Invasive Breast Research Articles

Retrospective Observational Study to Determine the Epidemiology and Treatment Patterns of Patients with Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) poses a serious therapeutic challenge due to the occurrence of frequently aggressive, heterogenic, and metastatic tumours. The absence of therapeutic targets for traditional therapies is a hindrance to establishing a standardised therapy for TNBC. There is limited TNBCs epidemiological and real-world data about TNBC treatment regimens in Poland. We retrospectively analysed clinical data from our hospital registry from 2015 and 2020. A total of 8103 individuals with breast cancer were admitted to the MSCI, while 856 (10.6%) were diagnosed with TNBC. Most of the early-stage or locally advanced TNBC individuals had underlying conditions, presented mostly poorly differentiated (G3) stage II tumours and featured a bi-modal age distribution. On average, one-third of all tested TNBCs carried BRCA mutations and its identification impacted surgery preference. We observed a significant increase in the use of systemic therapy among TNBCs, whereas carboplatin and dose-dense regimens showed the most prominent upsurge in the neoadjuvant setting. Moreover, the use of neoadjuvants was positively correlated with less invasive breast and lymph node surgeries. The presented data align with general trends observed in other countries and will contribute to expanding knowledge in the planning of treatment regimens and their outcomes.

Early prediction of pathological response to neoadjuvant chemotherapy of breast tumors: a comparative study using amide proton transfer-weighted, diffusion weighted and dynamic contrast enhanced MRI.

To examine amide proton transfer-weighted (APTw) combined with diffusion weighed (DWI) and dynamic contrast enhanced (DCE) MRI for early prediction of pathological response to neoadjuvant chemotherapy in invasive breast cancer. In this prospective study, 50 female breast cancer patients (49.58 ± 10.62 years old) administered neoadjuvant chemotherapy (NAC) were enrolled with MRI carried out both before NAC (T0) and at the end of the second cycle of NAC (T1). The patients were divided into 2 groups based on tumor response according to the Miller-Payne Grading (MPG) system. Group 1 included patients with a greater degree of decrease in major histologic responder (MHR, Miller-Payne G4-5), while group 2 included non-MHR cases (Miller-Payne G1-3). Traditional imaging protocols (T1 weighted, T2 weighted, diffusion weighted, and DCE-MRI) and APTw imaging were scanned for each subject before and after treatment. APTw value (APTw0 and APTw1), Dmax (maximum diameter, Dmax0 and Dmax1), V (3D tumor volume, V0 and V1), and ADC (apparent diffusion coefficient, ADC0 and ADC1) before and after treatment, as well as changes between the two times points (ΔAPT, ΔDmax, ΔV, ΔADC) for breast tumors were compared between the two groups. APT0 and APT1 values significantly differed between the two groups (p = 0.034 and 0.01). ΔAPTw values were significantly lower in non-MHR tumors compared with MHR tumors (p = 0.015). ΔDmax values were significantly higher in MHR tumors compared with non-MHR tumors (p = 0.005). ADC0 and ADC1 values were significantly higher in MHR tumors than in non-MHR tumors (p = 0.038 and 0.035). AUC (Dmax+DWI + APTw) = AUC (Dmax+APTw) > AUC (APTw) > AUC (Dmax+DWI) > AUC (Dmax). APTw imaging along with change of tumor size showed a significant potential in early prediction of MHR for NAC treatment in breast cancer, which might allow timely regimen refinement before definitive surgical treatment.

Inhibition of the membrane repair protein annexin-A2 prevents tumor invasion and metastasis

Cancer cells are exposed to major compressive and shearing forces during invasion and metastasis, leading to extensive plasma membrane damage. To survive this mechanical stress, they need to repair membrane injury efficiently. Targeting the membrane repair machinery is thus potentially a new way to prevent invasion and metastasis. We show here that annexin-A2 (ANXA2) is required for membrane repair in invasive breast and pancreatic cancer cells. Mechanistically, we show by fluorescence and electron microscopy that cells fail to reseal shear-stress damaged membrane when ANXA2 is silenced or the protein is inhibited with neutralizing antibody. Silencing of ANXA2 has no effect on proliferation in vitro, and may even accelerate migration in wound healing assays, but reduces tumor cell dissemination in both mice and zebrafish. We expect that inhibiting membrane repair will be particularly effective in aggressive, poor prognosis tumors because they rely on the membrane repair machinery to survive membrane damage during tumor invasion and metastasis. This could be achieved either with anti-ANXA2 antibodies, which have been shown to inhibit metastasis of breast and pancreatic cancer cells, or with small molecule drugs.

