ER Expression Research Articles

Examination of Tumor-related Biomarkers in Women with Breast Cancer Referred to Naft Hospital of Ahvaz, Southwest Iran

Introduction: Breast cancer is acknowledged as the most prevalent form of cancer in women. The aim of the present study was to examine tumor-related biomarkers such as ER, PR, HER2/neu, P53, Ki-67, CEA, CA-125, and CA 15-3 in women with breast cancer referred to Naft Hospital of Ahvaz, southwest Iran. Methods: This research was a cross-sectional investigation. A total of 100 Iranian women patients were selected who were diagnosed with breast cancer tumors (stage 3). All tumor markers like CEA, CA 15-3, and CA-125 were determined using a chemiluminescent instrument. In addition, the immunohistochemical technique was used to analyze the expression of ER, PR, HER/neu, Ki-67 antigen, and P53 protein. Results: The mean of patients' age was 48.53 ± 9.57 years. ER accounted for the highest frequency (73, or 73%) out of 100 cases. There were 69 (69%), 55 (55%), and 53 (53%) positive cases for PR, HER2/neu, and P53, respectively. The chi-square analysis revealed a significant difference for HER2/neu and P53 biomarkers (p = 0.027). The odds ratio was 2.625 [95% CI: (1.167-5.906)], and the positive correlation between them was 0.236 (p = 0.018). Moreover, a significant difference was found for ER and PR biomarkers (p < 0.0001), and the odds ratio was 18.25 [95% CI: (7.039- 47.319)]. There was a significant positive correlation (r = 0.908, p < 0.0001) between ER and PR. A remarkable positive correlation (r = 0.256, p = 0.010) was also observed between CEA and CA 15-3 biomarkers. Conclusion: Our findings indicated that the highest frequency of tumor-associated biomarkers was ER, and there was a positive correlation between ER and PR, HER2/neu, and P53, as well as CEA and CA 15-3.

All-trans-retinoic acid modulates glycolysis via H19 and telomerase: the role of mir-let-7a in estrogen receptor-positive breast cancer cells

BackgroundBreast cancer (BC) is the most commonly diagnosed cancer in women. Treatment approaches that differ between estrogen-positive (ER+) and triple-negative BC cells (TNBCs) and may subsequently affect cancer biomarkers, such as H19 and telomerase, are an emanating delight in BC research. For instance, all-trans-Retinoic acid (ATRA) could represent a potent regulator of these oncogenes, regulating microRNAs, mostly let-7a microRNA (miR-let-7a), which targets the glycolysis pathway, mainly pyruvate kinase M2 (PKM2) and lactate dehydrogenase A (LDHA) enzymes. Here, we investigated the potential role of ATRA in H19, telomerase, miR-let-7a, and glycolytic enzymes modulation in ER + and TNBC cells.MethodsMCF-7 and MDA-MB-231 cells were treated with 5 µM ATRA and/or 100 nM fulvestrant. Then, ATRA-treated or control MCF-7 cells were transfected with either H19 or hTERT siRNA. Afterward, ATRA-treated or untreated MDA-MB-231 cells were transfected with estrogen receptor alpha ER(α) or beta ER(β) expression plasmids. RNA expression was evaluated by RT‒qPCR, and proteins were assessed by Western blot. PKM2 activity was measured using an NADH/LDH coupled enzymatic assay, and telomerase activity was evaluated with a quantitative telomeric repeat amplification protocol assay. Student’s t-test or one-way ANOVA was used to analyze data from replicates.ResultsOur results showed that MCF-7 cells were more responsive to ATRA than MDA-MB-231 cells. In MCF-7 cells, ATRA and/or fulvestrant decreased ER(α), H19, telomerase, PKM2, and LDHA, whereas ER(β) and miR-let-7a increased. H19 or hTERT knockdown with or without ATRA treatment showed similar results to those obtained after ATRA treatment, and a potential interconnection between H19 and hTERT was found. However, in MDA-MB-231 cells, RNA expression of the aforementioned genes was modulated after ATRA and/or fulvestrant, with no significant effect on protein and activity levels. Overexpression of ER(α) or ER(β) in MDA-MB-231 cells induced telomerase activity, PKM2 and LDHA expression, in which ATRA treatment combined with plasmid transfection decreased glycolytic enzyme expression.ConclusionsTo the best of our knowledge, our study is the first to elucidate a new potential interaction between the estrogen receptor and glycolytic enzymes in ER + BC cells through miR-let-7a.

Apelin receptor modulation mitigates letrozole-induced polycystic ovarian pathogenesis in mice

AimsPolycystic ovarian syndrome (PCOS) is one of the most common (about 5–20%) reproductive disorders in women of reproductive age; it is characterized by polycystic ovaries, hyperandrogenism, and oligo/ anovulation. The levels and expression of ovarian adipokines are deregulated in the PCOS. Apelin is an adipokine that acts through its receptor (APJ) and is known to express in the various tissues including the ovary. It has also been suggested that apelin and APJ could be targeted as therapeutic adjuncts for the management of PCOS. However, no study has been conducted on the management of PCOS by targeting the apelin system. Thus, we aimed to evaluate its impact on combating PCOS-associated ovarian pathogenesis. MethodsThe current work employed a letrozole-induced-hyperandrogenism PCOS-like mice model to investigate the effects of apelin13 and APJ, antagonist ML221. The PCOS model was induced by oral administration of letrozole (1 mg/kg) for 21 days. A total of four experimental groups were made, control, PCOS control, PCOS + aplein13, and PCOS + ML221. The treatment of apelin13 and ML221 was given from day 22 for two weeks.Key findings: The letrozole-induced PCOS-like features such as hyperandrogenism, cystic follicle, decreased corpus luteum, elevated levels of LH/FSH ratio, and up-regulation of ovarian AR expression were ameliorated by apelin13 and ML221 treatment. However, the PCOS-augmented oxidative stress and apoptosis were suppressed by apelin 13 treatments only. ML221 treatment still showed elevated oxidative stress and stimulated apoptosis as reflected by decreased antioxidant enzymes and increased active caspase3 and Bax expression. The expression of ERs was elevated in all groups except control. Furthermore, the PCOS model showed elevated expression of APJ and apelin13 treatment down-regulated its own receptor. Overall, observing the ovarian histology, corpus luteum formation, and decreased androgen levels by both apelin13 and ML221 showed ameliorative effects on the cystic ovary. SignificanceDespite the similar morphological observation of ovarian histology, apelin13 and ML221 exhibited opposite effects on oxidative stress and apoptosis. Therefore, apelin13 (which down-regulates APJ) and ML221 (an APJ antagonist) may have suppressed APJ signalling, which would account for our findings on the mitigation of polycystic ovarian syndrome. In conclusion, both apelin13 and ML221 mediated mitigation have different mechanisms, which need further investigation.

The Expression of Adipogenic Marker Is Significantly Increased in Estrogen-Treated Lipedema Adipocytes Differentiated from Adipose Stem Cells In Vitro.

Lipedema is a chronic, idiopathic, and painful disease characterized by an excess of adipose tissue in the extremities. The goal of this study is to characterize the gene expression of estrogen receptors (ERα and ERβ), G protein-coupled estrogen receptor (GPER), and ER-metabolizing enzymes: hydroxysteroid 17-beta dehydrogenase (HSD17B1, 7, B12), cytochrome P450 (CYP19A1), hormone-sensitive lipase (LIPE), enzyme steroid sulfatase (STS), and estrogen sulfotransferase (SULT1E1), which are markers in Body Mass Index (BMI) and age-matched non-lipedema (healthy) and lipedema ASCs and spheroids. Flow cytometry and cellular proliferation assays, RT-PCR, and Western Blot techniques were used to determine the expression of ERs and estrogen-metabolizing enzymes. In 2D monolayer culture, estrogen increased the proliferation and the expression of the mesenchymal marker, CD73, in hormone-depleted (HD) healthy ASCs compared to lipedema ASCs. The expression of ERβ was significantly increased in HD lipedema ASCs and spheroids compared to corresponding healthy cells. In contrast, ERα and GPER gene expression was significantly decreased in estrogen-treated lipedema spheroids. CYP19A1 and LIPE gene expressions were significantly increased in estrogen-treated healthy ASCs and spheroids, respectively, while estrogen upregulated the expression of PPAR-ϒ2 and ERα in estrogen-treated lipedema-differentiated adipocytes and spheroids. These results indicate that estrogen may play a role in adipose tissue dysregulation in lipedema.

