Triple-negative Breast Cancer Subtype Research Articles

Abstract PO1-15-04: Young Black Women With Triple-Negative Breast Cancer Molecular Subtypes: Population-Specific Patterns and Batch Effect Considerations

Abstract Introduction: While triple-negative breast cancer (TNBC) molecular subtypes have been associated with biological differences and clinical outcomes, studies have overwhelmingly been conducted in populations of European or Asian ancestry. Data collected across diverse populations is required to better leverage clinical directions from translational studies in TNBC. Through females recruited through the Black Women: Etiology and Survival of Triple-Negative Breast Cancers (BEST) study, we sought to characterize subtypes and explore associations. Methods: Our study included Black women diagnosed with early-stage (I-III) invasive TNBC at < = age 50 from 2005 to 2016, with recruitment from Florida and Tennessee state cancer registries. Germline DNA, tumor RNA, clinical outcomes, and risk factor data were collected. TNBC status in the BEST study was based on immunohistochemistry from pathology reports, cancer registry data, and self-reported data. Analyses on banked tumor samples, extracted at multiple time points, were conducted through PAM50 (Nanostring) and whole-transcriptome RNA-seq. TNBC subtype was assessed using TNBCtype-4 (Lehmann et al. 2016). Sources of batch effect were evaluated using PCA and corrected via limma. Genetic ancestry based on OncoArray or MEGA genotyping data was inferred using 1000Genomes continental reference samples. Immune cell characterization was performed via CIBERSORT and ESTIMATE. Statistical significance in comparing differences across subtypes was assessed via Chi-square test. Kaplan-Meier estimates and log-rank test were used for survival analysis. Results: RNA-seq from 114 self-reported Black females with TNBC was analyzed. TNBC subtype distribution in the BEST study cohort included 31.6% basal-like 1 (BL1), 28.9% basal-like 2 (BL2), 16.7% luminal androgen receptor (LAR), and 21.9% mesenchymal (M). While results were largely consistent with prior studies in patients of European or East Asian ancestry, a higher percentage of BL2 subtype was observed (28.9% vs. 21.0%, p=0.053). Based on PAM50 subtyping, 100% of tumors with BL1 and M were basal, whereas 68.8% of LAR and 86.7% of BL2 subtypes were basal. Tumors without subtype decreased from 9.8% to 1.1% after batch correcting for location at which RNA was extracted (Moffitt vs. Vanderbilt) and time between RNA banking and sequencing. All TNBC subtypes in this population demonstrated monocyte/activated dendritic cell predominance, with no significant subtype-specific associations with immune cell patterns. A lower relative proportion of M subtype was found in tumors from BRCA1/2 carriers, but this was not statistically significant due to the small number of carriers (8% vs. 18%; p=0.22). With a median follow-up time of 9 years, there was no significant difference in 10-year overall survival by TNBC subtype (p=0.18). However, patients with M subtype appeared to have worse survival relative to other subtypes combined (p=0.036). Conclusion: Our study is among the largest to date that interrogates TNBC subtypes and associated molecular/clinical data in self-reported Black females with invasive breast cancer. Our findings suggest that established TNBC subtyping can be applied in patients of African ancestry. Furthermore, data to correct for lab-based confounders remains critical; in this study, it enabled us to use many samples that could not be subtyped initially. Several population-specific patterns were observed, including no difference in 10-year overall survival across TNBC subtypes (consistent with prior data), but worse initial outcomes in M subtype; a preliminary association between BRCA1/2 carriers and non-M subtypes (in contrast to prior data); and no significant difference in immune cell distributions (in contrast to prior data mainly from patients of European ancestry). Studies of associations between TNBC subtypes, additional clinical data, and treatment data (type and response rates) are ongoing. Citation Format: Padma Rajagopal, Sonya Reid, Run Fan, Lindsay Venton, Anne Weidner, Mya Roberson, Susan Vadaparampil, Xuefeng Wang, Sean Yoder, Marilin Rosa, Jibril Hirbo, Jennifer Whisenant, Jennifer Pietenpol, Fei Ye, Tuya Pal, Brian Lehmann. Young Black Women With Triple-Negative Breast Cancer Molecular Subtypes: Population-Specific Patterns and Batch Effect Considerations [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-15-04.

Abstract PO5-14-09: Does transcriptome based DEPTH2 score reflect intratumor heterogeneity in breast cancer patients?

Abstract Background: Intratumor heterogeneity (ITH) refers to the presence of distinct cancer cell populations with different clones within a single tumor mass. Tumors with high ITH are more prone to progression, exhibit resistance to therapies, and are associated with an unfavorable prognosis. Therefore, evaluation of ITH is anticipated to serve as a breakthrough biomarker for prognosis and treatment strategies. Several computational algorithms have been proposed to quantify ITH. However, with the exception of DEPTH2, all of these algorithms require both DNA and RNA-sequence data from the same tumor, which can be challenging. In this study, we investigated whether DEPTH2 which solely analyzes RNA-sequence data, accurately reflects the biological characteristics of ITH. Methods: We analyzed clinicopathological and gene expression data from a total of 6573 breast cancer patients from several large independent cohorts: METABRIC (n = 1903), GSE96058 (n = 3273), GSE21974 (n = 57), GSE28844 (n = 61), GSE87455 (n = 153), GSE180280 (n = 57), and The Cancer Genome Atlas (TCGA, n = 1069). Results: DEPTH2 score was high in Triple Negative Breast Cancer (TNBC) subtype (p < 0.001), but no consistent correlation was observed with lymph node or distal metastasis. Higher DEPTH2 score was associated with cell proliferation, as evidenced by higher Nottingham histological grade and Ki67 gene expression (both p < 0.001). In agreement, high DEPTH2 tumors enriched all cell proliferation-related Hallmark gene sets such as E2F targets, G2M checkpoint, MYC targets v1 and v2, and Mitotic Spindle (FDR < 0.25). Additionally, high DEPTH2 tumors exhibited reduced immune cell infiltrations, such as CD8, CD4, Th1, Th2, B cells, and macrophages, and increased infiltrations of stromal cells, such as adipocytes, fibroblasts, blood and lymphatic endothelial cells (all p < 0.05). In TCGA cohort, DEPTH2 score exhibited a similar trend to intratumoral heterogeneity, homologous recombination deficiency, fraction altered, silent and nonsilent mutations, as well as SNV and Indel neoantigens. However, the Pearson’s correlation coefficient was very weak (p < 0.05). DEPTH2 score decreased in the GSE21974 and GSE87455 cohorts after neoadjuvant chemotherapy (NAC), whereas no change was observed in the GSE28844 and GSE180280 cohorts. DEPTH2 high tumors showed a lower rate of pathologic complete response (pCR) after NAC in estrogen receptor (ER)+HER2- subtype across all cohorts, however, opposite pattern was observed in the HER2+ and TNBC subtypes in all cohorts. Lastly, high DEPTH2 score was associated with lower disease free and overall survival in METABRIC and GSE96058 (p < 0.05), but not in the TCGA cohort. Conclusions: Our findings suggest that DEPTH2 score may not completely capture the biological characteristics associated with intratumor heterogeneity. Citation Format: Kohei Chida, Rongrong Wu, Arya Mariam Roy, Takashi Ishikawa, Kazuaki Takabe. Does transcriptome based DEPTH2 score reflect intratumor heterogeneity in breast cancer patients? [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-14-09.

