ER-positive Breast Cancer Patients Research Articles

PREDICT validity for prognosis of breast cancer patients with pathogenic BRCA1/2 variants

We assessed the PREDICT v 2.2 for prognosis of breast cancer patients with pathogenic germline BRCA1 and BRCA2 variants, using follow-up data from 5453 BRCA1/2 carriers from the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA) and the Breast Cancer Association Consortium (BCAC). PREDICT for estrogen receptor (ER)-negative breast cancer had modest discrimination for BRCA1 carrier patients overall (Gönen & Heller unbiased concordance 0.65 in CIMBA, 0.64 in BCAC), but it distinguished clearly the high-mortality group from lower risk categories. In an analysis of low to high risk categories by PREDICT score percentiles, the observed mortality was consistently lower than the expected mortality, but the confidence intervals always included the calibration slope. Altogether, our results encourage the use of the PREDICT ER-negative model in management of breast cancer patients with germline BRCA1 variants. For the PREDICT ER-positive model, the discrimination was slightly lower in BRCA2 variant carriers (concordance 0.60 in CIMBA, 0.65 in BCAC). Especially, inclusion of the tumor grade distorted the prognostic estimates. The breast cancer mortality of BRCA2 carriers was underestimated at the low end of the PREDICT score distribution, whereas at the high end, the mortality was overestimated. These data suggest that BRCA2 status should also be taken into consideration with tumor characteristics, when estimating the prognosis of ER-positive breast cancer patients.

Abstract 5994: INX-315, a potent and selective CDK2 inhibitor, demonstrates robust antitumor activity in CCNE1-amplified cancers

Abstract Cyclin-dependent kinases (CDK) are a family of serine/threonine kinases that heterodimerize with regulatory subunits called cyclins to drive cell cycle progression. Uncontrolled cellular proliferation is a hallmark of cancer commonly driven by dysregulated kinase activity of specific CDK family members, including cyclin-dependent kinase 2 (CDK2). Aberrant CDK2 activity most frequently occurs through amplification of CCNE1 and/or overexpression of its protein product cyclin E1, which is a canonical binding partner of CDK2. Overexpression of cyclin E1 is observed in many solid tumors including in patients with high grade serous ovarian cancer (HGSOC), gastric cancer, and ER-positive breast cancer patients whose tumors have progressed on a prior CDK4/6 inhibitor regimen. Selective inhibition of CDK2 is thus a compelling therapeutic approach to regain cell cycle control. Here, we report preclinical data supporting the development of INX-315 for patients with cancers in which proliferation is CDK2-dependent. INX-315 is a potent inhibitor of CDK2/cyclin E (0.6 nM biochemical IC50) with high selectivity over other CDK family members in both biochemical and intracellular NanoBRET assays. In CCNE1-amplified human ovarian and gastric cancer cell lines, INX-315 potently inhibited Rb phosphorylation, induced a G1 cell cycle arrest, and inhibited proliferation. INX-315 also showed potent anti-proliferative activity in luminal breast cancer cell lines that had been cultured in CDK4/6 inhibitor (+/- anti-estrogen therapy) for prolonged periods to the point of developing drug resistance. While these cell lines did not readily respond to either CDK4/6 or CDK2 inhibition alone, combination treatment again suppressed Rb phosphorylation, accompanied by G1 arrest and a senescent-like phenotype. Lastly, in CCNE1-amplified xenograft models of ovarian and gastric carcinomas, INX-315 inhibited Rb-phosphorylation and induced tumor regression. These data demonstrate INX-315 to be a potent and selective CDK2 inhibitor that may benefit patients with CDK2/cyclin E driven cancers. Citation Format: Alec G. Trub, John E. Bisi, Catherine Dietrich, Michael Taylor, Jay C. Strum, Shom Goel, Patrick J. Roberts. INX-315, a potent and selective CDK2 inhibitor, demonstrates robust antitumor activity in CCNE1-amplified cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5994.

Abstract P2-10-02: Impact of neoadjuvant treatment on the accuracy of breast MRI and calliper in premenopausal ER-positive, HER2-negative, and node-positive breast cancer patients: Prospective phase III clinical trial

Abstract Background: Improved understanding of factors affecting the accuracy of breast MRI after NST can lead to more tailored use of MRI in deciding surgical extent after NST. Purpose: To investigate whether the accuracy of magnetic resonance imaging (MRI) and caliper predicting residual tumor extent is affected by neoadjuvant treatment in estrogen receptor (ER) positive and HER2-negative, lymph node-positive, premenopausal breast cancer breast cancer. Materials and Methods: We analyzed the imaging and clinicopathologic data of 123 patients in phase 3, randomized clinical trial (NCT01622361), which compared neoadjuvant chemotherapy (NCT) with neoadjuvant endocrine therapy (NET) in estrogen receptor-positive (ER+)/human epidermal growth factor receptor 2-negative (HER2-), lymph node (LN)-positive, premenopausal breast cancer patients. In this study, we compared the largest size of MRI and Caliper after neoadjuvant treatment with one of pathology. Results: In patient with discrepancy in predicting response to NST, the measurement (MRI or caliper) that predict as non-response (SD or PD) was more accurate in both of NCT and NET group (ICC, Intraclass correlation coefficient, MRI; 0.84, 95% CI 0.616-0.942, Caliper;0.75, 95% CI 0.303-0.931). The accuracy of MRI was better in NCT group than in NET group (ICC 0.86; 95% CI 0.626-0.956 for NCT, 0.47; 95%CI 0.133-0.708 for NET). The mean absolute discrepancies (largest size by MRI or caliper -largest size in pathology) for the MRI and Caliper were, respectively, -0.78cm and -1.04cm in NCT group and -0.44cm and -1.32cm in NET group (p > 0.05). The proportion of discrepancy >1cm or >0.5cm were not significantly difference between NCT and NET groups among MRI and Caliper. Conclusion: In ER-positive, HER2-negative, lymph node-positive breast cancer, both MRI and caliper tend to overestimate residual tumor size than pathology in NET group compare to NCT group. When MRI and caliper show discrepancy in predicting response to NST, the measurement (MRI or caliper) that predict as non-response (SD or PD) was more accurate in both of NCT and NET group. These factors should be considered for deciding the evaluation method of response and surgical decision after NST and surgical decision in this subgroup. REGISTRATION: ClinicalTrials.gov identifier: NCT01622361 Citation Format: Sungchan Gwark, Sei Hyun Ahn, Jong Won Lee, Byung Ho Son, Beom Seok Ko, Jisun Kim, Il-Yong Chung, Sae Byul Lee, Jin-Hee Ahn, Kyung Hae Jung, Sung-Bae Kim, Hee Jin Lee, Gyungyub Gong, Hee Jeong Kim. Impact of neoadjuvant treatment on the accuracy of breast MRI and calliper in premenopausal ER-positive, HER2-negative, and node-positive breast cancer patients: Prospective phase III clinical trial [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-10-02.

