samples collected after exemestane treatment and three after letrozole treatment had higher activities than baseline samples when assay was carried out with letrozole. Discussion. Our results suggest the AroER tri-screen to be a very sensitive method to estimate the overall estrogen-mediated activity in human samples. Significant higher levels of estrogenic activity in human serum were observed during exemestane than those during letrozole treatment. Our observations, that additional letrozole could reduce further the estrogen activity in the exemestane-treated samples, demonstrate probably residual aromatase activity during therapy with exemestane alone.In addition to distinguish the effects of exemestane and letrozole, our results also demonstrate that the assay can also potentially detect the effects of estrogenic mimics. Citation Format: Bahrami N, Chang G, Kanaya N, Sauer T, Gravdehaug B, Chen S, Geisler J. Total estrogenic activity during neoadjuvant therapy with letrozole and exemestane – An intra-patient cross-over comparison using the AroER tri-screen [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-04-16.

Abstract P2-06-14: The role of SLC7A5 (LAT1) in endocrine therapy-resistant breast cancer

Abstract Endocrine therapies are commonly used to treat estrogen receptor-positive (ER+) breast cancers, which comprise 70% of all new breast cancer cases. Unfortunately, emergence of resistance to these therapies presents a major clinical challenge. Cancer cells can adapt to the dysregulation of cellular metabolism induced by endocrine therapy in order to evade cell death.Central to this adaptation is the scavenging of free-formed amino acids from the tumor microenvironment. For example, we found 109 solute carrier (SLC) mRNAs to be differentially expressed between endocrine-sensitive and resistant cells. We began our mechanistic studies of these genes with SLC family 7 member 5 (SLC7A5 or LAT1). SLC7A5 is a key component of a transmembrane transporter, which can complex with CD98 and increase the uptake of large, neutral amino acids (such as leucine or tyrosine). We used a panel of endocrine therapy-resistant (LCC9) and sensitive (MCF7; LCC1) breast cancer cells. SLC7A5 expression was upregulated by estrogen in MCF7 and LCC1 cells; this induction was blocked by fulvestrant treatment. Basal expression of the SLC7A5 protein in the absence of estrogen was 2.75-fold higher in LCC9 cells compared with MCF7 cells; SLC7A5 mRNA expression was 71-fold higher. Fulvestrant treatment did not significantly alter SLC7A5 mRNA or protein expression in LCC9 cells. Inhibiting SLC7A5 function using either a pharmacological inhibitor (JPH203), or depleting expression using siRNA, led to significant suppression of LCC9 cell growth. Cell cycle analysis revealed that SLC7A5 depletion caused cells to accumulate in the G1-phase, with a concurrent reduction of cells in S-phase. In four publicly available datasets of ER+, tamoxifen treated breast cancer patients, high expression of SLC7A5 was significantly associated with poor relapse-free survival. This study uncovers a novel adaptive mechanism in endocrine therapy-resistant breast cancer cells that is facilitated by increased expression of SLC7A5, which enables them to supplement their increased metabolic needs and promoting cell growth. Blocking the functions of SLC7A5, perhaps in conjunction with inhibition of autophagy, may therefore offer a new avenue of potential therapeutic intervention against endocrine therapy-resistant breast cancers. Citation Format: Sevigny CM, Sengupta S, Luo Z, Jin L, Pearce D, Clarke R. The role of SLC7A5 (LAT1) in endocrine therapy-resistant breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-06-14.

