ERβ Expression Research Articles

Abstract PO4-03-08: Enhanced Assessment of Clinical Behavior of TNBC and TPBC using Quantified Levels of HER2, Estrogen and Progestin Receptor Proteins and Expression of Their Cognate Genes

Abstract Background: Renewed interest in clinical relevance of low-HER2 protein in breast cancer management prompted a retrospective investigation of a unique de-identified Database of quantified biomarkers associated with patient outcomes. Currently, IHC of ER, PR and HER2 proteins provides semi-quantitative results, at times complicated by variation in methods and interpretation (cf. ASCO/CAP Guidelines). Our goal was to ascertain relationships of these clinical analytes to predict cancer relapse when quantified for protein products and cognate gene expression. Methods: Database contained 2756 quantified HER2 results by ELISA (Oncogene Sciences) or by EIA (Triton Diagnostics) of which 198 patients had associated clinical outcomes. No definition of HER2-low has been accepted for IHC, although > 50% of biopsies may be categorized as HER2-low (cf. ASCP CE Programs). Microarray results obtained for ~ 22,000 genes using RNA purified from LCM-procured carcinoma cells of 247 primary breast biopsies were analyzed. ESR1, PGR and ERBB2 gene expression levels in 276 biopsies had been validated by RTqPCR. To assess distribution and dispersion of HER2 protein, cumulative relative frequency distribution analyses were employed, focusing on cut-offs within interquartile range. Permutations and combinations of the 3 biomarkers based on IHC definitions (cf. ASCO/CAP Guidelines) were examined. Tertiles and quartiles of data sets were used to explore association between biomarker levels and clinical outcomes. Box plots depicted protein and gene expression levels, Progression-Free Survival (PFS), or Overall Survival (OS) across ER/PR/HER2 categories and compared differences using a Kruskal-Wallis ANOVA Rank Test. Cox regressions were used to contrast hazard ratios among the ER/PR/HER2 groups. Kaplan-Meier plots visualized survival rates among groups and a log-rank test detected statistical discrepancies. We investigated differences between groups for categorical variables using chi-squared tests (Fisher's exact test for non-normally distributed data), while for ordinal variables, ANOVA tests were used (Kruskal-Wallis for non-normally distributed data). Categorical variables were summarized as count (%), and ordinal variables were summarized as median [IQR]. Analyses were performed using R version 4.2.2 (Innocent and Trusting). Results: Neither quantified ER or PR alone nor ER/PR collectively were related to HER2 oncoprotein levels. Influence of menopausal status on biopsy biomarker profiles was investigated. With a median cutoff for HER2 protein, patients with ER+/PR+/HER2+ (TPBC) exhibited increased OS, whereas those with TNBC had decreased OS, of 8 combinations possible. Using tertiles and quartiles, TPBC was exhibited by 34-37% of specimens compared to TNBC, which represented 9.1-12.7% of biopsies when biomarker proteins were quantified. After qPCR data were stratified by ER/PR/HER2 status with a 50% quantile cut for HER2, 39% were TPBC and 10% were TNBC. Using microarray data with the same cutoffs, only 14% were TPBC and 17% were TNBC. Once TPBC were identified by either microarray or by qPCR, increased ESR1, PGR and ERBB2 expression collectively was associated with increased PFS and OS of patients. In general, biopsies that were TNBC or other combinations of biomarker’s gene expression correlated with poorer prognosis and overall survival. Conclusions: Quantifying levels of ER, PR and HER2 proteins as well as of ESR1, PGR and ERBB2 expression were correlated with prediction of clinical outcomes of TNBC, TPBC and patients with other biomarker subsets. Results support quantitative assessment of these biomarkers in biopsies to assess utility of the HER2-low type in personalizing breast cancer management and prediction of risk of relapse. Citation Format: James Wittliff, Michael Daniels. Enhanced Assessment of Clinical Behavior of TNBC and TPBC using Quantified Levels of HER2, Estrogen and Progestin Receptor Proteins and Expression of Their Cognate Genes [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 PO4-03-08.

Abstract PO2-15-12: Novel Transcriptomic Classification System Unveils HER2-Low Breast Tumors

Abstract Breast cancers (BCs) are segregable into subtypes based on their intrinsic molecular features. When evaluating HER2 status, clinicians often rely on IHC, which is plagued by reproducibility issues. The widely used PAM50 transcriptomic system also does not include the HER2-low subtype. Addressing these limitations is particularly important given the recent approval of targeted therapy for HER2-low BCs. Here, we developed a unique transcriptomic classification system that can identify HER2-low BCs. Accurate identification of HER2-low BCs can help physicians navigate treatment selection and, in turn, allow patients to benefit from the new targeted therapy. We analyzed 6,361 BC samples with clinical and pathological annotation, including FISH and IHC data, from three public datasets (TCGA BRCA and SCAN-B [total n = 4,457]; METABRIC [n = 1,904]) for gene expression, differential expression, gene enrichment, copy number alterations, and mutations. An algorithm was applied to categorize samples from TCGA and SCAN-B into subtypes by customizing a gene set, employing UMAP dimensionality reduction, and utilizing clustering with the HDBSCAN algorithm. Each resulting cluster that was associated with a specific gene set was named and excluded from the next analysis. After four iterations, five subtypes were sequentially identified: Basal, HER2-high, HER2-low, Luminal B (LumB), and Luminal A (LumA). The Light GBM-based hierarchical classifier model was trained on the TCGA and SCAN-B cohorts and validated on the METABRIC dataset. Samples with high ERBB2 expression and copy number amplification were classified as HER2-high, and samples with similar PAM50 expression profiles but lacking ERBB2 expression were considered HER2-low. Luminal samples were grouped into two subtypes: LumA, characterized by low proliferation rates, and LumB, characterized by high proliferation rates. HER2-high, Basal, LumA, and LumB subtypes exhibited expected mutational and gene expression profiles. We observed ERBB2 copy number amplification and high gene expression in HER2-high samples from the TCGA and METABRIC cohorts, as confirmed by FISH and IHC in 97% and 93% of samples, respectively. Conversely, we did not see high ERBB2 expression or copy number amplification in the HER2-low samples, which was concordant with an absence of HER2 3+ expression by IHC. HER2-low samples had high mutational frequencies in PTEN, KMT2C, and ERBB2 (12%, 14%, and 8% respectively, adj. p < 0.005, chi-square test). HER2-low samples also showed increased EGFR (logFC = 2.8, adj. p < 0.001) and CLDN8 (logFC = 3.4, adj. p < 0.001) expression levels. The novel classifier identified 55% of the HER2-low samples as the luminal androgen receptor (LAR) Burstein subtype, as a result of higher AR expression than in HER2-high samples (logFC = 2.9, p = 0.04). Classification of SCAN-B luminal samples into the LumA and LumB subtypes also better predicted Ki-67 positivity (≥ 20% of Ki-67+ malignant cells by IHC, F1-score = 0.77) than the previously reported PAM50 classification (F1-score = 0.62). Our refined classifier effectively defined five breast cancer subtypes, including the HER2-low subtype, based on their molecular profiles. By determining HER2 status with transcriptomic data, our classifier may help minimize IHC-related reproducibility issues and, in turn, facilitate more precise treatment decision-making. Given its concordance with copy number amplification, IHC, and FISH data, our classification system can distinguish between HER2-high and HER2-low BCs. Since HER2-low BCs may represent a unique group that requires different treatment approaches, further optimization of our classifier may assist in identifying effective therapies for patients with HER2-low BCs. Citation Format: Polina Turova, Vladimir Kushnarev, Oleg Baranov, Anna Butusova, Sofia Menshikova, Sheila Yong, Anna Love, Konstantin Chernyshov, Nikita Kotlov. Novel Transcriptomic Classification System Unveils HER2-Low Breast Tumors [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 PO2-15-12.