Abstract P4-02-28: EMP1 low expressing tumor is associated with cell proliferation and poor overall survival of ER-positive breast cancer patients

Abstract Background: Epithelial membrane protein 1 (EMP1) belongs to PMP22 gene family and is reported to be expressed low in numerous gastrointestinal cancers. However, little is known about its role in breast cancer. EMP1 functions to promote cell proliferation. In this study, we hypothesized that low EMP1 expressing breast cancer is associated with high proliferative characteristics and poor survival. Material and Methods: The clinicopathological data and transcriptome data of breast cancer patients from two independent large publicly available databases, The Cancer Genome Atlas (TCGA, n = 1090) and The Molecular Taxonomy of Breast Cancer International Consortium (METARBRIC, n = 1904), were utilized in the current study. Survival analyses; Overall survival (OS), Disease-specific survival (DSS) and Disease-free survival (DFS) were performed by comparing the high and low expression groups. CYT score, xCell, and other immunological factors were used to evaluate intratumoral immune cell composition. Also, gene set enrichment analysis (GSEA) was performed between EMP1 high and low expression groups. Results: We divided each cohort into EMP1 expression high and low groups by utilizing median cutoff. The expression levels of EMP1 were not associated with clinical stage in any subtypes. However, interestingly lower expression of EMP1 was significantly associated with more advanced grades in ER positive/HER2 negative (ER+/HER2) subtype in both TCGA and METABRIC cohorts (p< 0.001 and p< 0.001, respectively). Also, low EMP1 expressing tumors demonstrated higher MKI67 expression levels in ER+/HER2- subtype consistently in both TCGA (p< 0.001) and METABRIC cohorts (p< 0.001). Furthermore, GSEA demonstrated that low EMP1 expressing tumors enriched the gene sets associated with cell proliferation such as MYC Targets, E2F signaling, and G2M Checkpoint signaling, compared with low EMP1 expressing tumors in ER+/HER2- of both TCGA and METABRIC cohorts. On the contrary, high EMP1 expressing tumors enriched the immune related gene sets such as Coagulation, Inflammatory_Response, Complement, and IL-6_JAK_STAT3 Signaling in ER+/HER2- of both TCGA and METABRIC cohorts. To this end, we further hypothesized that high EMP1 tumors were associated with favorable tumor immune microenvironment (TIME) and analyzed TIME utilizing xCell. However, to our surprise high EMP1 expressing tumors were not associated with favorable TIME. Low EMP1 expressing tumors demonstrated worse DFS, DSS, and OS compared with high EMP1 expressing tumors in ER+/HER2- breast cancer patients (p=0.013, p=0.003, and p=0.006) which was not the case in the other subtypes in METABRIC cohort. Conclusion: Low EMP1 expressing tumors were associated with improved OS, DSS, DFS in ER-positive breast cancer patients. Also, low EMP1 expressing tumors were found to associate with advanced grades and enriched gene sets related to cell proliferation and cell cycle, which may explain the poor survival of patients with low EMP1 expressing ER+/HER2- breast cancer. Citation Format: Junko Ukai, Yoshihisa Tokumaru, Masanori Oshi, Yoshimi Niwa, Ryutaro Mori, Kazuaki Takabe, Manabu Futamura. EMP1 low expressing tumor is associated with cell proliferation and poor overall survival of ER-positive breast cancer patients [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-02-28.

Abstract P4-09-11: Development of personalized medicine for breast cancer based on elucidation of resistance mechanisms to CDK4/6 inhibitors