Urokinase-type plasminogen activator (uPA) regulates invasion and matrix remodelling in colorectal cancer

BackgroundCancer cells remodel their local physical environment through processes of matrix reorganisation, deposition, stiffening and degradation. Urokinase-type plasminogen activator (uPA), which is encoded by the PLAU gene, is an extracellular proteolytic enzyme known to be involved in cancer progression and tumour microenvironment (TME) remodelling. Perturbing uPA therefore has a strong potential as a mechano-based cancer therapy. This work is a bioengineering investigation to validate whether 1) uPA is involved in matrix degradation and 2) preventing matrix degradation by targeting uPA can reduce cancer cell invasion and metastasis. MethodsTo this aim, we used an engineered 3D in vitro model, termed the tumouroid, that appropriately mimics the tumour’s native biophysical environment (3 kPa). A CRISPR-Cas9 mediated uPA knockout was performed to introduce a loss of function mutation in the gene coding sequence. Subsequently, to validate the translational potential of blocking uPA action, we tested a pharmacological inhibitor, UK-371,801. The changes in matrix stiffness were measured by atomic force microscopy (AFM). Invasion was quantified using images of the tumouroid, obtained after 21 days of culture. ResultsWe showed that uPA is highly expressed in invasive breast and colorectal cancers, and these invasive cancer cells locally degrade their TME. PLAU (uPA) gene knock-out (KO) completely stopped matrix remodelling and significantly reduced cancer invasion. Many invasive cancer gene markers were also downregulated in the PLAU KO tumouroids. Pharmacological inhibition of uPA showed similarly promising results, where matrix degradation was reduced and so was the cancer invasion. ConclusionThis work supports the role of uPA in matrix degradation. It demonstrates that the invasion of cancer cells was significantly reduced when enzymatic breakdown of the TME matrix was prevented. Collectively, this provides strong evidence of the effectiveness of targeting uPA as a mechano-based cancer therapy.

Deciphering the Role of p60AmotL2 in Epithelial Extrusion and Cell Detachment.

Preserving an accurate cell count is crucial for maintaining homeostasis. Apical extrusion, a process in which redundant cells are eliminated by neighboring cells, plays a key role in this regard. Recent studies have revealed that apical extrusion can also be triggered in cells transformed by oncogenes, suggesting it may be a mechanism through which tumor cells escape their microenvironment. In previous work, we demonstrated that p60AmotL2 modulates the E-cadherin function by inhibiting its connection to radial actin filaments. This isoform of AmotL2 is expressed in invasive breast and colon tumors and promotes invasion in vitro and in vivo. Transcriptionally regulated by c-Fos, p60AmotL2 is induced by local stress signals such as severe hypoxia. In this study, we investigated the normal role of p60AmotL2 in epithelial tissues. We found that this isoform is predominantly expressed in the gut, where cells experience rapid turnover. Through time-lapse imaging, we present evidence that cells expressing p60AmotL2 are extruded by their normal neighboring cells. Based on these findings, we hypothesize that tumor cells exploit this pathway to detach from normal epithelia and invade surrounding tissues.

Development of a Multilevel Model to Identify Patients at Risk for Delay in Starting Cancer Treatment

Delays in starting cancer treatment disproportionately affect vulnerable populations and can influence patients' experience and outcomes. Machine learning algorithms incorporating electronic health record (EHR) data and neighborhood-level social determinants of health (SDOH) measures may identify at-risk patients. To develop and validate a machine learning model for estimating the probability of a treatment delay using multilevel data sources. This cohort study evaluated 4 different machine learning approaches for estimating the likelihood of a treatment delay greater than 60 days (group least absolute shrinkage and selection operator [LASSO], bayesian additive regression tree, gradient boosting, and random forest). Criteria for selecting between approaches were discrimination, calibration, and interpretability/simplicity. The multilevel data set included clinical, demographic, and neighborhood-level census data derived from the EHR, cancer registry, and American Community Survey. Patients with invasive breast, lung, colorectal, bladder, or kidney cancer diagnosed from 2013 to 2019 and treated at a comprehensive cancer center were included. Data analysis was performed from January 2022 to June 2023. Variables included demographics, cancer characteristics, comorbidities, laboratory values, imaging orders, and neighborhood variables. The outcome estimated by machine learning models was likelihood of a delay greater than 60 days between cancer diagnosis and treatment initiation. The primary metric used to evaluate model performance was area under the receiver operating characteristic curve (AUC-ROC). A total of 6409 patients were included (mean [SD] age, 62.8 [12.5] years; 4321 [67.4%] female; 2576 [40.2%] with breast cancer, 1738 [27.1%] with lung cancer, and 1059 [16.5%] with kidney cancer). A total of 1621 (25.3%) experienced a delay greater than 60 days. The selected group LASSO model had an AUC-ROC of 0.713 (95% CI, 0.679-0.745). Lower likelihood of delay was seen with diagnosis at the treating institution; first malignant neoplasm; Asian or Pacific Islander or White race; private insurance; and lacking comorbidities. Greater likelihood of delay was seen at the extremes of neighborhood deprivation. Model performance (AUC-ROC) was lower in Black patients, patients with race and ethnicity other than non-Hispanic White, and those living in the most disadvantaged neighborhoods. Though the model selected neighborhood SDOH variables as contributing variables, performance was similar when fit with and without these variables. In this cohort study, a machine learning model incorporating EHR and SDOH data was able to estimate the likelihood of delays in starting cancer therapy. Future work should focus on additional ways to incorporate SDOH data to improve model performance, particularly in vulnerable populations.