Royal jelly and its hormonal effects in breast cancer: a literature review.

Breast cancer is the most common cancer in women which can be cured in most individuals with early-stage non-metastatic disease. Imbalance in estrogen signaling pathways and propagating levels of estrogens has important roles in breast cancer development. Targeting the estrogen receptor signaling pathway is linked to breast cancer treatment. Royal jelly is one of the bee products containing 10-hydroxy-2-decenoic acid, a structure similar to mammalian estrogen, allowing it to attach to estrogen receptors. It is considered as a general tonic and immunomodulator which may be helpful in reducing the side effects of cancer treatments. Currently, there are controversial data regarding the pros and cons of royal jelly in cancer. Here we provide an overview of the effects of royal jelly on sex hormones and its possible role in breast cancer. Electronic databases including PubMed, Scopus, and Web of Science were searched with the search terms royal jelly, cancer, and sexual hormones. All preclinical and clinical studies regarding the hormonal effects of royal jelly were included. According to the collected preclinical data, consumption of royal jelly at daily doses below 200mg/kg can be useful to decrease the risk of breast cancer since it reduces the serum level of estrogen; whereas increases progesterone, which subsequently decreases the expression of ERs on the ER-positive cells. Future clinical studies are essential to confirm the safe dose of royal jelly as an adjuvant therapy in breast cancer.

Abstract PO5-01-01: The comparison of HER2 low and HER2 0 in ductal carcinoma in situ (DCIS) for breast cancer

Abstract Background: Trastuzumab deruxtecan is a standard of care after prior chemotherapy for metastatic breast cancer with HER2 low. HER2 low recognized as a new category of breast cancer. The definition of HER2 low is IHC 1+ or IHC 2+/ISH- in invasive ductal components. We have already reported the frequency and prognosis of HER2 positive and low in invasive cancer (Breast Cancer. 2022;29:234-241), but the distribution and characteristics of HER2 low in ductal carcinoma in situ (DCIS) is unknown. It is also well known that HER2 expression is higher in DCIS than in invasive cancer. We focused on the frequency of HER2 low in DCIS, performed an analysis of its frequency and characteristics, and compared to DCIS with HER2 0. Methods: We retrospectively examined breast cancer patients with DCIS and invasive ductal carcinoma (IDC) who underwent surgery at our institution between 2005 and 2015. Regarding the expression status of HER2, we classified cases into three groups: HER2 0, HER2-low (defined as IHC 1+ or IHC 2+/ISH-), and HER2-positive (IHC 2+/ISH+ or IHC 3+), and investigated their frequency. We analyzed the relationship between HER2 expression and Van Nuys classification, as well as the relationship between HER2 expression and ER expression, using the chi-square test. Results: A total of 726 DCIS patients were included in this analysis. The patient demographics showed a median age of 52 years (range: 20-84), with 413 patients (56.9%) undergoing total mastectomy and 311 patients (42.8%) undergoing breast-conserving surgery (two cases had unknown surgical procedures). The expression of HER2 in DCIS patients was observed as HER2-0, low and positive in 100 (13.8%), 474 (65.3%) and 152 patients (20.9%), respectively. The expression of HER2 in IDC was observed as HER2 0, low and positive in 838 (17.1%), 3169 patients (64.4%) and 911 patients (18.5%) (Table 1). DCIS tended to have higher HER2 expression(P=0.048). Then we compared the characteristics of HER2 0 and HER2 low in DCIS. Table 2 shows the relation between Van Nuys classification and HER2 expression. Van Nuys classification was evaluated in 346 cases. Van Nuy grade 3 accounted for 4.8% of the HER2 0 cases and 15.8% of the HER2 low cases. HER2 low cases tend to have higher grades(P < 0.01). Table 3 shows the relation between ER expression and HER2 expression. ER negative accounted for 5% of HER2 0 cases and 8.4% of HER2 low cases. ER negatives tended to be more common in Her2 low cases(P < 0.01). Conclusion: HER2 low is not considered a molecular biological classification, but rather a classification of the target of treatment for Trastuzumab deruxtecan. In our study, comparing HER2 0 and HER2 low, HER2 low tended to have higher grade and lower ER expression. Further investigation including basic research is needed to clarify the biological meaning of her2 low. Table1: The frequency of HER2 expression in DCIS and IDC Table2: The relationship between Van Nuys classification and HER2 expression Table3: The relationship between ER status and HER2 expression Citation Format: Nari Kureyama, Maho Kusudo, Akira Nakakami, Rie Komaki, Yuka Endo, Kazuki Nozawa, Ayumi Kataoka, Haruru Kotani, Akiyo Yoshimura, Masaya Hattori, Masataka Sawaki, Hiroji Iwata. The comparison of HER2 low and HER2 0 in ductal carcinoma in situ (DCIS) for 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 PO5-01-01.

Abstract PS05-09: A single-arm Phase II clinical study of fulvestrant combined with chemotherapy in the neoadjuvant treatment of HR+/HER2- locally advanced breast cancer

Abstract Background: HR+/HER2- breast cancer has been found to be less sensitive to neoadjuvant chemotherapy (NCT), leading to an objective response rate (ORR) of approximately 65%. Endocrine therapy combined with chemotherapy may help improve the ORR in these patients. The objective of this Phase II study was to evaluate the efficacy and safety of adding fulvestrant to NCT in patients with HR+/HER2- locally advanced breast cancer(LABC). Additionally, the study aimed to investigate the association of 18F-FES PET/CT and metabolites with efficacy. Methods: In this single-arm Phase II study, eligible patients were females with histologically confirmed HR+/HER2- LABC (Stage IIB-IIIC). Patients received fulvestrant (500 mg, on days 0, 14, 28, then every 28 days thereafter, every 4 weeks for six cycles) plus AC-T regimen, followed by surgery. Premenopausal women were administered a concomitant GnRH analogue. Patients underwent 18F-FES PET/CT before the initiation of fulvestrant, and plasma samples were collected for LC-MS analysis at baseline. The primary endpoint was ORR. Secondary endpoints included pathological complete response (tpCR) and safety. Results: From December 2020 to September 2022, 36 patients were enrolled. The median age was 53 years (range35-67). 78% were ECOG PS of 1, 69% were postmenopausal, 92% were nodal involved, and 83% were in stage III. After neoadjuvant therapy, the ORR was 86.1%. All patients completed the surgery, with a tpCR rate of 8.3% (3/36). 80.6% (29/36) patients were classified as Miller-Payne (MP) grade ≥3. Grade ≥3 treatment-emergent adverse events (TEAEs) occurred in 22% (8/36) of the participants. The most common TEAEs were neutropenia (13.9%) and leukopenia (8.3%). The expression of ER, PR, and Ki-67 in postoperative pathology significantly decreased compared to baseline (p < 0.05). Additionally, the change in ER expression was significantly correlated with the regression volume of primary breast tumors (R = 0.56, p = 0.0044). The decrease in ER value in patients with MP ≥ 4 was significantly larger than in patients with MP ≤ 3 (p = 0.0054). Among the 36 patients, 24 completed the 18F-FES PET/CT scan before initiating fulvestrant.The average SUVmax in primary breast lesions was 4.17 (range 1.00-12.80). The SUVmax of breast lesions was significantly correlated with clinical efficacy (p = 0.0018) and with changes in ER expression before and after treatment (R = 0.68, p = 0.00023). Meanwhile, the SUVmax of patients with MP ≥ 4 grade was significantly higher than that of patients with MP ≤ 3 grade (p=0.018). A total of 25 plasma samples were available for metabolic analysis. Among these samples, 13 differential metabolites were identified between patients with MP ≥ 4 grade and MP ≤ 3 grade, which were markedly enriched in 19 metabolic pathways. Conclusions: The addition of fulvestrant to NCT showed manageable toxicity and promising antitumor activity for patients with HR+/HER2- LABC. 18F-FES PET/CT might serve as a tool to predict the effectiveness of neoadjuvant combination therapy. Altered metabolites or metabolic pathways might be associated with the response to this combined treatment approach. Keywords: breast cancer, HR+/HER2-, fulvestrant, neoadjuvant treatment, 18F-FES PET/CT Citation Format: Qing Shao, Jing Wu, Ningning Zhang, Xiaohua Zeng. A single-arm Phase II clinical study of fulvestrant combined with chemotherapy in the neoadjuvant treatment of HR+/HER2- locally advanced 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 PS05-09.