Abstract Background CDK4/6 inhibitors are the latest and highly specific CDK inhibitors approved for metastatic and early estrogen receptor (ER)-positive breast cancer patients, improving the survival outcomes. Prior preclinical research has indicated various aberrations of cell cycle regulation and activated oncogenic signaling pathway as the mechanisms of resistance to CDK4/6 inhibitors. However, there is currently no available biomarker except ER and human epidermal growth factor receptor 2 (HER2) in clinical practice. The aim of our study is to elucidate diverse mechanisms of acquired resistance of ER-positive breast cancers to CDK4/6 inhibitors and develop personalized medicine in ER-positive breast cancer patients. Materials and methods To generate resistant lines, the ER-positive human breast cancer cell lines MCF7 and T47D were treated individually with increasing concentrations of the CDK4/6 inhibitors palbociclib and ribociclib until a target concentration of 3μM for six months. Cells were maintained in RPMI 1640 containing 10% fetal bovine serum, 100 U/ml penicillin/streptomycin at 37°C in a 5% CO2 incubator. Western blot analysis was performed for whole cell extract proteins from resistant cells to CDK4/6 inhibitors and parental cells. Colony formation assay was done to observe cell viability. Results Western blot analysis showed elevated FGFR2, CDK6, pS6RP, and cyclin E1 in resistant cells compared to parental cells. Specifically, cyclin E1 and pS6RP were increased in MCF7 palbociclib- and ribociclib-resistant cells, while FGFR2 and CDK6 in T47D palbociclib- and ribociclib-resistant cells. Notably, CDK6 was upregulated in MCF7 ribociclib-resistant cells, whereas increased cyclin E1 was detected in T47D palbociclib-resistant cells, respectively. Colony formation assay revealed acquired ability of resistance to CDK4/6 inhibitors in generated resistant cell lines. Conclusions The tumors harboring resistance to CDK4/6 inhibitors have heterogenous mechanisms of therapeutic resistance to these agents. The novel combination treatment of targeted molecular therapy and CDK4/6 inhibition will lead development of personalized medicine for ER-positive breast cancer based on elucidation of diverse resistant mechanisms to CDK4/6 inhibition. Western blot analysis of lysates from MCF-7 cells treated for 24 to 48 hours with palbociclib (Palbo) and ribociclib (Ribo) Cyclin E1 and pS6RP were increased in MCF7 palbociclib- and ribociclib-resistant cells, whereas CDK6 was upregulated especially in MCF7 ribociclib-resistant cells. Western blot analysis of lysates from T47D cells treated for 24 to 48 hours with palbociclib (Palbo) and ribociclib (Ribo) FGFR2 and CDK6 were increased in T47D palbociclib- and ribociclib-resistant cells, whereas increased cyclin E1 was detected especially in T47D palbociclib-resistant cells. Viability in MCF7 ribociclib-resistant and parental cells by colony formation assay Colony formation assay revealed acquired ability of resistance to CDK4/6 inhibitors in MCF7 resistant-cells. Citation Format: Yoshie Kobayashi, Yusuke Motoi, Mutsumi Fujimoto, Hideo Shigematsu. Development of personalized medicine for breast cancer based on elucidation of resistance mechanisms to CDK4/6 inhibitors [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-09-11.

Abstract P3-11-02: CST1 Interaction with RAB1B Modulates Tamoxifen resistance of Breast Cancer by Regulating Autophagy

Abstract Background: Breast cancer is a prominent cause of cancer-related death among women worldwide. Approximately,70% of breast cancers (BC) are estrogen and progestogen receptor-positive (HR+) at diagnosis. Adjuvant endocrine therapy is a pivotal component of treatment for this type of patient. Tamoxifen (TAM), a widely recommended drug in endocrine therapy, is confronted with a high risk of recurrence after 5 years of treatment among breast cancer patients, which shows big clinical demands. Therefore, it is urgent to find new targets for tamoxifen resistance therapy. Recently, our team found that enhanced expression of Cystain SN (CST1) was detected in tissues that had undergone tamoxifen treatment appearing to recurrence and metastasis in ER-positive breast cancer patients. However, the molecular mechanisms of modulating tamoxifen resistance remain unknown. Therefore, our team suspected that there might be a correlation between the abnormal expression of CST1 and tamoxifen resistance. To investigate whether CST1 is involved in tamoxifen resistance and its molecular mechanism, we conducted further research. It is critical to elucidate the molecular mechanisms of tamoxifen resistance and to explore a novel effective therapeutic target. Method: Enhanced expression of CST1 was confirmed by immunohistochemistry in breast cancer tissue samples and by quantitative real-time PCR in Tamoxifen-resistant cancer cell lines MCF-7R/T47DR. To further investigate the molecular function of CST1, we knockdown its expression in MCF-7R/T47DR cells by transfecting with the lentiviral particles of shCST1 and corresponding negative control (Shanghai Genechem Co., Ltd.) and overexpressed CST1 in MCF-7/T47D cells using ove-GST-CST1, ove-HA-RAB1B plasmids. The effects of CST1 on cell viability and proliferation in breast cancer cells were detected by cell counting kit-8 and colony formation assays, respectively. The interaction between CST1 and RAB1B was validated by Co-immunoprecipitation and Western blot. Autophagosomes were found using transmission electron microscopy (TEM) along with Western blotting to detect the transformation of LC3-I to LC3-II protein (i.e., the LC3-II/LC3-I ratio) and P62 protein levels. Results: We found that aberrant expression of Cystatin SN (CST1) was identified in ER-positive breast cancer (BC) cells (Figure 1) and which is crucial in contributing to Tamoxifen resistance (Figure 2). The direct promotion of autophagy by RAPA can faciliate cells to endocrine resistance. And knockdown of CST1 not only suppressed autophagy in Tamoxifen-resistant cells but also attenuated the resistance, which determined the correlation of CST1 with autophagy in BC (Figure 3). Rab1b (Ras-related protein Rab-1B), a membrane protein essential for autophagosome formation, was verified to have interaction with CST1 through Coimmunoprecipitation (CO-IP) experiments (Figure 4). Meanwhile, RAB1B expression was also reduced by CST1 knockdown in the resistant cells. The interaction between CST1 and RAB1B induces autophagy which decreases Tamoxifen’s therapeutic efficacy. Furthermore, rescue experiments suggested that CST1 knockdown–induced sensitization in the efficacy of TAM Therapy and decreased autophagy could be restored via RAB1B overexpression.Conclusions: These results indicate that CST1 interacts with RAB1B to facilitate BC resistant to TAM through regulation of autophagy and CST1 might be a potential therapeutic target to overcome endocrine therapy resistance. Figure 1. Enhanced expression of CST1 in Tamoxifen-resistance breast cancer Figure 1. Enhanced expression of CST1 in Tamoxifen-resistance breast cancer Figure 2. CST1 expression is positively correlated with autophagy in ER-positive breast cancer Figure 2. CST1 expression is positively correlated with autophagy in ER-positive breast cancer Figure 3. CST1 promotes cells resistant to tamoxifen therapy by activating auophagy Figure 3. CST1 promotes cells resistant to tamoxifen therapy by activating auophagy Figure 4. CST1 interacts with RAB1B in cell autophagy Figure 4. CST1 interacts with RAB1B in cell autophagy Citation Format: li liu. CST1 Interaction with RAB1B Modulates Tamoxifen resistance of Breast Cancer by Regulating Autophagy [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-11-02.

