Outcome Of Triple-negative Breast Cancer Patients Research Articles

Integrated machine learning algorithms identify KIF15 as a potential prognostic biomarker and correlated with stemness in triple-negative breast cancer

Cancer stem cells (CSCs) have the potential to self-renew and induce cancer, which may contribute to a poor prognosis by enabling metastasis, recurrence, and therapy resistance. Hence, this study was performed to identify the association between CSC-related genes and triple-negative breast cancer (TNBC) development. Stemness gene sets were downloaded from StemChecker. Based on the online databases, a consensus clustering algorithm was conducted for unsupervised classification of TNBC samples. The variations between subtypes were assessed with regard to prognosis, tumor immune microenvironment (TIME), and chemotherapeutic sensitivity. The stemness-related gene signature was established and random survival forest analysis was employed to identify the core gene for validation experiments and tumor sphere formation assays. 499 patients with TNBC were classified into three subgroups and the Cluster 1 had a better OS than others. After that, WGCNA study was performed to identify genes important for Cluster 1 subtype. Out of all 8 modules, the subtype of Cluster 1 and the yellow module with 103 genes demonstrated the largest positive association. After that, a four-gene stemness-related signature was established. Based on the yellow module, the 39 potential pivotal genes were subjected to the random forest survival analysis to find out the gene that was relatively important for OS. KIF15 was confirmed as the targeted gene by LASSO and random survival forest analyses. In vitro experiments, the downregulation of KIF15 promoted the stemness of TNBC cells. The expression levels of stem cell markers Nanog, SOX2, and OCT4 were found to be elevated in TNBC cell lines after KIF15 inhibition. A stemness-associated risk model was constructed to forecast the clinical outcomes of TNBC patients. The downregulation of KIF15 expression in a subpopulation of TNBC stem cells may promote stemness and possibly TNBC progression.

CRISPR Screening of Transcribed Super-Enhancers Identifies Drivers of Triple-Negative Breast Cancer Progression.

Triple-negative breast cancer (TNBC) is the most therapeutically recalcitrant form of breast cancer, which is due in part to the paucity of targeted therapies. A systematic analysis of regulatory elements that extend beyond protein coding genes could uncover avenues for therapeutic intervention. To this end, we analyzed the regulatory mechanisms of TNBC-specific transcriptional enhancers together with their non-coding enhancer RNA (eRNA) transcripts. The functions of the top 30 eRNA-producing super-enhancers were systematically probed using high-throughput CRISPR-interference assays coupled to RNA-seq that enabled unbiased detection of target genes genome-wide. Generation of high resolution Hi-C chromatin interaction maps enabled annotation of the direct target genes for each super-enhancer, which highlighted their proclivity for genes that portend worse clinical outcomes in TNBC patients. Illustrating the utility of this dataset, deletion of an identified super-enhancer controlling the nearby PODXL gene or specific degradation of its enhancer RNAs led to profound inhibitory effects on target gene expression, cell proliferation, and migration. Furthermore, loss of this super-enhancer suppressed tumor growth and metastasis in TNBC mouse xenograft models. Single-cell RNA-seq and ATAC-seq analyses demonstrated the enhanced activity of this super-enhancer within the malignant cells of TNBC tumor specimens compared to non-malignant cell types. Collectively, this work examines several fundamental questions about how regulatory information encoded into eRNA-producing super-enhancers drives gene expression networks that underlie the biology of triple-negative breast cancer.

Restoration of TFPI2 by LSD1 inhibition suppresses tumor progression and potentiates antitumor immunity in breast cancer

Histone lysine-specific demethylase 1 (LSD1) is frequently overexpressed in triple negative breast cancer (TNBC), which is associated with worse clinical outcome in TNBC patients. However, the underlying mechanisms by which LSD1 promotes TNBC progression remain to be identified. We recently established a genetically engineered murine model by crossing mammary gland conditional LSD1 knockout mice with Brca1-deficient mice to explore the role of LSD1 in TNBC pathogenesis. Cre-mediated Brca1 loss led to higher incidence of tumor formation in mouse mammary glands, which was hindered by concurrent depletion of LSD1, indicating a critical role of LSD1 in promoting Brca1-deficient tumors. We also demonstrated that the silencing of a tumor suppressor gene, Tissue Factor Pathway Inhibitor 2 (TFPI2), is functionally associated with LSD1-mediated TNBC progression. Mouse Brca1-deficient tumors exhibited elevated LSD1 expression and decreased TFPI2 level compared to normal mammary tissues. Analysis of TCGA database revealed that TFPI2 expression is significantly lower in aggressive ER-negative or basal-like BC. Restoration of TFPI2 through LSD1 inhibition increased H3K4me2 enrichment at the TFPI2 promoter, suppressed tumor progression, and enhanced antitumor efficacy of chemotherapeutic agent. Induction of TFPI2 by LSD1 ablation downregulates activity of matrix metalloproteinases (MMPs) that in turn increases the level of cytotoxic T lymphocyte attracting chemokines in tumor environment, leading to enhanced tumor infiltration of CD8+ T cells. Moreover, induction of TFPI2 potentiates antitumor effect of LSD1 inhibitor and immune checkpoint blockade in poorly immunogenic TNBC. Together, our study identifies previously unrecognized roles of TFPI2 in LSD1-mediated TNBC progression, therapeutic response, and immunogenic effects.