Abstract P1-05-03: Isoform specific targeting of insulin receptor

Abstract The insulin receptor (InsR) exists in both an A and B isoform. InsR-B differs from InsR-A by the inclusion of exon 11, which encodes 12 amino acid residues at the C-terminus of the InsR alpha-subunit. Increased InsR-A expression is associated with mitogenic signaling pathways while InsR-B is linked to insulin-mediated metabolic functions. Predominant InsR-A expression may therefore be important in growth and fetal development of embryos, whereas predominant InsR-B expression has a role in metabolic insulin action in adult life. Increased InsR-A expression is seen in breast cancer. In endocrine resistant breast cancer, InsR-A is expressed at high levels (Gradishar, et al. Clin Cancer Res 22:301 2016 PMID: 26324738). Thus, developing InsR-A specific inhibitors could be a useful therapy for breast cancer. We have previously published InsR specific binders using a T7 phage gene 2 protein (Gp2), a small protein scaffold (Chan, et al. Mol Cancer Ther 16:1324 2017 PMID: 28468775), with the long-term goal of creating effective InsR inhibitors and diagnostics. Using yeast display and directed evolution, we identified three Gp2 variants (Gp2 #1, #5, and #10) with low nanomolar affinity and specific binding to cell surface InsR. We have shown that these Gp2 variants inhibited insulin-mediated monolayer proliferation in both endocrine-sensitive and resistant breast cancer, but did not downregulate InsR expression. To further characterize the specificity of Gp2 variants, we used two techniques. HEK293T cells were infected with lentiviral vectors expressing either InsR-A tagged with mCherry or InsR-B tagged with eGFP. Using these cells, we performed “mock panning” and showed the Gp2 #5 variant bound both InsR-A and InsR-B, but had higher affinity for InsR-B. We also incorporated Gp2 #5 into the capsid of a tropism-null adeno-associated virus (AAV). Using this Gp2-AAV, we infected HEK293T-InsR-A or InsR-B cells at a number of different multiplicities of infection. These data were consistent with panning data and showed specific Gp2-AAV infection of cells expressing high levels of InsR-B, but not InsR-A. Thus, our data show that Gp2 variants we created have a higher affinity for InsR-B than InsR-A. Despite this preferred affinity, these Gp2 binders have sufficient binding to InsR-A to disrupt the biological effects of insulin in breast cancer cells. Thus, even relatively low affinity binding to InsR-A can disrupt its function. Further development of InsR-A Gp2 binders may be developed and provide more specific targeting of the breast cancer specific isoform of InsR. Citation Format: LaPara K, Chan JY, Zdechlik A, Ljunggren K, Schmidt D, Hackel B, Yee D. Isoform specific targeting of insulin receptor [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-05-03.

Abstract P5-04-02: Progesterone receptor (PR) antagonism by telapristone acetate (TPA): A randomized, placebo-controlled phase IIB pre-surgical window trial in women with stage 0-II breast cancer

Abstract Background: In vitro and preclinical data indicate that TPA, a selective PR modulator, has activity against hormone-sensitive early breast cancer. We conducted a pre-surgical window trial of oral TPA in Stage 0-II breast cancer to assess the effect of TPA on suppression of cell proliferation (Ki67), and on differential gene expression in responsive and non-responsive tumors. Methods: We enrolled 70 pre and postmenopausal women into a 1:1 randomized, double-blind, placebo-controlled trial of oral TPA 12mg (Repros Therapeutics Inc.) for 2-10 weeks. The primary endpoint was Ki67 labelling, comparing diagnostic core needle biopsy to post-therapy surgical specimens. Ki67 changes were quantitated by dual immunohistochemistry (Ki67/pan-cytokeratin) and image analysis (Aperio ImageScope and Definiens Tissue Studio®). RNA-sequencing (using RNA extracted from the paraffin blocks) was performed with Illumina TruSeq RNA Coding Access method. Differential gene expression pre-post therapy was assessed, followed by Gene Set Enrichment Analysis for pathway analysis. Ki67 changes from baseline were tested with Paired signed-rank test. For gene expression analysis, p values were calculated by Wald test and adjusted for multiple comparisons by Benjamini-Hochberg method (adjusted p <0.05 and 2-fold gene expression cut-off). Results: Among 61 evaluable women, (29 placebo and 32 TPA) 97% of tumors were ER or PR positive and 91% were ER and PR positive (balanced across arms). A significant 6% decrease in mean %Ki67 was seen in the TPA arm (p= 0.003). When stratified by menopause, the significance held in premenopausal women (n= 22, p= 0.03) but not in postmenopausal women (n=10, p= 0.08). However, a Ki67 decrease (4%) was also observed in placebo group (p = 0.04); this was non-significant after pre- postmenopausal stratification. Overall, differential gene expression analysis showed no significant modulation of genes in either group. Using a pre-specified response parameter (50% relative reduction in Ki67), we identified 12/32 (38%) “responders” in the TPA, and 9/29 (31%) in the placebo arm. In sub-group analysis of these responders, we found 103 genes to be significantly modulated by treatment in the TPA “responders”, but saw no significant change in any gene expression in placebo “responders”. Gene set enrichment analysis for the 103 genes showed that TPA blocked the progression of cell cycle genes (PTTG1, PLK1, UBE2C, HIST1H3F, PSMD3, and etc.) and suppressed PGR and ERBB2 expression. In a pre-planned pooled analysis, these results will be combined with NCT02314156, reported in SABCS abstract 851790. Conclusions: An anti-proliferative (Ki67) signal of TPA was observed in early stage breast cancer patients, but interpretation was limited by placebo group changes. The TPA group demonstrated differential suppression of proliferation-related genes among Ki67 responders, but the placebo group did not. Ongoing analysis will examine signatures related to stemness, metastasis, and immune suppression (potentially better endpoints in trials targeting P signaling). These analyses may help us select the right population and the right biomarkers for future trials. Citation Format: Lee O, Sullivan ME, Xu Y, Shidfar A, Ivancic D, Zeng Z, Singhal H, Helenowski I, Jovanovic B, Hansen N, Bethke K, Gann P, Gradishar WJ, Clare SE, Khan SA. Progesterone receptor (PR) antagonism by telapristone acetate (TPA): A randomized, placebo-controlled phase IIB pre-surgical window trial in women with stage 0-II breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-04-02.

