Triple-negative Subtype Research Articles

Abstract PO4-24-10: Prognostic role of SOX2 and STAT3 expression on circulating T lymphocytes and CD44+/CD24neg cells in the locally advanced and metastatic breast cancer

Abstract Introduction: Breast cancer (BC) is associated with a continuous increase in incidence, with high mortality rates in several countries. There are four immunophenotypes of BC, luminal A, luminal B, triple negative (TN), and HER2+. Some proteins in tumor cells and the tumor microenvironment are part of tumorigenesis and metastases. The CD44 molecule is considered an important marker of the inflammatory response. CD44 is present in all leukocytes and on the surface of tumor stem cells, involving tumor invasion and metastasis. Some authors have demonstrated that the signal transducer and activator of transcription 3 (STAT3) and sex-determining region Y-related group box 2 (SOX2) are related to the regulation of somatic cell division and tumorigenesis and metastasis in BC. Aim: To evaluate SOX2 and STAT3 expression on circulating T lymphocytes and CD44+/CD24neg cells in locally advanced and metastatic breast cancer. Methods: A cross-sectional study was conducted at the Hospital de Cancer de Pernambuco (HCP) and the Translational Research Laboratory of the Instituto de Medicina Integral Prof. Fernando Figueira (IMIP) from March 2017 to April 2018. Sixty-five women diagnosed with breast cancer participated in this study, aged between 28 and 64. Of these, 51 women had stage III (locally advanced) breast tumors, and 14 with metastatic disease (stage IV). There were 24 healthy women aged 18 to 65 as a control group. The analysis of CD44, SOX2, and STAT3 expression was performed by flow cytometry. Results: elevated levels of CD44+/CD24neg cells and CD44+/CD24neg/SOX2+ cells were observed in the blood of BC patients with luminal B, HER2+, and TN compared to the control group (p< 0.0001). Low CD44+/CD24neg/STAT3+ levels in the patient's blood compared to the control group. Elevated CD44+/CD24neg and CD44+/CD24neg/STAT3+ cells were observed in locally advanced BC compared to metastatic group (p < 0.05). Patients with TNBC had elevated SOX2+T lymphocyte levels compared to luminal B and HER2+ subtypes groups (p< 0.05). HER2+ patients had low levels of T lymphocytes and SOX2+T cells compared to controls and luminal A groups (p < 0.05). High levels of total T cells (p=0.0007) and low SOX2+T lymphocytes (p=0.02) in locally advanced compared to metastatic disease (stage IV). Elevated levels of SOX2+T lymphocytes in patients with negative lymph nodes (N0) compared to N1 and N2 groups (p=0.004 and p < 0.0001, respectively). High levels of CD44+/CD24negSTAT3+ cells and T lymphocytes and low levels of STAT3+T lymphocytes in locally advanced TN subtype (stage III) compared to metastatic (stage IV). Conclusion: SOX2 and STAT3 expression on circulating T lymphocytes and CD44+/CD24neg cells in peripheral blood are prognostic roles in breast cancer. SOX2 and STAT3 expression are potential predictive biomarkers of disease progression in breast cancer regardless of tumor subtype. Investigating these biomarkers in peripheral blood has some advantages because peripheral blood collection is less invasive, easy to access, and allows evaluation at diagnosis, during treatment, and in cases of recurrence. Citation Format: Denise Viana, Victor Andrade, Marcelo Salgado, Carolina Vasconcelos, Leuridan Torres. Prognostic role of SOX2 and STAT3 expression on circulating T lymphocytes and CD44+/CD24neg cells in the locally advanced and 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 PO4-24-10.

Abstract PO2-16-12: Real world evidence of neoadjuvant chemotherapy for breast cancer treatment in a Brazilian multicenter cohort: correlation of pathological complete response and overall survival

