Receptor-positive Breast Cancer Patients Research Articles

Prognostic and Predictive Significance of HER2-low Expression in Metastatic Hormone Receptor Positive Breast Cancer Patients Receiving CDK4-6 Inhibitor Therapy

Objective: This study aimed to analyze the predictive and prognostic value of HER2-low expression in hormone receptor (HR) positive human epidermal growth factor receptor-2 (HER2) negative metastatic breast cancer patients receiving cyclin-dependent kinase-4/6 inhibitor (CDK4/6i) therapy. Methods: This retrospective study included patients who received CDK4/6i plus endocrine therapy (ET). The pathological and clinical characteristics and survival times of the patients were compared and analyzed. Results: Our study included 122 patients. There were HER2-zero 88(72%) and HER2-low 34 (28%) patients. The median progression free survival (mPFS) of all patients who received CDK4/6i+ET was 21 (95% confidence interval (CI),18.5–23.5) months, while mPFS was not reached in the HER2-zero group, and mPFS in the HER2-low group was 12 (95%CI, 6.8–17.1) months (p=0.001). The mPFS was shorter in patients with primary endocrine resistance (6 vs. 21 months, p=0.001). There was a change in the HER2-low status of 26(45%) patients with recurrence compared to the first biopsy. In the HER2-zero and HER2-low groups, 22(25%) and 24(71%) patients, respectively, progressed with CDK4/6i+ET (p=0.001). Estrogen receptor (ER) levels less than and greater than 50% resulted different mPFS (6 and 21 months, respectively) (p=0.025). Median PFS differed based on CDK4/6i+ET combination, treatment line, and best treatment response (all p=0.001). In multivariate analysis, HER2 status(p=0.018), chemotherapy status(p=0.006), best response status with CDK4/6i (p=0.001) for PFS, and best response status with CDK4/6i therapy (p=0.007) for OS were significant. Conclusions: In patients with HR+HER- metastatic breast cancer receiving CDK4/6i therapy, the duration of mPFS was lower in the HER2-low group than that in the HER2-zero group. HER2-low expression is a predictive biomarker of response to CDK4/6 inhibitor therapy.

Abstract PO4-04-10: Selection criteria for second-line treatments after Cyclin-dependent kinase 4/6 inhibitors failure in Hormone Receptor positive metastatic breast cancer patients: the preliminary experience of the HERMIONE-13 trial

Abstract BACKGROUND: Cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) represent the standard of care for hormone receptor-positive (HR+)/Human Epidermal Growth Factor 2 negative (HER2-) metastatic breast cancer (MBC); no consensus actually exists for second-line (2L) treatments. The HERMIONE-13 trial evaluates the adopted therapeutic options after CDK4/6i failure and the potential factors that influence these choices. METHODS: HERMIONE-13 is a retrospective and prospective multicentric observational trial involving 15 Italian Centers. Information about therapies after disease progression to CDK4/6i from January 2016 until December 2020 was collected for the present analysis, aiming at evaluating which are the clinical criteria guiding the choice of 2L therapy after CDK4/6i failure. RESULTS: 114 pts have been enrolled: 14 pts (12.3%) received neoadjuvant treatment, 80 (70.2%) adjuvant therapy, of whom 46 (57.5%) chemotherapy (CT)+ endocrine therapy (ET) and 33 (41.3%) ET alone. Among the 79 pts treated with adjuvant ET, 44 pts (55.7%) showed primary or secondary endocrine resistance. 39 pts (34.2%) received first-line (1L) CDK 4/6i in combination with Fulvestrant (F), the remaining with aromatase inhibitors (AIs). Median duration of 1L CDK4-6i was 10.4 months (interquartile range (IQR) 4.7-17.4). Median age at 2L start was 59.5 years (IQR 51-68). As 2L therapy, 79 pts (69.3%) received CT±targeted therapy (TT) and 35 (30.7%) ET±TT. Visceral metastases at the beginning of 2L were documented in 46 pts (40.4%). Comorbidities were present in 48 (42.9%) of the pts, mainly cardiac diseases (25, 22.3%); 71 pts (63.4%) showed a 1L-related adverse events (AEs) (NA: 2 pts), mainly hematological toxicity (60, 53.6%). Among 79 pts treated with adjuvant ET, 60 (76%) were addressed to 2L CT±TT, while 19 (24%) to an ET-based therapy. A longer duration of 1L was observed in pts treated with 2L ET±TT (12.5 months, 7.2-22.2) in comparison with those treated with CT±TT (8.7 months, 4.2-14.9). The majority of pts treated with 1L F+CDK 4/6i underwent 2L CT-based treatment, as summarized in Table 1. Analyzing the population treated with 2L CT±TT, we observed that median age was 58 years (49-67), slightly lower than that of pts treated with ET±TT (63, 54-69). Among pts with visceral metastases, 30 pts (65.2%) were addressed to 2L CT±TT and 16 pts (34.8%) to ET±TT; CT±TT was also the favorite choice for the majority of pts with concomitant disease (33, 68.8%) and with previous CDK4/6i-related toxicity (47, 66.2%). Among age, visceral metastases, comorbidities, adjuvant treatment, duration of 1L therapy, use of 1L F and 1L related AEs, univariate analysis revealed that previous use of adjuvant ET (p=0.021), 1L F (p< 0.001), duration of 1L treatment (p=0.043) and age at 2L start (p=0.053) are all factors influencing the choice of 2L therapy. At the multivariate logistic regression model, only the use of F in association with CDK4/6i as 1L treatment and age at 2L therapy were associated with the choice between ET±TT vs CT± TT, with a backward selection, as summarized in table 2. CONCLUSION: The preliminary results of our study suggest that previous F in combination with CDK4/6i is a statistically confirmed predictor of choice for subsequent CT, whereas the increase in age is significantly associated with the choice for ET. Further research is needed to investigate the optimal sequencing of treatments following CDK4/6i and to determine predictive factors of response to 2L therapies. Table 1 2L choices in 1L F+CDK 4/6i pts. Table 2 Statistical model with the significant variables. Citation Format: Marina Elena Cazzaniga, Emanuela Rossi, Antonella Turla, Massimo Ambroggi, Alice Baggi, Rossana Berardi, Fulvio Borella, Serena Capici, Federica Cicchiello, Luigi Coltelli, Ugo De Giorgi, Antonella Ferro, Ornella Garrone, Monica Giordano, Elisabetta Landucci, Marco Mazzotta, Gabriella Moretti, Raffaella Palumbo, Francesca Fulvia Pepe, Patrizia Vici, Fable Zustovich, Viola Cogliati. Selection criteria for second-line treatments after Cyclin-dependent kinase 4/6 inhibitors failure in Hormone Receptor positive metastatic breast cancer patients: the preliminary experience of the HERMIONE-13 trial [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-04-10.