Multi-omic analysis of dysregulated pathways in triple negative breast cancer.

The aggressive characteristics of triple-negative breast cancer (TNBC) and the absence of targeted medicines make TNBC a challenging clinical case. The molecular landscape of TNBC has been well-understood thanks to recent developments in multi-omic analysis, which have also revealed dysregulated pathways and possible treatment targets. This review summarizes the utilization of multi-omic approaches in elucidating TNBC's complex biology and therapeutic avenues. Dysregulated pathways including cell cycle progression, immunological modulation, and DNA damage response have been uncovered in TNBC by multi-omic investigations that integrate genomes, transcriptomics, proteomics, and metabolomics data. Methods like this pave the door for the discovery of new therapeutic targets, such as the EGFR, PARP, and mTOR pathways, which in turn direct the creation of more precise treatments. Recent developments in TNBC treatment strategies, including immunotherapy, PARP inhibitors, and antibody-drug conjugates, show promise in clinical trials. Emerging biomarkers like MUC1, YB-1, and immune-related markers offer insights into personalized treatment approaches and prognosis prediction. Despite the strengths of multi-omic analysis in offering a more comprehensive view and personalized treatment strategies, challenges exist. Large sample sizes and ensuring high-quality data remain crucial for reliable findings. Multi-omic analysis has revolutionized TNBC research, shedding light on dysregulated pathways, potential targets, and emerging biomarkers. Continued research efforts are imperative to translate these insights into improved outcomes for TNBC patients.

Comprehensive analysis of stereotactic Radiosurgery outcomes in triple-negative breast cancer patients with brain metastases: The influence of immunotherapy and prognostic factors

IntroductionBreast cancer stands as the second most common solid tumors with a propensity for brain metastasis. Among metastatic breast cancer cases, the brain metastasis incidence ranges from 10 % to 30 %, with triple-negative breast cancer (TNBC) displaying a heightened risk and poorer prognosis. SRS has emerged as an effective local treatment modality for brain metastases; however, data on its outcomes specifically in pure triple-negative subtype remain scarce. MethodWe retrospectively reviewed the electronic medical records of all brain metastasis (BM) TNBC patients treated with SRS. Patient, tumour characteristics and treatment details data were collected. This retrospective cohort study aimed to evaluate local control (LC), distant brain metastasis free survival (DBMFS), and overall survival (OS) outcomes in TNBC patients undergoing SRS for brain metastases while identifying potential prognostic factors. ResultForty-three patients with TNBC and brain metastases treated with SRS between January 2017 and 2023 were included. The study found rates of LC (99 % at 1 year) and DBMFS (76 % at 1 year) after SRS, with brain metastasis count (p = 0,003) and systemic treatment modality (p = 0,001) being significant predictors of DBMFS. The median OS following SRS was 19.5 months, with neurological deficit (p = 0.003) and systemic treatment modality (p = 0.019) identified as significant predictors of OS. ConclusionSRS demonstrates favourable outcomes in terms of local control and distant brain metastasis-free survival in TNBC. Neurological deficit and systemic treatment significantly influence overall survival, emphasizing the importance of personalized treatment approaches and (magnetic resonance imaging) MRI surveillance based on these factors.

Peposertib, a DNA-PK Inhibitor, Enhances the Anti-Tumor Efficacy of Topoisomerase II Inhibitors in Triple-Negative Breast Cancer Models.