Abstract Introduction: Breast cancer is the most common malignancy among women in Brazil and worldwide. Neoadjuvant chemotherapy (NAC) has been increasingly used for the treatment of breast cancer conditions ranging from locally advanced tumors to early-stage triple-negative and HER-2 positive tumors. Nowadays only 5% of breast cancer patients are enrolled in clinical trials. Real-world data is important to evaluate how clinical trial treatments behave in the real world with all the heterogeneous patient characteristics of those treated differently from the homogeneity of clinical trials. Objectives: To evaluate the rates of pathological complete response (pCR) in patients undergoing neoadjuvant chemotherapy (NAC) for the treatment of breast cancer (BC) To examine the differences in pathological complete response (pCR) rates between HER-2 positive breast cancer patients with and without trastuzumab treatment, and between triple-negative breast cancer (TNBC) patients with and without platinum-based treatment, Additionally, we aim to assess the impact of pCR on overall survival (OS) and disease-free survival (DFS) using real-world data (RWD). Methods: This was a retrospective, multicentric cohort study, that included female patients over 18 years of age, with a diagnosis of non-metastatic breast cancer undergoing NAC. As an exploratory real-world data study, no confirmatory hypothesis was established, so no corrections for multiple comparisons were necessary. Overall survival (OS) and disease-free survival (DFS) were estimated by the Kaplan-Meier method calculated at five years. Additionally, we conducted a multivariate analysis to identify significant associations with pathologic complete response (pCR) and overall survival (OS). We employed a forest plot to visually represent these associations Results: From 2011 to 2020, 1,891 patients were included in the study, and 421 (22,3%) achieved pCR (ypT0 ypN0) and considering the presence of residual in situ carcinoma (DCIS) the pCR was seen in 467 patients (23,5) without significant difference (p=0,567) . The pCR rate varied between the subtypes: luminal A 17,0%, luminal B 21,3%, luminal HER2 23,0%, TNBC 22,5% and HER2 28,5% (p = 0,057). The type of neoadjuvant chemotherapy (NAC) regimen was correlated with pathological complete response (pCR). Among HER2-positive patients, those who received Trastuzumab had a significantly higher rate of pCR compared to those who did not receive it (p < 0.0001). Similarly, in patients with TNBC, those who underwent treatment with platinum-based regimens also showed higher rates of pCR (p < 0.0001). There were 694 deaths, and the 5-year OS rate was 62.9%. OS was also grouped according to pCR status, and pCR OS rate was 88,3% and non-pCR 58.1% significant difference observed (p < 0.0001). Five-year DFS of pCR was 92.2% and non-pCR 64.3% (p < 0.0001). Conclusion: this real-world data study evaluated the rates of pathological complete response (pCR) in breast cancer patients undergoing neoadjuvant chemotherapy (NAC). The results revealed varying pCR rates among different breast cancer subtypes, with HER2-positive and triple-negative subtypes showing higher rates. The use of Trastuzumab in HER2-positive patients and platinum-based regimens in triple-negative patients correlated with significantly higher pCR rates. Furthermore, achieving pCR was associated with improved overall survival (OS) and disease-free survival (DFS). These findings emphasize the importance of considering individual tumor characteristics and tailoring treatment strategies for breast cancer patients to optimize outcomes. Clinicopathological characteristics of patients AJCC, American Joint Committee on Cancer; IDC, invasive ductal carcinoma; ILC, invasive lobular carcinoma; TNBC triple negative breast cancer. NAC schemas and correlation with pCR. AC-T, doxorubicin + cyclophosphamide – taxane; AC-TPlatinum, doxorubicin + cyclophosphamide – taxane+carboplatin; AC-TH, doxorubicin + cyclophosphamide – taxane+herceptin. Citation Format: Marcelo Antonini, Andre Mattar, Fernanda Grace Bauk Richter, Gabriel Pannain, Andressa Amorim, Marina Diogenes, Odair Ferraro, Luiz Gebrim, Reginaldo Coelho Lopes, Juliana Real. Real world evidence of neoadjuvant chemotherapy for breast cancer treatment in a Brazilian multicenter cohort: correlation of pathological complete response and overall survival [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 PO2-16-12.

The use of longitudinal CT-based radiomics and clinicopathological features predicts the pathological complete response of metastasized axillary lymph nodes in breast cancer

BackgroundAccurate assessment of axillary status after neoadjuvant therapy for breast cancer patients with axillary lymph node metastasis is important for the selection of appropriate subsequent axillary treatment decisions. Our objectives were to accurately predict whether the breast cancer patients with axillary lymph node metastases could achieve axillary pathological complete response (pCR).MethodsWe collected imaging data to extract longitudinal CT image features before and after neoadjuvant chemotherapy (NAC), analyzed the correlation between radiomics and clinicopathological features, and developed models to predict whether patients with axillary lymph node metastasis can achieve axillary pCR after NAC. The clinical utility of the models was determined via decision curve analysis (DCA). Subgroup analyses were also performed. Then, a nomogram was developed based on the model with the best predictive efficiency and clinical utility and was validated using the calibration plots.ResultsA total of 549 breast cancer patients with metastasized axillary lymph nodes were enrolled in this study. 42 independent radiomics features were selected from LASSO regression to construct a logistic regression model with clinicopathological features (LR radiomics-clinical combined model). The AUC of the LR radiomics-clinical combined model prediction performance was 0.861 in the training set and 0.891 in the testing set. For the HR + /HER2 − , HER2 + , and Triple negative subtype, the LR radiomics-clinical combined model yields the best prediction AUCs of 0.756, 0.812, and 0.928 in training sets, and AUCs of 0.757, 0.777 and 0.838 in testing sets, respectively.ConclusionsThe combination of radiomics features and clinicopathological characteristics can effectively predict axillary pCR status in NAC breast cancer patients.

Neoadjuvant radiotherapy in ER+, HER2+, and triple-negative -specific breast cancer based humanized tumor mice enhances anti-PD-L1 treatment efficacy.

Pre-operative radiation therapy is not currently integrated into the treatment protocols for breast cancer. However, transforming immunological "cold" breast cancers by neoadjuvant irradiation into their "hot" variants is supposed to elicit an endogenous tumor immune defense and, thus, enhance immunotherapy efficiency. We investigated cellular and immunological effects of sub-lethal, neoadjuvant irradiation of ER pos., HER2 pos., and triple-negative breast cancer subtypes in-vitro and in-vivo in humanized tumor mice (HTM). This mouse model is characterized by a human-like immune system and therefore facilitates detailed analysis of the mechanisms and efficiency of neoadjuvant, irradiation-induced "in-situ vaccination", especially in the context of concurrently applied checkpoint therapy. Similar to clinical appearances, we observed a gradually increased immunogenicity from the luminal over the HER2-pos. to the triple negative subtype in HTM indicated by an increasing immune cell infiltration into the tumor tissue. Anti-PD-L1 therapy divided the HER2-pos. and triple negative HTM groups into responder and non-responder, while the luminal HTMs were basically irresponsive. Irradiation alone was effective in the HER2-pos. and luminal subtype-specific HTM and was supportive for overcoming irresponsiveness to single anti-PD-L1 treatment. The treatment success correlated with a significantly increased T cell proportion and PD-1 expression in the spleen. In all subtype-specific HTM combination therapy proved most effective in diminishing tumor growth, enhancing the immune response, and converted non-responder into responder during anti-PD-L1 therapy. In HTM, neoadjuvant irradiation reinforced anti-PD-L1 checkpoint treatment of breast cancer in a subtype -specific manner. According to the "bench to bedside" principle, this study offers a vital foundation for clinical translating the use of neoadjuvant irradiation in the context of checkpoint therapy.