Abstract PO1-02-04: The efficacy of endocrine therapy during the waiting period for surgery for hormone receptor-positive breast cancer

Abstract Background: Although neoadjuvant endocrine therapy has been used to improve breast conservation rate, its prognostic relevance is unknown. The search for a valid prognostic factor equivalent to pCR in neoadjuvant chemotherapy is a current challenge in neoadjuvant endocrine therapy. In this study, we investigated the efficacy of short term neoadjuvant endocrine therapy utilizing the waiting period for surgery and the prognostic factor including Preoperative Endocrine Prognostic Index (PEPI) score. Methods: A total of 480 patients with StageⅠ-Ⅲ hormone receptor-positive, HER2-negative breast cancer was treated with endocrine therapy during the waiting period for surgery between October 2012 and December 2021. Premenopausal and postmenopausal patients received tamoxifen (TAM) and non-steroidal aromatase inhibitor (AI), respectively. The primary endpoint was change in tumor size by ultrasound and Ki67 expression before and after short term endocrine therapy. The secondary endpoint was prognosis of patients divided by PEPI score which was calculated using tumor size, lymph node metastasis, Ki67 expression, and ER Allred score. This study was approved by the institutional review board of Teikyo University. Results: Median age and tumor size of the entire 480 patients was 55 years (range, 31 - 89), 1.65 cm (range, 0.4 – 7.5), respectively. The median duration of endocrine therapy was 39 days (range, 1-90). 19 (0.3 %) pts were clinically node-positive. Of the entire 480 patients, 211 and 269 patients had TAM and AI, respectively. After short term endocrine therapy, the tumor size was significantly reduced from 1.83cm to 1.62cm in AI group (p=0.02), although TAM did not decrease the tumor size (p=0.89). The Ki67 expression was significantly decreased from 16.4% to 11.3% in TAM group (p=0.002), and from 15.9% to 6.98% in AI group (p < 0.001). There were significant differences in the distribution of PEPI scores between TAM and AI group (p < 0.05) (Table). At the median follow-up of 1012 days, patients with PEPI score ≥ 4 showed worse disease-free survival compared with patients with PEPI score < 4 in TAM group (Figure1). Overall survival is not available because there were no deaths in TAM group. At the median observation period of 833 days, patients with PEPI score ≥ 4 showed worse disease-free survival compared with patients with PEPI score < 4 in AI group (Figure2). In terms of mortality, patients with PEPI score ≥ 4 had worse overall survival than patients with PEPI score 0 and 1-3 (p=0.07). Conclusions: These results suggested that neoadjuvant endocrine therapy during the waiting period for surgery might be effective in reducing Ki67 expression level and PEPI score might be useful in predicting the prognosis of hormone receptor positive breast cancer patients. Table. Distribution of PEPI score Figure 1. Disease-free survival in patients treated with tamoxifen Figure 2. Disease-free survival in patients treated with non-steroidal aromatase inhibitor Citation Format: Yuka Maeda, Saki Naruse, Yuka Isono, Ayana Sato, Akiko Matsumoto, Tatsuhiko Ikeda, Hiromitsu Jinno. The efficacy of endocrine therapy during the waiting period for surgery for hormone receptor-positive breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-02-04.

Abstract PO1-04-14: PLK1 Inhibitor Onvansertib Extends the Response and Overcomes Resistance to Paclitaxel in Palbociclib-resistant HR+ Breast Cancer Patient-derived Xenografts