Abstract PO5-13-06: Classifying HER2-low breast cancer using a combination of ERBB2 mRNA expression and altered genes

Abstract Background It has been recently demonstrated that some HER2-low, defined as immunohistochemistry (IHC) 1+ or 2+ with no gene amplification by FISH, breast cancer (BC) patients respond to trastuzumab deruxtecan (T-DXd). Results of the DAISY trial also suggested clinical activity with T-DXd in some of those classified as HER2-0 cancers by IHC, indicating an unmet need to better identify cancers that may truly respond to T-DXd. We compared ERBB2 (HER2) mRNA expression obtained from whole-transcriptome sequencing with IHC/FISH assignment of HER2 status. Furthermore, we constructed a classifier using ERBB2 expression and alterations in other genes as a proof of concept to illustrate classification of BC samples into HER2-0 versus HER2-low status. Methods We chose consecutive HR positive/HER2-negative BC samples that were submitted for OncoExTraTM testing between April 2020 and October 2022. The OncoExTra test uses tumor-normal whole exome and whole transcriptome sequencing to detect somatic single base substitutions, indels, copy number alterations, gene fusions and alternative transcripts. In addition, we investigated the expression profile of select genes for this analysis. We utilized OncoExTra data to examine the relationship between HER2 IHC status and gene expression, mutation and copy number profiles of selected genes, and built a classifier to predict HER2 status. Results A total of 279 BC samples were analyzed, including 106 HER2-IHC 0, 120 HER2-IHC 1+ and 53 HER2-IHC 2+. Of these, 273 were primary and 6 were metastatic samples. The distributions of patient age and tumor fraction were similar among these categories (p=0.54 and p=0.41, respectively; one-way ANOVA). ERBB2 expression differed among all three HER2-IHC categories and was lowest in HER2-0 and highest in HER2-2+ (HER2-0 vs HER2-1+, p< 0.001; HER2-0 vs HER2-2+, p< 0.0001; HER2-1+ vs HER2-2+, p=0.011; HER2-0 vs HER2-low, p< 0.0001; Mann-Whitney U test in all comparisons). Five genes were mutated in more than 10% of samples: PIK3CA (45.5%), GATA3 (15.4%), CDH1 (12.5%), MAP3K1 (11.1%) and TP53 (10.8%). Mutations in two genes, GATA3 and PTEN, appeared to differ in frequency between the HER2-0 and HER2-low categories (Table 1). We selected 4 genes with mutations, 2 genes with copy number changes, and expression from ERBB2, ESR1, CD274, HLA-A, HLA-B and HLA-C to train a logistic regression classifier to predict HER2 IHC status. The classifier was trained on 163 samples, hyperparameter tuning was done using 55 samples, and testing was performed on 61 samples. Preliminary results indicated that of the 28 HER2-IHC 0 samples in the test set, 17 (65%) were classified as HER2-low. Further development and evaluation of the classifier using a blinded testing set is currently in progress. Conclusion In this cohort of BC samples, ERBB2 mRNA expression was positively associated with HER2 protein IHC status. Additional studies are required to determine whether a classifier including ERBB2 mRNA expression could be used to identify patients for T-DXd therapy. The relative enrichment of PTEN alterations in HER2-IHC 0 samples suggests that mTOR/AKT inhibitors may sometimes be appropriate in patients who are not eligible for T-DXd therapy, and further investigation should be considered. Table 1. Number (frequency) of altered genes by HER2 IHC status. Citation Format: Gargi Basu, Niru Chennagiri, Turgut Dogruluk, David Hall, Jess Hoag, Cynthia Flannery, Snehal Thakkar, Melanie Palomares, Frederick Baehner, Lajos Pusztai. Classifying HER2-low breast cancer using a combination of ERBB2 mRNA expression and altered genes [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 PO5-13-06.

Abstract PS09-01: Individual patient data meta-analysis of clinical and translational biomarkers for prediction of pathological complete response (pCR) after de-escalated therapy in HER2+ breast cancer in four trials of the West German Study Group