Determining individual suitability for neoadjuvant systemic therapy in breast cancer patients through deep learning.

The survival advantage of neoadjuvant systemic therapy (NST) for breast cancer patients remains controversial, especially when considering the heterogeneous characteristics of individual patients. To discern the variability in responses to breast cancer treatment at the individual level and propose personalized treatment recommendations utilizing deep learning (DL). Six models were developed to offer individualized treatment suggestions. Outcomes for patients whose actual treatments aligned with model recommendations were compared to those whose did not. The influence of certain baseline features of patients on NST selection was visualized and quantified by multivariate logistic regression and Poisson regression analyses. Our study included 94,487 female breast cancer patients. The Balanced Individual Treatment Effect for Survival data (BITES) model outperformed other models in performance, showing a statistically significant protective effect with inverse probability treatment weighting (IPTW)-adjusted baseline features [IPTW-adjusted hazard ratio: 0.51, 95% confidence interval (CI), 0.41-0.64; IPTW-adjusted risk difference: 21.46, 95% CI 18.90-24.01; IPTW-adjusted difference in restricted mean survival time: 21.51, 95% CI 19.37-23.80]. Adherence to BITES recommendations is associated with reduced breast cancer mortality and fewer adverse effects. BITES suggests that patients with TNM stage IIB, IIIB, triple-negative subtype, a higher number of positive axillary lymph nodes, and larger tumors are most likely to benefit from NST. Our results demonstrated the potential of BITES to aid in clinical treatment decisions and offer quantitative treatment insights. In our further research, these models should be validated in clinical settings and additional patient features as well as outcome measures should be studied in depth.

Impact of Radiation Therapy Modalities on Loco-regional Control in Inflammatory Breast Cancer

In inflammatory breast cancer, radiation therapy intensification is considered a standard of care by some teams, although the level of evidence remains low. We sought to analyze the impact of radiation therapy modalities on the risk of loco-regional and distant relapse. This retrospective multicenter study included patients with localized inflammatory breast cancer treated between 2010 and 2017. Standard postmastectomy radiation therapy consisted of daily fractions to a total dose of 50 Gy equivalent without a boost or bolus, while intensified radiation therapy referred to the use of a boost or bolus. The cumulative incidence curves of locoregional and distant recurrence were displayed using the competing risk method. Of the 241 included patients, 165 were treated with standard and 76 with intensified radiation therapy. There was significantly more nodal involvement in the intensified group. With a median follow-up of 40 months postradiation therapy, there was no difference between standard versus intensified radiation therapy regarding the cumulative incidence of locoregional (P = .68) or distant recurrence (P = .29). At 5 years, the risks of locoregional and distant recurrence were 12.1% (95% CI, 7.5; 17.7) and 29.4% (95% CI, 21.8; 37.3) for patients treated with standard radiation therapy and 10.4% (95% CI, 4.4; 19.3) and 21.4% (95% CI, 12.6; 31.9) for those treated with intensified radiation therapy. In multivariate analyses, triple-negative subtype and absence of complete pathologic response were associated with a higher risk of loco-regional recurrence. Radiation therapy intensification had no significant impact on locoregional and distant recurrence. For patients with a non-complete pathologic response (n = 172, 71.7%), no significant differences were observed between the 2 groups for loco-regional (P = .80) and distant (P = .39) recurrence. Severe toxicity rates were similar in both groups. Contrary to other important series, this large retrospective multicentric study did not show a locoregional or distant control benefit of intensified radiation therapy. Pooled prospective studies and meta-analyses of intensified radiation therapy are warranted to endorse this approach.

Investigation of efficacy of two different chemotherapy protocols used in neoadjuvant chemotherapy in clinical stages II-IV canine malignant mammary tumours.

The first aim of this study is to demonstrate the clinical efficacy and reliability of two different neoadjuvant chemotherapy (NAC) protocols consisting of doxorubicin/cyclophosphamide (AC) and paclitaxel in dogs with clinical stages II-IV canine malignant mammary tumours (CMTs). Secondly, to determine the Luminal A, Luminal B, HER2-positive and triple-negative molecular subtypes and their value in predicting clinical response to NAC in biopsy samples, and thirdly, to reveal the changes in Ki-67, human epidermal growth factor receptor type 2 (HER2), oestrogen receptor (ER), and progesterone receptor (PgR) expression levels induced by NAC. Thirty dogs with clinical stages II-IV CMTs (T1-3N0-1M0) according to the modified TNM system were included in the study. Dogs in group-1 (n = 15) AC combination and dogs in group-2 (n = 15) were administered paclitaxel. Partial response (PR) was the most common clinical response in both treatment groups (66.66% and 86.66%, respectively). There was no difference between the groups regarding clinical response parameters (p = .001). The rate of treatment responders was higher than the rate of non-responders in both groups (p < .001). The adverse effects observed in both groups were mostly limited to grades 1 and 2 and all were easy to manage. The most frequently detected molecular subtype was Luminal A (59.25%). Complete response (CR) was achieved in 33.33% of dogs with triple-negative CMT in the AC group and 14.29% of the Luminal A subtype in the paclitaxel group. Alterations in Ki-67, HER2, ER, and PgR expressions after chemotherapy were not statistically significant (p > .05). As a result, we have shown that these neoadjuvant chemotherapy protocols are effective and safe alternative treatment options for CMTs.