Abstract Paclitaxel is a first-line chemotherapy for hormone receptor positive (HR+) metastatic breast cancer patients after progression on a combination of endocrine and CDK4/6 inhibitor therapy. Response rate to paclitaxel ranges between 20-40%, and most patients progress due to intrinsic or acquired resistance, leaving them with limited treatment choices. Therapeutic strategies to overcome paclitaxel resistance and extend its clinical benefit are urgently needed. Paclitaxel-resistant tumors have increased expression of polo-like kinase 1 (PLK1), a serine-threonine-protein kinase that regulates mitosis and cell cycle progression. PLK1 has also been shown to mediate resistance to CDK4/6 inhibitor palbociclib in HR+ breast cancer. Onvansertib is an orally bioavailable, highly potent, and selective PLK1 inhibitor in clinical development. Onvansertib exhibited synergy in vitro and showed potent anti-tumor activity in vivo in combination with paclitaxel in ovarian cancer and triple negative breast cancer (TNBC) preclinical models. A phase 1b/2 clinical trial is ongoing to evaluate the safety and efficacy of onvansertib plus paclitaxel in advanced TNBC (NCT05383196). Here we investigated whether the potential of onvansertib and paclitaxel combination could be extended to HR+ breast cancer. Co-treatment with onvansertib and paclitaxel synergistically inhibited the viability of HR+ breast cancer cell lines (MCF-7, T-47D, EFM-19, CAMA-1, ZR-75-1). Compared to the single agents, the combination induced increased apoptosis. The combination was further tested in vivo in patient-derived xenograft (PDX) models. To this end, six HR+ breast cancer PDXs with intrinsic or acquired resistance to palbociclib were established from primary breast tumors (n=1) or metastatic bone biopsies (n=5). The PDX tumors were engrafted subcutaneously in nude mice and the effect of onvansertib (45 mg/kg, oral, 5 days a week) and paclitaxel (15-25mg/kg, IP, once a week) was investigated as monotherapy and in combination. The combination of onvansertib and paclitaxel was tolerated with minimal toxicity and showed enhanced anti-tumor activity compared to either agent alone in all 6 PDX models. Paclitaxel and onvansertib monotherapies had little to no anti-tumor activity in 4 out of 6 PDX models (HBCx124palboR25, HBCx-139 palboR5, HBCx-202 and HBCx-131). Importantly, the combination of onvansertib and paclitaxel induced strong anti-tumor activity in these 4 models. Tumor regression was observed in the 2 PDX models with acquired resistance to palbociclib, HBCx-124palboR25 and HBCx-139palboR5, with 62% (5/8 mice) and 54% (6/11 mice) complete response rates, respectively. The combination showed tumor stasis in HBCx-131 and induced tumor regression in HBCx-202, established from patient resistant to palbociclib. In 2 of the 6 PDXs, paclitaxel exhibited anti-tumor activity, inducing tumor regression in HBCx-137paboR26 and tumor stasis in HBCx-86. In comparison, the combination of onvansertib and paclitaxel had greater activity, inducing tumor regression in both models with a higher rate of complete response than the monotherapy. Complete response rates for paclitaxel versus the combination were 62% and 100% in the HBCx-137palboR26 model and 0% and 67% in the HBCx-86 model. Notably, the antitumor activity of the onvansertib and paclitaxel combination was very durable, showing a robust delay in tumor relapse after stopping therapy. Together, our data strongly support that combining paclitaxel with the PLK1 inhibitor onvansertib extends its benefit and overcomes paclitaxel resistance, and represents a promising therapeutic strategy for HR+ breast cancer patients after progression on a combination of endocrine and CDK4/6 inhibitor therapy. Citation Format: Maya Ridinger, Pierre Painsec, Sreeja Sreekumar, Dalia Gonzalez, Davis Klein, Laura Sourd, Elodie Montaudon, Paul Cottu, Tod Smeal, Elisabetta Marangoni. PLK1 Inhibitor Onvansertib Extends the Response and Overcomes Resistance to Paclitaxel in Palbociclib-resistant HR+ Breast Cancer Patient-derived Xenografts [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-04-14.

Abstract LB450: Mechanisms of acquired resistance to AKT inhibitor capivasertib in AKT1 mutant patient derived breast cancer models

Abstract Capivasertib, a pan-AKT inhibitor, has recently been approved in combination with fulvestrant to treat ER+ HER2- breast cancer patients with one or more of PI3KCA/AKT1/PTEN alterations. However, as with many targeted therapies acquired drug resistance remains a challenge. In this study, we investigated the mechanisms of acquired capivasertib resistance in AKT1 mutant breast cancer models. We established two estrogen receptor positive (ER+) AKT1 E17K mutant breast cancer patient derived organoids (PDOs), that were sensitive to capivasertib in vitro. PDOs were chronically exposed to 1µM capivasertib. PDOs were sequenced (DNA & RNA) at multiple timepoints of treatment (3 days, 4 and 9 weeks after development of resistance). Using CRISPR-Cas9 editing we also engineered CAMA1 (ER+) cells to express AKT1 E17K, and generated derived resistant AKT1 mutant and parental models in response to continuous capivasertib exposure. CAMA1 AKT1 mutant and parental resistant were screened with an siRNA Kinome library supplemented with genes from the PDO RNA-sequencing. Phosphoproteomics was performed on capivasertib resistant E17K models in the presence and absence of capivasertib. PDO models persisting after either 4 or 9 weeks of capivasertib treatment showed no acquisition of genetic driver events, or changes in AKT copy number. Resistant organoids at 9 weeks treatment showed increased expression of ERBB3 (HER3), EGFR3 and LPAR5 as well as increased expression of MAPK signalling components. In siRNA screens on CAMA1 derived resistant models, siRNA targeting ERBB2, CDK4, PLK4 and CHEK1, ESR1 and LPAR5 modulated sensitivity to capivasertib. Analysis of phosphoproteins in capivasertib resistant models revealed reactivation of mTORC1 signalling despite sustained AKT inhibition, with dramatic upregulation of PDK1 and mTORC2. Consistent with the siRNA screens, drug combinations identified fulvestrant as a synergistic agent with capivasertib in the resistant E17K models. Sensitivity to mTOR inhibition was maintained in capivasertib resistant models, with mTOR inhibition re-sensitizing cells to capivasertib. In these models, acquired resistance to capivasertib occurs through upregulation of ER signalling and rewiring of pathway signalling to bypass AKT inhibition and reactivate mTORC1. Targeting these mechanisms could prolong the use of capivasertib, with future work focusing on validating these mechanisms and re-sensitizing drug combinations in other AKT1 mutant models & PDOs. Citation Format: Sarah Mearns, Alex Pearson, Li-Xuan Sim, Rosalind Cutts, Prithika Sritharan, Heena Shah, Aditi Gulati, Stuart Williamson, Simon Barry, Elza De-Bruin, Nicholas C. Turner. Mechanisms of acquired resistance to AKT inhibitor capivasertib in AKT1 mutant patient derived breast cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB450.