Abstract PD7-07: Synergistic targeting of CDK4/6 and BCL-2 pathways in estrogen receptor positive breast cancer

Abstract Background: Despite incremental advances in chemotherapy and endocrine therapy, survival outcomes for patients with ER-positive (ER+) metastatic breast cancer (MBC) remain poor. The majority of relapsing tumors exhibit deregulation of the cyclin-dependent kinase 4 and 6 (CDK4/6)/cyclin D1 (CCND1)/Rb signaling pathway. CDK4/6 inhibitors (such as palbociclib) in combination with endocrine therapy have been shown to significantly improve progression free survival in patients who are in 1st or 2nd line relapse, although overall survival benefit has yet to be demonstrated. This may reflect their largely cytostatic mechanism of action, with minimal induction of tumor cell death. Thus, combinatorial strategies that also induce apoptosis could be beneficial. Notably, the pro-survival protein BCL-2 is overexpressed in the majority of ER+ tumors and the potent and specific BCL-2 inhibitor venetoclax (ABT-199) has been found to synergize with endocrine therapy in patient derived xenograft (PDX) models. Promising activity has also been observed in an early phase clinical trial. We therefore investigated dual targeting of the CDK4/6 and BCL-2 pathways in pre-clinical models of ER+ and BCL-2+ breast cancer. Results: We first examined endocrine sensitive or resistant cell-lines and found that pro-survival BCL-2 proteins were upregulated in resistant cells. BCL-2 family protein levels were also found to be elevated in palbociclib resistant cells, suggesting that BCL-2 could represent a therapeutic target. We next determined whether venetoclax improved response to dual therapy comprising the selective estrogen receptor degrader fulvestrant and palbociclib. In clonogenic assays of endocrine sensitive breast cancer cell lines, triple therapy containing venetoclax significantly reduced the number and size of colonies, when compared to double therapy. The addition of venetoclax to fulvestrant/palbociclib also augmented cell death in tumor organoid models derived from either ER+ BCL-2+ primary tumors or PDX models. Moreover, triple therapy improved tumor response and overall survival in mice bearing ER+ BCL-2+ PDX tumors. Mechanistically, this was accompanied by increased apoptosis and reduced cellular proliferation (as determined by cleaved caspase-3 and Ki67 levels, respectively). As CDK4/6 inhibitors have recently been shown to promote anti-tumor immunity, we evaluated immune modulation using the ER+ 67NR cell line in a syngeneic (BALB/c) mouse mammary tumor model. Similar to the PDX models, triple therapy comprising fulvestrant, palbociclib and venetoclax was more effective than double therapy comprising either fulvestrant/palbociclib or fulvestrant/venetoclax. Flow cytometric analysis of tumors revealed that this was accompanied by a reduced intratumoral FOXP3+:cytotoxic CD8 T-cell ratio. Conclusions: The addition of the BCL-2 inhibitor venetoclax to conventional therapy comprising endocrine therapy and a CDK4/6 inhibitor augments tumor response and elicits a favorable intratumoral immune profile. Collectively, these findings support investigation of combination therapy in the clinic for patients with ER+ BCL-2+ MBC. Citation Format: Whittle JR, Vaillant F, Policheni AN, Liu K, Pal B, Giner G, Fernandez K, Gray DH, Caldon CE, Smyth GK, Visvader JE, Lindeman GJ. Synergistic targeting of CDK4/6 and BCL-2 pathways in estrogen receptor positive breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD7-07.