Characteristics of Triple Negative Subtype Breast Cancer in Dr. Hasan Sadikin General Hospital for 5 Years (2019-2023): An Observational Study

Background: Triple-negative breast cancer has a percentage of 15% to 20% in breast cancer patients at the time of first diagnosis. Guidelines from St. Gallen guidelines, the American Society of Clinical Oncology, and the American College of Pathology state that triple-negative breast cancer is breast cancer with ER and PR expression &lt;1%. As an aggressive subtype, outcomes are poor when compared with hormone receptor-positive. Methods: This research is an observational study. The research subjects in this study were triple-negative breast cancer patients in Dr. Hasan Sadikin General Hospital, Surgical Oncology division, West Java, Indonesia, from October 2018 - November 2023. There were 702 research subjects who would be studied. Characteristics of triple-negative breast cancer were taken from medical records. Results: The results showed that the average age of the subjects was 47 years. Based on histology, the most common was Invasive Carcinoma of no special type (86.75%), followed by Invasive lobular carcinoma (7.26%), medullary carcinoma (1.7%), micropapillary carcinoma (0.99%), metaplastic carcinoma (2.36%) and the least amount is Pleomorphic invasive lobular carcinoma (0.71%). The side of the breast affected is not significant, and the highest grade is 3. The involvement of lymph node metastases is due to aggressive tumor subtypes and lymphovascular invasion. Lymphovascular invasion is known to be a negative prognostic factor. It is a late stage for metastasis and local recurrence. Conclusion: Triple-negative breast cancer is breast cancer that has histopathological heterogeneity; most of the tumors are grade 3 and have significant metastases in regional lymph nodes. There is no significant relationship between grading and regional lymph node metastases. there is a significant relationship between lymphovascular invasion and regional lymph nodes metastases.

Radiomics Analysis of Contrast-Enhanced Breast MRI for Optimized Modelling of Virtual Prognostic Biomarkers in Breast Cancer.

Breast cancer clinical stage and nodal status are the most clinically significant drivers of patient management, in combination with other pathological biomarkers, such as estrogen receptor (ER), progesterone receptor or human epidermal growth factor receptor 2 (HER2) receptor status and tumor grade. Accurate prediction of such parameters can help avoid unnecessary intervention, including unnecessary surgery. The objective was to investigate the role of magnetic resonance imaging (MRI) radiomics for yielding virtual prognostic biomarkers (ER, HER2 expression, tumor grade, molecular subtype, and T-stage). Patients with primary invasive breast cancer who underwent dynamic contrast-enhanced (DCE) breast MRI between July 2013 and July 2016 in a single center were retrospectively reviewed. Age, N-stage, grade, ER and HER2 status, and Ki-67 (%) were recorded. DCE images were segmented and Haralick texture features were extracted. The Bootstrap Lasso feature selection method was used to select a small subset of optimal texture features. Classification of the performance of the final model was assessed with the area under the receiver operating characteristic curve (AUC). Median age of patients (n = 209) was 49 (21-79) years. Sensitivity, specificity, positive predictive value, negative predictive value and accuracy of the model for differentiating N0 vs N1-N3 was: 71%, 79%, 76%, 74%, 75% [AUC = 0.78 (95% confidence interval (CI) 0.72-0.85)], N0-N1 vs N2-N3 was 81%, 59%, 24%, 95%, 62% [AUC = 0.74 (95% CI 0.63-0.85)], distinguishing HER2(+) from HER2(-) was 79%, 48%, 34%, 87%, 56% [AUC = 0.64 (95% CI 0.54-0.73)], high nuclear grade (grade 2-3) vs low grade (grades 1) was 56%, 88%, 96%, 29%, 61% [AUC = 0.71 (95% CI 0.63-0.80)]; and for ER (+) vs ER(-) status the [AUC=0.67 (95% CI 0.59-0.76)]. Radiomics performance in distinguishing triple-negative vs other molecular subtypes was [0.60 (95% CI 0.49-0.71)], and Luminal A [0.66 (95% CI 0.56-0.76)]. Quantitative radiomics using MRI contrast texture shows promise in identifying aggressive high grade, node positive triple negative breast cancer, and correlated well with higher nuclear grades, higher T-stages, and N-positive stages.

CORONet: A Cross-Sequence Joint Representation and Hypergraph Convolutional Network for Classifying Molecular Subtypes of Breast Cancer Using Incomplete DCE-MRI.