Abstract 3829: Identifying the genetic basis for resistance to aromatase inhibitors (AIs) in metastatic breast cancer (mBC) patients

Abstract Background: AIs, such as letrozole, anastrozole, and exemestane, are used to treat hormonal receptor positive (HR+) breast cancer (BC) patients. Many patients either respond poorly or relapse on AIs. A comprehensive view of the underlying genomic landscape of such patients may aid in a better understanding of the resistance mechanisms and the development of new targeted therapies. This study leveraged a real-world clinico-genomic dataset of BC patients to identify key biomarkers and the corresponding high frequency mutations in this relapse/refractory patient population. Methods: This retrospective study used ConcertAI’s Genome360TM BC dataset (N=3223). A cohort of HR+/HER2− mBC patients who were either refractory to or relapsed on AIs (N=863) were included. To identify key resistance genes/pathways, only genomic data (from NGS tests) from a biopsy performed after tumor progression on AIs was considered. Prevalence analysis was done only based on known pathogenic/likely pathogenic variants. Pathway analysis of identified biomarkers with the mechanism of action of AIs was performed to identify and validate key resistance pathways. Further, signatures of unique protein alteration were identified within the biomarkers of interest. Results: The median age of patients at the time of diagnosis was 54 years. Adenocarcinoma and Lobular/Ductal carcinoma were the two most prevalent histologies. 51% of patients in this cohort were treated with AIs in the first line. Table 1 lists the 20 most frequently altered genes observed in this cohort. Genes with a potential role in AI resistance based on pathway analysis are indicated. The most common mutations in selected biomarkers are also provided. These biomarkers were observed at a much lower frequency in patients who responded well to AIs. Conclusion: This study provides insights into the genetic basis of resistance to AIs in mBC patients, opening up possibilities for development of new treatments to overcome resistance. Table 1: Top 20 biomarkers and their variants found in AI relapse/refractory mBC Patients. Biomarker Prevalence (%) Potential Role in AI Resistance Key Variants ESR1 37.81 Yes D538G, Y537, Y537N, E380Q, Y537C PIK3CA 37.04 Yes H1047R, E545K, E542K, H1047L, E726K, N345K TP53 35.56 GATA3 14.25 Yes Frameshift Mutation (Mainly) CDH1 11.64 Yes Frameshift Mutation (Mainly) MAP3K1 9.35 KMT2C 9.02 PTEN 8.38 Yes Frameshift Mutation (Mainly) ARID1A 8.17 RB1 7.38 Yes Frameshift Mutation (Mainly) DNMT3A 5.31 ATM 5.30 TBX3 5.10 SPEN 4.90 NF1 4.82 Yes Frameshift Mutation (Mainly) AKT1 4.74 Yes E17K ERBB2 3.21 Yes L755S, V777L, D769Y KRAS 2.74 Yes G12V, G12D, G12A BRCA1/2 2.69 CDKN2A 1.78 Citation Format: Neeraj Singh, Smita Agrawal. Identifying the genetic basis for resistance to aromatase inhibitors (AIs) in metastatic breast cancer (mBC) patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3829.

Epiretinal membrane formation associated with letrozole therapy – Two case reports

Letrozole, an aromatase inhibitor, is being increasingly used for adjuvant endocrine therapy in hormone-positive postmenopausal breast cancer. Although aromatase inhibitors are thought to increase vitreoretinal traction by causing estrogen reduction, posterior segment complications of letrozole are sparsely reported. We report two cases of presumed epiretinal membrane formation in postmenopausal, hormone receptor–positive breast cancer patients on adjuvant letrozole therapy.

Quantitative expression of oestrogen receptor in breast cancer: Clinical and molecular significance

BackgroundOestrogen receptor (ER) positive breast cancer (BC) patients are eligible for endocrine therapy (ET), regardless of ER immunohistochemical expression level. There is a wide spectrum of ER expression and the response to ET is not uniform. This study aimed to assess the clinical and molecular consequences of ER heterogeneity with respect to ET-response. MethodsER expression, categorised by percentage and staining intensity in a large BC cohort (n = 7559) was correlated with clinicopathological parameters and patient ET response. The Cancer Genome Atlas Data BC cohort (n = 1047) was stratified by ER expression and transcriptomic analysis completed to better understand the molecular basis of ER heterogeneity. ResultsThe quantitative proportional increase in ER expression was positively associated with favourable prognostic parameters. Tumours with 1–9% ER expression were characteristically similar to ER-negative (<1%) tumours. Maximum ET-response was observed in tumours with 100% ER expression, with responses significantly different to tumours exhibiting ER at < 100% and significantly decreased survival rates were observed in tumours with 50% and 10% of ER expression. The Histochemical-score (H-score), which considers both staining intensity and percentage, added significant prognostic value over ER percentage alone with significant outcome differences observed at H-scores of 30, 100 and 200. There was a positive correlation between ER expression and ESR1 mRNA expression and expression of ER-regulated genes. Pathway analysis identified differential expression in key cancer-related pathways in different ER-positive groups. ConclusionET-response is statistically proportionally related to ER expression with significant differences observed at 10%, 50% and 100%. The H-score adds prognostic and predictive information.

Individual participant data from digital sources informed and improved precision in the evaluation of predictive biomarkers in Bayesian network meta-analysis

ObjectivesWe aimed to develop a network meta-analytic model for the evaluation of treatment effectiveness within predictive biomarker subgroups, by combining evidence from individual participant data (IPD) from digital sources (in the absence of randomized controlled trials) and aggregate data (AD). Study Design and SettingA Bayesian framework was developed for modeling time-to-event data to evaluate predictive biomarkers. IPD were sourced from electronic health records, using a target trial emulation approach, or digitized Kaplan-Meier curves. The model is illustrated using two examples: breast cancer with a hormone receptor biomarker, and metastatic colorectal cancer with the Kirsten Rat Sarcoma (KRAS) biomarker. ResultsThe model allowed for the estimation of treatment effects in two subgroups of patients defined by their biomarker status. Effectiveness of taxanes did not differ in hormone receptor positive and negative breast cancer patients. Epidermal growth factor receptor inhibitors were more effective than chemotherapy in KRAS wild type colorectal cancer patients but not in patients with KRAS mutant status. Use of IPD reduced uncertainty of the subgroup-specific treatment effect estimates by up to 49%. ConclusionUtilization of IPD allowed for more detailed evaluation of predictive biomarkers and cancer therapies and improved precision of the estimates compared to use of AD alone.

MCM-2 Levels as a Potential Biomarker for Predicting High-Risk Breast Cancer Patients According to TAILORx Classification.