Abstract PO1-25-11: Deciphering biological differences between normal breast tissues in men and women using single cell sequencing and in vitro modeling

Abstract Breast cancer in men is relatively rare but outcome from the disease is worse than in women. Although 87% breast cancers in men are Estrogen Receptor-positive (ER+), anti-estrogen therapy is less efficacious in men compared to women with similar breast cancer subtype. Recent genomic analysis of breast tumors in men and women have identified overlapping as well as unique genomic aberrations in breast tumors of men. It is unclear whether genomic aberrations enriched in breast cancers of men uniquely affect the biology and thus alter response to antiestrogen therapy. This lack of knowledge is mainly due to scarcity of model systems. To overcome this limitation, we collected ultrasound guided breast biopsies of healthy men and characterized breast epithelial cells grown from these biopsies. Biopsies were also subjected to single nuclei sequencing. Single nuclei breast atlas of men is currently being superimposed on single nuclei breast atlas generated from breast biopsies of healthy women to determine whether distinct cell types exist in men and to distinguish ductal and lobular breast epithelial cells. Breast epithelial cells of men expressed higher levels of ER compared to similarly propagated breast epithelial cells of women. Expression of ER in a subpopulation of cultured breast epithelial cells was confirmed by immunofluorescence. Among the pioneer factors that determine genome wide binding pattern of ER, TBX3 is expressed at a higher level but FOXA1 is expressed at a lower level in breast epithelial cells of men compared to women. Comparative RNA-seq analysis of breast epithelial cells of men and women revealed elevated non-genomic ER signaling in men compared to women, which could be responsible for limited response of breast tumors of men to anti-estrogen therapy. Additional pathways uniquely upregulated in breast epithelial cells of men include non-canonical Wnt, HIF1A, JUN, NF-kB, TGFb, and FOXO1 signaling. Expectedly, breast epithelial cells of men expressed 15 Y chromosome associated genes including the epigenetic regulators KDM5D and UTY, which have recently been shown to be responsible for sex-specific differences in outcome from other cancer types and loss of Y chromosome could be driver event in certain cancer types. We have created immortalized and transformed variants of breast epithelial cells of men to further aid in mechanistic investigation and drug discovery. Furthermore, our tissue bank currently has breast biopsies of ~30 healthy men to be used as controls for breast cancer studies in men. To our knowledge, this is the first model system to study breast cancer in men starting with breast tissues of healthy men. Citation Format: Poornima Bhat-Nakshatri, Benjamin Fischer, Aditi Khatpe, Adedeji Adebayo, Hongyu Gao, Yunlong Liu, Michele Cote, Harikrishna Nakshatri. Deciphering biological differences between normal breast tissues in men and women using single cell sequencing and in vitro modeling [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 PO1-25-11.

Abstract PO3-25-03: Can intratumoral microRNA-125a levels be a prognostic biomarker of a ER-positive breast cancer patients？

Abstract Background: MicroRNA-125a (miR-125a) has been shown to function as tumor suppressive miRNA by pre-clinical in vitro studies. However, to the best of our knowledge, there has been no study that investigated the clinical relevance of miR-125a in breast cancer patients. We hypothesized that miR-125a expression in breast cancer associate with its aggressiveness and can be a prognostic biomarker. Material and Methods: Non-tumorigenic epithelial cell line, MCF-10A and breast cancer cell lines, MCF-7, T47D, and MDA-MB231, were used to analyze the expression levels of miR-125a as well as cell viability. The data of 2042 patients were extracted from publicly available databases, The Molecular Taxonomy of Breast Cancer International Consortium (METARBRIC), The Cancer Genome Atlas (TCGA), and GSE57897. These databases included clinicopathological and survival data as well as transcriptome data. The survival analysis, gene set enrichment analysis (GSEA) were conducted comparing miR-125a high expressing and low expressing tumors, divided by the median cutoff. The association between the miR-125a expression and tumor immune microenvironment was assessed by xCell. Results: The expression levels of miR-125a were suppressed in breast cancer cell lines, MCF-7, T47D, and MDA-MB231compared with MCF-10A. This was concordant with human samples. The expression levels of miR-125a were lower in tumors compared with normal breast tissues in both TCGA and GSE57897 cohorts. These results were in agreement with the notion that it is a tumor suppressive miRNA (p< 0.001 and p< 0.001, respectively). ER-positive/HER2-negative (ER+/HER2-) showed the highest miR-125a expression among the subtypes in TCGA (p< 0.001). MiR-125a expression was not associated with cancer staging in any of the subtypes in neither TCGA nor METABRIC cohorts. To our surprise, high miR-125a tumors demonstrated worse disease free survival (DFS), disease specific survival (DSS), and overall survival (OS) compared with low expressing in ER+/HER2- breast cancer patients (p=0.008, p=0.005, and p=0.037) which was not the case for the other subtypes in METABRIC cohort. Interestingly, miR-125a expression was associated with Nottingham histological grade only in ER+/HER2- among the subtypes (p< 0.001). Also, high miR-125a tumors significantly demonstrated higher expression of MKI67, one of the most commonly used parameters for cell proliferation, correlated with miR-125a expression in ER+/HER2- (p=0.02), but not in other subtypes. Furthermore, high miR-125a tumors enriched four out of five cell proliferation-related gene sets in Hallmark collection, such as E2F Targets, G2M Checkpoint, Mitotic Spindle, and MYC Targets V2 in ER+/HER2- subtype, but not in TNBC. This was also the case in immune-related gene sets; interferon-alpha response and interferon-gamma response that enriched to high miR-125a tumors in ER+/HER2-, but not in TNBC. Anti-tumor immune cells, CD8 cells, T-helper type 1 cells, and M1 macrophages, were all elevated in high miR-125a tumors in ER+/HER2- subtype (all p< 0.03), but none in TNBC. Also, pro-cancerous immune cell such as T-helper type 2 cells were similarly highly infiltrated with high miR-125a tumors only in ER+/HER2- subtype (p=0.026). Given its association with the survival outcome, we cannot help but speculate that miR-125a expression is parallel with highly proliferative ER+/HER2- breast cancer, but its tumor suppressive effect is not enough to improve survival outcome. Conclusion: High miR-125a levels in tumors were associated with aggressive biology and worse survival, thus it can be a candidate for a prognostic biomarker in ER+/HER2- patients. Citation Format: Yoshihisa Tokumaru, Masanori Oshi, Mai Okawa, Saki Ando, Yoshimi Niwa, Ryutaro Mori, Kazuaki Takabe, Manabu Futamura. Can intratumoral microRNA-125a levels be a prognostic biomarker of a ER-positive breast cancer patients？ [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 PO3-25-03.

Abstract PO1-16-02: Correlation of Circulating Tumor Cells (CTC) with Clinical Characteristics, Pathological Factors, and Treatment Response in Patients with Metastatic Breast Cancer (MBC)