Breast cancer, the predominant malignancy among women, is characterized by significant heterogeneity, leading to the emergence of distinct molecular subtypes. Accurate differentiation of these molecular subtypes holds paramount clinical significance, owing to substantial variations in prognosis, therapeutic strategies, and survival outcomes. In this study, we propose a cross-sequence joint representation and hypergraph convolution network (CORONet) for classifying molecular subtypes of breast cancer using incomplete DCE-MRI. Specifically, we first build a cross-sequence joint representation (COR) module to integrate image imputation and feature representation into a unified framework, encouraging effective feature extraction for subsequent classification. Then, we fuse multiple COR features and applied feature selection to reduce the redundant information between sequences. Finally, we deploy hypergraph structures to model high-order correlation among different subjects and extracted high-level semantic features by hypergraph convolutions for molecular subtyping. Extensive experiments on incomplete DCE-MRIs of 395 patients from the TCIA repository showed a significant improvement of our CORONet over state of the arts, with the area under the curve (AUC) of 0.891 and 0.903 for luminal and triple-negative (TN) subtype prediction, respectively. Similar advantages of CORONet were also confirmed in partial complete DCE-MRIs of 144 patients, achieving an AUC of 0.858 and 0.832 for predicting luminal and TN subtypes of breast cancer, respectively. Nevertheless, both of these values were lower compared to the scenario where DCE-MRIs from all 395 patients were utilized. Our study contributes to the precise molecular subtyping using incomplete multi-sequence DCE-MRI, thereby offering promising prospects for future risk stratification of breast cancer patients.

A clinical analysis of clinicopathological features and prognostic factors of triple-negative breast cancer

Objectives. The aim of this study was to explore the clinicopathological characteristics, prognostic factors, recurrence patterns and survival analysis of triple negative breast cancer patients compared to non-triple negative breast cancer patients. Materials and methods. The cohort included 420 patients who were diagnosed with breast cancer. The patients were evaluated based on the molecular classification and grouped into TNBC and non-TNBC. Data was explored using SPSS Version 29.0.0.0. Patient and tumor characteristics were studied. Univariate and multivariate Cox Regression was used to analyze prognostic factors. Kaplan Meier method with the log-rank test was performed to observe DFS and OS. Outcomes. The triple negative subtype was observed in 57(13.6%) patients. Patients with TNBC had a greater proportion of grade 3 tumors compared to those with non-TNBC (43.9% vs. 5.5%, p&lt;.001). In the univariate analysis pathological type, tumor size, tumor grade, nodal invasion, lymphovascular invasion, perineural invasion and extra-nodal extension were identified as statistically significant prognostic factors. The mean time to relapse for TNBC was lower 60.0 (95% CI, 37.16 to 82.83) compared to the non-TNBC group with a mean time to relapse of 72.0 months (95% CI, 55.81 to 88.18), nevertheless distributions were not statistically significant X2(1)=1.524, p.217. Mean overall survival was 80.53 months (95% CI, 77.75 to 83.32) in non-TNBC compared to TNBC of 80.04 month (95% CI, 66.76 to 93.33) with a non-statistically significantly Log rank test X2(1)=1.252, p.263. Conclusions. Triple negative breast cancer represents a heterogenous molecular and prognostic group of tumors which needs further clinical trials in order to develop targeted therapies.

PFDN4 as a Prognostic Marker Was Associated with Chemotherapy Resistance through CREBP1/AURKA Pathway in Triple-Negative Breast Cancer.

Breast cancer is the most common malignancy and its incidence is increasing. It is currently mainly treated by clinical chemotherapy, but chemoresistance remains poorly understood. Prefolded proteins 4 (PFDN4) are molecular chaperone complexes that bind to newly synthesized polypeptides and allow them to fold correctly to stabilize protein formation. This study aimed to investigate the role of PFDN4 in chemotherapy resistance in breast cancer. Our study found that PFDN4 was highly expressed in breast cancer compared to normal tissues and was statistically significantly associated with stage, nodal status, subclasses (luminal, HER2 positive and triple negative), triple-negative subtype and disease-specific survival by TCGA database analysis. CRISPR knockout of PFDN4 inhibited the growth of 89% of breast cancer cell lines, and the triple-negative cell line exhibited a stronger inhibitory effect than the non-triple-negative cell line. High PFDN4 expression was associated with poor overall survival in chemotherapy and resistance to doxorubicin and paclitaxel through the CREBP1/AURKA pathway in the triple-negative MDAMB231 cell line. This study provides insightful evidence for the value of PFDN4 in poor prognosis and chemotherapy resistance in breast cancer patients.

Prognostic Importance of Axillary Lymph Node Response to Neoadjuvant Systemic Therapy on Axillary Surgery in Breast Cancer-A Single Center Experience.

Neoadjuvant systemic treatment (NST) is the standard treatment for HER2+, triple-negative (TN), and highly proliferative luminal HER2- early breast cancer. Pathologic complete response (pCR) after NST is associated with improved outcomes. We evaluated the predictive factors for axillary-pCR (AXpCR) and its impact on the extent of axillary node surgery. This retrospective study included 92 patients (median age of 50.4 years) with an initially node-positive disease. Patients were treated with molecular subtype-specific NST (4.3% were luminal A-like, 28.3% luminal HER2-, 26.1% luminal HER2+, 18.5% HER2+ non-luminal, and 22.8% TN). Axillary-, breast- and total-pCR were achieved in 52.2%, 48.9%, and 38% of patients, respectively. In a binary logistic regression model for the whole population, the only independent factor significantly associated with AXpCR was breast-pCR (OR 7.4; 95% CI 2.6-20.9; p < 0.001). In patients who achieved breast-pCR, aggressive subtypes (HER2+ and TN; OR 11.24) and clinical tumor stage (OR 0.10) had a significant impact on achieving AXpCR. Axillary lymph node dissection was avoided in 53.3% of patients. In conclusion, in node-positive patients with HER2+ and TN subtypes, who achieved breast-pCR after NST, de-escalation of axillary surgery could be considered in most cases.