Abstract P6-10-06: A preoperative window-of-opportunity study of imlunestrant in estrogen receptor-positive, HER2-negative early breast cancer: Results from the EMBER-2 study

Abstract Background: Imlunestrant is a novel, orally bioavailable selective estrogen receptor degrader (SERD) with pure antagonistic properties that result in sustained inhibition of estrogen receptor (ER)-dependent gene transcription and cell growth. In a phase 1 study, imlunestrant monotherapy showed favourable safety, pharmacokinetics (PK) and preliminary efficacy in heavily pre-treated ER-positive (ER+) advanced breast cancer patients (Jhaveri ASCO 2022). Here, we present pharmacodynamic (PD) data from the preoperative window of opportunity (WOO) study (EMBER-2, NCT04647487), evaluating the biological activity of imlunestrant monotherapy in ER+, HER2-negative (HER2-) early breast cancer (EBC). Methods: Post-menopausal women with stage I–III operable ER+ (>50%) or Allred score >5, HER2- untreated EBC ≥1 cm in diameter were randomized 1:1 to imlunestrant 400 mg once daily (QD) or imlunestrant 800 mg QD for 15 days (treatment window of -2 to +7 days) up to the surgery date. Pre- and on-treatment tumor samples were compared for changes in PD biomarkers. Primary study objective was change in ER expression (measured by IHC and quantified by H-score). Secondary objectives were change in progesterone receptor (PR) expression (measured by IHC and quantified by H-score) and Ki-67 (measured by IHC and expressed by percentage positive scoring) along with evaluation of safety and tolerability. Results: From Apr 28, 2021, to Mar 11, 2022, 58 patients were enrolled of which 54 were biomarker-evaluable for ER expression (400 mg: n = 28; 800 mg: n = 26). Patient demographics and tumor characteristics for all enrolled patients were similar across cohorts, with a median age of 64 years (50-83), 72% invasive ductal carcinoma (IDC), 28% invasive lobular carcinoma (ILC), 59% stage I, 36% stage II and 5% stage III disease. 91% of the patients had a compliance rate higher than 80%. Among biomarker evaluable patients, relative reduction in PD biomarkers after a median of 15 days (range 13 to 23 days) of treatment are presented in Table 1. There was no significant difference in PD biomarker modulation noted between the two imlunestrant doses (400 mg vs 800 mg) or based on tumor histology (IDC, ILC). Imlunestrant was well tolerated. There were no discontinuations due to adverse events (AEs). Treatment-related AEs (TRAEs) were mainly grade 1, most commonly: fatigue (10%), diarrhea (9%), hot flushes (7%), and nausea (5%). There were no TRAEs of diarrhea and nausea observed at the 400 mg dose. No grade 3 or higher TRAEs were reported. Conclusion: Imlunestrant demonstrated evidence of target engagement along with consistent biological activity across all evaluated dose levels and was well tolerated in an EBC population, further supporting continued adjuvant development in the ongoing EMBER-4 study. Additional biomarker analyses for the EMBER-2 study are also planned. Table 1. Relative reduction in PD biomarkers from Baseline to Day 15 Citation Format: Patrick Neven, Nicole Stahl, Maria Vidal, Miguel Martín, Nadia Harbeck, Peter A. Kaufman, Francois-Clement Bidard, Peter A. Fasching, Philippe Aftimos, Erika Hamilton, Stacey Carter, Peter Schmid, Duncan Wheatley, Manali Bhave, Kelly K. Hunt, Swati A. Kulkarni, Roohi Ismail-Khan, Claudia Karacsonyi, Shawn T. Estrem, Umut Ozbek, Bastien Nguyen, Eva Ciruelos. A preoperative window-of-opportunity study of imlunestrant in estrogen receptor-positive, HER2-negative early breast cancer: Results from the EMBER-2 study. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-10-06.

Abstract P6-01-24: Long-term outcome data using Endopredict® as risk stratification and chemotherapy decision biomarker in hormone receptor positive, HER2-negative early breast cancer