Abstract Background: Circulating Tumor Cells (CTC) can be isolated in 40-80% of patients with metastatic breast cancer (MBC). High levels of CTC, defined as ≥5/7.5 ml of blood are associated with shorter progression-free survival and overall survival. CTC can be tested for clinically relevant biomarkers, such as ER, and HER2 status, and have the potential to guide therapy as well as help with disease monitoring. We investigated CTC detection rate, its correlation with clinical characteristics, pathological characteristics, and response to treatment in patients with any type of metastatic breast cancer who progressed on at least one line of therapy. Methods: Eligible patients with MBC were enrolled in the IMAGE-II study (Individualized Molecular Analyses Guide Efforts in Breast Cancer) NCT02965755, in which we obtained archival tissue as part of the standard of care, and prospectively collected serial blood samples for CTC analysis. We compared CTC levels and biomarker status with clinical factors, tissue-based pathology, and molecular biomarkers. We assessed whether changes in CTC count correlated with treatment response. Blood samples were collected at baseline (Day 1), after 1-2 weeks of therapy, after 8-12 weeks, and at subsequent restaging (every 8-12 weeks). Medical records were reviewed every 3 months for ongoing treatment response, and death. Samples were processed at the Biocept CLIA-certified Laboratory. CTC were enumerated by the presence of CD45- and DAPI+ cells. Further CTC characterization was performed by antibody staining of specific protein biomarkers (ER, HER2). Fisher’s exact test was used to evaluate the association between baseline CTC < 5 and ≥5/7.5ml with patient characteristics. McNemar’s test was used to assess discordance between tissue-based and CTC-defined markers. Results: Between 1/26/2018 and 12/31/22 baseline samples were collected from 70 women with a median age of 56 (36-82) who were enrolled at four study sites. CTC was detected in 59 (84%) of participants. Baseline CTC counts were < 5/7.5 ml in 37/59 (63%) and ≥5/7.5 ml in 22/59 (37%) of participants. Differences between breast cancer subtype and CTC-receptor status were observed (Table 1). Among the 41 patients who were ER-positive, 31 (76%) were CTC ER-negative(p< 0.001). Among 29 patients who were ER-negative, 4 (14%) were CTC ER-Positive (P< 0.001). Among the 7 patients who were HER2 positive, 2(29%) were HER2 positive on CTC. Among 63 patients who were HER2 negative, 7(11%) had HER2 expression on their CTC. The discordance in the classification of HER2 wasn't statistically significant (p=0.77). Elevated CTC at baseline was more frequently detected in younger participants (< 50 years old) (55% vs 27%; P=0.22), in Black women compared to White (60 % vs 29 %, P=0.27), and in participants with visceral vs non-visceral metastasis (52% vs 28 %, P=0.40). Patients with CTC ≥5 vs < 5/7.5mL at baseline had a shorter duration of anti-HER2-based therapy (61.5 vs 836 days, p=0.04). There were no statistically significant differences among participants who received chemotherapy agents (316.5 vs 365.5 days, P=0.88), endocrine therapy (376 vs 490 days, P=0.89), or overall therapy (272 vs 390 days, P=0.21). Conclusions: We observed significant differences in the expression of ER between tumor tissue and CTC, which can be partly due to tumor evolution over time. Additionally, participants who are young, Black, and those who have visceral metastasis may have higher CTC counts. Higher CTC counts were associated with a shorter duration of anti-HER2 therapy. Although it didn’t meet statistical significance, a similar trend was observed in patients who received chemotherapy and endocrine therapy. Table 1: MBC Tissue-Based Subtype and CTC-receptor status at baseline Citation Format: Asnakech Bayable, Tingchang Wang, Amanda Blackford, Jessica Tao, Jenna Canzoniero, Seoho Lee, Faith Too, Barbara Blouw, Mary Wilkinson, Rima Couzi, Antonio Wolff, Christie Hiton, Ben Park, Vered Stearns, Cesar Santa-Maria. Correlation of Circulating Tumor Cells (CTC) with Clinical Characteristics, Pathological Factors, and Treatment Response in Patients with Metastatic Breast Cancer (MBC) [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 PO1-16-02.

Abstract PO2-14-08: Development and characterization of breast cancer organoids representing epithelial heterogeneity and drug response

Abstract Organoids serve as an important preclinical model in multiple cancers including breast cancer, demonstrating preservation of biological features and feasibility of drug screening, with reasonable cost and high scalability. It has been reported that patient-derived organoids (PDOs) can be robustly derived from both primary and metastatic breast tumors, with high resemblance to source tissues in histological, genomic, and transcriptomic features, as well as consistency in drug response compared to xeno-transplantations or patients (Sachs et al, 2018). Since 2018, the Institute for Precision Medicine (IPM) has developed a panel of breast cancer organoids. The current inventory now consists of 128 PDOs from 94 primary tumors, 12 metastases, 12 rapid-autopsy tumors, and 10 normal breast tissues with a 60% success rate of culture; as well as 33 organoids from animal models including 11 patient-derived xenograft organoids (PDXOs), 15 rat (RDO) and 7 mouse (MDO) tumor organoids with 85% success rate. For PDOs, the protocol includes in-lab processing of fresh, deidentified tissue within 60 minutes of surgical operation, thanks to close collaboration with surgeons, consented patients, and an institutional biospecimen core. BC organoids have been comprehensively characterized in terms of morphology, histology, genomics, transcriptomics, and functional experiments. Key biological features are preserved. PDOs (n=56) exhibit a mutational spectrum consistent with human breast cancers. ER expression is detectable in a subset of cultures with robust estradiol response indicated by GREB1 expression; and PDOs from invasive lobular carcinoma (ILC) mostly showed more discohesive structures than PDOs from no special type (NST), with clear E-cadherin loss under immunofluorescence. We have also successfully constructed transcriptionally and genetic modified BC PDOs with shRNA and CRISPR, which showed replicable performance in proliferation assay, drug response, and signaling by western blot. Of note, longitudinal tracking of PDOs with single cell RNA-sequencing (scRNA-seq) revealed well-preserved heterogeneity compared to primary tumor, while transcriptomic clonality was mildly decreased in later passages from pilot data. Future work will include expansion of current collection from various sources, longitudinal characterization with scRNA-seq and DNA sequencing, as well as more functional studies. We aim to construct a robust resource of well-characterized and reliably-usable model bank for preclinical breast cancer research. Citation Format: Daniel Brown, Kai Ding, Fangyuan Chen, Jian Chen, Steffi Oesterreich, Peter Lucas, Priscilla McAuliffe, Jennifer Atkinson, ADRIAN LEE. Development and characterization of breast cancer organoids representing epithelial heterogeneity and drug response [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 PO2-14-08.

Abstract PO1-26-03: Activity of alpelisib in a panel of breast XPDX models harboring hotspot and uncommon PIK3CA mutations

Abstract Background: Therapies targeting mutated phosphoinositide 3-kinase (PI3K) p110α catalytic subunit (PIK3CA) cancers remain an active field of research. Alpelisib was approved in combination with fulvestrant in ER+/HER2- breast cancer patients harboring PIK3CA mutations on hotspot amino acids E542, E545 Q546 or H1047. While mutation at these sites have been widely studied, they only comprise ~25% of identified variants in this protein. Recent studies have reported other mutations, including those at the c-terminus, which can lead to constitutive activation of PIK3CA; however, whether these variants are sensitive to agents like alpelisib is unclear. To better understand these variants and their role in PIK3CA-mediated breast cancer, we established a panel of PIK3CA-mutated ER+ and ER- breast XPDX models and evaluated each in vivo with single agent alpelisib. Methods: Ninety previously developed XPDX models representing PIK3CA-mutated ER+ and ER- breast cancer were evaluated in this study. Models were grown subcutaneously in female athymic nude mice supplemented with estradiol in drinking water when necessary and ER expression confirmed at multiple passes; PIK3A variants were determined by WES and RNAseq. For in vivo studies, alpelisib was administered once daily by oral gavage at 35 mg/kg through study completion. Endpoints included tumor volume and time from treatment initiation with %T/C values and tumor regression reported at study completion; a T/C of ≤ 20% versus control was considered sensitive. Tumor regression (%T/C< 0%) versus Day 0 tumor volume was also reported. Results: 60% of PIK3CA-mutated models were found ER+ with 40% also HER2+ while 20% of ER- PIK3CA-mutated models were also HER2+. H1047X mutations accounted for 40% of all PIK3CA variants, 75% of which were single or dual PIK3CAH1047R. Several hotspot-mutated models were sensitive to alpelisib some with regressions including ST986 (E542K), ST1097 (E454K) and STF040 (H1047R), as well as ST1245C (R88Q/N1044H), ST2076 (102-103ins/R357Q) and ST4176 (E726K/H1047R). HER2+ models were less sensitive to alpelisib although STM148D (H1047L) reported tumor regressions. Interestingly, ST1799/PBR (E542K/H1065L), a palbociclib resistant clone of the ER+ parent ST1799 model was resistant to alpelisib while the parent was found sensitive. Conclusion: We have benchmarked a panel of PIK3CA-mutated breast XPDX models with alpelisib and compared activity of models harboring hotspot versus uncommon PIK3CA mutations with ER and HER2 expression. This data is a valuable tool in further developing PIK3CA inhibitors and identifying new variant targets in breast cancer. Citation Format: Mercedes Castaneda, Alyssa Simonson, Johnnie Flores, Maci DeBoer, Jim Lund, Tahmi Rouzbahan, Kyriakos P. Papadopoulos, Amy Lang, Gladys Rodriguez, Maryam Elmi, Arthur Rosenthal, Drew Rasco, Victor Moreno, Amita Patnaik, Tatiana Hernandez, Emiliano Calvo, Lon Smith, Manish Sharma, Muralidhar Beeram, Michael Wick. Activity of alpelisib in a panel of breast XPDX models harboring hotspot and uncommon PIK3CA mutations [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 PO1-26-03.