Abstract Background Introduction of anti-HER2 therapies substantially improved outcomes for HER2-positive early breast cancer (HER2+ eBC). However, a considerable proportion of patients (pts) may still be overtreated with systemic chemotherapy (CTx) combinations. Establishing safe de-escalation strategies to avoid toxicities requires precise patient selection. Therefore, we set out to determine predictors for efficacy and survival in the unique setting of four de-escalation trials investigating short (12-week) neoadjuvant treatments (NAT) in HER2+ eBC. We investigated the prognostic ability of clinical and translational biomarkers for pCR to identify pts with the best prognosis after de-escalated systemic CTx-free NAT. Methods 756 pts who received de-escalated NAT were analyzed: trastuzumab + pertuzumab (T + P, n=92) and T + P + paclitaxel (pac, n=42) in ADAPT-HR-/HER2+ (NCT01817452); trastuzumab emtansine (T-DM1, n=118), T-DM1 + endocrine therapy (ET, n=125), and T + ET (n=129) in ADAPT-HR+/HER2+ (NCT01779206), T + P + pac (n=107) and T + P + ET (n=100) in TP-II (NCT03272477); and T + P + pembrolizumab (n=43) in Keyriched-1 (NCT03988036) in HER2-enriched (HER2-E) subtype by PAM50. The primary endpoint of each trial was pCR (ypT0/is ypN0). Survival was a secondary endpoint in all trials but Keyriched-1; survival data for TP-II are pending. Baseline gene expression was analyzed by BC360 assay; intrinsic molecular subtypes were determined using the PAM50 predictor. Stromal tumor infiltrating lymphocytes (sTILs) were evaluated at baseline and at week 3 of NAT. sTILs were categorized using a 30% threshold and the low cellularity category ( < 500 invasive tumor cells) at week 3. Prognostic markers for pCR were identified with univariate and multivariable logistic regression models with backstep algorithm excluding variables with p≥0.1; considered variables included age, sTILs (baseline, 3-weeks), cT, cN, grade, hormone receptor and HER2 status, intrinsic subtype, and standardized ERBB2 and ESR1 gene expression. These analyses were performed separately for NAT involving systemic CTx (i.e. containing pac, n=149) and systemic CTx-free NAT (n=607). Results Overall pCR rate was 39.1% (CTx: 66.4%; CTx-free: 32.5%). Multivariable analysis identified predictors of pCR after CTx-free NAT: low cellularity at week 3 (vs < 30% sTILs: OR 3.21, 95%CI 1.85-5.57), ERBB2 (OR 1.90, 95%CI 1.41-2.55), HER2 3+ (vs 1+/2+ and ISH+: OR 8.01, 95%CI 1.65-38.99), LumA/LumB/Basal subtype (vs HER2-E: OR 0.57, 95%CI 0.34-0.97), cT2 (vs 1: OR 0.54, 95%CI 0.33-0.89), and cN1-3 (vs 0, OR 0.46, 95%CI 0.27-0.78). In CTx-containing NAT, only ERBB2 (OR 2.08, 95%CI 1.28-3.40) and additionally ESR1 (OR 0.38, 95%CI 0.23-0.61) were prognostic for pCR. Updated analysis including expanded gene expression data from ADAPT-HR+/HER2+ and a prognostic score for pCR after CTx-free NAT developed using machine learning methods will be presented at the meeting. Conclusions This pooled analysis demonstrated a 66% pCR rate after a short (12-week) de-escalated NAT in HER2+ eBC. In one-third of patients (with mainly stage I cancer, higher HER2 expression by immunohistochemistry and gene expression analysis, and low cellularity at 3 weeks), pCR can be achieved without systemic CTx. Identified biomarkers could pave the way for developing new patient selection strategies to spare CTx-associated acute and late toxicities in a clinically meaningful number of patients. Citation Format: Monika Graeser, Oleg Gluz, Christine zu Eulenburg, Sherko Küemmel, Raquel von Schumann, Matthias Christgen, Rachel Wuerstlein, Enrico Pelz, Hans-Heinrich Kreipe, Peter Schmid, Marc Thill, Michael Braun, Jochem Potenberg, Claudia Schumacher, Joke Tio, Andreas Hartkopf, Marianne Just, Christian Schem, Kerstin Lüdtke-Heckenkamp, Eva-Maria Grischke, Felix Hilpert, Angela Kentsch, Ronald Kates, Ulrike Nitz, Nadia Harbeck. Individual patient data meta-analysis of clinical and translational biomarkers for prediction of pathological complete response (pCR) after de-escalated therapy in HER2+ breast cancer in four trials of the West German Study Group [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 PS09-01.

Abstract PO3-14-08: Metaplastic Breast Cancer: Whole Genome Sequencing Analysis and Emerging Therapeutic Targets

Abstract Background: Metaplastic breast cancer (MpBC) is a rare histologic subtype of triple negative breast cancer (TNBC) with an aggressive behavior and poor outcome. Previous studies by whole-exome and targeted sequencing showed that MpBCs display a dominant mutational signature 3, associated to homologous recombination (HR) deficiency (HRD). Tumors displaying HRD are associated with response to therapies causing double strand breaks. Nonetheless, MpBCs consistently display significantly lower rates of response to neoadjuvant chemotherapy than non-metaplastic TNBCs and effective therapeutic alternatives are an unmet clinical need. Here we sought to assess the HR features of MpBCs using whole-genome sequencing (WGS), and to evaluate the expression in MpBCs of antigens that are targets of novel antibody drug conjugates (ADCs) in common forms of breast cancer. In addition, we sought to assess the efficacy of ADCs and drugs targeting the DNA damage response (DDR) using preclinical MpBC models. Materials and Methods: We subjected 25 primary MpBC to WGS. HRDetect, the ‘gold standard’ tool for assessment of HRD, was utilized. Mutational signatures were inferred using Signal. 31 MpBCs were subjected to RNA-sequencing to assess the gene expression levels ADC targets compared to non-MpBCs from The Cancer Genome Atlas (TCGA). Efficiency of ADCs and DDR drugs was evaluated using the MpBC cell models HCC1806, BT549 and Hs578T in vitroand/or using HCC1806 xenografts in athymic mice in vivo. Results: Although the majority of MpBCs (13/25) displayed a dominant SBS mutational signature 3, only a small subset of cases (3/25) were classified as HRD by HRDetect. These three bona fide HRD cases were found to harbor bi-allelic inactivation of BRCA1 (n=2) or RAD51C (n=1), and displayed various genomic features of HRD, including deletions with microhomology, enrichment in tandem duplications, rearrangement signatures 3 and 5, and high large scale state transitions (LST) and telomeric allelic imbalance (NtAI) scores. Most MpBCs (22/25), however, were not classified as HRD by HRDetect displaying an incomplete HRD phenotype, with only partial genomic features of HRD. 52% of MpBCs (13/25) displayed APOBEC mutagenesis exposure, which was present in the three bona fide HRD MpBCs. RNA-sequencing analyses revealed detectable ERBB2 and TROP2 expression in all MpBCs interrogated, although at lower levels than in non-MpBCs from TCGA. The HCC1806, BT549 and Hs578T MpBC cell models were found to be sensitive to Sacituzumab Govitecan and Trastuzumab Deruxtecan (T-DXd) in cell viability and colony formation assays, and to T-DXd in vivo. These MpBC cell models were also found to be sensitive to DDR compounds, such as ATR, WEE1 and PKMYT1 inhibitors in vitro, alone and in combination with T-DXd or Sacituzumab Govitecan. Conclusions: Most MpBCs display an incomplete HRD genomic scar, except for those cases harboring bi-allelic inactivation of HRD genes. APOBEC mutagenesis is operative in MpBCs and co-exists with HRD. MpBCs express ERBB2 and TROP2, and MpBC cell models are sensitive to T-DXd and Sacituzumab govitecan, as well as DDR compounds, targeting the incomplete HRD phenotype of MpBCs, alone and in combination. Taken together, these findings warrant further studies in patients with MpBCs testing the therapeutic efficacy of agents targeting the incomplete HRD phenotype, as well as the expression of ADC targets in these aggressive rare forms of TNBCs. Citation Format: Fresia Pareja, Andrea Gazzo, Higinio Dopeso, David Brown, Pier Selenica, Yingjie Zhu, Juan Blanco-Heredia, Laxmi Gusain, Xin Pei, Giacomo Montagna, Nour Abuhadra, Hanna Y Wen, Edi Brogi, Nadeem Riaz, Sarat Chandarlapaty, Maurizio Scaltriti, Larry Norton, Simon Powell, Jorge Reis-Filho, Britta Weigelt. Metaplastic Breast Cancer: Whole Genome Sequencing Analysis and Emerging Therapeutic Targets [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 PO3-14-08.