High Levels of SII and PIV are the Risk Factors of Axillary Lymph Node Metastases in Breast Cancer: A Retrospective Study.

To investigate the predictive value of systemic immune-inflammation-index (SII) and Pan-Immune-Inflammation-value (PIV) for axillary lymph node (ALN) metastasis in patients with breast cancer. We retrospectively collected data of 247 patients with invasive breast from the Affiliated Hospital of Jiangnan University. The state of axillary lymph node (ALN) metastasis was confirmed by pathological diagnosis. Clinicopathological data (age, ER, PR, HER2, Ki67 expression levels, diapause status, weight, histological grade, vascular invasion, and state of axillary lymph node) were compared between differences of SII and PIV groups and an association between clinical indexes and ALN metastasis was evaluated. The cut-off values of SII and PIV were 320.04 and 92.01, respectively. The significant difference between vascular invasion (P=0.023) and axillary lymph node metastases (P<0.001) in the high and low SII levels. Significant differences were observed in tumor size (p=0.024), PR expression level (P=0.033) and the status of axillary lymph node metastases (p<0.001) between the high PIV group and the low PIV group. Univariate analysis showed that vascular invasion, tumor size, Ki67 expression level, SII, and PIV were significantly correlated with axillary lymph node metastases (p<0.05). Then, multivariate analysis revealed that the vascular invasion (p<0.001), HER2 expression levels (p<0.047), SII (p<0.001) and PIV (p<0.030) were risk factors for axillary lymph node metastases. High levels of SII, PIV, LVI, and HER2 are the risk factors for axillary lymph node metastases in breast cancer patients.

Hematologic toxicities of chemotherapy in breast and ovarian cancer patients carrying BRCA1/BRCA2 germline pathogenic variants. A single center experience and review of the literature

BRCA1 and BRCA2 play a central role in DNA repair and their germline pathogenic variants (gBRCA) confer a high risk for developing breast and ovarian cancer. Standard chemotherapy regimens for these cancers include DNA-damaging agents. We hypothesized that gBRCA carriers might be at higher risk of developing chemotherapy-related hematologic toxicity and therapy-related myeloid neoplasms (t-MN). We conducted a retrospective study of women newly diagnosed with invasive breast or ovarian cancer who were screened for gBRCA1/gBRCA2 at Geneva University Hospitals. All patients were treated with (neo-)adjuvant chemotherapy. We evaluated acute hematologic toxicities by analyzing the occurrence of febrile neutropenia and severe neutropenia (grade 4) at day 7–14 of the first cycle of chemotherapy and G-CSF use during the entire chemotherapy regimen. Characteristics of t-MN were collected. We reviewed medical records from 447 patients: 58 gBRCA1 and 40 gBRCA2 carriers and 349 non-carriers. gBRCA1 carriers were at higher risk of developing severe neutropenia (32% vs. 14.5%, p = 0.007; OR = 3.3, 95% CI [1.6-7], p = 0.001) and of requiring G-CSF for secondary prophylaxis (58.3% vs. 38.2%, p = 0.011; OR = 2.5, 95% CI [1.4–4.8], p = 0.004). gBRCA2 carriers did not show increased acute hematologic toxicities. t-MN were observed in 2 patients (1 gBRCA1 and one non-carrier). Our results suggested an increased acute hematologic toxicity upon exposure to chemotherapy for breast and ovarian cancer among gBRCA1 but not gBRCA2 carriers. A deeper characterization of t-MN is warranted with the recent development of PARP inhibitors in frontline therapy in gBRCA breast and ovarian cancer.

Radiotherapy Deserts: The Impact of Race, Poverty, and the Rural-Urban Continuum on Density of Providers and the Use of Radiation Therapy in the US

Prior efforts to characterize disparities in radiotherapy access and receipt have not comprehensively investigated interplay between race, socioeconomic status, and geography relative to oncologic outcomes. We sought to define these complex relationships at the US county level for prostate (PC) and invasive breast (BC) cancer in order to build a tool that facilitates identification of "radiotherapy deserts"-regions with mismatch between radiotherapy resources and oncologic need. We constructed an ecologic study model using national databases to evaluate 3141 US counties. Radiotherapy resources and utilization densities were operationalized as physician (PPR) and utilization (UPR) per person at risk: number of attending radiation oncologists and Medicare beneficiaries per 100,000 persons at risk, respectively. Oncologic need was defined by "hot zone" counties with ≥2 standard deviations (SD) above mean incidence and death rates. Uni- and multivariable logistic regressions examined links between PPR and UPR densities, epidemiologic variables, and hot zones for oncologic outcomes. Reported statistics are p<.05. Mean (SD) PPR and UPR densities were 2.1 (5.9) and 192.6 (557.6) for PC and 1.9 (5.3) and 174.4 (501.0) for BC, respectively. Counties with high PPR and UPR densities were predominately Metro [odds ratio (OR) 2.9-4.4], generally with higher %Black Non-Hispanic population (OR 1.5-2.3). Incidence and death rate hot zones were largely Non-Metro (OR 0.3-0.6), generally with higher %Black Non-Hispanic constituents (OR 3.2-6.3). Lower PPR density was associated with death rate hot zones for both cancers (OR 0.8-0.9); UPR density was generally not linked to oncologic outcomes on multivariable analysis. Mismatch between oncologic need with PPR and UPR disproportionately affects Non-Metro communities with higher %Black Non-Hispanic population. We developed an interactive web platform (bit.ly/densitymaps) to visualize "radiotherapy deserts" and drive targeted investigation of underlying barriers to care in areas of highest need, with the goal of reducing health inequities in this context.