Correlation of shear-wave elastography stiffness and apparent diffusion coefficient values with tumor characteristics in breast cancer

We aimed to investigate the correlation between shear-wave elastography (SWE) and apparent diffusion coefficient (ADC) values in breast cancer and to identify the associated characteristics. We included 91 breast cancer patients who underwent SWE and breast MRI prior to surgery between January 2016 and November 2017. We measured the lesion’s mean (Emean) and maximum (Emax) elasticities of SWE and ADC values. We evaluated the correlation between SWE, ADC values and tumor size. The mean SWE and ADC values were compared for categorical variable of the pathological/imaging characteristics. ADC values showed negative correlation with Emean (r = − 0.315, p = 0.002) and Emax (r = − 0.326, p = 0.002). SWE was positively correlated with tumor size (r = 0.343–0.366, p < 0.001). A higher SWE value indicated a tendency towards a higher T stage (p < 0.001). Triple-negative breast cancer showed the highest SWE values (p = 0.02). SWE were significantly higher in breast cancers with posterior enhancement, vascularity, and washout kinetics (p < 0.02). SWE stiffness and ADC values were negatively correlated in breast cancer. SWE values correlated significantly with tumor size, and were higher in triple-negative subtype and aggressive imaging characteristics.

Abstract 1500: Breast cancer-derived extracellular vesicles modulate the endocytic metabolism and morphodynamics of the endothelial cells of the blood-brain barrier

Abstract Breast cancer, particularly the triple-negative subtype, is the primary cause of brain metastasis in women, affecting 10-20% of breast cancer patients. Breast cancer patients who develop brain metastasis have few treatment options and a dismal prognosis with a median survival time of 6-16 months. We demonstrated, for the first time, that EVs derived from brain-seeking breast cancer cells (BrEVs) breach the intact blood-brain barrier (BBB) via transcytosis and cause a significantly increased incidence of breast-to-brain metastasis. Within this context, we are now studying the interactions between BrEVs and the endothelial cells (ECs) of the BBB. BrEVs were isolated by ultracentrifugation, characterized following the latest guidelines published by the International Society of Extracellular Vesicles (MISEV 2018) and tested in vitro using primary human brain ECs and in vivo using our mouse model of breast-to-brain metastasis. ECs were treated with BrEVs and analyzed using antibody-based assays, impedance measurements, genetic engineering and fixed and live cell imaging. The imaging results were analyzed using novel machine learning approaches to detect and describe, for the first time, EV-mediated morphodynamic changes at the brain EC barrier level. In vivo, we administered BrEVs intravenously to our models of breast-to-brain metastasis, followed by the analysis of mouse brains and their cerebral microvessels to examine microscopic structure and protein expression. Our studies show that (1) BrEVs target BBB ECs both in vitro and in vivo, causing significant variations of multiple proteins involved in the intracytoplasmic vesicles' long-recycling loop, (2) BrEVs are responsible for specific EC barrier function and morphodynamic changes and (3) BrEVs mediate, at least in part, their effect(s) through the delivery of specific micro RNAs. Taken together, our studies demonstrate that BrEVs have a significant impact on brain EC endocytic metabolism and morphodynamics, promoting transcytosis and sustaining breast-to-brain metastasis. Our findings increase our understanding of the early events that facilitate brain metastasis and have the potential to identify key regulators of brain metastasis formation. These results also inform and contribute to the development of EV-based therapeutic and diagnostic strategies for brain metastasis. (This work was supported by NIH R21 CA253051-01, the Breast Cancer Research Foundation, NIH T32 5T32HL007917-22 and the Nile Albright Research Foundation.) Citation Format: Sara Busatto, Golnaz Morad, Marsha A Moses. Breast cancer-derived extracellular vesicles modulate the endocytic metabolism and morphodynamics of the endothelial cells of the blood-brain barrier [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 1500.

Abstract 3354: Capturing tumor heterogeneity to personalize therapy in young patients with hormone receptor positive breast cancer