Abstract PO4-24-08: Genomic Landscape of Malignant Phyllodes Tumors Identifies Subsets for Targeted Therapy

Abstract Introduction: Phyllodes tumors (PT) are rare fibroepithelial tumors of the breast, comprising ~1% of all breast cancers, a subset of which have considerable malignant potential. Malignant phyllodes tumors (MPTs) have aggressive biological behavior and high local and distant recurrence rates. Distant metastases of MPTs have a poor prognosis given the limited treatment options available. Surgery remains the primary treatment modality for these patients; however, initial investigations suggest a potential for targeted therapies in managing this disease. The identification of targetable opportunities and a comprehensive understanding of the mutational profiles of MPTs could greatly enhance treatment options. Therefore, we aimed to assess the molecular landscape of these tumors. Methods: Phyllodes tumor samples underwent molecular profiling at Caris Life Sciences through DNA (592-gene panel or whole exome) and RNA sequencing (whole transcriptome). The MPTs were classified into primary and metastatic based on the specimen biopsy site. PD-L1+ expression was tested by IHC (SP142; ≥2+, ≥5%). Tumor mutational burden (TMB)-High was defined as ≥10 mutation/Mb. Immune cell fractions in the tumor microenvironment (TME) were estimated using quanTIseq (Finotello, 2019). Mann-Whitney U, Chi-square, and Fisher-Exact tests were applied where appropriate. Results: We identified 57 analyzed MPTs, all from female patients, with a median age of 53 years (range: 19-88). Approximately half of samples (53%, n=30) were labeled as from primary breast sites, and the remaining sites were metastatic (47%, n= 27). Of metastatic sites, lung was the most common location (74.1%, n=20/27), followed by bone, sacrum, pancreas and small bowel. Alterations in MPT include TERT promoter (56.8%, n=21/37), MED12 (51.6%, n=16/31), TP53 (40.0%, n=22/55), and NF1 (31.8%, n=14/44) mutations, with less frequent mutations of EGFR (10.5%, n=6/57), PIK3CA (7.0%, n=4/57), and BRAF (3.5%, n=2/57). As compared to primary MPT, metastatic MPT had roughly 2-fold higher prevalence of NF1 (45.5%: n=10/22 vs 18.2% n=4/22, p=0.05), KMT2D (25.9%: n=7/27 vs 10.7%: n=3/28, p=0.18), and RB1 (25.0%: n=6/24 vs 9.5%: n=2/21, p=0.25) though not significant. Multiple gene mutations were observed exclusively in lung metastases, as compared to non-lung sites including: TERT promoter (64.3%: n=9/14 vs 0.0%, p=0.08), MED12 (60.0%: n=6/10 vs 0.0%, p =0.044), and RB1 (35.3%: n=6/17 vs 0.0%, p=0.01). Conversely, NF1 (43.8%: n=7/16 vs 50.0%: n=3/6, p=1.0) and KMT2D (20.0%: n=4/20 vs 42.9%: n=3/7, p=0.32) mutation prevalence was higher in other metastatic sites. PD-L1+ expression was observed in 13.3% (n=7) of MPT overall, with similar prevalence among local and metastatic sites. No dMMR/MSI-H was observed, while low LOH (those with genomic LOH in < 16% of segment analyzed) was seen across MPT. Compared to a large cohort of breast adenocarcinoma samples (n=9,926) representing both HER2+ and HER2- tumors, MPT had low ERBB2 expression comparable to HER2- samples. B cells, M2 macrophages and neutrophils had the highest median cell fractions in the TME of MPT. Additionally, one MPT sample harbored a pathogenic NTRK1 fusion (TPM4:NTRK1), and treatment with larotrectinib for over 16 months suggests a clinical response to therapy. Conclusions: Our study demonstrates the importance of employing next generation sequencing (NGS) in MPTs to detect actionable genomic alterations. To effectively analyze fusions, an NGS panel that include RNA sequencing is recommended, considering the occurrence of NTRK1 fusion reported herein. Although HER2 transcriptional expression was low, further investigations examining HER2 IHC in PTs are still necessary. These finding therefore highlight the importance of NGS in phyllodes tumors research, as it may uncover potential targeted treatment options for patients. Citation Format: Rani Bansal, Tolulope Adeyelu, Andrew Elliott, Antoinette Tan, Jane Meisel, Matthew Oberley, Stephanie Graff, Jorge Reis-Filho, George Sledge Jr, Sarah Sammons, Laura Rosenberger. Genomic Landscape of Malignant Phyllodes Tumors Identifies Subsets for Targeted Therapy [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 PO4-24-08.

Changes in ovarian tissue structure and distribution of oestrogen receptors in Huanghuai goats at different ages.

To observe developmental changes in the ovarian tissue structure and distribution characteristics of oestrogen receptors (ERs) in the ovaries of Huanghuai goats at different ages, we selected healthy Huanghuai goats ewes and divided them into five groups (i.e. 3-, 30-, 60-, 90- and 120-day-old groups), with 10 animals in each group. The serum was separated after blood collection through the jugular vein, and the contents of oestrogen (E) and progesterone (P) in the serum of Huanghuai goats at each age were determined. Three goats were randomly selected from each group and sacrificed after anaesthesia, and the ovarian tissue was quickly obtained and placed in 4% paraformaldehyde fixative to prepare the tissue sections. Using HE, oestrogen receptors were immunohistochemically stained and observed. These results showed many primordial follicles and occasional secondary follicles in the ovaries of 3-day-old Huanghuai goats. Ovarian reticular structures were observed in 30-day-old ovarian medulla, with occasional near-mature growing follicles. Mature follicles and corpus luteum were occasionally detected in 60-day-old ovarian cortex. The 90-120-day-old ovarian cortices contained growing and mature follicles, and the number of mature follicles and corpora lutea increased, implying a significant luteal involution period. The E and P contents in the 120-day-old group were significantly higher than those in the 3-, 30-, 60- and 90-day-old groups. The levels of ERα and ERβ in the 3- and 30-day-old groups were mainly distributed in the granulosa cells of ovarian reproductive epithelial cells, primordial follicles, atretic follicles, and primary and secondary follicles. The ERα and ERβ levels of the 60-, 90- and 120-day-old groups were also distributed in the granulosa cells and luteal cells of mature follicles, especially in the 120-day-old endometrial cells of mature follicles, where ERβ was distributed significantly. The overall expression of ERβ in the ovary was higher than that of ERα. The results of this study provide basic data on the ovarian development and the specific expression of ERs and PRs in the ovaries of Huanghuai white goats, which play an important role in ovarian development and precocity.