The minichromosome maintenance protein-2 (MCM-2) is a more sensitive proliferation marker than Ki-67. This study aimed to evaluate the relationship between MCM-2 and Oncotype DX recurrence score (ODX-RS) and determine an MCM-2 cutoff value in high-risk patients according to TAILORx risk categorization. Hormone receptor (HR) positive HER-2 negative early-stage breast cancer patients (pT1-2, pN0-N1, M0) who had ODX-RS were included in the study. According to the TAILORx trial, patients were divided into two groups with high (ODX-RS ≥26) and low risk (ODX-RS <26) in terms of ODX-RS. Formalin-fixed-paraffin-embedded tissues of patients were re-evaluated, and 3 µm sections were prepared for MCM-2 immuno-histochemical staining. The relationship between ODX-RS and the percentage of MCM-2 staining was evaluated in two groups. The ROC curve analysis was performed to determine the MCM-2 cut-off value for the TAILORx high-risk group (ODX-RS ≥26). The mean MCM-2 value was significantly higher in the high-risk group [(60.2 ± 11.2 vs 34.4 ± 13.8, p < 0.001)]. In the multivariate analysis, MCM-2 (OR: 1.27, 95% CI: 1.08-1.49, p = 0.003) and progesterone receptor (PR) levels ≤10% (OR: 60.9, 95% CI: 4.1-89.7, p = 0.003) were found to be independent factors indicating a high-risk group. A one-unit increase in MCM-2 level increased the likelihood of being in the high-risk group by 1.27 times. In the ROC curve analysis, the optimal MCM-2 cut-off level was 50 (AUC: 0.921, sensitivity: 86.7%, specificity: 96.0%, p < 0.001). Our study is the first study in the literature to investigate the relationship between ODX-RS and MCM-2 levels in HR-positive HER-2 negative early breast-cancer patients. In this study, MCM-2 was an independent risk factor in identifying high-risk patients according to TAILORx risk classification. MCM 2 cut-off value (50) may help the decision on adjuvant chemotherapy in patients where the Oncotype DX test cannot be performed.

18 F-FES Uptake in Radiation Pneumonitis.

18 F-FDG uptake in radiation pneumonitis is well documented; however, the same is less so for 18 F-floroestradiol (FES), which specifically binds to the estrogen receptors in vivo. We observed increased FES uptake in the right lung of an estrogen receptor positive breast cancer patient who had undergone right modified radical mastectomy followed by radiation therapy to chest wall. The possibility of FES uptake in radiation pneumonitis must therefore be kept in mind while interpreting FES PET.

A multi-dimensional nomogram to predict non-sentinel lymph node metastases in T1-2HR+ breast cancer.

Axillary lymph node dissection (ALND) could be omitted for T1-2 breast cancer patients with 1-2 positive sentinel lymph node (SLN) after breast-conserving surgery when radiation is planned. However, whether ALND could be replaced by radiation in patients with 1-3 positive SLNs when no more non-SLN metastasis were observed after mastectomy are still controversial. The aim of our study was to develop and validate a nomogram for predicting the possibility of non-SLN metastasis in T1-2 and hormone receptor (HR) positive breast cancer patients with 1-3 positive SLNs after mastectomy. We retrospectively reviewed and analyzed the data including the basic information, preoperative sonographic characteristics, and pathological features in breast cancer patients with 1-3 positive SLNs in our medical center between Jan 2016 and Dec 2021. The Chi-square, Fisher's exact test, and t test were used for comparison of categorical and qualitative variables among patients with or without non-SLN metastasis. Univariate and multivariate logistic regression were used to determine the risk factors for non-SLN metastasis. These predictors were used to build the nomogram. The C-index and area under the receiver operating characteristic curve (AUC) was calculated to assess the accuracy of the model. A total of 49 in 107 (45.8%) patients were identified with non-SLN metastasis. In multivariate analysis, four variables including younger age, lower estrogen receptor (ER) expression, higher histological score, and cortex thickening of the lymph nodes were determined to be significantly associated with non-SLN metastasis. An individualized nomogram was consequently established with a favorable C-index of 0.822 and verified via two internal validation cohorts. The current study developed a nomogram predicting non-SLN metastasis for T1-2 and HR+ breast cancer with 1-3 positive SLNs after mastectomy and found that patients in the high-risk group exhibited worse relapse-free survival. The novel nomogram may further help surgeons to determine whether ALND could be omitted when 1-3 positive SLNs were observed in T1-2 and HR+ breast cancer patients.

Estrogen-Induced LncRNA, LINC02568, Promotes Estrogen Receptor-Positive Breast Cancer Development and Drug Resistance Through Both In Trans and In Cis Mechanisms.

Endocrine therapy is the frontline treatment for estrogen receptor (ER) positive breast cancer patients. However, the primary and acquired resistance to endocrine therapy drugs remain as a major challenge in the clinic. Here, this work identifies an estrogen-induced lncRNA, LINC02568, which is highly expressed in ER-positive breast cancer and functional important in cell growth in vitro and tumorigenesis in vivo as well as endocrine therapy drug resistance. Mechanically, this work demonstrates that LINC02568 regulates estrogen/ERα-induced gene transcriptional activation in trans by stabilizing ESR1 mRNA through sponging miR-1233-5p in the cytoplasm. Meanwhile, LINC02568 contributes to tumor-specific pH homeostasis by regulating carbonic anhydrase CA12 in cis in the nucleus. The dual functions of LINC02568 together contribute to breast cancer cell growth and tumorigenesis as well as endocrine therapy drug resistance. Antisense oligonucleotides (ASO) targeting LINC02568 significantly inhibits ER-positive breast cancer cell growth in vitro and tumorigenesis in vivo. Furthermore, combination treatment with ASO targeting LINC02568 and endocrine therapy drugs or CA12 inhibitor U-104 exhibits synergistic effects on tumor growth. Taken together, the findings reveal the dual mechanisms of LINC02568 in regulating ERα signaling and pH homeostasis in ER-positive breast cancer, and indicated that targeting LINC02568 might represent a potential therapeutic avenue in the clinic.

Assessment of Intratumoural and Stromal Infiltrating Lymphocytes In The Various Subtypes of Breast Carcinoma Patients who have Received Neoadjuvant Chemotherapy.