Abstract PO2-06-08: Suppression of GATA2/3-FOXA1-HER3 Axis by Histone Deacetylase (HDAC) Inhibitors shows Antitumor Activity in Basal-like Breast Cancer

Abstract Introduction: Basal-like breast cancer (BLBC) represents an aggressive subtype of triple-negative breast cancer (TNBC) that exhibits a high risk of recurrence and resistance to treatment. Histone deacetylase (HDAC) inhibitors (HDACis), including panobinostat and romidepsin, have obtained approval for the treatment of hematopoietic malignancies and demonstrated effectiveness in TNBC cells. We recently found that panobinostat and romidepsin potently induced TNBC cell growth inhibition and apoptosis via downregulation of HER3 through suppression of forkhead box protein A1 (FOXA1), a pioneering transcription factor. Nonetheless, the underlying mechanism through which HDACis suppress FOXA1, thereby inhibiting HER3 expression and its downstream signaling in FOXA1/HER3 co-expressing TNBC remains elusive. Methods: Colony formation, MTS, and LIVE/DEAD cell staining assays were used to detect cell viability. Apoptosis was detected by flow cytometry assays. QRT-PCR, western blots, and immunohistochemistry were performed to determine the expression and activation of genes and/or proteins. Co-immunoprecipitation was performed to assess the interaction between proteins. Lentivirus vectors containing cDNA or shRNAs were used to overexpress or knockdown gene expression. Chromatin immunoprecipitation-quantitative PCR and dual luciferase reporter assays were performed to elucidate the regulatory role of gene transcription. Results: Elevated expression of FOXA1 was observed in BLBC specimens and cell lines tested. FOXA1 was co-expressed with HER3 and transcriptionally activated the HER3 expression in BLBC cells. HER3 formed heterodimers with epidermal growth factor receptor (EGFR), activating downstream signaling pathways in BLBC cells. Treatment with panobinostat and romidepsin resulted in growth inhibition and apoptosis in BLBC cells by downregulating FOXA1 expression and inhibiting HER3 signaling. Notably, the combination of HDACis with an EGFR inhibitor (gefitinib) or an anti-HER3 antibody synergistically enhanced the anti-survival effects on BLBC cells. Additionally, gene expression profiling datasets revealed a significant positive correlation between FOXA1 expression and the transcription factors GATA-Binding Protein 2/3 (GATA2/3). Patients with high GATA2/3-FOXA1 expression exhibited worse Relapse-Free Survival (RFS) compared to those with low expressions in BLBC via Kaplan-Meier analysis. Further investigations showed that GATA2/3 directly activated FOXA1 transcription by binding to its promoter. Moreover, ectopic expression of GATA2/3 not only restored FOXA1 expression downregulated by HDACis but also attenuated the anti-survival effects of HDACis on BLBC cells. Conclusion: HDACis exhibit potent inhibitory effects on BLBC cells via downregulation of GATA2/3-mediated repression of FOXA1 gene transcription, which in turn suppresses HER3 expression and signaling. Our findings indicate that epigenetic targeting of the GATA2/3-FOXA1-HER3 axis may be an effective therapeutic strategy for the eradication of BLBC tumors. Keywords: FOXA1, GATA2/3, HER3, HDAC inhibitors, Basal-like breast cancer Citation Format: Bolin Liu, Congcong Tan, Hui Lyu. Suppression of GATA2/3-FOXA1-HER3 Axis by Histone Deacetylase (HDAC) Inhibitors shows Antitumor Activity in Basal-like 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 PO2-06-08.

Abstract PO1-05-14: Outcomes of first-line chemotherapy for visceral crisis in metastatic breast cancer

Abstract Background: Visceral crisis (VC) in metastatic breast cancer (BC) is defined as severe organ dysfunction, as assessed by signs and symptoms, laboratory tests, and rapid disease progression. The management of this condition is currently based on limited retrospective evidence and expert opinions, since VC has been a common exclusion criterion in clinical trials. There is a paucity of real-world data regarding the management of BC VC. We aim to evaluate the survival outcomes and prognostic factors of first-line palliative chemotherapy for VC in a tertiary cancer center in Brazil. Methods: Data were retrospectively collected from patients with metastatic BC diagnosed with VC between 2017 and 2022 in a single cancer center. Our analysis focused on patients receiving first-line chemotherapy for VC. Patients with hormone receptor-positive BC could have been previously treated with endocrine therapy for metastatic disease. CDK4/6 inhibitors were not available in the cancer center during the study period. Prognostic factors associated with survival were evaluated through univariate and multivariable analyses, using the Cox regression model. Results: A total of 106 patients with VC were evaluated. Among them, 58.5% (62 patients) had not received any previous line of chemotherapy in the metastatic setting before the diagnosis of VC (1st line cohort). Median overall survival (mOS) in the total population was 1.7 months, significantly distinct from the 1st line cohort, which had a mOS of 4.9 months (p < 0.001). Focusing on the 1st line cohort, the most common type of VC was pulmonary (40.3%), followed by hepatic (24.2%), and medullary infiltration (21%). 66.1% of patients had HR+HER2- (mOS 9.3 months), 19.3% HER2+ (mOS 4.5 months), and 11.3% triple-negative BC (TNBC) (mOS 1.5 months). Most patients were treated for VC based on systemic therapy combinations (59.7%), while 32.2% received monochemotherapy, and 8.1% were managed with best supportive care (BSC) alone. In the population receiving multidrug therapy, the mOS was 9.3 months, compared to 4.9 months with monochemotherapy and 0.7 months in patients undergoing BSC. Nevertheless, the type of treatment received was not associated with OS in the multivariable analysis. In the multivariable analysis, the prognostic factors associated with worse mOS were TNBC subtype, hepatic VC), and ECOG-PS > 2. (Table). Among patients with hepatic VC, 73.7% died during the same hospitalization and 60.5% received chemotherapy within 30 days prior to death. Conclusions: Patients with VC due to BC have a poor prognosis, even in the context of first-line chemotherapy. Factors such as ECOG-PS, BC subtype, and VC type should be taken into account when discussing the expected outcomes of this life-threatening condition. Taking these factors into account can help physicians to differentiate patients who are more likely to benefit from systemic oncologic treatment from those for whom such therapy would be futile or potentially harmful. Median overall survival according to baseline characteristics and multivariable analysis (1st line cohort, n=62) Citation Format: Matheus de Oliveira Andrade, Vitor Hugo Felix, Renata Colombo Bonadio, Laura Testa. Outcomes of first-line chemotherapy for visceral crisis in 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 PO1-05-14.