Abstract PO1-28-04: PILHLE-001: neoadjuvant pyrotinib combined with chemotherapy in HR-positive, HER2-low (IHC 2+/FISH-negative) early breast cancer

Abstract Background: Hormone receptor (HR)-positive/HER2-low (IHC 2+/FISH negative or IHC 1+) breast cancers (BCs) represent the largest proportion of breast carcinomas. However, its response to neoadjuvant chemotherapy or endocrine therapy is the lowest, with a residual cancer burden (RCB) 0/I rate around 15%. Previous studies have demonstrated that pyrotinib (P), a pan-HER tyrosine kinase inhibitor, exhibits excellent response in HER2-positive early or advanced BC (E/ABC). In-vitro experiments have shown that pyrotinib can effectively inhibit colony formation in HER2-low (IHC 2+/FISH negative) BC cells. This PILHLE-001 study (NCT05165225) aimed to evaluate the efficacy and safety of neoadjuvant pyrotinib plus chemotherapy in HR-positive/HER2-low (IHC 2+/FISH negative) EBC patients with potential biomarkers. Methods: PILHLE-001, as a single-arm, single-center phase Ⅱ trial, enrolled patients with previously untreated HR-positive (ER or PR > 1%) and HER2-low (IHC 2+/FISH negative) invasive EBC (TNM stage II-III) to receive pyrotinib 320mg QD and four Q3W of epirubicin-cyclophosphamide followed by four Q3W of docetaxel. MammaPrint/BluePrint at baseline (t0) was assessed. Breast magnetic resonance imaging (MRI) was conducted at t0, the end of cycle 2 neoadjuvant therapy (t1), and the end of all neoadjuvant therapy. Immune cell subpopulations assay and gene sequencing of tumor tissues with whole blood control samples were performed at t0 and surgery (t2). Additional core biopsy was administered to reassess Ki67 and tumor infiltrating lymphocytes (TILs) at t1. The primary outcome was RCB 0/I rate. The short-term secondary outcomes included pathological complete response (pCR, ypT0/is ypN0) rate, objective response rate (ORR), breast conservation surgery (BCS) rate, safety, and exploratory biomarkers analysis. Results: From July 2021 to March 2023, 48 patients were enrolled and treated. The median age of the patients was 48 years (range 28-66), 43 (90%) had cT1-2 tumors, 30 (63%) showed no node involvement, 37 (77%) had tumours with TNM stage II, and 47 (98%) had ER expression ≥ 50%. Twenty-eight (61%) of 46 patients had MammaPrint high risk. Out of the 48 patients, 26 (54.2%) achieved RCB 0/I, with a 95% confidence interval of 39.2% to 68.6%. The RCB 0/I rate was particularly higher in tumors with no lymph node involvement (73.3%), TNM stage II (64.9%), or abundant tumor-infiltrating lymphocytes (66.7%). Three (6.3%) patients obtained a pCR. The rate of BCS was 60.4% (29/48). The ORR was 45.8% (22/48) at t1 and 81.3% (39/48) at the end of all neoadjuvant therapy, respectively. No patients had progressive disease. All treatment-related adverse events (AEs) were mostly of grade 1 or 2 severity. Grade ≥ 3 AEs occurred in 21 (44%) patients, with the most prevalent being diarrhea [10 (21%)]. All AEs were reversible with symptomatic treatment and no treatment-related deaths occurred. Patients with MammaPrint risk-low BC had a lower RCB 0/I rate than those with risk-high BC (60.7% vs. 44.4%), although the difference was not statistically significant. Baseline specific immune cell subpopulations (lower PD-1, higher CD3, etc.) or the presence of non-altered PIK3CA were significantly associated with a higher likelihood of achieving RCB 0/I. Similarly, objective response, greater declines in specific MRI quantitative parameters (Ktrans and Kep), greater declines in Ki67, or more increased TILs at t1 were also significantly associated with a higher rate of RCB 0/I. Conclusions: The PILHLE-001 trial first revealed that neoadjuvant pyrotinib plus chemotherapy has encouraging efficacy and acceptable toxicity in patients with HR-positive/HER2-low (IHC 2+/FISH negative) EBC, and this regimen warrants further investigation in phase III randomized controlled trials. Citation Format: Chang Gong, Yuan Xia, Yingying Zhu, Yaping Yang, Wenqian Yang, Louis W.C. Chow, Li Ling, Yunjie Zeng, Jiajie Zhong, Ziliang Cheng, Jun Shen, Qun Lin, Yinduo Zeng, Qiang Liu, Erwei Song. PILHLE-001: neoadjuvant pyrotinib combined with chemotherapy in HR-positive, HER2-low (IHC 2+/FISH-negative) early 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 PO1-28-04.

Abstract PO3-24-08: Establishment of a selective culture method for breast cancer stem cells derived from patient tissue and a subgroup analysis of cancer stem cells

Abstract Purpose Cancer stem cells are considered to cause recurrence and metastasis due to their resistance to drug and radiation therapy. Breast cancer stem cells are therefore the true target for the treatment of breast cancer. In this study, we established a method to selectively culture breast cancer stem cells from patient-derived breast cancer tissue, and we analyzed their characteristics using immunostaining to identify gene expression. Method Breast cancer tissues from ER+, HER2- and drug-naive patients were fragmented and cultured using low-adherent plates (spheroid culture). We investigated the stemness of the breast cancer cells obtained by the spheroid culture by immunostaining the breast cancer stem cell markers and examining their proliferation and differentiation in a mouse transplantation model. The diversity of the cells was further examined by means of the expression of the EMT markers and genetic analysis. Results In 48 cases, the proportion of CD44+/CD24- breast cancer cells increased from 13.8% to 61.6% in the spheroid culture. In addition, even small numbers of cells obtained by the spheroid culture were transplanted into mice, and the transplanted cells subsequently differentiated in the mice, demonstrating their stemness. ER expression was negatively changed in 52.1% of cases, and the expression of EMT markers, Twist, Snail, and Vimentin, was increased from 43.8% to 75.0%, 12.9% to 58.1%, and 7.71% to 37.7%, respectively. A DNA microarray with 14 cases showed that breast cancer stem cells obtained from ER+ and HER2- breast cancer tissue were classified into two groups. Conclusion We demonstrated that breast cancer stem cells were selectively cultured from patient-derived breast cancer tissue in a spheroid culture. Furthermore, breast cancer stem cells have different characteristics from breast cancer cells in primary tumors and are diverse at an EMT level, and in genetic analyses. Table. Expression of primary tumors and spheroids Citation Format: Satoshi Sueoka, Takayuki Kadoya, Kai Azusa, Shinsuke Sasada, Akiko Emi, Morihito Okada, Yukino Kobayashi, Koh Nakayama. Establishment of a selective culture method for breast cancer stem cells derived from patient tissue and a subgroup analysis of cancer stem cells [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 PO3-24-08.