266 Breast Cancer in Women Under 30: A Multidisciplinary Approach in a Unique Cohort

Abstract Aim Breast cancer in younger women is relatively rare, tends to be more aggressive and is associated with unique challenges. We sought to examine our practice in managing cancer in patients under 30. Method A retrospective case-note review was performed on patients under 30 years of age diagnosed with invasive breast through our pathology database since 2007. Clinical and pathological data was collected on patients’ cancers, management and follow up as well family history, genetic testing, and fertility management. Results Forty-five patients were identified. Median age was 28 years (range 20 to 30). Mean tumour size was 30mm (range 4–65mm). Seventeen (38%) of cancers were ER negative, 16 (36%) were HER2 positive. Twenty-three patients (51%) had positive lymph node metastases. SIx patients (13%) had a strong family history and 9 (20%) were BRCA 1 or 2 gene positive. Seven patients (15%) underwent contra-lateral risk-reducing mastectomy. Twenty-5 patients (55%) had no children at diagnosis. Fertility management was discussed with &amp;gt;90% of patients and 12 patients (26%) underwent ovum or embryo preservation. Over a median follow up of 40.5 months (range 4.5 to 128 months), 8 patients (18%) developed local or distant recurrence and 6 patients (13%) died (due to metastasis). Conclusion In our experience, breast cancer patients under 30 had more aggressive cancers with a relatively high rate of axillary metastases at presentation. Young patients with breast cancer face unique management challenges such as fertility, genetic testing and survivorship issues that are best addressed in a multidisciplinary setting.

Development of a Methodology to Adapt an Equilibrium Buffer/Wash Applied to the Purification of hGPN2 Protein Expressed in Escherichia coli Using an IMAC Immobilized Metal Affinity Chromatography System

Protein purification is a complex and non-standardized process; the fact that proteins have different structural types making it difficult to create a standard methodology to obtain them in a pure, soluble, and homogeneous form. The present study shows the selective development of a buffer suitable for proteins of interest that allows high concentrations of hGPN2 protein to be obtained with low polydispersion and high homogeneity and purity. By taking the different reagents used in the construction of different buffers as a basis and performing purifications using different additives in different concentrations to determine the optimal amounts, the developed process helps to minimize the bonds, maintain solubility, release the proteins present in inclusion bodies, and provide an adequate environment for obtaining high concentrations of pure protein. GPN proteins are of unknown function, have not been purified in high concentrations, and have been found as part of the RNA polymerase assembly; if they are not expressed, the cell dies, and overexpression of certain GPN proteins has been linked to decreased survival in patients with invasive ductal carcinoma breast cancer types ER+ and HER2+. The results of the present study show that the use of the buffer developed for recombinant hGPN2 protein expressed in Escherichia coli could be manipulated in order to isolate the protein in a totally pure form and without the use of protease inhibitor tablets. The resulting homogeneity and low polydispersion was corroborated by studies carried out using dynamic dispersion analysis. Thanks to these properties, it can be used for crystallography or structural genomics studies.

Clinicopathological Features of 166 Cases of Invasive Ductal Breast Carcinoma and Effect of Primary Tumor Location on Prognosis after Modified Radical Mastectomy.

Objective To investigate the clinicopathological features of 166 cases of invasive ductal carcinoma (IDC) of the breast and to analyze the effect of the location of the primary tumor on the prognosis of modified radical mastectomy. Materials and Methods The clinical data of 166 patients with IDC who underwent modified radical mastectomy in our hospital from May 2015 to May 2017 were retrospectively analyzed. The clinicopathological features of IDC patients were recorded. Univariate analysis and the multivariate logistic regression model were used to analyze the relationship between the location of the primary tumor and the prognosis of IDC patients after modified radical surgery. The effect of primary tumor location on the prognosis of modified radical resection was used with Survival curve analysis. Results Among the patients in the central region, 13.33% had tumors >5 cm in diameter, which was higher than those in the other four groups. Among the patients in the upper inner quadrant, 59.38% received hormone therapy after operation, which was higher than those in the other four groups (P < 0.05). There were no significant differences in age, menopause, histological grading, molecular typing, lymph node metastasis, vascular invasion, radiation therapy, and chemotherapy among different groups (P > 0.05). Univariate analysis showed that molecular typing, lymph node metastasis, vascular invasion, and location of the primary tumor were all related to the prognosis of IDC patients after modified radical surgery, and the differences were statistically significant (P < 0.05). Logistic regression analysis showed that molecular typing, lymph node metastasis, vascular invasion, and primary tumor location were all independent influencing factors for prognosis of IDC patients after modified radical surgery (P < 0.05). As of 31 May 2021, there were 11 patients with recurrence and metastasis and 20 patients with death. The median survival time in the outer upper quadrant group was 80 months, which was higher than that in the outer lower quadrant group by 72 months, the median survival time in the central region group by 71 months, the median survival time in the inner upper quadrant group by 67 months, and the median survival time in the inner lower quadrant group by 61 months. The log-rank test showed all P < 0.001. Conclusion Patients with primary tumors located in the central area have larger tumor diameters. Patients located in the central area, upper inner quadrant, and lower inner quadrant are more likely to have lymphatic metastasis, have a more serious condition, and have a shorter prognosis survival time. Unluminal type, multiple lymph node metastases, vascular invasion, and the location of the primary tumor in the inner quadrant are all independent risk factors for prognosis in patients after modified radical surgery for IDC.