Abstract PO1-13-12: Mechanistic Analysis and Potential Therapeutic Implications in Breast Cancer with Fibrotic Focus

Abstract A fibrotic focus (FF) is a scar-like region that forms in the center of carcinoma due to excessive tumor stroma formation. In our earlier studies, we found FF as an independent poor prognostic factor for breast cancers. However, the underlying mechanisms of how the presence of FF contributed to a poor prognosis are still unknown. To improve our understanding of FF in breast cancer, we have investigated genes associated with FF and their impacts on breast cancer prognosis. Analysis of data from The Cancer Genome Atlas Program (TCGA) indicated that Bone Morphogenetic Protein 8A (BMP8A) was significantly upregulated in breast cancers with FF. To further validate this finding, immunohistochemistry (IHC) on BMP8A was performed on a local cohort of breast cancers (N= 900). Of these samples, 147 (16.3%) were found to be BMP8A positive, and there was a significant association between the presence of FF and high expression of BMP8A (p-value=0.026). We also found that high expression of BMP8A was positively correlated with apocrine phenotype, necrosis, increased Ki67 expression, and triple-negative breast cancer subtype; and most of these are poor prognostic factors for breast cancer. Kaplan-Meier survival estimate analysis revealed that high expression of BMP8A was significantly associated with poor overall survival (p-value=0.035). Gene ontology (GO) analysis showed that "Extracellular matrix organization" was the prominent biological process associated with BMP8A. KEGG pathway analysis suggested that BMP8A was significantly related to "Protein digestion and absorption", "ECM-receptor interaction", and "Focal adhesion" pathways, further validating its correlation with stromal FF. In cellular studies using MCF7 and MDA-MB-231 cell lines, overexpression of BMP8A was found to enhance breast cancer cell proliferation, invasion, and migration. Similar tumor-promoting phenotypes were exhibited when stimulating the cell lines with recombinant human BMP8A. In conclusion, our findings suggested that BMP8A was associated with FF in breast cancer, contributing at least partly to the aggressive features of these cancers. Our findings also provided new insights into the precision treatment and diagnosis of breast cancer. Further exploration of BMP8A as a therapeutic target and prognostic factor may contribute to improved management of breast cancer patients. Citation Format: Canbin Fang, Julia Y. S. Tsang, Gary M.K. Tse. Mechanistic Analysis and Potential Therapeutic Implications in Breast Cancer with Fibrotic Focus [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-13-12.

Abstract PO3-16-07: Strategies to Enact for Equitable and Unbiased Care of Black, Triple Negative Breast Cancer Patients

Abstract Background: Black women are at higher risk for early onset and increased breast cancer mortality, especially from the sub-type Triple-Negative Breast Cancer (TNBC), than their white peers. A patient experience survey has not been implemented or published for Black women diagnosed with TNBC. Tigerlily Foundation (TLF) conducted a TNBC survey of BIack women to understand the patient experience throughout the cancer continuum of care. Methods: The survey instrument received an IRB exception and included 40 questions organized by the following themes: Demographics, Self-Detection/Early Assessment, Screening to Diagnosis, Diagnosis to Treatment, Post Treatment and Palliative Care/Survivorship, Emotional Trauma and Mental Health, and Trust/Bias. Data collection occurred between December 2022 and January 2023.  Two methodological approaches influenced this innovative study design: 1) a Health Literate and Culturally Sensitive approach and 2) a Trusted Outreach approach. The survey was sent to leaders of patient-based organizations who shared the TLF survey with their constituents. Results: All participants (N = 106) racially identified as Black women diagnosed with TNBC. The age of participants ranged from 25 – 71. Participants were not always given information to manage their expectations before or after the screening process, as 38% reported they did not receive such information, while 45% reported they did. The number of times participants required diagnostic imaging ranged from only once (24%), two-three times (41%), four-five times (17%), or greater than six times (11%). Biopsies also varied among participants from one (25%), two-three times (40%), four-five times (15%), or greater than six times (8%). Biomarker testing was not equitably offered to all participants, as 30% reported they were not given the opportunity, while 48% were given the choice. While most participants reported a good understanding of their prognosis and treatment options (59%), other participants shared that the information they received could have been better (26%). Others stated they were given zero information to work with (15%). Many participants experienced a diagnosis change, and 57% were initially diagnosed with a different breast cancer subtype, while 24% were initially diagnosed with TNBC. Conclusions: Prospective implementation science is needed to ensure equitable care standards are sustainably provided to Black women. It is imperative to understand the cancer care continuum from the perspective of the patients, what they think of the care received. Equally important, it is necessary to know what education, resources and care the patients would have liked to receive before or upon diagnosis. Health equity is not achieved based on EHR, county-level beneficiary data, or SEER data alone. Gathering information regarding the patients' experience is a factor in achieving health equity. TLF successfully reached Black TNBC patients to share their authentic experiences and provided a framework for other research institutions and patient advocacy groups to do the same. Citation Format: Virginia Leach, Jeanne Regnante, Maimah Karmo, Shanda Cooper, Krista Peoples, Lizzie Wittig. Strategies to Enact for Equitable and Unbiased Care of Black, Triple Negative Breast Cancer Patients [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-16-07.