Abstract Background: The Endopredict test is used for estimating risk of distant recurrence for women presenting with early-stage breast cancer with a positive estrogen receptor (ER) and negative human epidermal growth factor receptor 2 (HER2) status. The current ASCO Guideline Update on biomarkers confirms the value of the Endopredict test to guide decisions of adjuvant endocrine and chemotherapy. This study shows prospective long-term outcome data of early breast cancer patients whose chemotherapy decision was guided by the Endopredict test result (EPclin). Methods: ER-positive and HER2-negative early breast cancer patients with 0-3 positive lymph nodes treated between March 2012 and March 2015 were included in this single institution study. The Endopredict® test was carried out on all tumour samples. Demographic, clinical and pathological data were assessed for each patient at baseline. Treatment compliance, local recurrence, distant metastases and survival was evaluated. Risk estimates were obtained by the Kaplan-Meier method and cumulative risk functions in case of competing risks. Group comparisons were performed by Cox proportional hazards regression models and quantified through hazard ratios. Median Follow-Up was estimated by the inverse Kaplan-Meier method. Exploratory hypothesis testing was conducted at two-sided 5% significance levels. Results: In a cohort of 368 consecutive cases the median follow-up time was 8.2 years. Endopredict allocated 238 pts (64%) in the EPclin low risk and 130 pts (34%) in the EPclin high risk group. The 5-year distant metastasis free survival (DMFS) in the EPclin low risk group was 96.6% (95% CI 0.943-0.989) and 85.5% (95% CI 0.796-0.920) in the EPclin high risk group. With a hazard ratio (HR) of 2.21 (95% CI: 1.27-3.88; p=0.005) the risk for distant metastasis in EPclin high risk patients was more than two-fold higher in comparison with EPclin low risk patients. 87 pts. (66.9%) of the EPclin high risk group underwent chemotherapy (compliant), whereas 43 pts (33.1 %) opposed the recommended chemotherapy (non-compliant). Kaplan-Meier plots in the EPclin high risk subgroups compliant vs non-compliant showed a significant disease-free survival (DFS) benefit towards the patients following the chemotherapy recommendation (HR 0.46; 95%CI 0.23-0.95; p=0.036). The 5-year DFS for the high risk compliant subgroup was 89.1% (95% CI: 0.827-0.961) vs. the high risk non-compliant subgroup with 68.9% (95% CI: 0.562-0.845). Regarding the subgroups pre- and postmenopausal, patients with a EPclin high risk test result were at significant higher risk of experiencing distant metastases than patients with a EPclin low risk test result in both subgroups (premenopausal: HR 3.55; 95%CI 1.17-12.32; p=0.025; postmenopausal: HR 1.19; 95%CI 0.99-3.7; p=0.054). We analyzed the EPclin categorization in context of the ki67 subtypes luminal A (low; 0-10%) and luminal B (high; 25-100%). The EPclin-based risk stratification was significantly associated with improved DFS in both ki67 subtypes (ki67 low: HR 4; 95%CI 1.25-12.04; p=0.021 and ki67 high: HR 3.77; 95%CI 1.19-18.93; p=0.022). 33.3% (21 pts) of all tumor samples classified as luminal B (63 pts), were reclassified towards the low risk group via Endopredict, sparing chemotherapy recommendation. Contrary 19.2% (14 pts) of all luminal A (73 pts) were categorized to high risk EPclin. Conclusion: These first long term prospective outcome data confirm, that Endopredict can guide decisions on adjuvant chemotherapy in early ER positive, HER2 negative breast cancer. Pts categorized as EPclin high risk benefitted from an adjuvant chemotherapy. Our results show that Endopredict risk stratification is also applicable in premenopausal women. Furthermore the Endopredict test showed a better classification accuracy in comparison to ki67 subtypes, resulting in a more precise estimation of prognosis. Citation Format: Evelyn Klein, Adriana Josipovic, Aurelia Noske, Sophie Anders, Carolin Mogler, Wilko Weichert, Alexander Hapfelmeier, Marion Kiechle, Johannes Ettl. Long-term outcome data using Endopredict® as risk stratification and chemotherapy decision biomarker in hormone receptor positive, HER2-negative early breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-01-24.

Abstract P2-03-11: Differential Long-Term Benefit from Adjuvant Tamoxifen Therapy in Estrogen Receptor (ER)-Positive/Human Epidermal Growth Factor Receptor 2 (HER2)-Negative Premenopausal and Postmenopausal Breast Cancer Patients

Abstract Background: Tamoxifen is a standard endocrine therapy for both pre- and postmenopausal ER-positive breast cancer patients. Patients with ER-positive disease have a long-term risk of distant recurrence, thus, long-term follow-up studies are essential to understand true treatment benefit. Clinically used tumor characteristics are prognostic 5-10 years after primary diagnosis, however, whether these characteristics are predictive of long-term tamoxifen benefit is largely unexplored. Therefore, we aimed to determine the long-term tamoxifen therapy benefit by the clinically used tumor characteristics in pre- vs postmenopausal patients in the Stockholm tamoxifen (STO)-trials with 20-years complete follow-up. Methods: Secondary analysis of 1242 ER-positive/HER2-negative patients from the STO-trials, randomized to at least 2 years of 40 mg tamoxifen vs no endocrine therapy (control). Premenopausal lymph node-positive patients were allocated to chemotherapy as standard of care and postmenopausal high-risk patients were further randomized to chemotherapy vs radiotherapy. Tumor immunohistochemical analysis was recently conducted. Complete 20-year follow-up was obtained from Swedish high-quality registries. Long-term distant recurrence-free interval (DRFI) was assessed by multivariable Cox proportional hazard regression and time-varying analysis using flexible parametric modelling. Results: Premenopausal patients showed significantly improved long-term DRFI from tamoxifen vs control if they were lymph node-negative (Hazard Ratio [HR]=0.46; 95% CI, 0.24-0.87), PR-positive (HR=0.61; 95% CI, 0.41-0.91), or of genomic low risk (HR=0.47; 95% CI, 0.26-0.85), see Table. In postmenopausal patients, significantly improved long-term DRFI from tamoxifen vs control was seen for all good prognosis tumor characteristics, i.e. small tumor size (pT≤20mm: HR=0.55; 95% CI, 0.39-0.77), tumor grade 1-2 (HR=0.55; 95% CI, 0.41-0.73), lymph node-negative (HR=0.44; 95% CI, 0.30-0.64), PR-positive (HR=0.60; 95% CI, 0.44-0.80), Ki-67-low (< 15%: HR=0.51; 95% CI, 0.38-0.68), and genomic low risk (HR=0.53; 95% CI, 0.37-0.74), see Table. Also, postmenopausal patients with large tumor size (pT>20mm: HR=0.64; 95% CI, 0.44-0.94) and PR-negative tumors (HR=0.51; 95% CI, 0.32-0.81) showed significant long-term tamoxifen benefit. Time-varying analysis in premenopausal patients indicated that tamoxifen therapy benefit diminished over time. Significant tamoxifen benefit until year 5, 10, and 15 after primary diagnosis was observed for PR-positive, lymph node-negative, and genomic low-risk patients, respectively. Postmenopausal patients had a significant long-term tamoxifen benefit if they had tumors of small or large tumor size, tumor grade 1-2, lymph node-negative status, PR-positive status, low Ki-67 levels, or genomic low risk. Conclusions: This study suggests a differential long-term tamoxifen therapy benefit in pre- vs postmenopausal patients. Clinically defined low-risk postmenopausal patients have long-term tamoxifen benefit, whereas the benefit is absent or diminish over time for premenopausal patients. Improved long-term prognostic and endocrine therapy predictive markers in premenopausal breast cancer patients with poor prognosis and long life-expectancy is needed, which could involve molecular tools. Long-term tamoxifen benefit in premenopausal and postmenopausal breast cancer patients by the clinically used tumor characteristics. Table Multivariable Cox proportional hazard regression analysis of 20-year distant recurrence-free interval (DRFI) for patients with ER-positive/HER2-negative tumors, comparing patients randomized to tamoxifen vs patients randomized to no endocrine therapy (control). Adjusted for age, randomization year, tumor size, tumor grade, lymph node status, PR status, Ki-67 status, chemotherapy, radiotherapy, and type of surgery. Citation Format: Annelie Johansson, Huma Dar, Anna Nordenskjöld, Gizeh Perez-Tenorio, Christina Yau, Christopher C. Benz, Laura J. Esserman, Laura Van’t Veer, Bo Nordenskjöld, Olle Stål, Tommy Fornander, Linda S. Lindström. Differential Long-Term Benefit from Adjuvant Tamoxifen Therapy in Estrogen Receptor (ER)-Positive/Human Epidermal Growth Factor Receptor 2 (HER2)-Negative Premenopausal and Postmenopausal Breast Cancer Patients [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-03-11.