Abstract 1160: Multi-scale deep learning feature distributions distinguish invasive ductal and lobular breast cancer slides

Abstract Advances in digital pathology have streamlined use of deep learning methodologies to connect morphology and phenotype in hematoxylin and eosin(H&amp;E) stained images. Whole slide images (WSIs) are gigantic, necessitating a tile-based analysis: a WSI is broken into smaller images called tiles, each tile is analyzed separately, and tile level information are then combined to get a slide level prediction. Context aware models integrating tiles of different sizes have been used to combine morphological features present at different scales, and attention-based pipelines have successfully been used to combine tile level information. Pipelines that combine attention-based and context aware learning are less studied. While both methodologies have shown utility when used individually, models that combine both tend to suffer from a large number of parameters to estimate, which frustrates reliable training. Here we show percentiles of deep learning features extracted at different scales serve as a crude multiscale attention mechanism. The proposed model separates TCGA-BRCA invasive ductal and lobular carcinoma FFPE WSIs (AUC=0.85±0.02), which improves upon the performance of the fully tile-based model of [1] (AUC=0.80 ±0.04). The proposed approach enjoys higher average AUC and lower standard deviation across multiple random train/test splits of data. Our results suggest percentile-based feature construction is an interesting alternative when reliable training of fully deep learning models is challenging.

Abstract 2690: Loss of E-cadherin induces IGF1R activation revealing a targetable pathway in invasive lobular breast carcinoma

Abstract Invasive Ductal Carcinoma (IDC) and Invasive Lobular Carcinoma (ILC) are two major subtypes of breast cancer with significant differences in their histological and molecular underpinnings. ILC has a unique loss of E-cadherin (CDH1) which we have previously demonstrated as a negative regulator of the Insulin-like Growth Factor 1 receptor (IGF1R) through a comprehensive analysis of cell line models and tumor samples. We propose that loss of E-cadherin in ILC sensitizes cells to growth factor signaling and thus alters their sensitivity to growth factor signaling inhibitors. To investigate this, we used CRISPR to generate CDH1 knockout (KO) IDC cell lines (MCF7, T47D, ZR75.1) to uncover the mechanism by which E-cadherin loss activates the IGF pathway while also assessing its targetability. CDH1 KO cells exhibited anchorage independent growth in suspension culture and altered p120 catenin localization as observed in ILC tumors. Through in vitro studies, we show increased signaling sensitivity to IGF/insulin ligands and enhanced signaling duration in CDH1 KO cells. In addition, we observed a higher migratory potential of CDH1 KO cells compared to wild type (WT) cells, which was further enhanced as a chemotactic response to IGF1 or serum. Further, this phenotype could be reversed with an IGF1R inhibitor, BMS-754807. We additionally identified an increase in Collagen I haptotaxis in the CDH1 KO cells, which was also translated into a novel invasive phenotype towards serum in the T47D CDH1 KO cells. Despite no significant differences in membranous IGF1R levels between WT and CDH1 KO cells, higher ligand-receptor interaction was observed with CDH1 KO cells, demonstrating an increased ligand-receptor complex formation upon stimulation. Our results suggest that loss of CDH1 results in an increase in IGF1 receptor availability for ligand binding which in turn allows for an enhanced downstream signaling activation. Interestingly, a physical repression of E-cadherin on IGF1R could not be demonstrated, suggesting spatial changes on the membrane following E-cadherin loss may control ligand binding. Critically, increased sensitivity to IGF1R, PI3K, AKT and MEK inhibitors was observed in CDH1 KO cells suggesting that these targets should be further explored in ILC and that CDH1 loss may be exploited as a biomarker of response, or for patient stratification to inhibitors targeting these pathways. Citation Format: Ashuvinee Elangovan, Laura Savariau, Megan E. Yates, Jagmohan Hooda, Alison M. Nagle, Steffi Oesterreich, Jennifer M. Atkinson, Adrian V. Lee. Loss of E-cadherin induces IGF1R activation revealing a targetable pathway in invasive lobular breast carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2690.