Breast cancer comprises a highly heterogeneous subset of tumours that respond well to Neoadjuvant Chemotherapy (NAC). Tumour Infiltrating Lymphocytes (TIL) act as a means to an end by shedding light on the treatment response as well as predictive factors to the clinicopathological features for the same. Therefore, this article attempts to shift the attention to the relevance of TIL in the aforementioned aspects by bringing to notice the contrasting traits displayed by them in the different immunohistochemical subtypes of breast carcinoma. 75 triple-negative breast cancer (TNBC) patients, 25 human epidermal growth factor receptor (HER2BC) positive patients and 77 hormone receptor (HRBC) positive breast cancer patients were included in this study who received NAC before surgical excision of the tumour which was then stained using routine Haematoxylin and Eosin techniques. Standardised guidelines were used to evaluate TIL in the stroma and the tumour. In TNBC, a significant association between Intratumoural (IT) TIL (p=0.0288) and Intrastromal (IS) TIL (p=0.0250) with pathological complete response (pCR). IS TIL and age at operation (p=0.0494) showed significant values but no correlation was found with IT TIL. In HER2BC, IS TIL revealed a significant association with the tumour response(p=0.0229). A strong association was found between IT TIL and the age of menopause(p=0.0441). In HRBC, no significant associations were found between IT and IS TIL scores and the clinicopathological features. The predictive factors of TIL and complete response post-neoadjuvant chemotherapy can be a strong indicative factor for immunohistochemical markers. It also helps throw light on further studies which can be carried out to determine the clinicopathological features and TIL correlation in the various subtypes of breast carcinoma.

Glucose-dependent effect of insulin receptor isoforms on tamoxifen antitumor activity in estrogen receptor-positive breast cancer cells.

Breast cancer is the most common malignancy in women, and it is linked to several risk factors including genetic alterations, obesity, estrogen signaling, insulin levels, and glucose metabolism deregulation. Insulin and Insulin-like growth factor signaling exert a mitogenic and pro-survival effect. Indeed, epidemiological and pre-clinical studies have shown its involvement in the development, progression, and therapy resistance of several cancer types including breast cancer. Insulin/Insulin-like growth factor signaling is triggered by two insulin receptor isoforms identified as IRA and IRB and by Insulin-like growth factor receptor I. Both classes of receptors show high homology and can initiate the intracellular signaling cascade alone or by hybrids formation. While the role of Insulin-like growth factor receptor I in breast cancer progression and therapy resistance is well established, the effects of insulin receptors in this context are complex and not completely elucidated. We used estrogen-dependent insulin-like growth factor receptor I deleted gene (MCF7IGFIRKO) breast cancer cell models, lentivirally transduced to over-express empty-vector (MCF7IGFIRKO/EV), IRA (MCF7IGFIRKO/IRA) or IRB (MCF7IGFIRKO/IRB), to investigate the role of insulin receptors on the antiproliferative activity of tamoxifen in presence of low and high glucose concentrations. The tamoxifen-dependent cytotoxic effects on cell proliferation were determined by MTT assay and clonogenic potential measurement. Cell cycle and apoptosis were assessed by FACS, while immunoblot was used for protein analysis. Gene expression profiling was investigated by a PCR array concerning genes involved in apoptotic process by RT-qPCR. We found that glucose levels played a crucial role in tamoxifen response mediated by IRA and IRB. High glucose increased the IC50 value of tamoxifen for both insulin receptors and IRA-promoted cell cycle progression more than IRB, independently of glucose levels and insulin stimulation. IRB, in turn, showed anti-apoptotic properties, preserving cells' survival after prolonged tamoxifen exposure, and negatively modulated pro-apoptotic genes when compared to IRA. Our findings suggest that glucose levels modify insulin receptors signaling and that this event can interfere with the tamoxifen therapeutic activity. The investigation of glucose metabolism and insulin receptor expression could have clinical implications in Estrogen Receptor positive breast cancer patients receiving endocrine treatments.

Abstract P5-03-04: PIK3CA mutation prevalence in hormone receptor positive breast cancer patients in United Arab Emirates

Abstract Background: The Phosphatidylinositol-4,5-bisphosphonate 3-kinase catalytic subunit alpha (PIK3CA) gene is mutated in about 30-40% of hormone receptor positive/human epidermal growth factor receptor 2 negative (HR+/HER2−) breast cancer (BC) patients. For HR+/HER2- advanced breast cancer patients with disease progression following endocrine-based therapy, the NCCN guideline recommends testing for PIK3CA mutations with tumour or liquid biopsy to identify suitable patients for targeted therapy with alpelisib, an oral α-specific PI3K inhibitor in combination with fulvestrant. The primary objective of this study was to evaluate the proportion and distribution of PIK3CA mutational landscape of HR+ve BC patients at the largest cancer centre in the United Arab Emirates (UAE). Material and methods: Retrospective review of consecutive HR+ve BC patients at Tawam Hospital for whom PIK3CA testing was requested. DNA was extracted from the samples and a targeted resequencing assay was used for mutation detection in exons 7, 9 and 20 of the PIK3CA gene. Sequencing was carried out using the Next Generation Sequencing platform Ion GeneStudio S5 Prime System with a detection limit of 2-5% of the mutant allelic content. Results: 124 patients with HR+ve BC were enrolled in the present study. The pathology samples were considered unsuitable/unsatisfactory in 18 cases. The median age was 51.5 years (range 31-90). All patients were female, 54% were post-menopausal and 49% presented with de-novo metastatic disease. Of the 106 eligible patients, PIK3CA mutations were detected in 33 (31%) patients, the most common being H1047R (45%) and E545K (30%) mutations in exons 20 and 9, respectively. Other less common mutations included C420R mutations (6%) in exon 7, E542 (6%) in exon 9 and H1047Y (3%) in exon 20. 9% of patients had more than one hotspot mutations, primarily in exons 9 and 20. Of the 12 HER2 +ve patients tested, 3 had PIK3CA mutations, most commonly the H1047R mutation in exon 20. Conclusion: The prevalence of PIK3CA mutations and the presence of most common hotspot mutations in exons 20 and 9 was consistent with prior published studies. The clinical relevance of PIK3CA mutations in HER2 +ve BC patients needs further assessment. Citation Format: Fathi Azribi, Mohammad Hourani, Sulaman Magdub, Aydah Alawadhi, Ali Yousif, Emad Dawoud, Khaled Al Qawasmeh, Mouza Al Ameri, Diaeddine Trad, Nouri Bennini, Mohamed Ahmed, Jawaher Ansari. PIK3CA mutation prevalence in hormone receptor positive breast cancer patients in United Arab Emirates [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 P5-03-04.