Sex-Specific Regulation of Local Gonadal Hormone Signaling During Pathological Cardiac Remodeling in Spontaneously Hypertensive Rats

Hypertensive heart disease is characterized by cardiac fibrosis in the left ventricle (LV). Angiotensin II (Ang II) promotes cardiac fibrosis through direct actions on cardiac fibroblasts. Gonadal hormones are known to impact cardiac fibrosis either through direct actions on fibroblasts or through modulation of Ang II receptors. We have previously shown that Ang II infusion alters collagen gene expression in a sex-specific manner. The present study seeks to further understand our observed sex difference by investigating the impact of Ang II on gonadal hormone regulation in the left ventricle (LV) and cardiac fibroblasts (CFs) following a fibrogenic stimulus. For whole LV analysis, male and female SHR (15-week-old) were infused with Ang II (400ng/kg/min, s.c.) or vehicle for 2 weeks via an osmotic mini pump (n=8-11 per group per sex). For CF-specific analysis, primary CFs were isolated from adult male and female SHR LV and cultured on CytoSoft cell culture plates (8 kPa stiffness) coated with Type I Collagen. CFs were brought to P1 for expansion and treated with TGFb1 (10ug/uL) or vehicle (PBS + 0.1% BSA) for 48 hours (n=7-8 per group per sex). Col1a1, AT1aR, estrogen receptors α and β (ER-α, ER-β), androgen receptor (AR), 5α-reductase (5α-R), and aromatase gene expression in the left ventricle was assessed via RT-qPCR and data analyzed by Two-Way ANOVA. Ang II infusion significantly increased ER-α gene expression in male, but not female LV (sex x Ang II interaction p=0.0303). ER-β gene expression was also significantly increased in the male LV (p=0.0066), but not females. AR gene expression remained unchanged in the Ang II-infused male LV but was significantly reduced in female LV when compared to control (sex x Ang II interaction p=0.0084). Ang II significantly increased 5α-R gene expression in male (p=0.0024), but not female LV. Aromatase gene expression tended to increase in both male and female Ang II-infused animals when compared to control. Ang II-induced changes in LV gonadal hormone gene expression did not significantly correlate with Col1a1 gene expression. In vitro stimulation of CFs with TGFb1 results in a significant reduction in ER-α (p<0.0001) and AR (p<0.0001) in both males and females. ER-β tended to decrease in males stimulated with TGFb1, but not females, relative to control. Taken together, the opposing findings between LV and CF may indicate cell-specific regulation of gonadal hormone receptors. It may be that upregulation of local estrogen signaling in the remodeling LV in males serves to offset the pathological effects of Ang II stimulation. Future studies will evaluate the consequences of changes in gonadal hormone receptor levels at the cellular and tissue level. NHLBI R01HL153112. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Tipping the balance — the move of aging females towards higher ERβ:ERα ratio and away from estrogen mediated vascular protection

Objective: The menopausal transition, marked by the cessation of endogenous estrogen production, is a hallmark of vascular aging in women, as the vascular function radically declines in the years hereafter (Parker et al. 2010). Primarily based on animal and in-vitro studies (Novella et al. 2012; Novensà et al. 2011; Park et al. 2017) it has been hypothesized that this is linked to menopause-dependent changes in the balance between the two dominant estrogen receptors, ERα and ERβ; as previous studies suggest that ERα favors NO-bioavailability whereas ERβ mediates increases in oxidative stress. However, it remains unresolved how aging actually influences changes in the balance between ERα and ERβ in skeletal muscle of females and how changes are related to downstream protein targets such as eNOS and NADPH oxidases. Study design: We measured skeletal muscle protein expression of estrogen receptors, androgen receptors, eNOS, SOD2, GPX1, gp91phox/-NOX2 and NOX4 in 82 females ranging from 20-70 yrs. of age. For age comparisons, data from the muscle biopsies were grouped by 5-year intervals. Statistical analyses were conducted by on one-way ANOVA and Pearson’s product moment correlations Results: The ERβ:ERα ratio was significantly higher in the 65+ yrs. compared to all other groups (p<0.01) and correlation analysis revealed that the ERβ/ERα ratio increased with aging (p=0.02, R2=0.07). The 65+ yrs. group presented a lower eNOS expression (p<0.05) than the 20-54 yrs. groups and correlation analysis showed a decrease in eNOS expression with aging (p=0.0002 R2=0.16). Gp91phox/-NOX2 expression correlated with ERβ and the expression of gp91phox/NOX2 (p=<0.0001 R2=0.4) was lower in the three middle groups, 45-59 yrs., compared with both the younger and older groups (p<0.05). ERα correlated with the antioxidants SOD2 and GPX1 (p=0.0001 R2=0.18 and p=<0.0001 R2=0.21, respectively). Conclusions: This study is the first to show that skeletal muscle expression of ERβ:ERα increases with age and that the expression of eNOS in skeletal muscle declines as a function of age in females. Our data also provides indirect support for that aging is associated with a shift from a more prominent ERα coupled antioxidant effect, through - SOD2/GPX1 related mechanisms, towards a more pro-oxidant environment through an ERβ mediated upregulation of Gp91phox/-NOX2. This shift, combined with the observation of an age induced decline in eNOS expression, could be mechanisms underlying reduced NO bioavailability in aging females. Further analyses are ongoing to throw light in these mechanisms and additional studies are warranted to fully elucidate the mechanistic pathways involved. Funding The study was funded by The Danish Ministry of Culture Fund for Research in Sports, The Independent Research Fund Denmark, Eva and Henry Frænkel Memorial Fund, and the Toyota Foundation. The study is part of The Copenhagen Women Study for which fi- nancial support was provided by the University of Copenhagen Excellence Programme for Interdisciplinary Research. The study was also supported by The Danish Ministry of Culture: Sports Research. Financial support is greatly appreciated from the Aarhus University Research Foundation (AUFF); the Hartmann Foundation; the A.P. Møller Foundation; the Toyota-Fonden, Denmark; and the Augustinus Foundation and Team Denmark. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Concordance between ER, PR, Ki67, and HER2-low expression in breast cancer by MammaTyper RT-qPCR and immunohistochemistry: implications for the practising pathologist.