Expression analysis elucidates the roles of Nicastrin, Notch4, and Hes1 in prognosis and endocrine-therapy resistance in ER-positive breast cancer patients.

Although some proposed mechanisms responsible for tamoxifen resistance have already been present, further study is needed to determine the mechanisms underlying tamoxifen resistance more clearly. The critical role of Notch signaling has been described in promoting resistance in therapeutics, but there is little information about its role in tamoxifen resistance progression. In the present study, the expression of Notch pathway genes, including Notch4, nicastrin and the Notch downstream target Hes1 was evaluated using quantitative RT-PCR in 36 tamoxifen-resistant (TAM-R) and 36 tamoxifen-sensitive (TAM-S) patients. Expression data were correlated with the clinical outcome and survival of patients. mRNA levels of Notch4 (fold change = 2.7), nicastrin (fold change = 6.71), and Hes1 (fold change= 7.07) were significantly higher in TAM-R breast carcinoma patients compared to sensitive cases. We confirmed all these genes were co-expressed. Hence, it seems that Notch signaling is involved in tamoxifen resistance in our TAM-R patients. Obtained results showed that Hes1, nicastrin, and Notch4 mRNA upregulation was correlated with the N stage. The extracapsular nodal extension was associated with nicastrin and Notch4 overexpression. Moreover, nicastrin overexpression was correlated with perineural invasion. Hes1 upregulation was also associated with nipple involvement. Finally, the Cox regression proportional hazard test revealed that overexpression of nicastrin was an independent worse survival factor. Presumably, upregulation of the Notch pathway may be involved in tamoxifen resistance in breast cancer patients.

Clinical Treatment Score Post-5 Years as a Tool for Risk Estimation of Late Recurrence in Thai Patients With Estrogen-Receptor-Positive, Early Breast Cancer: A Validation Study.

The risk of late distant recurrence (LDR) of estrogen receptor (ER)-positive breast cancer continues even after 5 years of endocrine treatment. Clinical Treatment Score after 5 years (CTS5) was developed and validated as a tool to assess the risk of LDR using data from Tamoxifen, Arimidex Alone or in Combinations (ATAC) and Breast International Group 1-98 (BIG1-98) trials. This study aimed to externally validate CTS5 in a real-world cohort of patients treated at an academic center in Thailand. The study was a retrospective analytical research study of early-stage, ER-positive breast cancer patients. The primary endpoint was LDR. The risk of LDR was determined using the CTS5 calculator. Cox regression model and Kaplan-Meier survival analysis were applied for prognostic validation of CTS5. Calibration was performed by comparing observed LDR to expected LDR using the Hosmer-Lemeshow (H-L) test. A total of 323 women were included with a median follow-up period of 11.6 years. The rate of LDR was 10.8%. The CTS5 was prognostic for LDR. C-index of the area under the ROC curve was 0.672. There was no significant difference between actual and expected numbers of LDR with an observed (O) LDR events to expected (E) number of LDR events ratio of 0.99 (0.86-1.12) (H-L P = .79) indicating a proper calibration in this cohort. Our study validated that CTS5 is accurate in predicting the risk of LDR in ER-positive breast cancer cases in Thai patients. Its performance seemed to be better in postmenopausal patients. CTS5 could be applied in routine clinical practice to improve decisions regarding prolonged endocrine therapy, particularly in resource-limited countries where molecular profiling are inaccessible.

Classification of breast cancer subtypes based on RNA profiling and immunohistochemical methods: clinical and biological aspects: A review

Transcriptome analysis provided a tool to identify expression subtypes of breast cancer (BC). A significant part of BCs are carcinomas that differ in the expression of luminal mammary ductal epithelium markers and estrogen signaling cascade genes (luminal subtypes A and B). Another group of BCs is characterized by the expression of ductal basal lining genes. Another subtype of BC has genes typically expressed in HER2-induced tumors. Immunohistochemical (IHC) examination is reliable in identifying the tumor subtype. Tumors with high IHC-expression of estrogen (ER) and progesterone (PR) receptors with no signs of HER2 gene activation and low proliferative activity should be referred to as luminal type A. The absence of ER, PR, and HER2 expression should be considered a sign of basal tumor subtype. However, the IHC classification cannot reliably distinguish HER2-positive tumors and HER2-enriched subtypes, which do not reflect the biological features of some BCs. For instance, a significant number of ER-positive breast cancer patients included in the MONALEESA ribociclib clinical study had HER2-subtype transcriptional signatures in the absence of HER2 receptor expression, and these were the ones who demonstrated a pronounced response to treatment.