Abstract P2-03-19: The efficacy of neoadjuvant endocrine therapy during the waiting period for surgery in postmenopausal hormone receptor positive breast cancer

Abstract Background: Although neoadjuvant endocrine therapy has been used to improve breast conservation rate, its prognostic relevance is unknown. The search for a valid prognostic factor equivalent to pCR in neoadjuvant chemotherapy is a current challenge in neoadjuvant endocrine therapy. In this study, we investigated the efficacy of short term neoadjuvant endocrine therapy utilizing the waiting period for surgery and the prognostic factor including Preoperative Endocrine Prognostic Index (PEPI) score. Patients and Methods: A total of 269 postmenopausal women with hormone receptor-positive, HER2-negative breast cancer was treated with endocrine therapy with non-steroidal aromatase inhibitor during the waiting period for surgery between October 2012 and November 2021. Of the entire 269 patients, 92 and 177 patients had anastrozole and letrozole, respectively. The primary endpoint was change in tumor size by ultrasound and Ki67 before and after short-term endocrine therapy. The secondary endpoint was prognosis of patients divided by PEPI score which was calculated using tumor size, lymph node metastasis, Ki67, and ER Allred score. This study was approved by the institutional review board of Teikyo University. Results: Median age was 68 years old (range, 41-89). ER and PgR was positive in 266 (98%) and 232 (86%) of the entire 269 patients, respectively. Median tumor size was 1.65 cm (range, 0.4-7.5). Seventeen (6.3%) pts were clinically node-positive. Patients with histological grade I tumor were 190 (70.6%). The median duration of endocrine therapy was 39 days (range, 2-88). Average pretreatment Ki67 expression was 10% (range, 0-90). Tumor diameter was significantly decreased to 1.43cm (range,0.45-5.83) after short-term endocrine therapy (p=0.01). The Ki67 expression was significantly decreased to 3.0% (range, 0-85) after endocrine therapy (p&amp;lt; 0.01) and only five patients (1.9%) showed marked increase in Ki-67 expression. PEPI score 0, 1-3 and ≥ 4 was found in 83 (30.9%),147 (54.7%) and 39 patients (14.5%), respectively. After the median observation period of 928 days, patients with PEPI score ≥ 4 showed worse disease-free survival (Figure) compared with patients with PEPI score 0 and 1-3 (p=0.06). In terms of mortality, patients with PEPI score ≥ 4 had worse overall survival than patients with PEPI score 0 and 1-3 (p=0.07). Conclusions: These results suggested that neoadjuvant endocrine therapy during the waiting period for surgery might be effective in reducing the size and Ki67 expression level and PEPI score might be useful in predicting the prognosis of postmenopausal hormone receptor positive breast cancer patients. Citation Format: Yuka Maeda, Saki Naruse, Yuka Isono, Ayana Sato, Miki Yamada, Akiko Matsumoto, Tatsuhiko Ikeda, Hiromitsu Jinno. The efficacy of neoadjuvant endocrine therapy during the waiting period for surgery in postmenopausal hormone receptor positive 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 P2-03-19.

Abstract PD10-10: PD10-10 Single cell characterization of longitudinal biopsies from breast cancer patients treated with the aromatase inhibitors letrozole and exemestane in sequence

Abstract Background About 70% of breast cancer cases are hormone receptor positive, indicating that cancer cells exploit estrogens for their growth. Postmenopausal estrogen receptor positive breast cancer patients are currently often treated with aromatase inhibitors suppressing serum and tumor tissue estradiol levels by &amp;gt;90%. Two widely used aromatase inhibitors are letrozole, a nonsteroidal inhibitor and exemestane, a steroidal aromatase inactivator. While the mechanisms of action of these two drugs are well studied, their effects on the tumor immune microenvironment and mechanisms of resistance to these drugs are still not sufficiently elucidated. Study design The NEOLETEXE trial1 was a neoadjuvant, randomized, open-label, intra-patient, cross-over, single center clinical trial aiming at treating postmenopausal patients with locally advanced breast cancer defined primarily as large T3/T4 and or N2/N3. However, patients with large T2 tumors were also eligible. Patients were randomized to neoadjuvant therapy with either letrozole (2.5 mg daily) or exemestane 25 mg daily for about 3 months, followed by a cross-over to the alternative drug for another 3 months prior to surgery. 102 patients were enrolled in NEOLETEXE. Methods Pre-treatment, on-treatment (3 months) and end-of-treatment (6 months of therapy) biopsies were subjected to single-cell transcriptome, T cell receptor and B cell receptor profiling using the Chromium Single-Cell v2 5′ Chemistry (10x Genomics). Libraries were paired-end sequenced on a NovaSeq6000. Single cell gene expression matrices were analyzed with the Seurat package (v4.0.2). After filtering out stressed/dying cells or cells with low quality sequencing, gene expression of the remaining good quality cells was normalized and scaled to construct principal components and further cluster cells. Results We clustered 362.762 cells from 26 pre-treatment, 20 on-treatment and 19 end-of-treatment biopsies and identified 8 main cell types to be present: T cells, B cells, epithelial cells, fibroblasts, endothelial cells, macrophages, mast cells and dendritic cells. To further identify specific and specialized cell subtypes, we clustered the cells belonging to the above-mentioned cell types independently and annotated the clusters obtained using validated marker genes. Finally, we use statistical methods and algorithms to characterize how the proportion of the different cell types changes in tumors under treatment pressure. Specifically, we show changes in the proportion of CD8 effector memory cells under treatment pressure, with a significant increase in cytotoxic T cell proportions after two months of treatment with aromatase inhibitor. Conclusions We use single cell profiling to obtain a high-resolution map of the cell types found in tumor biopsies of the NEOLETEXE trial to characterize the effects of aromatase inhibitors on the tumor microenvironment and to identify the cancer cell signatures during treatment with letrozole or exemestane. 1. Bahrami N., Sauer T., Engebretsen S., Aljabri B., Bemanian V.,Lindstrøm J., Lüders T., Kristensen V.N., Lorentzen A., Loeng M., Ødegård H.P., Kvaløy J.Ø., Vestøl I.B., Geisler S.B., Gravdehaug B., Gundersen J.M., Geisler J. The NEOLETEXE trial: a neoadjuvant cross-over study exploring the lack of cross resistance between aromatase inhibitors. Future Oncology, 15 (32), 3675-3682, 2019. Citation Format: Salim Ghannoum, Chloé Steen, Marie Fongård, Marius Bjørnstad, Laurens Reitsma, Stephanie Geisler, Manouchehr Seyedzadeh, Unn-Cathrin Buvarp, Marie Loeng, Torben Lüders, Diether Lambrechts, Marianne Lyngra, Vessela Kristensen, Jürgen Geisler, Xavier Tekpli. PD10-10 Single cell characterization of longitudinal biopsies from breast cancer patients treated with the aromatase inhibitors letrozole and exemestane in sequence [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 PD10-10.