There are limited data on the role of multigene tests and their correlation with immunohistochemistry (IHC), especially on core biopsy. MammaTyper is a quantitative conformite Europeeanne (CE) marked, National Institute for Health and Care excellence (NICE) approved, in in vitro diagnostic quantitative real-time polymerase chain reaction (RT-qPCR) test for assessment of mRNA expression of four biomarkers (ESR1, PGR, ERBB2, MKI67). We evaluated the concordance of MammaTyper with oestrogen receptor (ER), progesterone receptor(PR), HER2, and Ki67 by IHC on 133 core needle biopsies of breast cancer. HER2 was positive if IHC 3+ or 2+ and fluorescence in situ hybridization (FISH)-amplified. Global and hotspot Ki67 expression was analysed using a cutoff of ≥20% assessed manually and by digital image analysis. Agreements were expressed as overall percent agreement (OPA), positive percent agreement (PPA), negative percent agreement (NPA), and Cohen's kappa. RT-qPCR results of ESR1 were highly concordant with IHC with OPA of 94.7% using 1% cutoff and 91.7% when the low ER-positive category was included. The PPA and NPA between RT-qPCR and IHC for PR was 91.5% and 88.0%, respectively, when using the 1% cutoff. For ERBB2/HER2, the OPA was 95% and the PPA was 84.6%. 40 of 72 HER2 IHC score 0 tumours were classified as ERBB2 low. Best concordance between MKI67 by MammaTyper and Ki67 IHC was achieved using hotspot digital image analysis (OPA: 87.2%, PPA: 90.6%, NPA: 80%). RT-qPCR-based assessment of the mRNA expression of ESR1, PGR, ERBB2, and MKI67 showed high concordance with IHC, suggesting that the MammaTyper test on core needle biopsies represents a reliable, efficient, and reproducible alternative for breast cancer classification and refining HER2 low categorisation.

25-hydroxycholesterol promotes proliferation and metastasis of lung adenocarcinoma cells by regulating ERβ/TNFRSF17 axis.

Lung adenocarcinoma is the main type of lung cancer in women. Our previous findings have evidenced that 25-hydroxycholesterol (25-HC) promotes migration and invasion of lung adenocarcinoma cells (LAC), during which LXR as a 25-HC receptor plays an important role. Estrogen receptor beta (ERβ) is a receptor of 27-hydroxycholesterol that is structurally analogous to 25-HC, but its role in the functional actions of 25-HC remained largely unknown. In this study, we demonstrated that 25-HC treatment triggered ERβ expression in LAC. Knockdown of ERβ inhibited 25-HC-mediated proliferation, migration and invasion, and reduced 25-HC-induced LAC metastasis in vivo. Further investigation revealed that ERβ knockdown restrained the expression of TNFRSF17 (BCMA). In vivo experiments also confirmed that ERβ knockdown blocked 25-HC-induced TNFRSF17 expression. TNFRSF17 knockdown also restrained 25-HC-induced proliferation, migration and invasion. Bioinformatic analysis showed that the levels of ERβ and TNFRSF17 were elevated in lung adenocarcinoma, and were closely related to tumor stages and nodal metastasis status. These results suggested that 25-HC promoted the proliferation and metastasis of LAC by regulating ERβ/TNFRSF17 axis.

The Role of Estrogen and Estrogen Receptors in Head and Neck Tumors.

Head and neck squamous cell carcinoma (HNSCC) is the most common histological form of head and neck tumors (HNTs), which originate from the epithelium of the lips and oral cavity, pharynx, larynx, salivary glands, nasal cavity, and sinuses. The main risk factors include consumption of tobacco in all forms and alcohol, as well as infections with high-risk human papillomaviruses or the Epstein-Barr virus. Regardless of the etiological agent, the risk of developing different types of HNTs is from two to more than six times higher in males than in females. The reason for such disparities probably lies in a combination of both biological and psychosocial factors. Therefore, it is hypothesized that exposure to female sex hormones, primarily estrogen, provides women with protection against the formation and metastasis of HNTs. In this review, we synthesized available knowledge on the role of estrogen and estrogen receptors (ERs) in the development and progression of HNTs, with special emphasis on membrane ERs, which are much less studied. We can summarize that in addition to epidemiologic studies unequivocally pointing to the protective effect of estrogen in women, an increased expression of both nuclear ERs, ERα, and ERβ, and membrane ERs, ERα36, GPER1, and NaV1.2, was present in different types of HNSCC, for which anti-estrogens could be used as an effective therapeutic approach.

Mendelian randomization analysis identifies druggable genes and drugs repurposing for chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a prevalent condition that significantly impacts public health. Unfortunately, there are few effective treatment options available. Mendelian randomization (MR) has been utilized to repurpose existing drugs and identify new therapeutic targets. The objective of this study is to identify novel therapeutic targets for COPD. Cis-expression quantitative trait loci (cis-eQTL) were extracted for 4,317 identified druggable genes from genomics and proteomics data of whole blood (eQTLGen) and lung tissue (GTEx Consortium). Genome-wide association studies (GWAS) data for doctor-diagnosed COPD, spirometry-defined COPD (Forced Expiratory Volume in one second [FEV1]/Forced Vital Capacity [FVC] <0.7), and FEV1 were obtained from the cohort of FinnGen, UK Biobank and SpiroMeta consortium. We employed Summary-data-based Mendelian Randomization (SMR), HEIDI test, and colocalization analysis to assess the causal effects of druggable gene expression on COPD and lung function. The reliability of these druggable genes was confirmed by eQTL two-sample MR and protein quantitative trait loci (pQTL) SMR, respectively. The potential effects of druggable genes were assessed through the phenome-wide association study (PheWAS). Information on drug repurposing for COPD was collected from multiple databases. A total of 31 potential druggable genes associated with doctor-diagnosed COPD, spirometry-defined COPD, and FEV1 were identified through SMR, HEIDI test, and colocalization analysis. Among them, 22 genes (e.g., MMP15, PSMA4, ERBB3, and LMCD1) were further confirmed by eQTL two-sample MR and protein SMR analyses. Gene-level PheWAS revealed that ERBB3 expression might reduce inflammation, while GP9 and MRC2 were associated with other traits. The drugs Montelukast (targeting the MMP15 gene) and MARIZOMIB (targeting the PSMA4 gene) may reduce the risk of spirometry-defined COPD. Additionally, an existing small molecule inhibitor of the APH1A gene has the potential to increase FEV1. Our findings identified 22 potential drug targets for COPD and lung function. Prioritizing clinical trials that target these identified druggable genes with existing drugs or novel medications will be beneficial for the development of COPD treatments.

Estrogen receptors alpha and beta expression in different canine cancer types with an emphasis on hematopoietic malignancies.