Abstract Background: Breast cancer (BC) in young women (Y) is a distinct entity in terms of prognosis. Although triple negative and HER2 positive subtypes are enriched in Y patients, the hormone receptor (HR) positive subtype (luminal, LBC) remains the most common. A thorough understanding of LBC biology in Y women has not been reached yet, also hampered by lack of appropriate models that faithfully recapitulate these tumors in molecular and preclinical studies. Methods: To investigate molecular determinants of progression and therapeutic resistance, we collected a large panel of fresh specimens (N=226) from Y (&amp;lt; 40 years) and nY (&amp;gt;55 years) LBC patients, characterized by targeted sequencing and used to generate Patient-Derived Organoids (PDO) and Xenografts (PDX). A selected panel of Y and nY LBC patients (N=96), sharing similar clinical-pathological features, was used for whole exome sequencing (WES) and transcriptomic analyses. Results: We generated stable LBC PDO (N=32, 13 from Y patients), with a success rate of 50% and stable LBC PDX (N=15, 11 from Y patients), with a success rate 17%. Our targeted panel of NGS, covering most mutated genes in LBC at high coverage (1000x), revealed a significantly higher number of Y patients displaying mutations in PIK3CA and in PIK3CA+TP53. However, these data were not observable in WES analyses (200x), likely because many of those mutations displayed a VAF &amp;lt; 5% (20% of PIK3CA and 30% of TP53). The transcriptomic analysis showed an enrichment in signatures of aggressiveness in the Y LBC subgroup. The EGF family member AREG (Amphiregulin) was the most enriched gene in tumors from pre- versus post-menopausal patients. Following validation in a larger panel of LBC, both at RNA and protein level, we tested the impact of AREG in LBC cell lines. AREG expression was timely induced by growth factor and hormonal stimulation. Furthermore, stimulation using amphiregulin induced the activation of EGFR and its downstream pathway, creating a positive feedback loop. Relevant to the Y LBC phenotype, a consistent and time dependent induction of AREG transcription was induced following estrogen and progesterone stimulation, indicating that, in LBC, AREG might act as a transcriptional target of ESR1. Finally, high levels of amphiregulin reduced response to tamoxifen and CDK4/6 inhibitors in LBC cells and PDOs. Conclusions and Perspectives: Our study provides valuable molecular and biological insights into LBC in Y women. We identified distinct genetic and transcriptomic profiles, including PIK3CA and TP53 mutations and AREG overexpression, which may be exploited to plan more targeted and efficient treatments. The establishment of a living biobank of PDO and PDX from LBC patients of Y age will enable preclinical trials, facilitating discovery of biomarkers and therapeutic strategies tailored to the unique biology of Y LBC. Citation Format: Ilenia Segatto, Maria Chiara Mattevi, Alessandra Dall'Acqua, Lorena Musco, Federica Ruggiero, Gabriele Di Giustino, Andrea Favero, Gian Luca Rampioni Vinciguerra, Nicole Crestan, Luca Pellizzari, Riccardo Spizzo, Riccardo Bomben, Filippo Vit, Andrea Vecchione, Lorenzo Gerratana, Fabio Puglisi, Samuele Massarut, Tiziana Perin, Gustavo Baldassarre, Barbara Belletti. Capturing tumor heterogeneity to personalize therapy in young patients with hormone receptor positive breast 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 3354.

Abstract 6951: The role of estrogen-dependent activation of p38 MAPK signaling pathway in inflammatory breast cancer

Abstract Inflammatory breast cancer (IBC) is a rare and aggressive type of breast cancer in which the lymphatic vessels become blocked by cancer cells in the breast tissue. The five-year survival rate for this type of cancer is around 40%, one of the worst among breast cancer. Most IBCs are estrogen receptor (ER) and progesterone receptor (PR) negative and HER2 positive, with some presenting an amplification of HER2 while others are ER, PR, and HER2 negative, formally known as the triple-negative (TNBC) subtype. However, it has been reported that treatment with 17β-Estradiol (E2) of ER-negative IBC and non-IBC cells can exert rapid non-genomic signaling mediated by ERα-36, GPR30, and/or estrogen receptor beta, activating MAPK pathways involved in oncogenic phenotypes. Previous data generated in our lab showed that when cells were treated with estradiol it led to increased phosphorylation levels of p38. However, the specific role p38 MAPK signaling takes on in IBC is unknown. Thus, this study centers on examining the role of estradiol and p38 regulation in the triple-negative IBC cell line model, SUM149PT. We hypothesize that upon estradiol treatment p38 signaling pathway is activated resulting in the over-expression of pro-inflammatory cytokines which regulate pro-tumorigenic phenotypes including the growth and survival of IBC cells. Inflammatory cytokines can promote pro-oncogenic features by promoting cell proliferation, angiogenesis, and immune suppression. We treated SUM149 IBC cells with 10nM of estradiol and measured the levels of human cytokines using a Human Cytokine Antibody Array. In addition, treatment using SB202190, a potent p38 inhibitor, caused a significant decrease in cell viability of several breast cancer cell lines in a dose-dependent manner. Ongoing studies include assessing the effects of p38 inhibition proliferation, invasion, and migration. In summary, a better characterization of the effect of E2 and p38 activation in ER-negative cell lines may provide great insight into novel targeted therapies for IBC. Citation Format: Keyla V. Ramirez, Adrian D. Bonilla-González, Jeisac Guzman-Rivera, Esther A. Peterson-Peguero. The role of estrogen-dependent activation of p38 MAPK signaling pathway in inflammatory breast 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 6951.

Abstract 2521: New predictive markers for dasatinib responsiveness in triple-negative breast cancer: A biomarker discovery study

Abstract Triple negative breast cancer is the most heterogeneous and aggressive type of breast tumor type and is characterized by the lack of expression of clinical biomarkers (ER, PR, and HER2) and targeted therapies. In this regard, different clinical trials have failed, unable to stratify these patients to find an effective response to specific therapy. This study aimed to identify biomarkers exclusively expressed in the basal mammary compartment that can help us to subclassify the triple-negative breast cancer subtype. Based on computational analysis from single-cell RNA sequencing data, we have identified a list of basal identity-associated genes (BC-markers). Subsequent histological validation confirmed the expression pattern of these markers in a cohort of 50 samples, which has allowed us to stratify triple-negative breast cancer patients into BC-TNBC and luminal-like subtypes. Importantly, the expression of these markers correlated with poorer prognosis in TNBC patients. Functional analysis revealed that TAGLN played a significant role in cell proliferation and migration, potentially impacting tumor aggressiveness. Moreover, the expression of these BC-markers is associated with a mesenchymal phenotype which corresponds to the previously described Lehmannn’s mesenchymal-like signature. In this study, TAGLN appeared to influence dasatinib sensitivity, suggesting its potential as a predictive biomarker for dasatinib response in TNBC patients. Citation Format: Daniel Ortega-Álvarez, David Tebar, Marta Casado, Elena Castillo, Cristina Guardia, David Olivares-Osuna, Eva Musulén, Elisabetta Mereu, Ginés Luengo, Eva M. Galán-Moya, Veronica Rodilla. New predictive markers for dasatinib responsiveness in triple-negative breast cancer: A biomarker discovery study [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 2521.