Abstract PO5-06-01: Loss of hormone receptor expression in breast cancer is associated with increased brain tropism and accelerated progression of leptomeningeal disease

Abstract The four breast cancer subtypes—luminal A, luminal B, HER2-overexpressing, and triple-negative breast cancer (TNBC) – are classified based on gene expression profiles of hormone receptors (HRs) (estrogen receptor (ER) and progesterone receptor (PR)) and human epidermal growth factor receptor 2 (HER2/ERBB2). Negative hormone receptor status is known to be associated with more aggressive breast cancer. Compared to other breast cancer subtypes, the HR-negative TNBC and HER2-overexpressing subtypes are characterized by high rates of recurrence and a notable increase in brain metastases and in particular, leptomeningeal disease (LMD). LMD – the most aggressive form of central nervous system (CNS) metastasis, in which cancer cells infiltrate leptomeninges of the brain and spine – carries a dismal prognosis due to a rapid progression, poor detection, and no effective treatment. Several studies that investigated the link between HR status and LMD suggest that HR-negative breast cancers have a shorter time to LMD diagnosis. This shortened timeline is consistent with higher migratory capabilities reported for TNBC and HER2 breast cancer cells. However, as it is known that breast cancers exhibit subtype-specific metastatic tropism (i.e., luminal subtypes tend to metastasize to bone and liver, while TNBCs metastasize to the CNS), the longer time between primary breast cancer diagnosis and LMD diagnosis for HR-positive breast cancers may also reflect a longer metastatic route that first includes a non-CNS location. In this study, we performed a retrospective analysis on 141 breast cancer patients with LMD treated at Stanford Hospital between 2008-2020. The Stanford LMD cohort was analyzed using linear regression analysis, Cox regression analysis, and Chi-squared analysis. HR status was recorded (when available) for local recurrences and distant metastases, and was included in our regression models as a time-dependent HR covariate. We tested if: (1) HR-negative breast cancers had shorter time between primary and LMD diagnosis as well as worse survival after LMD diagnosis versus HR-positive breast cancers, and if (2) HR loss (i.e., HR status changing from positive to negative) increased likelihood of the metastasis to CNS. In agreement with published studies, the Stanford LMD cohort showed that HR-negative cancers had shorter time to development of LMD (p = 0.0004 for ER, p = 0.0001 for PR) and increased mortality following LMD (p = 0.010 for ER, p = 0.009 for PR). We found that HR-negative status at primary breast cancer diagnosis increased risk of first metastasis to CNS versus non-CNS locations (p = 0.021 for ER, p = 0.043 for PR). HR status switch (also known as hormone discordance) between primary diagnosis and metastasis occurred in 15% of patients in Stanford cohort, consistent with other studies. Importantly, accounting for HR status switch strengthened the association between HR-negative status and the risk of CNS metastasis [Hazard Ratio = 2.506 (1.395 - 4.500) for “baseline ER status” versus Hazard Ratio = 3.509 (1.890 - 6.515) for “time-dependent ER status”, Hazard Ratio = 2.420 (1.310 - 4.471) for “baseline PR status” versus Hazard Ratio = 3.407 (1.815 - 6.395) for “time-dependent PR status”]. In conclusion, HR-negative status was associated with shorter time to development of LMD, higher CNS tropism, and shorter survival following LMD diagnosis. Taking into consideration hormone discordance between the primary tumor and metastatic sites strengthened this association. Our results suggest a potential value in testing HR status at all recurrences and advanced CNS monitoring following HR loss. Citation Format: Sophia Pribus, Maxine C. Umeh-Garcia, Bo Gu, Bryanna Godfrey, Summer Han, Sophia Chernikova, Melanie Hayden Gephart. Loss of hormone receptor expression in breast cancer is associated with increased brain tropism and accelerated progression of leptomeningeal disease [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-06-01.

Abstract PO3-03-10: Race and neoadjuvant treatment of triple negative breast cancer (TNBC) in the US community oncology setting

Abstract Background: TNBC is an aggressive subtype of breast cancer with limited targeted therapies. Black women have twice the incidence of TNBC compared to White women and within TNBC, Black women have worse outcomes than White women. Differences in both tumor biology of TNBC and social determinants of health (SDOH) play a role in observed disparities. The focus of this study is to evaluate SDOH factors associated to differences in the use of neoadjuvant chemotherapy (NC) in Black and White women in the US community oncology setting. Methods: This study was a retrospective, observational, cross-sectional design and used iKnowMed SM EHR data from the The US Oncology Network. Patients diagnosed with TNBC between 03/31/2017 and 09/30/2021 with stages II-III disease of tumor size ≥ 2cm (T2 or higher) were included. The index date was date of TNBC diagnosis and records were assessed from 6 months pre- to 6 months post-diagnosis for NC initiation. Univariable and multivariable logistic regression was conducted to evaluate impact of self-reported race on NC. Age, stage, BMI, insurance, area deprivation index (ADI), size of practice represented by physician count and geographical region, were studied in their mediating and moderating effects1 on the relationship between race and NC treatment. Results: Of the 3321 patients with TNBC identified in this study, 1969 self-reported white and 494 self-reported black (80% and 20% respectively). In multivariable regression accounting for all variables, Black race (OR = 1.57, 95% CI [ 1.02, 2.44], p=0.04), younger age, more advanced stage, obesity, non-Medicaid insurance, and West region, were all found to be positively associated with use of NC. Higher physician count was nominally associated, (p = 0.07). ADI was not found to be associated to NC. Mediator and moderator analysis for the relationship between race and NC showed that only stage and ADI variable inclusion in the model are required for the positive association between black race and NC. This is driven by a significant interaction between ADI and stage (p = 0.027) which differed for Black and White women. Mainly, for Black women ADI is positively associated to NC treatment in stage IIA, but negatively associated in all other stages (interaction term, p = 0.033). For White women, ADI is negatively associated to NC treatment in all stages. Only Black women with stage IIA tend to be treated more instead of less with NC in areas of greater deprivation (greater ADI). BMI presents nominal interactions with both race and stage, where obesity was more strongly associated with NC treatment in Black women (p = 0.088) and in stage IIA (p = 0.086). All other variables presented association to NC treatment without mediation or moderation of the race effect and without detectable differences across stages. Conclusions: Black women with stage IIA appear to be a special subgroup of TNBC patients for whom treatment with NC increases with ADI. Increased ADI generally results in less standard of care treatment; this is observed in this study for White women overall and for Black women in stages > IIA. Black women in areas of greater deprivation with stage IIA disease, may be perceived to be presenting with more aggressive disease and thus offered NC preferentially. This result lends support to the hypothesis that young black women present with a distinct, more aggressive subtype of TNBC in the early stages. Mediator and moderator analyses are important tools in elucidating the relationships between factors underlying racial disparities in both care and outcomes. 1. Hayes AF, Rockwood NJ. Regression-based statistical mediation and moderation analysis in clinical research: Observations, recommendations, and implementation. Behav Res Ther. 2017 Nov;98:39-57. Odds ratios for multivariable analysis –insert figure image– Race, stage and ADI –insert figure image– Race and BMI in stage IIA –insert figure image– Citation Format: Paola Raska, Joseph Dye, Nicholas J Robert, Angel Kidd, Michael Danso. Race and neoadjuvant treatment of triple negative breast cancer (TNBC) in the US community oncology setting [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-03-10.