Triple-negative breast cancer (TNBC) remains the most lethal subtype of breast cancer, characterized by poor response rates to current chemotherapies and a lack of additional effective treatment options. While approximately 30% of patients respond well to anthracycline- and taxane-based standard-of-care chemotherapy regimens, the majority of patients experience limited improvements in clinical outcomes, highlighting the critical need for strategies to enhance the effectiveness of anthracycline/taxane-based chemotherapy in TNBC. In this study, we report on the potential of a DNA-PK inhibitor, peposertib, to improve the effectiveness of topoisomerase II (TOPO II) inhibitors, particularly anthracyclines, in TNBC. Our in vitro studies demonstrate the synergistic antiproliferative activity of peposertib in combination with doxorubicin, epirubicin and etoposide in multiple TNBC cell lines. Downstream analysis revealed the induction of ATM-dependent compensatory signaling and p53 pathway activation under combination treatment. These in vitro findings were substantiated by pronounced anti-tumor effects observed in mice bearing subcutaneously implanted tumors. We established a well-tolerated preclinical treatment regimen combining peposertib with pegylated liposomal doxorubicin (PLD) and demonstrated strong anti-tumor efficacy in cell-line-derived and patient-derived TNBC xenograft models in vivo. Taken together, our findings provide evidence that co-treatment with peposertib has the potential to enhance the efficacy of anthracycline/TOPO II-based chemotherapies, and it provides a promising strategy to improve treatment outcomes for TNBC patients.

Abstract PO1-24-08: Targeting of HER3 potentiates the antitumor activity of paclitaxel against triple negative breast cancer

Abstract Background: Triple negative breast cancer (TNBC) is characterized by the absence of expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) overexpression. This aggressive form of breast cancer carries a higher risk of recurrence compared to other subtypes, presenting a significant clinical challenge. Although chemotherapy is commonly used as the primary treatment for a substantial number of TNBC patients, it is often accompanied by drug resistance and frequent tumor recurrence. Urgent efforts are needed to identify novel molecular targets specific to TNBC and develop effective therapies to combat this aggressive disease. The ultimate objective is to discover treatments capable of overcoming drug resistance and improving the overall survival of patients with TNBC. Methods: Immunohistochemistry was performed to examine the expression of HER3 in TNBC samples. Western blots were used to assess protein expression and activation. Cell proliferation and viability were determined by cell growth (MTS) assays. TCGA databases were analyzed to correlate HER3 mRNA expression with the clinical outcomes of TNBC patients. Specific shRNA was used to knockdown HER3 expression. LIVE/DEAD Cell Imaging was to detect live and dead cells. Orthotopic tumor models were established in nude mice to determine the capability of TNBC cells forming tumors and to test if our newly developed anti-HER3 monoclonal antibody (mAb) 4A7 could potentiate the antitumor activity of paclitaxel in vivo. Results: The chemo-resistant subline HCC1806-TR was obtained by subjecting TNBC HCC1806 cells to prolonged treatment with paclitaxel. In comparison to the parental cell line HCC1806-P, HCC1806-TR exhibited enhanced HER3 expression and activation. It is noteworthy that approximately half of the tested TNBC specimens and cell lines displayed elevated expression of HER3. Analyzing TCGA databases revealed that TNBC patients with high levels of HER3 mRNA expression in their tumors had significantly poorer overall survival (OS) and relapse-free survival (RFS) compared to those with low HER3 expression. Specifically knockdown of HER3 markedly inhibited TNBC cell proliferation and mammosphere formation in vitro and tumor growth in vivo. Blockade of HER3 signaling with our mAb (4A7) in combination with paclitaxel exerted significant antitumor activity against TNBC in vitro and in vivo. Conclusions: Our data demonstrate that increased HER3 is an effective therapeutic target for TNBC and our anti-HER3 mAb (4A7) may enhance the efficacy of paclitaxel in TNBC. Keywords: HER3, Anbibody, Chemotherapy, Triple Negative Breast Cancer Citation Format: Hui Lyu, Sanbao Ruan, Congcong Tan, Ann Thor, Bolin Liu. Targeting of HER3 potentiates the antitumor activity of paclitaxel against triple negative 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 PO1-24-08.

Abstract PO2-13-07: SMARCA2 compensation of SMARCA4 loss mediates adaptive resistance to neoadjuvant systemic therapy in triple-negative breast cancer: paired transcriptomic analysis of pre- and post-treatment samples from NeoSTOP and NeoPACT