Abstract PO5-16-03: RT LAMP assay for the subtyping of breast cancer

Abstract Introduction Our previous study showed that direct reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay can be employed as a substitute in order to detect tumor involvement of lymph nodes within breast cancer patients. In this research, we aimed to investigate the potential applicability of RT-LAMP for the classification of breast cancer subtype. Method Breast Cancer Cell line RNA extraction Total RNA from the cell lines was isolated from cultured cells using RNAiso Plus (TaKaRa, Shiga, Japan) according to the manufacturer’s instructions; MCF10A, MCF7, T47D, BT474, SKBR3, MB468, HCC1806, MB231 Primer design For the Primer design, ER, PR, HER2, Ki 67, CK7 and GATA were targeted. LAMP primers targeted at ER were created between exons 5 and 6, PR were created between exons 6 and 7, HER2 were created between exons 17 and 18, KI67 were created between exons 2 and 3, CK7 were created between exons 5 and 6, and GATA3 were created between exons 2 and 3 utilizing Primer Explorer V4 (http://primereplorer.jp/elamp4.00/index.html). RT-LAMP assays RT-LAMP assays were carried out within a 25 µL reaction which included 12.5 µL of 2 × reaction buffer, 2µL of each primer, 1 µL of enzyme mixture (16U Bst DNA polymerase and 120U M-MLV reverse transcriptase) 8 µL of DEPC-treated water, and 2 µL of the template RNA. DEPC-treated water was also employed for purposes of a negative control. The reaction mixture was conducted within qRT-PCR. Results ER, PR, HER2, Ki-67, CK7, GATA3 expression level were checked for each breast cancer cell line using RT-LAMP. The luminal cell line exhibited elevated levels of ER and PR expression, while the her2 cell line demonstrated a high level of expression for her2. In the RT LAMP assay, there is a direct correlation between shorter detection time and higher expression levels of the substance. Specifically, in the luminal type, ER/PR was detected within a timeframe of 10 to 12 seconds, while HER2 was detected within a timeframe of 15 to 16 seconds. The expression levels of ER, PR, and HER2 were compared between MCF7 (luminal A) and MDA-MB-231 (TNBC) cell lines. RT-LAMP was performed to examine the RNA expression levels, while RT-PCR was applied to confirm the cDNA expression levels. MCF7 and MDA-MB-231 were mixed at 1:9, 5:5 and 9:1 ratios, respectively, and the difference in ER/PR/HE2 expression level was confirmed (Table1, Figure1). The presence of ER was detected in the MCF7 cell line at 14.44 minutes, and PR was observed at 18.84 minutes. HER2 was detected at a similar time (approximately 15 minutes) in both the MCF7 and MB 231 cell lines. Conclusion This study demonstrated that ER, PR, and HER2 can be quantified using RT-LAMP. Ongoing experiments are being conducted to classify breast cancer using the RT-LAMP method, aiming to enhance sensitivity in the quantification of ER, PR, and HER2. Additionally, new primers are being developed specifically for HER2 detection. RT-LAMP offers the advantages of being a point-of-care test that can be conducted on-site and reducing the discrepancies between pathologists. Citation Format: In Hee Lee, Byeongju Kang, Jeeyeon Lee, Jin Hyang Jung, Ho Yong Park, Nora Jee-Young Park, Ji-Young Park, Eun Ae Kim, Jieun Kang, Yee Soo Chae, Soo Jung Lee. RT LAMP assay for the subtyping of 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 PO5-16-03.

Abstract LBO1-05: Impact of age and ovarian function suppression (OFS) on endocrine response to short preoperative endocrine therapy (ET): Results from the multicenter ADAPTcycle trial (n=4,334)

Abstract Background: In HR+/HER2- early breast cancer (EBC), short preoperative endocrine therapy (ET) is a promising tool for ET-efficacy assessment based on Ki67-decrease after 2-4 weeks of ET. Low post-endocrine Ki67 (Ki67post) is associated with good prognosis in large prospective trials. WSG-ADAPT demonstrated that ET-response is a valid criterion for decisions in uncertain adjuvant chemotherapy indications, e.g., in premenopausal patients (pts) with N0 and Recurrence Score (RS, Oncotype DX®) RS 16-25 or with N1 and RS ≤25. Preliminary results from the phase III ADAPTcycle trial indicated higher efficacy of preoperative ET in premenopausal pts. if ovarian function suppression (OFS) was used together with tamoxifen (TAM) or aromatase inhibitors (AI). In the final screening population, we are now able to confirm these results and investigate the influence of age subgroups, RS, individual biological markers, and OFS on ET-response. Methods: In ADAPTcycle (n=5,290 screened, 1,670 randomized by 06/23; 84 sites in Germany), N0-1 pts with RS >25 or N2-3 pts with RS ≤25 and ET-response were randomized to (neo)adjuvant chemotherapy (CT) followed by ET vs. ribociclib + AI (premenopausal: +GnRH). In premenopausal pts, participation with N1-disease and RS ≤25 or N0 and RS 16-25 was allowed irrespective of ET-response, but randomization recommended only for ET-responders; in the preoperative phase, OFS+TAM or AI for ET-response assessment was protocol-recommended. Results: This analysis includes all pts with complete baseline characteristics (including RS) and ET-response data (n=4,334); ET-response was defined as Ki67post ≤10% (central pathology) after 2-4 weeks (if OFS: 4 weeks) of therapy. ER, PR, and HER2-levels were analyzed by IHC and mRNA. Median age was 56 years (22-87y). 55% had cT2-4 tumors; 28% had cN+ disease; 42% were G3 (central lab). Median RS was 20, median baseline Ki67 25%. n=1,368 were ≤50y and premenopausal (“≤50y”): n=314 (23.0%) and 255 (18.6%) were treated by AI+OFS and TAM+OFS, respectively. 2,966 were >50y or postmenopausal (“ >50y”), of whom 2,565 received AI (86.5%). ET-response occurred in 48.2% of “≤50y” vs. 72.7% in “ >50y” pts (p< 0.001). We observed significant differences in ET-response rates between “≤50y” vs. “ >50y” with TAM alone (34.7% vs. 46.4%); In “≤50y” pts, 55.7% ET-response occurred with TAM+OFS vs. 76.4% with AI+OFS, comparable to 76.8% in “ >50y” patients with AI. Moreover, OFS led to higher ET-response rates (by uni- and multivariable analysis) even in the small (n=78) group of premenopausal pts >50y. Remarkably, even in pts with RS >25, substantial ET-response rates were observed with AI: 66.7% (“≤50y”, AI+OFS), 55.6% (“ >50y”, AI) vs. lower rates with TAM alone (>50y: 18.2%; < 50y: 15.8%). ET-response rate in “≤50y” pts was associated with ET type, RS, and baseline Ki67 in both uni- and multivariable analysis, but not age subgroup (≤40 vs. 41-50y). ER, PR, and HER2 expression by IHC or RT-PCR were associated with ET-response by univariable (but not multivariable) analysis. In “ >50y” pts, by uni- and multivariable analysis, again ET-type, RS, and baseline Ki67, as well as ER-expression (by IHC) and ESR1, PR, and HER2-expression (by RT-PCR) were associated with higher ET-response rates. Conclusions: With about 10,000 pre- and postmenopausal pts, ADAPT and ADAPTcycle provide the largest international database regarding ET-response. ADAPTcycle confirms ADAPT ET-response rates in pts receiving TAM or AI; adding OFS to either improves ET-response, irrespective of RS. ET-response measurement adds value in therapy decisions, particularly in ≤50y N0-1 pts. ET-response might provide key information even in pts with high RS; outcome data of ADAPTcycle will reveal potential clinical consequences. Optimal preoperative ET, especially in premenopausal pts, might even enable sparing chemotherapy in additional pts. Citation Format: Oleg Gluz, Matthias Christgen, Ulrike Nitz, Sherko Küemmel, Michael Braun, Marc Thill, Rachel Wuerstlein, Pauline Wimberger, Andreas Hartkopf, Christian Schem, Matthias Zaiss, Vesna Bjelic-Radisic, Marianne Just, Kristina Veselinovic, Myriam Vincent, Monika Graeser, Katja Krauss, Oliver Hoffmann, Kerstin Lüdtke-Heckenkamp, Ronald Kates, Christine zu Eulenburg, Katarzyna Jozwiak, Sandy Burmeister, Rick Baehner, Peter Schmid, Hans-Heinrich Kreipe, Nadia Harbeck. Impact of age and ovarian function suppression (OFS) on endocrine response to short preoperative endocrine therapy (ET): Results from the multicenter ADAPTcycle trial (n=4,334) [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 LBO1-05.