Abstract OT1-01-01: The NEO-LET-EXE-trial: An intra-patient cross-over trial to explore the "lack of cross-resistance" between steroidal and non-steroidal aromatase inhibitors

Abstract Background. The aromatase inhibitor letrozole and the aromatase inactivator exemestane currently belong to the most widely used antihormonal drugs for breast cancer worldwide. Both compounds are strongly suppressing estradiol levels in postmenopausal patients with breast cancer. However, in the metastatic setting, these drugs may be used after another, causing new responses in selected patients following switching to the alternative drug after progressing on the first choice. This well-known “lack of cross resistance” has been recognized for some time and is documented by several trials. However, the precise explanation for this clinical observation is still unknown. The solution may potentially lead us to a novel strategy to treat hormone-sensitive breast cancer. Trial design. NEO-LET-EXE is a neoadjuvant, randomized, open-label, intra-patient cross-over trial . Eligibility criteria. Postmenopausal patients suffering from estrogen receptor (ER) positive (>50%), HER-2 negative, locally advanced breast cancer, suitable for neoadjuvant/presurgical antihormonal therapy, may be enrolled. Age: 18+ (no upper limit). Specific aims. To explain the phenomenon of a lack of cross-resistance between steroidal and non-steroidal aromatase inhibitors in vivo. Sequential tumor biopsies and blood samples, obtained at baseline and following 2 months of therapy with each drug given in sequence, will be used to perform a comprehensive exploration of the consequences of each drug therapy. The influence on plasma and tissue steroids (estrogens, androgens, etc.) will be compared. In addition, whole genome sequencing, whole exome sequencing, epigenetics, proteomics and plasma analysis (cytokines, tumor DNA fragments, etc.) will be performed. Statistical methods. Data will be analyzed using mixed effects models. Present accrural and target accrural. 49 out of planned 100 patients have been enrolled so far. The last patient is expected to enter the trial in Q4 2019. Citation Format: Bahrami N, Sauer T, Loeng M, Gravdehaug B, Engebretsen SS, Aljabri B, Bemanian V, Lindstrøm JC, Lüders T, Kristensen VN, Geisler J. The NEO-LET-EXE-trial: An intra-patient cross-over trial to explore the "lack of cross-resistance" between steroidal and non-steroidal aromatase inhibitors [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr OT1-01-01.

Tamoxifen and pregnancy: an absolute contraindication?

Breast cancer is the most common malignancy among young women of reproductive age. Adjuvant treatment with tamoxifen reduces the risk of recurrence in hormone-sensitive breast cancer. However, the use of tamoxifen is considered contraindicated during pregnancy, because of a limited number of case reports demonstrating potential adverse effects on the fetus. The objective of this report is to give a more broad overview of the available data on the effect of tamoxifen exposure during pregnancy. A literature review was performed using PubMed and the databases of the Netherlands Pharmacovigilance Centre Lareb and of the International Network on Cancer, Infertility, and Pregnancy. A total of 238 cases of tamoxifen use during pregnancy were found. Of the 167 pregnancies with known outcome, 21 were complicated by an abnormal fetal development. The malformations described were non-specific and the majority of cases concerned healthy infants despite exposure to tamoxifen. There seems to be an increased risk of fetal abnormalities when taking tamoxifen during pregnancy (12.6% in contrast to 3.9% in the general population), but the evidence is limited and no causal relationship could be established. The possible disadvantage of postponing or discontinuing tamoxifen for the maternal prognosis is unclear. Patients should be counseled about the use of tamoxifen during pregnancy instead of presenting it as being absolutely contraindicated.

Liver Kinase B1-A Potential Therapeutic Target in Hormone-Sensitive Breast Cancer in Older Women.