Estrogen-Inducible LncRNA BNAT1 Functions as a Modulator for Estrogen Receptor Signaling in Endocrine-Resistant Breast Cancer Cells.

Recent advances in RNA studies have revealed that functional long noncoding RNAs (lncRNAs) contribute to the biology of cancers. In breast cancer, estrogen receptor α (ERα) is an essential transcription factor that primarily promotes the growth of luminal-type cancer, although only a small number of lncRNAs are identified as direct ERα targets and modulators for ERα signaling. In this study, we performed RNA-sequencing for ER-positive breast cancer cells and identified a novel estrogen-inducible antisense RNA in the COL18A1 promoter region, named breast cancer natural antisense transcript 1 (BNAT1). In clinicopathological study, BNAT1 may have clinical relevance as a potential diagnostic factor for prognoses of ER-positive breast cancer patients based on an in situ hybridization study for breast cancer specimens. siRNA-mediated BNAT1 silencing significantly inhibited the in vitro and in vivo growth of tamoxifen-resistant ER-positive breast cancer cells. Notably, BNAT1 silencing repressed cell cycle progression whereas it promoted apoptosis. Microarray analysis revealed that BNAT1 silencing in estrogen-sensitive breast cancer cells repressed estrogen signaling. We showed that BNAT1 knockdown decreased ERα expression and repressed ERα transactivation. RNA immunoprecipitation showed that BNAT1 physically binds to ERα protein. In summary, BNAT1 would play a critical role in the biology of ER-positive breast cancer by modulating ERα-dependent transcription regulation. We consider that BNAT1 could be a potential molecular target for diagnostic and therapeutic options targeting luminal-type and endocrine-resistant breast cancer.

POR overexpression induces tamoxifen-resistance in breast cancer through the STAT1/c-Myc pathway.

Breast cancer is the most common cancer in women worldwide. Althoughtamoxifen (TAM), a selective estrogen receptor (ER) modulator, is widely used to treat ER-positive breast cancers, resistance to TAM remains a major clinical problem. NADPH-dependent cytochrome P450 reductase (POR) is known to participate in drug metabolism and steroid metabolism. Recent studies showed that high POR expression was correlated with poor outcomes in triple-negative breast cancer (TNBC), and POR might be a prognostic biomarker in TNBC. However, the role of POR in TAM resistance is still elusive. In this study, we found that high POR expression was associated with poor prognosis of ER-positive and TAM-treated breast cancer patients. In addition, COX analysis showed that POR expression was an independent prognostic biomarker for ER-positive as well as TAM-treated breast cancer patients. Furthermore, our results suggested that POR overexpression promoted TAM resistance by activating the STAT1/c-Myc pathway in ER-positive breast cancer cells. Immunohistochemical analysis showed that high POR/STAT1 expression was correlated with poor prognosis in TAM-treated breast cancer patients. Notably, combined treatment with TAM and a specific STAT1 inhibitor Fludarabine was more effective for inhibiting TAM-resistant breast cancer cells. Altogether, our findings suggested that POR overexpression induced TAM resistance through STAT1/c-Myc pathway and might serve as an independent prognostic biomarker in TAM-treated breast cancer patients. Combining TAM and STAT1 inhibitors might be an effective strategy for treating POR-induced TAM-resistant breast cancer.

241 (PB-065) Poster - Single nucleotide polymorphisms of ABCB1 (rs1128503) and ABCC2 (rs145008610) genes and its clinical impact in ER & PR positive breast cancer patients in a tertiary care hospital of India

Background: Inter-individual differences in drug response are frequent clinical challenge due to genetic variation. ATP-binding cassette (ABC) transporters are crucial determinants of drug disposition and have been studied extensively in response to chemotherapeutic regimen and tamoxifen treatment. But no major findings were established till date in Indian scenario that correlates the effect of drug in ABC polymorphism.

Methylation-Mediated Silencing of RBP7 Promotes Breast Cancer Progression through PPAR and PI3K/AKT Pathway.

Retinoid-binding protein7 (RBP7) is a member of the cellular retinol-binding protein (CRBP) family, which is involved in the pathogenesis of breast cancer. The study aims to illustrate the prognostic value and the potential regulatory mechanisms of RBP7 expression in breast cancer. Bioinformatics analysis with the TCGA and CPTAC databases revealed that the mRNA and protein expression levels of RBP7 in normal were higher compared to breast cancer tissues. Survival analysis displayed that the lower expression of RBP7, the worse the prognosis in ER-positive (ER+) breast cancer patients. Genomic analysis showed that low expression of RBP7 correlates with its promoter hypermethylation in breast cancer. Functional enrichment analysis demonstrated that downregulation of RBP7 expression may exert its biological influence on breast cancer through the PPAR pathway and the PI3K/AKT pathway. In summary, we identified RBP7 as a novel biomarker that is helpful for the prognosis of ER+ breast cancer patients. Promoter methylation of RBP7 is involved in its gene silencing in breast cancer, thus regulating the occurrence and development of ER+ breast cancer through the PPAR and PI3K/AKT pathways.

Adherence to adjuvant hormonal therapy in localised breast cancer.