Abstract PS12-07: Phase II clinical trial of palbociclib and binimetinib in advanced triple-negative breast cancer (TNBC) with hyperactivation of ERK and/or CDK4/6

Abstract Background: We previously demonstrated that a kinase-based taxonomy of TNBC was most parsimonious than next-generation sequencing in defining TNBC subtypes associated with prognostic categories in early disease. The most aggressive TNBC variants were driven by a heterogeneous set of genetic aberrations that converged in the increased activity of 6 kinases: KIT, PNKP, PRKCE, P70S6K, ERK and CDK6 (Nat Commun; 9:3501-18). The combined inhibition of these kinases in pairs led to potent tumor regression in preclinical models, being the most powerful combination that one directed against CDK6 and ERK. This prompted us to design a phase II trial testing the combination of palbociclib (against CDK6) and binimetinib (against the ERK upstream kinase MEK, since no ERK inhibitor was available at that moment outside phase I trials) in advanced TNBC. Hyperactivation of CDK6 and/or ERK was selected as entry criterion. Trial design: This was a single-arm, prospective, multicentric, open-label, phase II investigator-initiated trial. CDK6 and phosphor-ERK levels were measured in tumor samples by immunohistochemistry and normalized with a reference sample collection to a Z-score. Patients with scores for either kinase above the median were candidates for the trial. Key inclusion criteria included metastatic >18year-old TNBC, adequate organ function, measurable disease, and progression to 1-2 prior treatment lines (including immunotherapy, and a PARP inhibitor in case of germline BRCA1/2 mutation). Patients started continuous oral binimetinib at 45 mg/BID and palbociclib 100 mg/day from days 1 to 21, in 28-day cycles. Patients experiencing ≤ grade 1 tolerable side effects as the greatest toxicity were escalated to palbociclib to 125 in cycle 2. RECIST 1.1 and NCI CTC AE V 5.0 were used for assessing disease control (q8 weeks) and toxicity. The primary aims were to assess the efficacy and toxicity of this combination, and the secondary one to detect biomarkers of activity. At the time of trial design, in absence of available Sacituzumab for prescription, the reference PFS to beat in advanced lines for physician’s choice in TNBC was 1.7 month (NEJM; 384:1529-41, 2021). With alpha and beta errors of 0.05 and 0.2, the minimum number of patients to demonstrate a 30% improvement in PFS to 2.5 months was 25. Results: From November 2020 to April 2023, 69 patients were screened and 24 entered the trial (5 positive for phosphor-ERK; 2 for CDK6; 17 for both). Toxicity was generally mild and included grade 1-2 diarrhea (33% of the patients), grade 1-2 asthenia (50%), grade 1-3 neutropenia (75%), grade 2 retinal toxicity (8.3%) and grade 3 rash (4.2%); no grade 4/5 toxicities were observed. Median PFS was 1.83 months (range 0.3 to 11.3+). Phospho-ERK and CDK6 levels were not correlated (Pearson’s R= -0.089; P=0.68); CDK6 levels did not show association with PFS time (R = -0.120; P=0.58). Interestingly, however, phosphor-ERK levels in the baseline tumor sample showed correlation with PFS time (R = 0.428; P=0.037). Conclusion: The combination of palbociclib and binimetinib was generally safe, and PFS time showed correlation with baseline phosphorylation levels of ERK. However, the trial did not meet its primary endpoint. Citation Format: Rodrigo Sánchez-Bayona, Alfonso Cortes, Juan Miguel Cejalvo, Luis Manso, Serafin Morales, Jose A Garcia-Saenz, Jorge Silva, Juan A Guerra, Diego Malón-Giménez, Silvana Mouron, Eduardo Caleiras, Miguel Quintela-Fandino. Phase II clinical trial of palbociclib and binimetinib in advanced triple-negative breast cancer (TNBC) with hyperactivation of ERK and/or CDK4/6 [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 PS12-07.

The prognostic role of circulating tumor DNA across breast cancer molecular subtypes: a systematic review and meta-analysis

ObjectiveCirculating tumor DNA (ctDNA) is increasingly being used as a potential prognostic biomarker in cancer patients. We aimed to assess the prognostic value of ctDNA in different subtypes of breast cancer patients throughout the whole treatment cycle. Materials and methodsPubMed, Web of Science, Embase, Cochrane Library, Scopus, and clinical trials.gov databases were searched from January 2016 to May 2022. The following search terms were used: ctDNA OR circulating tumor DNA AND breast cancer OR breast carcinoma. Only studies written in English languages were included. The following pre-specified criteria should be met for inclusion: (i) original articles, conference abstracts, etc.; (ii) patients with breast cancer; (iii) ctDNA measurement; and (iv) clinical outcome data such as recurrence-free survival (RFS) and overall survival (OS). Random-effect model was preferred considering the potential heterogeneity across studies. The main outcomes are ctDNA detection rate and postoperative long-term outcomes (RFS, OS). ResultsA total of 24 studies were screened. At every measurement time, the ctDNA detection rate of the HR+ subgroup was similar to that of the HR- subgroup (P=0.075; P=0.458; P=0.744; and P=0.578), and the ctDNA detection rate of the HER2+ subgroup was similar to that of the HER2- subgroup (P=0.805; P=0.271; P=0.807; and P=0.703). In the HR+ subgroup, RFS and OS of ctDNA positive patients were similar to those of ctDNA negative patients (P=0.589 and P=0.110), while RFS and OS of the ctDNA positive group was significantly shorter than those of the ctDNA negative patients in the HR- subgroup (HR=4.03, P<0.001; HR=3.21, P<0.001). According to HER grouping, the results were the same as above. In the triple negative breast cancer (TNBC) subgroup, the RFS and OS of ctDNA-positive patients was significantly shorter than of the ctDNA negative patients before and after surgery. ConclusionsctDNA was more predictive of recurrence-free survival and overall survival in the HR- subgroup than in the HR+ subgroup, and the same result was showed in the HER2- subgroup vs. HER2+ subgroup. The prognosis of the TNBC subtype is closely related to ctDNA before and after surgery.