Background: The role of liver kinase B1 (LKB1), a serine/threonine kinase, has been described in the development of PeutzJagher’s syndrome, where a proportion (~45%) of patients have developed breast cancer in their lifetime. Cell line studies have linked LKB1 with oestrogen receptors (ER) and with the Adenosine monophosphate-activated protein kinase (AMPK) pathway for energy metabolism. However, limited studies have investigated protein expression of LKB1 in tumour tissues and its intracellular relationships. This study aimed to investigate the intracellular molecular relationships of LKB1 in older women with early operable primary breast cancer and its correlation with long-term clinical outcome. Methods: Between 1973 and 2010, a consecutive series of 1758 older (≥70 years) women with T0-2N0-1M0 breast carcinoma were managed in a dedicated facility. Of these, 813 patients underwent primary surgery, and 575 had good quality tumour samples available for tissue microarray construction. LKB1 was assessed in 407 cases by indirect immunohistochemistry (IHC). Tumours with 30% or more of cells with cytoplasmic LKB1 expression were considered positive. LKB1 expression was compared with tumour size, histological grade, axillary lymph node stage, ER, PgR, EGFR, HER2, HER3, HER4, BRCA1&2, p53, Ki67, Bcl2, Muc1, E-Cadherin, CD44, basal (CK5, CK5/6, CK14 and CK17) and luminal (CK7/8, CK18 and CK19) cytokeratins, MDM2 and MDM4, and correlated with long-term clinical outcome. Results: Positive LKB1 expression was seen in 318 (78.1%) patients, and was significantly associated with high tumour grade, high Ki67, over-expression of HER2, VEGF, HER4, BRCA2, MDM2 and negative expression of CD44 (p < 0.05). There was no significant correlation with tumour size, axillary lymph node status, ER, PgR, p53, basal or luminal cytokeratins, Bcl2, Muc1, EGFR, HER3, MDM4, E-cadherin and BRCA1. LKB1 did not show any significant influence on survival in the overall population; however, in those patients receiving adjuvant endocrine therapy for ER positive tumours, those with positive LKB1 had significantly better 5-year breast cancer specific survival when compared to those without such expression (93% versus 74%, p = 0.03). Conclusion: LKB1 expression has shown association with poor prognostic factors in older women with breast cancer. However, LKB1 expression appears to be associated with better survival outcome among those patients receiving adjuvant endocrine therapy. Further research is required to explore its potential role as a therapeutic target.

Overexpression of the steroidogenic acute regulatory protein in breast cancer: Regulation by histone deacetylase inhibition

Dysregulation of steroid biosynthesis has been implicated in the pathophysiology of a variety of cancers. One such common malignancy in women is breast cancer that is frequently promoted by estrogen overproduction. All steroid hormones are made from cholesterol, and the rate-limiting step in steroid biosynthesis is primarily mediated by the steroidogenic acute regulatory (StAR) protein. Whereas the involvement of StAR in the regulation steroid hormone biosynthesis is well established, its association to breast cancer remains obscure. Herein, we report that estrogen receptor positive breast cancer cell lines (MCF7, MDA-MB-361, and T-47D) displayed aberrant high expression of the StAR protein, concomitant with 17β-estradiol (E2) synthesis, when compared their levels with normal mammary epithelial (MCF10A and MCF12F) and triple negative breast cancer (MDA-MB-468, MDA-MB-231, and BT-549) cells. StAR was identified as a novel acetylated protein in MCF7 cells, in which liquid chromatography-tandem mass spectrometry analysis identified seven StAR acetyl lysine residues under basal and in response to histone deacetylase (HDAC) inhibition. A number of HDAC inhibitors were capable of diminishing StAR expression and E2 synthesis in MCF7 cells. The validity of StAR protein acetylation and its correlation to HDAC inhibition mediated steroid synthesis was demonstrated in adrenocortical tumor H295R cells. These findings provide novel insights that StAR protein is abundantly expressed in the most prevalent hormone sensitive breast cancer subtype, wherein inhibition of HDACs altered StAR acetylation patterns and decreased E2 levels, which may have important therapeutic implications in the prevention and treatment of this devastating disease.

Associations of adult-attained height and early life energy restriction with postmenopausal breast cancer risk according to estrogen and progesterone receptor status.