Abstract 4060: Identification of anti-neoplastic compounds on a triple negative breast cancer cell line from T. violacea

Abstract Background: The incidence of Invasive triple negative breast cancer (TNBC) is higher than average in South Africa, with 90% of patients being Black South Africans. Current treatments for breast cancer are associated with severe side effects. Additionally, TNBC is resistant to hormone therapy commonly used to treat other forms of breast cancer. Tulbaghia violacea, a plant native to Southern Africa, is a medicinal herb used in traditional medicine. This study aims to establish the impact of water soluble extracts from T. violacea on TNBC cancer cell lines and identify any anti-cancer compounds present in this extract. Methods: Following the determination of the IC50 value for this extract. The anti-proliferative and anti-metastatic activities of the extract on the TNBC cell line were determined using scratch assays and cell invasion and migration assays, performed using real time cell analysis. The pro-apoptotic action of the extract on the TNBC cell line was determined using flow cytometry cell death assays. Once the anti-cancer activity of the extract was confirmed, the composition of the extract was determined in order to establish what known or possible anti-cancer compounds are present within it. This was performed using gas chromatography-mass spectroscopy (GC-MS). The structure of these identified compounds was then confirmed using Nuclear Magnetic Resonance (NMR). Results: The IC50 value for the T. violacea water soluble extract against MDA MB 231 cells was determined to be ~350 µg/mL. The extract was also shown to have anti migration and invasion as well as pro-apoptotic effects on these cells. Fourteen known anti-cancer compounds were identified in this water soluble extract. In addition to this multiple other compounds were shown to also be present in the extract. Discussion: The water soluble T. violacea leaf extract has cytotoxic effects and the ability to inhibit proliferation and metastasis in a TNBC cell line. The identification of fourteen compounds with known anti-cancer activity may explain the effects this extract has on these TNBC cells, however, the contribution made by any of the other compounds to this activity remains to be determined. Conclusion: These findings suggest that one or more of the compounds present in T. violacea may serve as lead compounds for the development of new treatments for TNBC. Citation Format: Zodwa Dlamini, Clement Penny, Rodney Hull, Mohammed Alouna. Identification of anti-neoplastic compounds on a triple negative breast cancer cell line from T. violacea [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4060.

Abstract 3682: Quadruple negative breast tumors in African American women express factors associated with worse prognosis compared to triple negative tumors

Abstract Introduction: Quadruple negative breast cancer (QNBC) is defined by the absence of androgen (AR), estrogen (ER), progesterone (PR) receptors and human epidermal growth factor (HER2) expression. This subset of triple negative breast cancer (TNBC) has been described as unique to African American (AA) women and represents about 67-90% of all TNBC. Yet, there are limited clinical and therapeutic studies focused on this new subset of breast cancer (BCa) that is highly aggressive and portends a worse survival compared to AR positive Bca subtypes. The objectives of our study were to 1) determine the molecular and clinical characteristics of QNBC and compare them with other BCa subtypes and 2) determine the association of QNBC with clinicopathological factors and prognostic markers. Materials and Methods: Tissue arrays were constructed from FFPE tumor blocks of invasive ductal breast carcinomas in 202 AAs, diagnosed at Howard University from 2000 to 2010. Two separate tissue cores of IDC represented each surgical case in the TMA and was linked to an Institutional Review Board-approved database of demographic and clinical data. Using a microtome, 5-µm sections were cut from the TMA blocks and mounted onto Superfrost Plus microscope slides. Sections were stained with a mouse monoclonal antibody against AR (Clone AR 441, DAKO). Immunohistochemistry for AR was considered positive if ≥10% of tumor cells showed nuclear staining. Bivariate analysis was performed via χ2 analysis and survival data was calculated via Kaplan-Meier curves (SPSS v28.0.0). Results: The most prevalent breast cancer subtype was luminal A (ER+ or PR+, HER2−; 43.5%), followed by TNBC (33.7%). Of the 67 TNBCs on the array, 63 had AR data; 33 (52%) of the TNBCs were AR negative. Greater than 70% of all tumors were stage I and II; and Grade 3 comprised 67.4% of the tumors. QNBC tumors were more likely to be higher grade (p&amp;lt;0.001) and larger in size (p=0.05) compared to other subtypes. There was also a trend for increased metastases in QNBCs (Luminal and HER2 tumors=11.2%; TNBC=13.3%; AR negative= 18.8%; and QNBC=20.8% (p=0.15)). Compared to TNBCs, QNBCs were larger (p=0.048), more likely to express markers associated invasion (Fascin, p=0.004) and reduced survival (Vimentin, p=0.014). There was a trend for increased expression of markers associated with metastases: Mammaglobin (expressed in 30.8% of TNBCs compared to 52.1% of QNBCs; p=0.17) and CD105 (expressed in 6.7% of TNBCs compared to 27.1% of QNBCs; p=0.097). There was no association between QNBC and stage, recurrence-free survival, or overall survival. Conclusion: In our study of AAs, a statistically significant association was noted between QNBC tumors and poorer prognosis. The high prevalence of AR negativity of TNBCs in AAs could explain observed worse outcomes and supports the existence of a unique subtype of Bca known as QNBC, worthy of scientific attention. Citation Format: Oluwadamilola Oladeru, Yasmine Kanaan, Tammy Naab, Luisel J. Ricks-Santi. Quadruple negative breast tumors in African American women express factors associated with worse prognosis compared to triple negative tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3682.