Long-term oncologic outcomes of unselected triple-negative breast cancer patients according to BRCA1/2 mutations

Triple-negative breast cancer (TNBC) patients are more likely to have BRCA1/2 mutations, with a prevalence rate of about 10–20%. Although several studies have analyzed the oncologic outcomes between BRCA1/2 carriers and non-carriers, the impact on breast cancer patients is still unclear. A retrospective review was performed to determine the long-term outcomes of TNBC patients, focusing on the impact of BRCA1/2 mutations. A total of 953 TNBC patients who underwent primary breast cancer surgery from June 2008 to January 2016 were included. We examined long-term outcomes, including contralateral breast cancer (CBC) incidence, recurrence patterns, and survival rates over a median follow-up of 80.9 months (range 3–152 months). 122 patients (12.8%) had BRCA1/2 mutations. BRCA1/2 mutation carriers were significantly younger at diagnosis and more likely to have a family history of breast/ovarian cancer. CBC incidence at 60, 120, and 150 months was significantly higher in BRCA1/2 mutation carriers compared to non-carriers (P = 0.0250, 0.0063, and 0.0184, respectively). However, there were no significant differences in disease-free survival, overall survival, breast cancer-specific survival, or distant-metastasis-free survival between the two groups. BRCA1/2 mutation status was a significant risk factor for CBC (HR = 6.242, P < 0.0001). Interestingly, among 29 patients with CBC recurrence, 24 patients (82.8%) had recurring TNBC subtype and among the CBC recurrence patients, 19 patients (65.5%) resumed chemotherapy. In the TNBC subtype, appropriate genetic testing and counseling are pivotal for surgical decisions like risk-reducing mastectomy (RRM). Furthermore, long-term surveillance is warranted, especially in BRCA1/2 carriers who did not receive RRM.

Comprehensive Molecular Analyses of an M2-Like Tumor-Associated Macrophage for Predicting the Prognosis and Immunotherapy in Breast Cancer.

The involvement of M2-like tumor-associated macrophages (TAMs) in the advancement and treatment of cancer has been widely documented. This study aimed to develop a new signature associated with M2-like TAMs to predict the prognosis and treatment response in individuals diagnosed with breast cancer (BC). Weighted gene co-expression network analysis (WGCNA) was used to identity for M2-like TAM-related modular genes. The M2-like TAM-related modular subtype was identified using unsupervised clustering. WGCNA identified 722 M2-like TAM genes, 204 of which were associated with recurrence-free survival (RFS). Patients in cluster 1 exhibited upregulated cancer-related pathways, a higher proportion of triple-negative breast cancer (TNBC) subtypes, lower expression of immune checkpoints, and worse prognosis. Cluster 2 was characterized by upregulated immune-related pathways, a higher proportion of luminal A subtypes, and higher expression of immune checkpoints. A prognostic signature was created and confirmed using an independent dataset. A well-built nomogram can accurately forecast the survival outcomes for every individual. Furthermore, patients classified as low-risk exhibited a more favorable outlook, elevated tumor microenvironment (TME) score, and superior reaction to immunotherapy. In conclusion, we discovered 2 different types of M2-like TAMs and developed a prognostic signature revealing the diversity of M2-like TAMs in BC and their correlation with immune status and prognosis. This feature can predict the prognosis and immunotherapeutic effects of BC and offer novel concepts and approaches for tailoring BC treatment.

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.

Abstract P49: PROTAC A1874: A Potential Targeted Therapy for Breast Cancer

Abstract Background PROteolysis TArgeting Chimera (PROTAC) A1874 is a heterobifunctional structured compound consisting of a BRD4-recognizing ligand and an MDM2-recognizing ligand that functions to degrade BRD4 via the ubiquitin-proteasome system. A1874 also plays a dual role in downregulating downstream BRD4 regulator, MYC, and stabilizing p53 within nanomolar concentrations. This PROTAC thus presents itself as a potential therapy for both estrogen-receptor positive (ER+) and triple-negative breast cancer (TNBC) subtypes, which typically harbors wild-type p53 and are associated with MYC overexpression respectively. Thus, this study aims to 1) investigate the anti-oncogenic effects and mechanism of A1874 and 2) its effect in combination with existing therapies in several breast cancer (BC) cell lines. Methods A panel of BC cell lines harboring different p53 status were tested for their sensitivity to A1874. Several functional assays were conducted to determine the anti-oncogenic effects of A1874. Bliss scores were calculated using SynergyFinder to determine the interaction of drugs in combination with A1874. Results BC cell lines with p53 WT status showed significantly lower IC50 sensitivity to A1874 in the nanomolar range compared to those with p53 mutation, which exhibited sensitivity in the micromolar range regardless of subtypes. Moreover, it is effective in Tam-resistant ER+, p53 WT cells. A1874 exclusively degrades BRD4 in p53 WT cells and causes G1 arrest, inhibition of colony formation, proliferation, and migration. Knockdown of MDM2 or TP53, and lentiviral transduction of p53 mutant plasmid resulted in significant reduction in sensitivity to A1874 in p53 WT cell lines and abrogate its BRD4-degrading effect. Combination of A1874 with the mutant p53 reactivating drug, PRIMA-1, synergistically resensitize mutant p53 cells to the cytotoxic effect of A1874 across subtypes. Conclusions Our results highlight a promising potential of PROTAC A1874 as a therapeutic drug in breast cancer, specifically the ER+ subtype which typically retains its p53 WT status. Citation Format: Charlie Marvalim, Arpita Datta, Serena Bee Kee Seah, Edward Kai-Hua Chow, Soo Chin Lee. PROTAC A1874: A Potential Targeted Therapy for Breast Cancer [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P49.