Hormonal therapies are the cornerstone of systemic adjuvant treatment of oestrogen receptor (ER) positive breast cancer. The full benefit of this treatment is obtained with long-term adherence. However, discontinuation of hormonal therapy is common. Factors associated with non-compliance to therapy are complex and worth of detailed evaluation. A retrospective analysis of medical records of 284 early ER-positive breast cancer patients prescribed adjuvant hormonal therapy during a 5-year period in a single centre was undertaken. Characteristics of the patients and their disease as well as adherence to therapy and continuation at 1 and 3years were recorded. The group of patients that were on treatment at 3years and the group that had discontinued therapy before 3years were compared to identify differences predicting lack of adherence. The discontinuation rate of hormonal therapy at 1year was 13%, and the discontinuation rate at 3years was 21.2%. Patient age and menopause status were not associated with hormone therapy adherence at 3years. The type of hormonal therapy (aromatase inhibitor or tamoxifen) was also not associated with adherence. In contrast, patients that received adjuvant chemotherapy before starting hormonal therapy had a higher adherence to hormonal therapy (86.9% at 3years vs. 75.7% in patients that had not received adjuvant chemotherapy, χ2 p= 0.04). Among co-morbidities, patients with a concomitant diagnosis of psychiatric disease at the time of breast cancer diagnosis were at increased risk of hormone therapy non-adherence. Progression-free survival and overall survival were inferior in the non-adherent group compared with the patients who continued their hormonal therapy at 3years. Adjuvant chemotherapy is associated with better subsequent adherence to hormonal therapy in early breast cancer patients. On the other hand, psychiatric co-morbidities are associated with worse adherence. De-escalation of adjuvant therapy guided by genomic tests leads to a significant percentage of early ER-positive breast cancer patients not receiving chemotherapy. Non-adherence to hormonal therapy would leave a subset of these patients with no adjuvant systemic therapy. The current results will guide efforts to increase compliance to hormonal therapies in specific groups of patients.

Genome engineering for estrogen receptor mutations reveals differential responses to anti-estrogens and new prognostic gene signatures for breast cancer

Mutations in the estrogen receptor (ESR1) gene are common in ER-positive breast cancer patients who progress on endocrine therapies. Most mutations localise to just three residues at, or near, the C-terminal helix 12 of the hormone binding domain, at leucine-536, tyrosine-537 and aspartate-538. To investigate these mutations, we have used CRISPR-Cas9 mediated genome engineering to generate a comprehensive set of isogenic mutant breast cancer cell lines. Our results confirm that L536R, Y537C, Y537N, Y537S and D538G mutations confer estrogen-independent growth in breast cancer cells. Growth assays show mutation-specific reductions in sensitivities to drugs representing three classes of clinical anti-estrogens. These differential mutation- and drug-selectivity profiles have implications for treatment choices following clinical emergence of ER mutations. Our results further suggest that mutant expression levels may be determinants of the degree of resistance to some anti-estrogens. Differential gene expression analysis demonstrates up-regulation of estrogen-responsive genes, as expected, but also reveals that enrichment for interferon-regulated gene expression is a common feature of all mutations. Finally, a new gene signature developed from the gene expression profiles in ER mutant cells predicts clinical response in breast cancer patients with ER mutations.

Absolute Lymphocyte Count Is an Independent Prognostic Factor for ER-positive HER2-negative Advanced Breast Cancer Patients Treated With CDK4/6 Inhibitors.

The aim of this study was to elucidate the clinical significance of peripheral blood biomarkers, including absolute lymphocyte count (ALC), neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR) and C-reactive protein (CRP) in patients with estrogen receptor-positive human epidermal growth factor receptor 2-negative advanced breast cancer treated with the CDK4/6 inhibitors, abemaciclib and palbociclib. A total of 83 patients treated with fulvestrant plus abemaciclib or palbociclib were included in this study. Progression-free survival (PFS) and overall survival (OS) were compared in relation to baseline levels of ALC, NLR, PLR and CRP. The cut-off values of ALC, NLR, PLR, and CRP for PFS were determined from the receiver operating characteristic curve using the Youden index for area under the curve and set at 1,212/μl, 1.964, 170 and 0.220 mg/dl, respectively. In the abemaciclib-treated group, ALC-high patients showed significantly better PFS than ALC-low patients (p=0.0151) and multivariate analysis revealed that ALC was an independent prognostic factor for PFS (p=0.0085). In the palbociclib-treated group, there was no significant relationship between any peripheral blood biomarkers and PFS. In both treatment groups, ALC-high patients showed significantly better OS than ALC-low patients (p=0.0169 and 0.0290, respectively). Multivariate analysis revealed ALC was an independent prognostic factor for OS in both abemaciclib- and palbociclib-treated groups (p=0.0112 and 0.0202, respectively). ALC is an independent prognostic factor for estrogen receptor-positive human epidermal growth factor receptor 2-negative advanced breast cancer patients treated with the CDK4/6 inhibitors abemaciclib and palbociclib.

Adjuvant endocrine therapy in patients with estrogen receptor-low positive breast cancer: A prospective cohort study

BackgroundLittle is known about the benefits of adjuvant endocrine therapy (ET) in low ER-positive breast cancer (1%–10%) patients. We analyzed the association between ET and breast cancer-specific survival (BCSS) in these patients with respect to the regimen and the duration of ET. MethodsPatients were classified into three groups based on the regimen and duration of ET. The regimens included aromatase inhibitor (AI) monotherapy or sequential tamoxifen followed by an AI (AI/T + AI), or only tamoxifen and no ET. The duration of ET included 2–3 years and >3 years. Multivariate Cox regression analysis was employed to calculate the hazard ratios (HRs) with 95% confidence intervals (CIs). ResultsOf the 10,696 patients diagnosed with breast cancer between 2010 and 2020, 407 women were identified with ER-low positive disease and met the inclusion criteria. During a median follow-up of 5.2 years, patients who received ET improved BCSS. Of them, those with AI/T + AI had increased BCSS compared to patients without ET, after adjusting for demographics and tumor characteristics, especially in ER-low/HER-2-positive breast cancer. After additional adjustment for treatment mode, the association maintained a similar trend. Patients who received >3 years of ET was associated with a better DFS. There was no significant difference in BCSS between patients with 2–3 years and >3 years of ET. ConclusionFor ER-low patients, findings suggest that ET with AI/T + AI may be a reasonable treatment alternative. This effect should be assessed in randomized studies.