Abstract PS03-07: Morphometric signature identifies ductal carcinoma in situ of the breast with low risk of progression to invasive breast cancer

Abstract Background. Ductal carcinoma in situ (DCIS) is a frequently found precursor of invasive breast cancer (IBC). However, the majority of DCIS will never progress to IBC. As we cannot distinguish yet which DCIS will remain indolent (‘harmless’) from those that might progress in the future to IBC, almost all women with DCIS are intensively treated by surgery, often followed by radiotherapy. This brings an urgent clinical need to learn distinguishing harmless from potentially progressive DCIS to save many women with indolent DCIS the burden of unnecessary overtreatment. Aim. We aimed to investigate if the geometry and spatial configuration of DCIS ducts in Hematoxylin-Eosin (H&E) stained tissue sections are related to the risk of progression of DCIS to ipsilateral IBC (iIBC). Methods. We obtained data from a population-based cohort of women diagnosed with primary DCIS between 1989 and 2004 in the Netherlands, treated with breast conserving surgery (BCS) only and a median follow-up time of 12 years. A nested case-control study (n=689) was designed in which patients diagnosed with iIBC recurrences during follow-up were considered as “cases” (n= 226) and those with no subsequent iIBC as “controls” (n=463). The DCIS and stroma regions were digitally annotated on H&E-stained whole slide images (WSIs) by a pathologist as ground truth for the deep learning neural network of HALO AI module (IndicaLabs). We developed a computational pipeline to automatically detect and measure stroma areas, DCIS ducts, and the nucleus of their cells. We validated the accuracy of DCIS detection in H&Es WSIs from an external study, in which DCIS regions were digitally annotated by an independent pathologist (Translational Breast Cancer Research Consortium, TBCRC). We classified cases and controls according to morphological measurements using logistic ridge regression with double-loop cross-validation, followed by hierarchical clustering. The risk of subsequent iIBC after primary DCIS diagnosis was evaluated by multivariate Cox proportional hazards models. Results. The accuracy of DCIS detection performed by the computational pipeline was compared with pathologist annotations in 20 slides from the TBCRC study. Results showed a satisfactory DCIS overlap area agreement of 0.76 (0.68 – 0.83). We applied the DCIS computational pipeline on the case-control series. We obtained 15 morphological measurements for each DCIS duct, such as duct area, cell density, distance between ducts, average nucleus area, etc. We calculated 8 distribution parameters from each measurement in each WSI, including median and range. After leaving out redundant variables, 55 unique morphometric variables were obtained, representing the heterogeneity of DCIS ducts per WSI. The c lassifier revealed a median area-under the curve (AUC) of 0.66 (0.55-0.77) to predict 5-years free of iIBC, 0.59 (0.50-0.67) to predict 10-years and 0.60 (0.52-0.68) to predict 15-years. The 30 variables with the highest association with outcome were used to build four morphometric signatures. Signature number 1, which is characterized by lesions with small-sized ducts, a lower number of cells and a lower DCIS/stroma area ratio, showed a significant lower risk of developing iIBC compared to the other three signatures in a multivariate Cox regression model including grade, ER, COX-2 and HER2 expression: HR = 0.56 (0.28-0.78 95%CI). Conclusion. We developed a computational pipeline able to detect and measure DCIS ducts in H&E WSIs with high accuracy and reproducibility. DCIS lesions presenting the morphometric signature of small-sized DCIS ducts have a very low chance to progress to invasive breast cancer. After successful validation, our morphometric method will serve as a robust and easy to implement biomarker for de-escalation strategies in DCIS, and as such, could limit unnecessary overtreatment in the near future. Citation Format: Marcelo Sobral-Leite, Simon Castillo, Shiva Vonk, Xenia Melillo, Noomie Lam, Brandi de Bruijn, Yeman Hagos, Joyce Sanders, Mathilde Almekinders, Lindy Visser, Emilie Groen, Carolien Van der Borden, Petra Kristel, Ercan Caner, Leyla Azarang, Yinyin Yuan, Renee Menezes, Esther Lips, Jelle Wesseling, Grand Challenge PRECISION Consortium. Morphometric signature identifies ductal carcinoma in situ of the breast with low risk of progression to invasive 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 PS03-07.

Abstract GS01-01: Biomarker Results in High-risk Estrogen Receptor Positive, Human Epidermal Growth Factor Receptor 2 Negative Primary Breast Cancer Following Neoadjuvant Chemotherapy ± Nivolumab: an Exploratory Analysis of CheckMate 7FL

Abstract Background: CheckMate 7FL (CM 7FL; NCT04109066) is a prospective, phase 3, randomized, multicenter, double-blind trial investigating nivolumab (NIVO) in combination with neoadjuvant chemotherapy (NACT) and adjuvant endocrine therapy (ET) in patients (pts) with high-risk, estrogen receptor-positive (ER+), human epidermal growth factor receptor 2-negative (HER2−) primary breast cancer (BC). The primary endpoint (pathological complete response, pCR) was met, resulting in a statistically significant improvement with added NIVO to NACT; residual cancer burden (RCB) 0–1 rate was meaningfully improved. NIVO benefit was larger in pts with PD-L1+ tumors (SP142 assay, % immune cells ≥ 1%). This analysis focuses on additional biomarkers to explore associations with NIVO treatment effect. Methods: The study enrolled newly diagnosed pts, stages T1c–2 N1–2 or T3–4 N0–2, grade 2 (ER 1–‍10%) or 3 (ER ≥ 1%). Pts were randomized 1:1 to NACT + NIVO 360 mg Q3W/NIVO 240 mg Q2W (arm A) or NACT + placebo (PBO; arm B). Clinical efficacy data included pCR (ypT0/is ypN0) and RCB 0–1 rates in the modified ITT (mITT) population and the SP142 PD-L1+ subgroup. Baseline PD-L1 expression from core biopsies was evaluated with the Dako 28-8 assay using the combined positive score (CPS) algorithm. Various cutoffs (CPS ≥ 1, 10, and 20) were used for this exploration. Concordance between PD-L1 SP142 and 28-8 CPS was evaluated, with assays performed on different slides from the same tumor biopsy blocks. Results of efficacy by ER and/or progesterone receptor (PR) expression, as well as Ki67 index and stromal tumor-infiltrating lymphocytes, will be presented at the meeting. Results: Overall, 830 pts were screened, 521 randomized, and 517 treated, with the primary efficacy population (mITT; n = 510) including all randomized pts but excluding 11 pts from Russian sites with insufficient follow-up. A total of 510 and 349 pts (68.4% of mITT population) were evaluated by PD-L1 SP142 and 28-8 CPS assays, respectively. The prevalence of PD-L1+ by CPS was balanced in arms A and B (52% vs 50% had CPS ≥ 1; 19% vs 16% had CPS ≥ 10, and 11% vs 9% had CPS ≥ 20, respectively). NIVO effect was larger in pts with tumors with increasing PD-L1 expression, with a ∆pCR rate (unweighted rate difference between arms A and B) of 16.6% in CPS ≥ 1 (40.4% vs 23.8% in arms A/B; 95% CI, 2.8 to 29.4), 32.4% in CPS ≥ 10 (65.7% vs 33.3% in arms A/B; 95% CI, 7.3 to 52.3), and 52.3% in CPS ≥ 20 (78.9% vs 26.7% in arms A/B; 95% CI, 18.6 to 72.4). In comparison, the ∆pCR rate was 10.7% in the mITT population (24.5% vs 13.8% in arms A/B; 95% CI, 3.9 to 17.4) and 5.7% in pts with CPS < 1 (14.0% vs 8.2% in arms A/B; 95% CI, -4.0 to 15.5; refer to the Table for additional details). Unweighted rate differences between arms A and B for RCB 0–1 (∆RCB 0–1 rate) followed a similar trend and were observed to be 17.0% in CPS ≥ 1, 34.4% in CPS ≥ 10, and 52.3% in CPS ≥ 20. In comparison, the ∆RCB 0–1 rate was 9.4% in the mITT population and 3.3% in pts with CPS < 1 (refer to the Table for additional details). Further biomarker data and cutoffs will be presented at the meeting. Conclusions: Greater PD-L1 expression was associated with higher pCR and RCB 0–1 rates, suggesting that pts with PD-L1+, high-risk, ER+/HER2− primary BC can achieve substantial pCR rates with the addition of NIVO to NACT. Table. Summary pCR and RCB 0–1 rates by PD-L1 SP142 and 28-8 CPS at various cutoffs. Note: CPS data with the cutoff at 5 will be presented at the meeting. Citation Format: Sherene Loi, Giuseppe Curigliano, Roberto Salgado, Roberto Iván Romero Díaz, Suzette Delaloge, Carlos Ignacio Rojas García, Marleen Kok, Cristina Saura, Nadia Harbeck, Elizabeth Mittendorf, Denise Yardley, Lajos Pusztai, Alberto Suárez Zaizar, Andrei Ungureanu, Felipe Ades, Rajalakshmi Chandra, Raheel Nathani, Misena Pacius, Thomas Spires, Jenny Wu, Heather McArthur. Biomarker Results in High-risk Estrogen Receptor Positive, Human Epidermal Growth Factor Receptor 2 Negative Primary Breast Cancer Following Neoadjuvant Chemotherapy ± Nivolumab: an Exploratory Analysis of CheckMate 7FL [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 GS01-01.