Unchartered waters: Significance of fall in Ki67 index after short-term preoperative endocrine therapy in early breast cancers.

Endocrine treatment for breast cancer acts largely by inhibiting tumor cell proliferation. The biomarker Ki67 is linked to the proliferative index of the tumour. To identify the factors affecting the fall in Ki67 value in early-stage hormone receptor (HR) positive breast cancer patients receiving short-term preoperative endocrine therapy in an Indian cohort. Women with hormone receptor positive, invasive, nonmetastatic, and early breast cancer (<T2, <N1) were assigned to short-term preoperative tamoxifen 20 mg daily (pre-menopausal women) or Letrozole 2.5 mg daily (post-menopausal women) for a minimum of 7 days after noting the baseline Ki67 value from the diagnostic core biopsy specimen. The postoperative Ki67 value was estimated from the surgical specimen, and the factors determining the extent of fall were evaluated. The short-term preoperative endocrine therapy resulted in a reduction in the median Ki67 index, which was significantly greater among postmenopausal women who received Letrozole (63.25 (31.94-80.5)) than among premenopausal women who received Tamoxifen (0 (-28.99-62.25)) (p-value 0.001). The fall in Ki67 value was particularly marked for patients with low-grade tumors with high Estrogen and progesterone receptor expression (p-value < 0.05). The duration of treatment (<2 week or 2-4 week or >4 week) did not affect the fall in Ki67. Preoperative therapy with Letrozole resulted in a more significant fall in Ki67, as compared to therapy with Tamoxifen. Determining the fall in Ki67 value in response to preoperative endocrine therapy could provide an insight into the response to endocrine therapy in luminal breast cancer.

ESR1-мутації як предиктор прогресії та метастазування гормонозалежного раку молочної залози

The relevance of the work is due to the need for additional research to better understand the prevalence of ESR1 mutations at different stages of recurrent disease and their prognostic implications. The purpose of the study was to determine the incidence of ESR1 mutations in ER-positive breast cancer, its prognostic value in the choice of treatment. Materials and methods. A systematic review of quality studies, which were taken from PubMed and Thomas Reuters Web of Science databases, published between 2007 and 2019 was performed. Search terms included ESR1 mutations, estrogen receptor, breast cancer, recurrence, metastasis, aromatase inhibitors, fulvestrant and tamoxifen. Only full-text studies in English on the development of ESR1 mutations and their outcomes on disease progression were included. Studies were selected using predefined data fields, taking into account the quality of the study. This prospective clinical study is conducted by means of molecular genetic analysis, namely, gene polymorphism analysis by polymerase chain reaction. This study is carried out on the basis of the Kyiv City Clinical Oncology Center and the Department of Oncology of the Bogomolets National Medical University. A retrospective cohort of hormone receptor positive breast cancer patients experiencing either local or metastatic recurrence was collected from available archival specimens. All clinical data were obtained from the patients' clinical records by an expert breast oncologist. This included age, TNM stage, grade, immunohistochemistry scores for estrogen receptor, progesterone receptor, human epidermal growth factor receptor 2, and treatment lines. Results and discussion. Estrogen receptor and progesterone receptor positivity was determined based on local pathology practice (&gt; 1% positively stained cells). The article demonstrated that ESR1 mutations often occur during AI therapy in the setting of metastasis and may play a role in metastasis progression. Advances in DNA sequencing technology have led to more sensitive detection of ESR1 mutations in clinical samples, and there are now several studies using sequencing and ddPCR techniques to track ESR1 and other mutations during treatment and progression. Targeted DNA sequencing and ddPCR technologies have shown that ESR1 mutations may pre-exist in approximately 5% of primary tumors and are significantly enriched by 30-40% in the setting of metastasis. The ctDNA analysis provides a simple, non-invasive and relatively inexpensive method for monitoring mutations that may arise during treatment, which can ultimately be used to guide treatment decisions. Conclusion. Importantly, ESR1 mutation monitoring alone is not clinically predictive of treatment; however, monitoring the acquisition of stem cell or other mutations may predict response and/or progression of treated cancers. Currently, patients with tumors expressing ESR1 mutations are best treated with the combination of fulvestrant and palbociclib, as this combination has significantly improved PFS in patients with most identified ESR1 mutations. Ongoing clinical trials using fulvestrant with specific PI3K-alpha inhibitors are showing promising clinical results, but analysis of whether patients with specific ESR1 mutations will benefit from this treatment has not yet been published