Retrospective descriptive analysis of the demographic and clinicopathological presentation of breast cancer patients in Kasr Al-Ainy Hospital over 5 years

Background Breast cancer is the most common type of cancer and the second leading cause of mortalities among women. Early-stage diagnosis of breast cancer increases the chances of survival, and therefore, reduces mortality rates. A national initiative was implemented in Egypt in 2019 when women above the age of 18 years were granted free screening at 3538 healthcare units, and 114 hospitals nationwide. Objective To describe the sociodemographic factors and clinicopathological presentation of breast cancer among the patients presented to Cairo University Hospital over 5 years. Patients and methods We conducted a descriptive historical study at Kasr Al-Ainy Teaching Hospital of Cairo University, we retrospectively assessed all women with primary breast cancer diagnosed over 5 years during the period from the July 1, 2017 to the July 1, 2022. Data was collected from medical records, operative notes, radiology reports, and pathology reports from the database system of the Oncology Department of Cairo University Hospitals (Kasr Al-Ainy). The patient data is put into a spreadsheet that focuses on the demographic and clinicopathological characteristics of the patients (age, size of the tumor, side of breast affected, histopathology findings, imaging characteristics, immunohistochemistry, and TNM staging of breast cancer). Results We enrolled a total of 509 female patients who were screened for early detection of breast cancer during the past 5 years in general surgery, radio diagnosis, and oncology departments, Kasr Al-Ainy Teaching Hospital; they showed a mean age of 53.7±11.7 years and ranged between 26 and 88 years. In the current study molecular subtyping showed that 24.4% were Luminal A, 34% were Luminal B1, 19.4% were Luminal B2, 9.2% were HER2-enriched and 13% were triple-negative breast cancer subtype. We found that neoadjuvant therapy was prescribed for 190 (37.3%) patients, 36 (19%) of those patients achieved pCR, while the remaining patients had residual disease in the postoperative specimen. Conclusion We concluded that in our center, Egyptian females are diagnosed with breast cancer earlier compared with developed countries; however, the current study reports approximately the same percentages of molecular subtypes, rates of pCR, and metastatic disease at the time of presentation compared with developed countries.

Abstract 1409: The role of liver receptor homolog 1&amp;lt;LRH-1&amp;gt;in regulating breast cancer progression by modulating the immune response

Abstract Breast cancer remains the second leading cause of cancer-related death among American women, with the majority of morality being associated with metastatic disease. At this stage, immunotherapy is only approved for a small subset of patients whose tumors are of the triple negative subtype &amp;lt;TNBC&amp;gt; that stain positive for PD-L1. However, the majority of these tumors prove refractory. Thus, it is imperative to develop novel therapeutic targets to improve the immune response in breast cancer. Since cholesterol metabolism is important for myeloid immune cell function, we launched studies to identify proteins involved in cholesterol homeostasis that are amenable to small molecule intervention. Liver Receptor Homolog 1 &amp;lt;LRH-1/NR5A2&amp;gt; is highly expressed in myeloid cells, particularly neutrophils. We have found that elevated LRH-1 expression in breast cancer patients is associated with increased overall and recurrence-free survival rate. Thus, we hypothesized that LRH-1 acts as an immune modulator in myeloid cells, with subsequent effects on breast cancer progression. We initiated a series of experiments to characterize the role of LRH-1 in important aspects of neutrophil function: migration, NETosis, phagocytosis and influence on T cells. Neutrophils and neutrophil NETosis has previously been implicated in breast cancer progression including reemergence from dormancy and metastatic recurrence. Interestingly, we found that LRH-1 inhibits neutrophil migration towards cancer cells, a finding that may have important implications regarding infiltration of immune-suppressive myeloid cells. Furthermore, neutrophils treated with an LRH-1 agonist demonstrated decreased NETosis, while an antagonist resulted in increased NETosis. On the other hand, LRH-1 agonist treatment strongly decreased the neutrophil phagocytotic ability, while treatment with an antagonist did not significantly impact this process. Finally, T cells had increased expansion when cocultured with neutrophils that had been previously treated with an LRH-1 agonist, and reduced proliferation in the presence of neutrophils pretreated with an LRH-1 antagonist or inverse agonist. This was especially apparent in CD4+ helper T cells and later divisions. Ongoing work is aimed at determining the pathological relevance of these findings. Collectively, our data indicate that LRH-1 plays important roles in regulating different neutrophils functions, including migration, NETosis, phagocytosis, and T cell expansion. Overall, these findings suggest that LRH-1 in neutrophils regulates the immune response, and therefore can be a potential therapeutic target for cancer patients. Funding: National Cancer Institute (ERN: R01CA234025) Department of Defense (ERN: BCRP Era of Hope Award) Citation Format: Yu Wang, Bryan Duong, Natalia Krawczynska, Shruti V. Bendre, Claire P. Schane, Erin Weisser, Lara Kockaya, Yifan Fei, Anasuya Das Gupta, Hashni E. Vidana Gamage, Adam T. Nelczyk, Erik R. Nelson. The role of liver receptor homolog 1&amp;lt;LRH-1&amp;gt;in regulating breast cancer progression by modulating the immune response [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 1409.