Enhancing therapeutic efficacy in luminal androgen receptor triple-negative breast cancer: exploring chidamide and enzalutamide as a promising combination strategy

Extensive exploration of the molecular subtypes of triple-negative breast cancer (TNBC) is critical for advancing precision medicine. Notably, the luminal androgen receptor (LAR) subtype has attracted attention for targeted treatment combining androgen receptor antagonists and CDK4/6 inhibitors. Unfortunately, this strategy has proven to be of limited efficacy, highlighting the need for further optimization. Using our center’s comprehensive multiomics dataset (n = 465), we identified novel therapeutic targets and evaluated their efficacy through multiple models, including in vitro LAR cell lines, in vivo cell-derived allograft models and ex vivo patient-derived organoids. Moreover, we conducted flow cytometry and RNA-seq analysis to unveil potential mechanisms underlying the regulation of tumor progression by these therapeutic strategies. LAR breast cancer cells exhibited sensitivity to chidamide and enzalutamide individually, with a drug combination assay revealing their synergistic effect. Crucially, this synergistic effect was verified through in vivo allograft models and patient-derived organoids. Furthermore, transcriptomic analysis demonstrated that the combination therapeutic strategy could inhibit tumor progression by regulating metabolism and autophagy. This study confirmed that the combination of histone deacetylase (HDAC) inhibitors and androgen receptor (AR) antagonists possessed greater therapeutic efficacy than monotherapy in LAR TNBC. This finding significantly bolsters the theoretical basis for the clinical translation of this combination therapy and provides an innovative strategy for the targeted treatment of LAR TNBC.

Abstract LB006: Cell marker-based classification of TNBC subtypes and patient outcomes

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive BC subtype characterized by significant molecular and clinical heterogeneity, influenced by genetic, phenotypic, and environmental factors, as well as interactions of the tumor with its surrounding milieu. In the recent past, gene expression-based subtyping methods have provided a functional understanding for the observed molecular diversity/heterogeneity. In this study, we propose an approach to reclassifying TNBC while considering the cellular heterogeneity of the tumor as well as the tumor milieu. Methods: RNA-Sequencing data for TNBC cohort of 250 diverse women [European (EA, self-reported race, SRR): 114 and African (AA, SRR): 136)], was obtained from our collaborators at LSUHSC. 38/250 were labeled ERhi via IHC but histologically confirmed to be TNBC patients. We utilized 261 breast specific cell type markers to classify our cohort via hierarchical (HC) and consensus (CC) clustering. Differentially expressed genes (DEGs) were identified at p-adj ≤ 0.05. Endotype specific networks were generated using protein-protein interactions, DEGs, and genes regulated by transcription factor. Survival was assessed via log-rank tests. Genetic admixture was performed on 227/250 patients using method described by Pakstis, A. J. et al. Results: We identified three distinct mechanistic subtypes (endotype, CC1-3) containing nine distinct groups (HC1-9), revealing substantial heterogeneity in cell-type and tissue-state within TNBC. Each subtype exhibited unique biomarkers, reflecting diverse immune, extracellular matrix, adipogenesis, and inflammatory signatures. Endotype specific networks revealed patients in CC1 exhibited upregulation of CD8 T cell activation, interferon and interleukin markers, while CC2 showed upregulation of extracellular matrix genes, and CC3, the subtype containing ERhi patients, showed upregulation of fatty acid regulation, hormone sensitivity, and pro-inflammatory genes. Survival analysis demonstrated favorable outcomes for CC1 and less favorable outcomes for CC2. Additionally, SRR-inferred survival differences highlighted poorer outcomes for CC2 groups. We found SRR-inferred prognostic biomarkers such as TTC6, ANKRD30A, and KRT86 associated with distinct prognostic profiles in CC2. Genetic admixture showed a comparable distribution of SRR and genetic ancestry across subtypes. We developed a user-friendly Shiny App for our subtyping method and validated it using TCGA TNBC data. Conclusion: TNBC subtyping using cellular heterogeneity of biopsied samples provides a mechanistic approach to patient classification. Subtype-specific mechanisms suggest tailored therapeutic strategies and provide survival outcomes specific to each subtype. Our method can be applied to other cohorts using the application developed in this work. Citation Format: Zahra Mesrizadeh, Kavitha Mukund, Fokrul Hossain, Jovanny Zabaleta, Denise Danos, Luis Del Valle, Xiao-Cheng Wu, Chindo Hicks, Yuan Chun Ding, Augusto Ochoa, Lucio Miele, Victoria L. Seewaldt, Shankar Subramaniam. Cell marker-based classification of TNBC subtypes and patient outcomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB006.

Abstract LB312: Subtype- and race-specific differences in immune landscape of breast cancer

Abstract Tumor mass comprises of not just the tumor cells but a variety of stromal cells. Immune cells residing within the tumor microenvironment modulate the growth and behavior of the tumor cells through a bi-directional crosstalk and affect the outcomes of cancer therapies. Breast cancer (BC) is a highly heterogeneous disease having four major molecular subtypes. Triple-negative breast cancer (TNBC) is the most aggressive subtype and has limited available therapeutic options. Further, it disproportionately affects African American (AA) women, with nearly two times higher incidence as compared to Caucasian American (CA) women. It is associated with an earlier onset and overall higher mortality of BC. Here, we investigated the differences in the immune landscape between TNBC and non-TNBC subtypes and examined if there existed any race-specific patterns. A total of 81 breast cancer samples with a distribution of 55 non-TNBC (28 CA and 27 AA) and 26 TNBC (12 CA and 14 AA) were selected after histopathologic analysis. Tissues were analysis using the NanoString nCounter® pancancer immune panel to determine tissue-specific immune cell populations. We observed a significantly higher presence of tumor-infiltrating lymphocytes in TNBC compared to non-TNBC, with a higher immune score for CD45+ (total lymphocytes), T-cells, B-cells and tumor-associated macrophages. Specifically, among the T-cell population, Treg cells, Th1 cells, and CD8+ cells were higher in TNBC samples. The pathway prediction indicated 'primary immunodeficiency' and 'cancer immunotherapy by PD-1 blockade' pathways being significantly up-regulated in TNBC samples. Overexpression of CTLA4, IDO1, PDL1 and PDL2 as well as Treg marker genes, such as FOXP3 and LAG3, which are involved in immunosuppressive pathways, was observed in TNBC. In race-wise comparisons of non-TNBC and TNBC sample between CA and AA patients, we found that AA non-TNBC tumors had higher Treg cell scores. On the other hand, CA TNBC samples had higher Th1 and CD8+ T-cell scores compared to AA TNBC. The pathway prediction from differentially expressed genes between CA and AA non-TNBC samples showed complement activation pathway to be down-regulated in AA patients, which could be associated with an increased presence of Treg cells. Further, when comparing CA vs AA TNBC samples, we found significant up-regulation of oncogene BMI1 and down-regulation of genes involved in T-cell activation such as CD47, CD44, FOS, and S100A8 in AA TNBC, supporting their more aggressive nature. In conclusion, our data show that TNBC patients have a higher infiltration of immune cells, which remain suppressed due to the presence of Treg cells and PD1 signaling pathway. The presence of higher Treg cells in AA non-TNBC and lower Th1 and CD8+ T-cells in AA TNBC samples suggests an overall immune suppressed microenvironment in AA patients compared to CA patients. Overall, our data suggest that TNBC could be good candidate for immune therapy. Citation Format: Amod Sharma, Kunwar Somesh Vikramdeo, Sarabjeet Kour Sudan, Mohammad Aslam Khan, Mohammad Tahir, James Elliot Carter, Cindy Nelson, Ajay P Singh, Seema Singh. Subtype- and race-specific differences in immune landscape of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB312.