Abstract PO2-24-10: Anti-Tumorigenic Immuno-Microenvironment by AB-329, a Potent and Selective AXL inhibitor, combined with paclitaxel in Triple-Negative Breast Cancer Preclinical Model

Abstract BACKGROUND The Gas6/AXL signaling pathway plays a substantial role in cancer cell survival, proliferation, immunosuppression, and metastasis. Elevated AXL expression is associated with advanced cancer stages and decreased overall survival rates, underscoring its potential as a therapeutic target for metastatic cancers. We investigated this possibility by utilizing AB-329, a selective small molecule AXL kinase inhibitor, to treat triple-negative breast cancer (TNBC). TNBC, typified by the lack of ER, PR, and HER2 expression, frequently displays mesenchymal characteristics and carries a significant risk of metastasis and an immunosuppressive tumor microenvironment. METHODS We assessed AXL protein expression in 28 human and 11 mouse breast cancer cell lines using Western blotting. The Microarray database was utilized to examine AXL gene expression in 40 human breast cancer cells. The Sulforhodamine B proliferation and soft agar assays measured the antiproliferative effect of AB-329 in TNBC cell lines. The in vitro anti-metastatic impact of AB-329 was evaluated through migration and invasion assays. We appraised the anti-tumor effects of AB-329 in combination with paclitaxel using 4T1 and E0771-LMB mouse TNBC xenograft models. We used FACS and CIBERSORTx to perform immune cell type profiling in E0771-LMB xenograft tumor tissues. To understand AB-329’s immunomodulatory effects in tumors, single-cell RNA sequencing was conducted in a humanized mouse model with SUM149 human TNBC xenografts. RESULTS Microarray data revealed higher AXL expression in TNBC cells compared to non-TNBC cells. Western blotting supported this, showing higher AXL protein expression, particularly in basal and mesenchymal subtypes of TNBC cells, compared to luminal breast cancer cells. AB-329 presented moderate antiproliferative effects as a monotherapy in the TNBC cell lines tested, with half-maximal inhibitory concentrations exceeding 5 µM. Nevertheless, combining AB-329 with paclitaxel resulted in significant growth inhibition in all tested TNBC cell lines (p < 0.05). Importantly, AB-329 alone could inhibit TNBC migration and invasion at a concentration of 1 µM (p < 0.01) and showed enhanced inhibition rates when paired with paclitaxel (p < 0.05). The combination of AB-329 and paclitaxel considerably reduced tumor growth in the 4T1 and E0771-LMB xenograft models compared to paclitaxel or AB-329. Total RNA sequencing and CIBERSORTx analysis indicated that the AB-329 and paclitaxel combination increased Natural Killer (NK) cells’ infiltration into E0771-LMB mouse TNBC xenograft tumors compared to single agent-treated tumors. A nearly 2.5-fold increase in NKT cell infiltration was also detected with AB-329 treatment compared to Control in SUM149 human TNBC xenografts. CONCLUSIONS Our preclinical data suggest that AB-329, an AXL inhibitor, could synergize effectively with paclitaxel in TNBC treatment, notably inhibiting tumor cell proliferation and migration while enhancing NK cell infiltration. Intriguingly, we observed an increased infiltration of NKT cells into tumors following AB-329 treatment, which may promote an anti-tumorigenic immuno-microenvironment. These results underscore the necessity for further investigation into the role of AB-329 in modulating the tumor immuno-microenvironment through AXL pathway inhibition and accentuate the potential for combining AB-329 with immunotherapy. Citation Format: Dileep Reddy Rampa, Jon Fuson, Huey Liu, Max Pan, Naoto Ueno, Jangsoon Lee. Anti-Tumorigenic Immuno-Microenvironment by AB-329, a Potent and Selective AXL inhibitor, combined with paclitaxel in Triple-Negative Breast Cancer Preclinical Model [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 PO2-24-10.

Abstract PO4-18-01: International Validation of a Staging Model for de novo Metastatic Breast Cancer

Abstract Background Given the heterogeneity in outcomes for de novo metastatic breast cancer (dnMBC), a staging system was recently developed that refines prognostic estimates for patients presenting with distant metastases (JCO 2023;10:2546). This staging model was developed using the National Cancer Database and stratified patients into 4 subgroups, termed IVA, IVB, IVC, and IVD based on 3-year overall survival (OS): IVA >70%, IVB 50-70%, IVC 25-< 50%, and IVD < 25%; the primary factors affecting higher stage grouping were negative for ER, PR, and HER2 expression and a higher number of involved organ sites. Here, we aim to validate this model using an international data set. Methods Data for dnMBC patients were obtained from the Netherlands Cancer Registry (NCR, hosted by IKNL), Epidemiological Strategy and Medical Economics (ESME) by Unicancer, British Columbia Cancer (BCCan), Instituto Nacional de Cancerología, México (INCan), and Grupo Español de Investigación en Cáncer de Mama (GEICAM); the diagnosis years included varied by organization (range 2002-2021). Stage groups were assigned based on previously published criteria, defined by T-category, grade, ER, PR, HER2, histology, organ system site of metastases (bone-only, brain-only, visceral), and number of organ systems involved. For each cohort, followup time and OS were estimated using the reverse Kapan-Meier and Kaplan-Meier method, respectively. Median rates were estimated across all cohorts by weighting on cohort sample size or number at risk. Cox proportional hazards models were used to estimate the association of stage with OS after adjustment for age at diagnosis (treatment data not available for all cohorts). For the ESME cohort, a subgroup multivariable analysis was used to estimate the association of stage with OS after adjustment for age, local (surgery or radiotherapy) and first line systemic treatments. Results The final validation cohort was comprised of N=11,199 patients from 5 international organizations: n=5063 (45.2%, IKNL), n=4139 (37.0%, ESME), n=774 (6.9%, BCCan), n=757 (6.8%, INCan), and n=466 (4.2% GEICAM). Median followup across all cohorts was 77.5 months (95% CI 74.3-80.2). Median followup (in months) for the IKNL, ESME, BCCan, INCan, and GEICAM cohorts was 45.3 (95% CI 43.6-47.1), 77.5 (95% CI 74.3-80.2), 105.0 (95% CI 95.9-106.5), 93.0 (95% CI 86.5-102.1), and 49.5 (95% CI 46.5-53.4), respectively. Patients were stratified into stage groups: IVA, n=603 (5.4%); IVB, n=5704 (50.9%); IVC, n=3356 (30.0%); IVD, n=1536 (13.7%). For all cohorts combined, the weighted average OS rates consistently decreased with increasing stage group; similar findings were noted for individual cohorts (all p< 0.001; Table). On multivariable subgroup analysis including age at diagnosis, stage group was significantly associated with OS, and the risk of death increased with increasing stage (all p< 0.001; Table). On multivariable subgroup analysis for n=4139 patients (ESME cohort), stage group remained significantly associated with OS [IVA: reference; IVB: HR 1.51 (95% CI 1.17-1.93); IVC: HR 2.11 (95% CI 1.64-2.72); IVD: HR 3.60 (95% CI 2.75-4.70)] after adjusting for age and treatment. Conclusions These findings provide external validation of the previously published staging guidelines for dnMBC. This may guide future revisions of the AJCC staging guidelines for patients with dnMBC and provide patients and providers valuable information in planning therapy and goals of care as they approach the end-of-life. Table. Citation Format: Jennifer Plichta, Samantha Thomas, Sabine Siesling, Linda de Munck, Amélie Lusque, Thomas Grinda, Lovedeep Gondara, Stephen Chia, Paula Cabrera-Galeana, Nancy Reynoso-Noverón, Sara López-Tarruella, Isabel Álvarez López, Stephen Edge, Gabriel Hortobagyi. International Validation of a Staging Model for de novo Metastatic 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 PO4-18-01.