Abstract 81: In depth single cell profiling of a case of breast cancer bone metastases with associated organoid models reveal a precision medicine approach to treatment

Abstract Occurring in 65-80% of metastatic breast cancer (BC), bone metastasis (BoM) is the major cause of BC related mortality. Lack of understanding of BoM evolution and heterogeneity at patient specific levels and precise application of targeted therapies are major challenges of managing BoM. In this study, we described in depth histologic and molecular characterization of a case of invasive lobular breast cancer (ILC) bilateral metastasis to bone, and identified and tested potential targets for treating BoM. H&amp;E/IHC staining, whole exome sequencing (WES), and RNAseq were performed on FFPE primary tumor and pelvis and tibia BoMs collected from our study case. Organoids were derived from the two BoMs and single cell RNA sequencing (scRNAseq) undertaken on the organoids and their originating tumors. H&amp;E/IHC demonstrated evolution of the disease from an ER+ primary ILC to ER- BoM with mixed lobular and ductal carcinoma features. WES revealed two druggable mutations including PIK3CA (E545K) and BRCA1 (D1813H/H399P). RNAseq revealed upregulation of TGF-β, Wnt/beta-catenin and PI3K pathways, epithelial to mesenchymal transition (EMT) and angiogenesis in BoM compared to primary tumor, representing promising targets for BoM. scRNAseq revealed 5 major cell types including epithelial, fibroblasts, immune, osteoclasts and endothelial cells, and pronounced intracellular heterogeneity. Six epithelial clusters were identified, featuring high TNF-α signaling, high partial EMT signatures regulated by PRRX1/2, TWIST1/2, and FOXS1, high proliferation, and endocrine resistance signatures. In fibroblasts, 9 clusters were identified representing ECM remodeling, angiogenesis, osteoclast-like, MSC, IFN response and myofibroblasts. Immune cells majorly composed of monocytes/macrophages, CD4+, CD8+ and Treg T cells, and NK cell. WES and scRNAseq analysis demonstrated that organoids preserved mutational landscape and cellular heterogeneity of matched BoMs. Consistent with the BRCA1 and PIK3CA mutations, organoids were responsive to a PARP (Talazoparib: IC50 1.3uM) and PI3K (Alpelisib: IC50 4-9uM) inhibitors. In summary, we have identified potential therapeutic targets from understanding evolution and heterogeneity of BC BoM, and evaluated these in patient-specific organoids, thereby providing insights for the design of a precision medicine based clinical treatment strategy. Citation Format: Kai Ding, Fangyuan Chen, Nolan Priedigkeit, Daniel D. Brown, Tanya Heim, Rebecca Watters, Kurt Weiss, Peter C. Lucas, Jennifer M. Atkinson, Steffi Oesterreich, Adrian V. Lee. In depth single cell profiling of a case of breast cancer bone metastases with associated organoid models reveal a precision medicine approach to treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 81.

Abstract 3135: Postmenopausal obese mammary adipocytes promote breast tumorigenesis via the LCN2/IL-6/STAT3 signaling axis

Abstract Obesity is associated with a substantially increased risk (~50%) and a poor prognosis of breast cancer (BC) in postmenopausal (PM) women. The mechanism(s) underlying obesity-related BC are not clearly understood and, to date, most studies focus on the systemic effects of subcutaneous or visceral adipocytes on BC. We hypothesized that the increased local presence of ‘obese’ mammary adipocytes within the breast microenvironment promotes the acquisition of an invasive and vascular breast tumor cell phenotype and thereby markedly accelerates tumor proliferation and progression. We first asked whether local interactions between mammary adipocytes and BC cells might promote tumor growth and if so, what the underlying mechanism(s) might be. BC cells (ER- and ER+), when treated with the secretome of mammary adipocytes from obese women (ObAdCM), upregulated potent angiogenic/inflammatory factors, including VEGF, bFGF, lipocalin-2 (LCN2), IL-6 and significantly suppressed the angiogenesis inhibitor Tsp-1. ObAdCM stimulated endothelial cell (EC) recruitment by BC cells and increased EC proliferation, indispensable steps in tumor neovascularization. PM ObAdCM significantly stimulated BC cell proliferation, migration and invasion. Both IL-6 and LCN2-neutralization resulted in significant reduction of the ObAdCM-stimulated BC cell migration and proliferation, suggesting that these factors play a role in obesity-mediated BC progression. ObAdCM induced pSTAT3/pAkt signaling in BC cells and IL-6 and LCN2 could independently stimulate pSTAT3 in BC cells. STAT3 inhibition abrogated the ObACDM-mediated effects on BC cell proliferation and migration, suggesting that the pro-tumorigenic effects of PM obese mammary adipocytes are mediated by LCN2/IL-6/STAT3 signaling. Comparative RNAseq analyses identified &amp;gt;30 differentially expressed genes (DEGs) in PM compared to pre-menopausal (Pre-M) obese mammary adipocytes. Furthermore, GO and KEGG enrichment analysis of DEGs suggested an upregulation of PI3K/Akt signaling, ECM-receptor interactions, focal adhesions and lipid/fatty acid metabolism in PM mammary adipocytes. Taken together, our results indicate that obese mammary adipocytes in the breast tumor microenvironment can promote migration, proliferation, invasion and the vascular phenotype of BC cells and suggest that targeting the LCN2/IL-6/STAT3 signaling axis may be a useful strategy in the treatment of obesity-driven breast tumorigenesis. [Supported by NIH RO1CA185530, the Breast Cancer Research Foundation, the Karp Family Foundation and the Nile Albright Research Foundation] Citation Format: Roopali Roy, Emily Man, Katherine Gonzalez, Lauren Merritt, Cassandra Daisy, Rama Aldakhallah, Liang Sun, Biju Isaac, Shira Rockowitz, Margaret Lotz-Bousvaros, Susan Pories, Marsha A. Moses. Postmenopausal obese mammary adipocytes promote breast tumorigenesis via the LCN2/IL-6/STAT3 signaling axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3135.