Adult-attained height is a marker for underlying mechanisms, such as cell growth, that may also influence postmenopausal breast cancer (BC) risk, perhaps specifically hormone-sensitive BC subtypes. Early life energy restriction may inhibit these mechanisms, resulting in shorter height and a reduced postmenopausal BC risk. Women (62,573) from the Netherlands Cohort Study completed a self-administered questionnaire in 1986 when 55-69 years old, and were followed-up for 20.3 years (case-cohort: Nsubcohort = 2,438; Ncases = 3,354). Cox multivariable-adjusted hazard ratios (HR) and 95% confidence intervals (CI) were estimated for BC risk overall and by estrogen and progesterone receptor subtypes in relation to height and early life energy restriction during the Hunger Winter, War Years, and Economic Depression. Although energy restriction can only influence longitudinal growth in women exposed before and/or during the growth spurt, it may also influence BC risk when occurring after the growth spurt, possibly through different growth processes. Therefore, Cox analyses were additionally conducted according to timing of energy restriction in relation to the growth spurt. Height was associated with an increased BC risk (HRper 5cm = 1.07, 95%CI:1.01-1.13), particularly hormone receptor-positive BC. Energy restriction before and/or during the growth spurt was associated with a decreased hormone receptor-positive BC risk. Energy restriction during the Hunger Winter increased the estrogen receptor-negative BC risk regardless of the timing of energy restriction. In conclusion, height and energy restriction before and/or during the growth spurt were both associated with hormone receptor-positive BC risk, in the direction as expected, indicating critical exposure windows for hormonal growth-related mechanisms.

Retraction: Aromatase Inhibitor-mediated Downregulation of INrf2 (Keap1) Leads to Increased Nrf2 and Resistance in Breast Cancer.

Aromatase inhibitors (AIs) are effective drugs that reduce or eliminate hormone sensitive breast cancer. However, despite their efficacy, resistance to these drugs can occur in some patients. The INrf2 (Keap1):Nrf2 complex serves as a sensor of drug/radiation-induced oxidative/electrophilic stress. INrf2 constitutively suppresses Nrf2 by functioning as an adapter protein for the Cul3/Rbx1-mediated ubiquitination/degradation of Nrf2. Upon stress, Nrf2 dissociates from INrf2, is stabilized, translocates to the nucleus, and coordinately induces a battery of cytoprotective gene expression. Current studies investigated the role of Nrf2 in AI resistance. RT-PCR and immunoblot assays showed that AI-resistant breast cancer LTLTCa and AnaR cells express lower INrf2 and higher Nrf2 protein levels, as compared to drug sensitive MCF-7Ca and AC1 cells, respectively. The increase in Nrf2 was due to lower ubiquitination/degradation of Nrf2 in AI-resistant cells. Higher Nrf2-mediated levels of biotransformation enzymes, drug-transporters and anti-apoptotic proteins contributed to reduced efficacy of drugs and aversion to apoptosis that led to drug resistance. shRNA inhibition of Nrf2 in LTLTCa (LTLTCa-Nrf2KD) cells reduced resistance and sensitized cells to AI exemestane. Interestingly, LTLTCa-Nrf2KD cells also showed reduced levels of aldehyde dehydrogenase, a marker of Tumor-Initiating Cells and significantly decreased mammosphere formation, as compared to LTLTCa-Vector control cells. The results together suggest that persistent AI treatment down-regulated INrf2 leading to higher expression of Nrf2 and Nrf2 regulated cytoprotective proteins that resulted in increased AI drug resistance. These findings provide a rationale for the development of Nrf2 inhibitors to overcome resistance and increase efficacy of AI.

Pharmacokinetic evaluation of a transdermal anastrozole-in-adhesive formulation.