Estrogen receptors (ERs) are located in both healthy and neoplastic tissues. The type of estrogen receptor expressed varies depending on its location, tumor type, and species. Estrogen action is mediated by binding to ER and activating the transcriptional and signaling processes that result in the control of gene expression. There are two main types of estrogen receptors: ER alpha (ERα) and ER beta (ERβ). Both receptors are functionally different, they may act antagonistically and are distributed in different tissues but their structure is similar - as they are composed of 5 different domains: A/B, C, D, E, and F. The signaling pathway and hence regulation of the gene expression by ERs is a complex and multifactorial process that involves both genomic and nongenomic actions. In the human reproductive tract, both ERα and β are present, with predominant expression of ERβ, while there are no satisfactory data distinguishing the type of ERs expressed in the canine reproductive tract. In mammary gland neoplasia, a decreased or lacking ERα expression in humans is associated with a poorer prognosis. This is similar to dogs, where higher ERα expression intensity was noted in benign tumors than in carcinomas. In human hematopoietic malignancies, ERβ is a predominant receptor. Selective and non-selective ERβ agonists have an antiproliferative and pro-apoptotic effect on human lymphoma cell lines and may be effective in the therapy of ERβ positive lymphomas and leukemias. In canine lymphoma tissues, none or only marginal expression of ERs was detected over the decades. Considering available data, we conducted preliminary studies proving that, in contrast to humans, the dominant ER expressed in canine hematopoietic tumors is ERα.

Empagliflozin and liraglutide ameliorate HFpEF in mice via augmenting the Erbb4 signaling pathway.

Heart failure with preserved ejection fraction (HFpEF) is closely associated with metabolic derangement. Sodium glucose cotransporter-2 inhibitors (SGLT2i) and glucagon-like peptide-1 receptor agonists (GLP-1RA) exert anti-HFpEF effects, but the underlying mechanisms remain unclear. In this study, we explored the anti-HFpEF effects of empagliflozin and liraglutide and the underlying molecular mechanisms in a mouse model of HFpEF. This model was established by high-fat diet (HFD) feedingplus Nω-nitro-L-arginine methyl ester (L-NAME) treatment. The mice were treated with empagliflozin (20 mg·kg-1·d-1, i.g.) or liraglutide (0.3 mg·kg-1·d-1, i.p.) or their combination for 4 weeks. At the end of the experimental protocol, cardiac function was measured using ultrasound, then mice were euthanized and heart, liver, and kidney tissues were collected. Nuclei were isolated from frozen mouse ventricular tissue for single-nucleus RNA-sequencing (snRNA-seq). We showed that administration of empagliflozin or liraglutide alone or in combination significantly improved diastolic function, ameliorated cardiomyocyte hypertrophy and cardiac fibrosis, as well as exercise tolerance but no synergism was observed in the combination group. Furthermore, empagliflozin and/or liraglutide lowered body weight, improved glucose metabolism, lowered blood pressure, and improved liver and kidney function. After the withdrawal of empagliflozin or liraglutide for 1 week, these beneficial effects tended to diminish. The snRNA-seq analysis revealed a subcluster of myocytes, in which Erbb4 expression was down-regulated under HFpEF conditions, and restored by empagliflozin or liraglutide. Pseudo-time trajectory analysis and cell-to-cell communication studies confirmed that the Erbb4 pathway was a prominent pathway essential for both drug actions. In the HFpEF mouse model, both empagliflozin and liraglutide reversed Erbb4 down-regulation. In rat h9c2 cells, we showed that palmitic acid- or high glucose-induced changes in PKCα and/or ERK1/2 phosphorylation at least in part through Erbb4. Collectively, the single-cell atlas reveals the anti-HFpEF mechanism of empagliflozin and liraglutide, suggesting that Erbb4 pathway represents a new therapeutic target for HFpEF. Effects and mechanisms of action of empagliflozin and liraglutide in HFpEF mice. HFpEF was induced with a high-fat diet and L-NAME for 15 weeks, and treatment with empagliflozin and liraglutide improved the HFpEF phenotype. Single nucleus RNA sequencing (snRNA-seq) was used to reveal the underlying mechanism of action of empagliflozin and liraglutide.

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 &amp; 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 &amp; 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.

The Estrogenic Effect of Lysiphyllum strychnifolium (Craib) A. Schmitz Leaf Water Extract in MCF-7 Cells

Lysiphyllum strychnifolium (Craib) A. Schmitz (LS) has been widely used as traditional medicine in the northeast region of Thailand to stimulate the production of breast milk. There are no known studies on the estrogenic action of LS on the stimulation of breast milk production. Hence, the present study aimed to investigate the estrogenic effect of LS leaf water extract compared with quercetin, one of the compounds in LS, in human breast cancer cells (MCF-7). The effect of LS leaf water extract and quercetin on cell viability of MCF-7 cells was studied by MTT assay at a concentration range of 0 to 500 µg/mL. The expression of estrogen-dependent genes, the pS2, ERα, and ERβ was also examined by real-time RT-PCR, and the expression of ERα protein was detected by Western blotting. The quercetin content in the LS water extract was 285.67 ± 0.11 µg/g of dry extract. MCF-7 cells treated with LS leaf water extract (20 µg/mL) showed an upregulation of pS2 gene expression similar to that of the treatment with E2 (10−9 M) compared with that of the untreated control. The ERα gene expression was found to be upregulated by quercetin (0.16 µg/mL) and E2 (10−5 M) compared with the untreated control. In addition, quercetin (0.16 µg/mL) and LS extract (0.8, 4, and 20 µg/mL) decreased the phosphorylation of ERα at Ser167. LS extract (20 µg/mL) decreased ERα, but there was no significant effect on the ERα at Ser118 protein expression. This study provided scientific evidence for the potential estrogenic activities of LS leaf water extract. This indicates that ER-dependent pathways in MCF-7 cells served as mediators, facilitating the estrogenic properties of LS and its compounds to function effectively. Since LS induced pS2 gene transcription, it was confirmed that this could affect the transcription of estrogen-responsive genes, causing estrogenic effects. HIGHLIGHTSThis is the first study to demonstrate the estrogenic effect of Lysiphyllum strychnifolium leaf water extract. L. strychnifolium leaf water extract induced pS2 gene transcription, and it was confirmed that this could affect the transcription of estrogen-responsive genes which cause estrogenic effects. The results of this study support the mechanism and scientific evaluation on the use of L. strychnifolium for stimulation of breast milk in folk medicine. GRAPHICAL ABSTRACT

Regulatory role of miR-125a expression with respect to its target genes LIFR, ERBB2 and STAT3 in the pathogenesis of recurrent pregnancy losses.