Abstract 2461: Integrated lifetime and spectral phasor imaging of biomarker expression and metabolism in hormone receptor positive breast cancer models

Abstract In HR+ (hormone receptor positive) breast cancer, endocrine therapies that directly antagonize estrogen receptor or reduce synthesis of its ligand can be highly effective, but endocrine-resistance occurs frequently. Metabolic dysregulation is a mechanistic driver of endocrine-resistant HR+ breast cancer, but most existing methods cannot be multiplexed with assays for biomarker expression. Spectral phasor imaging of multiplex immunofluorescence (mIF) staining for expression of hormone receptors and other biomarkers combined with phasor lifetime analysis of NADH to quantify metabolic state will provide integrated spatial maps of metabolism and biomarker expression in endocrine-resistant HR breast cancer. We used the HCI-013 (013) and HCI-013EI (013EI) patient-derived xenograft (PDX) system, derived from a HR+, invasive lobular metastatic breast cancer which harbors the Y537S ESR1 mutation. Tumors were stained with a multiplex immunofluorescence (mIF) panel for pan-cytokeratin, ER, PR, HER2, and Ki67, detected by spectrally separate OPAL dyes. Hyperspectral phasor fluorescence spectra were obtained using successive single photon excitation at 488 nm, 561 nm and 640 nm and using multiphoton excitation at 1024 nm. Single photon excitation excites specific dyes, and multiphoton excites the entire panel of dyes simultaneously. The emission was collected using a Zeiss 880 laser scanning microscope equipped with a 32 channel spectral detector. Phasor coordinates of each component were resolved into fractional intensities of the five genes. In the same tissues and sections, phasor fluorescence lifetime imaging (FLIM) of NADH was used to quantify metabolic heterogeneity. NADH is present in two forms: free and protein-bound NADH. They have different fluorescence lifetimes and the ratio of these two species are correlated with NAD+/NADH ratio. Free NADH is associated with increased glycolysis. We observed greater metabolic heterogeneity in 013EI PDX samples, with similar levels of free NADH (glycolysis) at the tumor border, but significantly increased bound NADH (oxidative phosphorylation) in the tumor core. Spectral phasor data show differential expression of PR and PanCK (for 640 nm excitation), and HER2 and ERalpha (for 561 nm excitation) in the edge and center of cancer in 013 and 013EI variants. Our results show how a combination of spectral phasor and phasor-FLIM can be used to define metabolic changes in endocrine-resistant, HR+ PDX models and correlate them with biomarker expression. Ongoing work is quantitatively mapping these measurements at single-pixel resolution, and applying this imaging workflow to RNAscope in situ hybridization assays for gene expression. Combining multiphoton spectral (RNAscope) and FLIM (NADH) will allow pixelwise correlation of the gene expression and metabolic changes. Citation Format: Belen Torrado, Alexander Vallmitjana, Ayodeji Olukoya, Shaymaa Bahnassy, Hillary Stires, Aaron Rozeboom, Rebecca B. Riggins, Suman Ranjit. Integrated lifetime and spectral phasor imaging of biomarker expression and metabolism in hormone receptor positive breast cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2461.

Correlation of Her-2 Expression with Clinicopathological Characteristics in Invasive Ductal Breast Cancer Patients

Breast cancer is the most common cancer in the world and a leading cause of death in women. This cancer is the major cause of mortality and morbidity in Indonesian women. This study aims to investigate the correlation of Her-2 expression with a clinicopathological profile in invasive ductal breast cancer. This study was retrospective in design. Histopathological diagnosis and immunohistochemical ER, PR, and Her2 from 95 patients with invasive ductal breast cancer were included during the period January 2017 to June 2018.