Background and objectiveAnastrozole is a well-established active pharmaceutical ingredient (API) used for the treatment of hormone-sensitive breast cancer (BC) in postmenopausal women. However, treatment with the only available oral formulation is often associated with concentration-dependent serious side effects such as hot flashes, fatigue, muscle and joint pain, nausea, diarrhea, headache, and others. In contrast, a sustained-release system for the local application of anastrozole should minimize these serious adverse drug reactions.MethodsAnastrozole-in-adhesive transdermal drug delivery systems (TDDS) were developed offering efficient loading, avoidance of inhomogeneity or crystallization of the drug, the desired controlled release kinetics, storage stability, easy handling, mechanical stability, and sufficient stickiness on the skin. In vitro continuous anastrozole release profiles were studied in Franz diffusion cells. In vivo, consecutive drug plasma kinetics from the final anastrozole transdermal system was tested in beagle dogs. For drug analysis, a specific validated liquid chromatography– mass spectrometry method using fragment ion detection was developed and validated.ResultsAfter efficient drug loading, a linear and sustained 65% drug release from the TDDS over 48 h was obtained. In vivo data showed a favorable anastrozole plasma concentration–time course, avoiding side effect-associated peak concentrations as obtained after oral administration but matching therapeutic plasma levels up to 72 h.ConclusionThese results provide the basis for establishing the transdermal application of anastrozole with improved pharmacokinetics and drug safety as novel therapeutic approach and promising option to treat human BC by decreasing the high burden of unwanted side effects.

337P - First-line treatment for endocrine sensitive bone-only metastatic breast cancer: Is more always better?

337P - First-line treatment for endocrine sensitive bone-only metastatic breast cancer: Is more always better?

Effects of Radiotherapy in Early-Stage, Low-Recurrence Risk, Hormone-Sensitive Breast Cancer.

Radiotherapy after breast conservation has become the standard of care. Prior meta-analyses on effects of radiotherapy predated availability of gene expression profiling (GEP) to assess recurrence risk and/or did not include all relevant outcomes. This analysis used GEP information with pooled individual-level data to evaluate the impact of omitting radiotherapy on recurrence and mortality. We considered trials that evaluated or administered radiotherapy after lumpectomy in women with low-risk breast cancer. Women included had undergone lumpectomy and were treated with hormonal therapy for stage I, ER+ and/or PR+, HER2- breast cancer with Oncotype scores no greater than 18. Recurrence-free interval (RFI), type of RFI (locoregional or distant), and breast cancer-specific and overall survival were compared between no radiotherapy and radiotherapy using adjusted Cox models. All statistical tests were two-sided. The final sample included 1778 women from seven trials. Omission of radiotherapy was associated with an overall adjusted hazard ratio of 2.59 (95% confidence interval [CI] = 1.38 to 4.89, P = .003) for RFI. There was a statistically significant increase in any first locoregional recurrence (P = .001), but not distant recurrence events (P = .90), or breast cancer-specific (P = .85) or overall survival (P = .61). Five-year RFI rate was high (93.5% for no radiotherapy vs 97.9% for radiotherapy; absolute reduction = 4.4%, 95% CI = 0.7% to 8.1%, P = .03). The effects of radiotherapy varied across subgroups, with lower RFI rates for those with Oncotype scores of less than 11 (vs 11-18), older (vs younger), and ER+/PR+ status (vs other). Omission of radiotherapy in hormone-sensitive patients with low recurrence risk may lead to a modest increase in locoregional recurrence event rates, but does not appear to increase the rate of distant recurrence or death.

CYP2D6*3 (A2549del), *4 (G1846A), *10 (C100T) and *17 (C1023T) genetic polymorphisms in Iranian breast cancer patients treated with adjuvant tamoxifen.

There is controversy regarding the efficacy of tamoxifen in breast cancer patients who are carriers of cytochrome P450 2D6 (CYP2D6) gene polymorphisms. Poor metabolizer genotypes may not fully convert tamoxifen to its active metabolite endoxifen and thus have less exposure to anti-estrogen therapy. The present study was conducted to identify the prevalence of CYP2D6 genotypes among Iranian breast cancer patients. A total of 84 estrogen receptor-positive breast cancer patients treated at a referral center in the north of Iran were examined. A peripheral blood sample was obtained from each patient to determine the presence of *3, *4, *10 and *17 single nucleotide polymorphisms of the CYP2D6 gene by polymerase chain reaction-based restriction fragment-length polymorphism analysis. Of the four genotypes assessed, CYP2D6*4 was the most common variant and was identified in 41 (48.8%) patients as heterozygous (G/A) and 3 (3.6%) as homozygous (A/A) alleles. CYP2D6*10 heterozygous mutated alleles (C/T) were also a common genotype that presented in 22 (26.2%) of the study subjects. Variant *17 was less common and was detected only as heterozygous (C/T) in 3 patients (3.6%). No CYP2D6*3 heterozygous or homozygous mutated alleles were observed. In conclusion, the frequency of the CYP2D6 nonfunctional alleles *4 and *10 appeared relatively high in Iranian patients with hormone-sensitive breast cancer. This finding may affect the selection of an optimal hormone therapy, as patients with low CYP2D6 pathway activity may not sufficiently convert tamoxifen to its active metabolite endoxifen.