Studies have investigated miR-125a for its predictable role in recurrent pregnancy loss (RPL) cases to regulate many biological events required for the maintenance of pregnancy by regulating its confirmed target genes LIFR, ERBB2 and STAT3. The present study included 40 cases of women with at least two RPLs in ≤20 weeks of gestation against 40 healthy multiparous women without a previous history of abortion. Expression analysis of ERBB2, LIFR, STAT3 and miR-125a was conducted by quantitative real-time PCR (qPCR). The expression of miR-125a was significantly lower in the plasma of RPL cases (P = 0.0001) and showed a significantly increased mean expression level in product of conception (2.56-fold, P < 0.0001). Among the target gene of miR-125a, ERBB2 and STAT3 gene expression level was significantly increased (2.58-fold, P = 0.04; 1.87-fold, P = 0.025), respectively in RPL cases while the LIFR gene revealed comparable expression (P = 0.64). Furthermore, expression analysis of ERBB2 gene with respect to its regulatory miR-125a cases depicted a significant association (P = 0.0005). Kaplan-Meier survival analysis revealed cases with low miR-125a expression had significantly shorter time to miscarriages, (log-rank P = 0.02). Also, decreased expression of miR-125a significantly conferred >2-fold increased risk for RPL (HR = 2.34: P < 0.05). The overall conclusion of the study was that altered miR-125a expression may cause deregulation in target genes LIFR, ERBB2 and STAT3 resulting in adverse consequence in the outcome of pregnancy.

Abstract 1139: Unravelling tumor heterogeneity in patients with HER2-low hormone receptor-positive breast cancer using spatial transcriptomics

Abstract Background: The current binary distinction between HER2-positive and HER2-negative breast cancer (BC) has recently been challenged by the emergence of the HER2-low entity. Antibody-drug conjugates targeting HER2 have shown efficacy in both HER2-positive and HER2-low BC, while some preliminary evidence showed efficacy also in patients with HER2-zero BC. Here, by combining spatial transcriptomics (ST) and morphological annotation, we aimed to investigate the inter- and intra-tumor heterogeneity of ERBB2 expression in relation to breast cancer IHC subtypes. Methods: We performed ST (Visium 10X Genomics) on frozen tumor samples from patients with hormone receptor positive (HR+) ductal BC with HER2 IHC staining and FISH. Hematoxylin/eosin slides were morphologically annotated integrating manual and machine learning-based approaches reaching single-cell resolution (QuPath software). The relative histomorphological categories composition of each spot across the ST slide was computed as percentage of pixels. Spots were defined as tumoral if the percentile of pixels in that spot surpassed the 80th percentile of all the tumor spots in the cohort. Results: Our cohort consisted of 56 HER2-zero (IHC 0), 30 HER2-low (IHC 1+) and 20 HER2-positive (IHC 2+/FISH positive or IHC 3+) samples. HER2-zero samples contained a significantly higher percentage of acellular stroma spots compared to HER2-low (pvalue=0.04), while no other morphological annotation showed a difference, including tumor spots. Only tumor spots were retained for downstream analysis. A difference in the ERBB2 gene expression was found at the spot level, with a higher expression in HER2-low samples compared to HER2-zero samples (median expression 0.4, 0.7 and 3.1 in HER2-zero, HER2-low and HER2-positive, respectively). Inter-sample heterogeneity was observed, with 27% of the HER2-zero samples having equal or higher mean ERBB2 expression that the mean ERBB2 expression of the HER2-low cohort. Variability within individual samples was observed, revealing two peaks in ERBB2 expression, one characterized by low levels and the other by medium-high levels. This pattern was frequently observed in samples classified as HER2-zero and HER2-low, indicating the existence of tumor spots with diverse ERBB2 expression. Conclusions: Our results revealed inter- and intra-tumor heterogeneity of ERBB2 expression in HER2-zero and HER2-low BC. The use of spatial transcriptomics can provide more details of the spatial distribution of ERBB2. Further validation is warranted. Citation Format: Danai Fimereli, Matteo Serra, Mattia Rediti, Frederic Lifrange, Nicola Occelli, Laetitia Collet, Delphine Vincent, Ghizlane Rouas, Ligia Craciun, Denis Larsimont, David Venet, Miikka Vikkula, Francois P. Duhoux, Françoise Rothé, Christos Sotiriou. Unravelling tumor heterogeneity in patients with HER2-low hormone receptor-positive breast cancer using spatial transcriptomics [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 1139.

Abstract 6264: Role of Nuclear ErbB3 localization in regulating ligand-dependent ErbB2-ErbB3 heterodimer activation and downstream targets in prostate cancer

Abstract Objective: Prostate cancer (PCa)-specific nuclear expression of ErbB3, a receptor tyrosine kinase of the epidermal growth factor receptor (EGFR) family, has long been recognized. While an 80kDa nuclear variant of ErbB3 has been identified, full-length 185 kDa ErbB3 also translocates to the nucleus in PCa in a bone microenvironment and androgen status-dependent manner. Here, we studied potential mechanisms by which nuclear ErbB3 may regulate PCa progression. Methods: ErbB3 localization was investigated in vitro (human prostate cancer cell lines, LNCaP, C4-2, 22Rv1) using amiloride or heregulin-1β (ErbB3-specific ligand). ErbB3 expression and subcellular localization were analyzed using immunofluorescent microscopy and subcellular fractionation followed by immunoblot. Physiological readouts included viability, migration and reporter gene assays. Results: Here we show that ErbB3 can occupy a nuclear/perinuclear location in prostate cancer cells. The hormone sensitive PCa line LNCaP expressed higher levels of nuclear ErbB3 compared to castration resistant lines C4-2 and 22Rv1. The diuretic amiloride was previously shown to inhibit ErbB3 internalization by macropinocytosis thus we investigated whether it could also modulate ErbB3 nuclear localization in PCa cells. In LNCaP cells, amiloride dose dependently prevented ErbB3 nuclear transport, whereas in C4-2 and 22Rv1, no such effect was observed. Further analysis revealed that amiloride promoted ErbB3 expression in the membrane, where ErbB3 underwent ligand-dependent phosphorylation, formed ErbB2/ErbB3 dimers, and in turn phosphorylated the downstream targets AKT and ERK1/2 (p42/p44MAPK). On the other hand, EGFR phosphorylation and dimerization were unaffected by amiloride. Our results suggest that ErbB3 is stored in the nuclear region in an inactive form, whereas upon stress induction, including androgen deprivation, ErbB3 is translocated to the plasma membrane, where it is activated by ligand binding, and causes downstream signaling. Conclusion: We previously showed that the EGFR/ErbB2 dual inhibitor lapatinib failed to affect PCa - we now show that this may be due to modulation of nuclear and cytoplasmic ErbB3 expression. Amiloride prevented translocation (and thus expression of) ErbB3 to the nucleus and retained this RTK in the membrane, resulting in the formation of active ErbB2/ErbB3 dimers. Lapatinib was then able to inhibit and inactivate its specific target, ErbB2, thereby inducing significant apoptosis. The above indicates a novel role for nuclear ErbB3 in PCa that can be regulated by the combination of amiloride and lapatinib. Citation Format: Maitreyee K. Jathal, Maria M. Mudryj, Paramita M. Ghosh. Role of Nuclear ErbB3 localization in regulating ligand-dependent ErbB2-ErbB3 heterodimer activation and downstream targets in prostate cancer [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 6264.