Elevated level of neuroserpin is an indication for the resistance to gambogic acid-induced apoptosis and oxidative stress in triple-negative breast cancer cells.

The triple-negative breast cancer (TNBC) subtype, characterized by loss of HER2, estrogen, and progesterone receptors, displays aggressive phenotype and poor prognosis compared to other BC subtypes. Since the TNBC cells are devoid of receptors, endocrine therapy is an ineffective option for TNBC patients, necessitating canonical chemotherapy strategies to treat TNBC. It is crucial to use alternative and natural agents to support chemotherapy in TNBC. To clarify the molecular mechanism of the tumorigenic effects of gambogic acid (GA) on TNBC cells with different epithelial character since GA has a wide spectrum of anticancer activity for most cancer types. We determined the cytotoxic dose of GA incubation of TNBC cells (MDA-MB-231 and BT-20 cells) for 24 h. We performed the MTT test and toluidine blue (TB) staining protocol for TNBC cells. We analyzed E-cadherin, N-cadherin, Bax, and neuroserpin mRNAs in both cells by qPCR. We evaluated apoptosis using DAPI staining and assessed the ROS using the 2',7'-dichlorofluorescin diacetate (DCFH-DA) method. We determined the IC50 concentrations of GA in MDA-MB-231 and BT-20 cells to be 315.8 nM and 441.8 nM, respectively. TB staining showed that BT-20 cells survive at excessive cytotoxic doses of GA, while most of the MDA-MB-231 cells were killed. Also, we found that BT-20 cells are more resistant to GA-induced apoptosis and oxidative stress than the MDA-MB-231 cells. qPCR results showed that GA upregulated neuroserpin, an oxidative stress-relieving factor in the BT-20 cells, but not in the MDA-MB-231 cells. The elevated level of neuroserpin could be a predictive marker to determine the development of resistance to chemotherapeutic agents.

Targeting Interleukin-13 Receptor α2 and EphA2 in Aggressive Breast Cancer Subtypes with Special References to Chimeric Antigen Receptor T-Cell Therapy.

Breast cancer (BCA) remains the leading cause of cancer-related mortality among women worldwide. This review delves into the therapeutic challenges of BCA, emphasizing the roles of interleukin-13 receptor α2 (IL-13Rα2) and erythropoietin-producing hepatocellular receptor A2 (EphA2) in tumor progression and resistance. Highlighting their overexpression in BCA, particularly in aggressive subtypes, such as Her-2-enriched and triple-negative breast cancer (TNBC), we discuss the potential of these receptors as targets for chimeric antigen receptor T-cell (CAR-T) therapies. We examine the structural and functional roles of IL-13Rα2 and EphA2, their pathological significance in BCA, and the promising therapeutic avenues their targeting presents. With an in-depth analysis of current immunotherapeutic strategies, including the limitations of existing treatments and the potential of dual antigen-targeting CAR T-cell therapies, this review aims to summarize potential future novel, more effective therapeutic interventions for BCA. Through a thorough examination of preclinical and clinical studies, it underlines the urgent need for targeted therapies in combating the high mortality rates associated with Her-2-enriched and TNBC subtypes and discusses the potential role of IL-13Rα2 and EphA2 as promising candidates for the development of CAR T-cell therapies.

Abstract 812: Association of obesity with the early onset and diagnosis of triple negative breast cancer in black women

Abstract Breast cancer (BC) continues to be the most common cancer and the second leading cause of cancer-related death in American women. Furthermore, it affects Black women more adversely than their White counterparts, who experience an early disease onset, diagnosed more often with aggressive triple-negative breast cancer (TNBC) molecular subtype, and face greater mortality. The reasons for these disparities; however, are largely unknown. Here, using a retrospective dataset, we examined the association of body mass index (BMI) with BC early onset and diagnosis with specific molecular subtype in race-based distributions. Study cohort included 1085 BC patients, who visited University of South Alabama Health clinics between October, 2017 and March, 2022. Patients were divided into three BMI [(normal weight, NW (&amp;lt;24.9), overweight, OW (25-29.9), and obese, Ob (&amp;gt;30.0)], three age (&amp;lt;45 years, 45-65 years, &amp;gt;65 years), and four molecular subtypes [Luminal A (HR+, HER2-), Luminal B (HR+, HER2+), HER2 enriched (HR-, HER2+), and TNBC (HR-, HER2-) categories. The data show a higher prevalence of BC in Ob women (46%) than OW (27%) and NW (27%) women of all age groups and its incidence was much greater in Ob Black women (61%) than Ob White women (39%). Further, Black women had nearly twice higher diagnosis at an early age than White women. Moreover, Ob Black women had over 7- and 1.5-folds higher incidence of early onset BC than NW and OW Black women, respectively, whereas, early onset BC did not exhibit a correlative pattern in White women. Luminal A and TNBC subtypes were the most common in women with early onset BC and Black women exhibited more than twice incidence of TNBC (32%) compared to the White women (14%) of all ages. In contrast, incidence of luminal A subtype was less prevalent in Black (57.4%) than in White (72.6%) women. Interestingly, both luminal A and TNBC subtypes were also more prevalent in Ob women; however, the risk of being diagnosed with TNBC increased dramatically in Ob Black women compared to Ob White women. Together, these findings establish obesity as a major underlying cause of early onset BC and TNBC diagnosis in Black women, which could be targeted to diminish the BC disparity gaps. Citation Format: Sarabjeet Kour Sudan, Amod Sharma, Kunwar Somesh Vikramdeo, Wade Davis, Cindy Nelson, Karan Singh, Ajay Pratap Singh, Seema Singh. Association of obesity with the early onset and diagnosis of triple negative breast cancer in black women [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 812.