Abstract B10: HDAC inhibition modulates immune checkpoint pathway in triple-negative breast cancer

Abstract Background: Breast cancer remains one of the most serious and common diseases, second only to lung cancer as a leading cause of cancer death in women, accounting for 30% of new diagnoses. In particular, hormone-sensitive metastatic breast cancer and triple-negative breast cancer (TNBC) remains a therapeutic challenge. Despite aggressive therapeutic options, resistance develops in almost all patients and survival remains poor, prompting the need for novel approaches. This project focuses on the characterization of the molecular mechanisms underlying epigenetic modifiers and in particular Histone deacetylase inhibitors (HDACi) as priming modulators of immunotherapy, mainly focusing on the diversity between TNBC and hormone responsive breast cancer. HDACi represent a new class of anticancer agents that can affect multiple cancer pathways, including reverse hormone therapy resistance, resulting in prolonged antitumor responses in patients. In addition to their effects on tumor signaling, HDACi have immune modulatory functions, including modulation of regulatory T-cells, Foxp3 expression, changes in tumor-infiltrating lymphocyte composition and induction of PD-L1 expression. The PD-1/PD-L1 pathway is an immune checkpoint inhibitor pathway that under normal conditions down-regulates cytotoxic T-cell activity to maintain immune homeostasis. Cancer cells take advantage of this pathway to suppress immune control and inhibit the anti-tumor immune response by deactivating cytotoxic T-cells in the tumor microenvironment. Breast cancer is considered a less immunogenic cancer compared to melanoma and lung cancer. PD-L1 expression is mainly found in TNBC, HER2+, ER- and PR- tumors. Increased PD-L1 expression correlates with higher tumor infiltrating lymphocytes and those data together correlate with better response in breast cancer patients. PD-1/PD-L1 pathway represents one of the primary immunosuppressive drivers in multiple types of cancer. Thus, inhibiting PD-1/PD-L1 interactions may prevent T-cell suppression and reactivate immune-surveillance. Methods and Results: Evaluating PD-L1 basal expression in a different set of human and mouse breast cancer cell lines by Western blotting and real-time PCR, we found TNBC and HER2+ cells express the highest PD-L1 levels. Testing different epigenetic modifiers, we found that HDACi were able to up-regulate PD-L1 mRNA and protein expression in a time-dependent manner up to 72 hours in the TNBC MDA-MB231 cells. Co-culturing tumor cells with human peripheral blood mononuclear cell (PBMCs), we performed comprehensive flow cytometric immunophenotyping to define the role of epigenetic priming in promoting immune cell activation. We found that HDACi upregulate PD-L1 and HLA-DR expression on MDA-MB231 tumor cells independently from PBMCs presence. Opposite effects were observed with hormone responsive ER+ MCF-7 cells, where PD-L1 was not significantly increased and HLA-DR was down-regulated upon HDACi treatment. These effects were associated with reduced Foxp3 expression on CD4+ Tregs in vitro after HDACi treatment. Importantly, HDACi significantly enhanced the in vivo response to PD-1 and CTLA-4 blockade in the triple-negative 4T1 breast cancer mouse model. This was associated with increased tumor infiltrating T cells, increased survival, and tumor growth inhibition in vivo. Conclusion: Overall, this study suggests that the combination of HDACi with immune checkpoint inhibitors identify a novel therapeutic strategy and warrants further clinical evaluation for the treatment of TNBC. Citation Format: Manuela Terranova Barberio, Scott Thomas, Niwa Ali, Nela Pawlowska, Jeenah Park, Michael D. Rosenblum, Alfredo Budillon, Pamela N. Munster. HDAC inhibition modulates immune checkpoint pathway in triple-negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B10.