Abstract Objectives/Rationale: Triple-negative breast cancer (TNBC) patients with residual disease (RD) after neoadjuvant systemic therapy (NAST) are at high risk for disease recurrence and death. While multiple prognostic biomarkers associated with response to NAST have been identified, adaptive resistance mechanisms to NAST remain poorly understood. Identifying and characterizing adaptive resistance mechanisms can illuminate novel therapeutic approaches and improve outcomes for TNBC patients with RD. Methods: Total RNA was isolated from pre-treatment and paired surgical specimens (for patients with RD) from TNBC patients treated with chemotherapy on the NeoSTOP (NCT02413320) or chemoimmunotherapy on the NeoPACT (NCT03639948) neoadjuvant trials and was subjected to RNA exome sequencing. Comparative marker selection analysis was performed by computing the two-sided paired samples t-test for each gene followed by pre-ranked gene set enrichment analysis (GSEA) amongst 23,797 annotated gene sets. Gene sets with false-discovery rate (FDR) corrected P values < 0.001 were determined to be significant. Results: Pre-treatment sequencing data were available for N=200 patients and the overall pathologic complete response (pCR) rate was 56.5%. Paired pre- and post-treatment sequencing data were available for N=58 patients with RD (N=27 from NeoSTOP and N=31 from NeoPACT). 165/23,797 gene sets were significantly enriched in post-treatment compared to pre-treatment samples. SHEN_SMARCA2_TARGETS_UP (M29) was among the top five significantly enriched gene set and was selected for further analysis based on the known role of the SWI/SNF complex in cell survival and the availability of clinically viable SMARCA2 degraders. Pre-treatment SMARCA4 expression was associated with pCR, with lower median expression noted in patients with RD compared to patients who achieved pCR (P=0.04). Pre-treatment SMARCA2 expression was not associated with pCR (P=0.48). On paired sample analysis there was pronounced downregulation of SMARCA4 in post-treatment compared to pre-treatment samples in all patients (mean Z=-1.46, P< 0.0001), in patients treated with chemotherapy (mean Z=-1.65, P< 0.0001), and in patients treated with chemoimmunotherapy (mean Z=-1.28, P< 0.0001). Although there was no difference in SMARCA2 expression between paired pre- and post-treatment samples (mean Z=0.03, P=0.83), there was enrichment of SMARCA2 target expression on GSEA analysis (NES=2.63, FDR q< 0.00001) indicating that SMARCA2 activity is compensating for therapy-induced SMARCA4 loss. Conclusions: We identified 165 gene sets that were significantly enriched in paired post-treatment compared to pre-treatment TNBC samples. Altered activity of the SMARCA2 subunit of the SWI/SNF chromatin remodeling complex was identified as one of the top hits. SMARCA2 and SMARCA4 are mutually exclusive subunits of SWI/SNF, and inactivation of both SMARCA2 and SMARCA4 is synthetic lethal. We observed an association of lower pre-treatment SMARCA4 expression with resistance to NAST and pronounced therapy-induced downregulation of SMARCA4 and induction of SMARCA2 target genes in post-treatment compared to pre-treatment samples, suggesting that SMARCA2 compensates for adaptive SMARCA4 loss. SMARCA2 degraders are in early-phase clinical trials and are known to induce synthetic lethality in SMARCA4-deficient cells. Our results identify adaptive loss of SMARCA4 and synthetic lethal targeting of compensatory SMARCA2 as an attractive therapeutic target in TNBC patients with RD after neoadjuvant chemotherapy or chemoimmunotherapy. Citation Format: Shane Stecklein, Rachel Yoder, Joshua Staley, Zachary Schmitt, Anne O'Dea, Lauren Nye, Deepti Satelli, Gregory Crane, Rashna Madan, Maura O'Neil, Andrew Godwin, Harsh Pathak, Qamar Khan, Joyce O'Shaughnessy, Priyanka Sharma. SMARCA2 compensation of SMARCA4 loss mediates adaptive resistance to neoadjuvant systemic therapy in triple-negative breast cancer: paired transcriptomic analysis of pre- and post-treatment samples from NeoSTOP and NeoPACT [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 PO2-13-07.

Biotin-stapled mixed micelles of lapatinib: Unleashing apoptosis power in triple-negative breast cancer

The treatment options currently offered for triple-negative breast cancer (TNBC) encounter numerous obstacles, including disappointing overall survival rates, unavoidable side effects, and the development of drug resistance. Additionally, there is a growing need for nutrients, like vitamins (e.g., biotin), to sustain the unregulated growth of TNBC cells. Hence, targeted therapy (LPT B-MM) for enhancing the treatment of TNBC was explored which encompassed hydrophobized [using α-TOS (α-Tocopheryl succinate] Pluronic-based mixed micelles having inherent sodium-dependent multivitamin transporter (SMVT) targeting (biotin-stapled) ability. LPT B-MM had optimal size (210.72± 25.88 nm), PDI (0.272± 0.023), and enhanced drug loading capacity (∼13.31± 2.12%). Transmission electron microscopy validated the spherical shape of these micelles. In vitro release studies demonstrated drug release in a sustained manner without the risk of hemolysis. Importantly, LPT B-MM displayed heightened cellular uptake, increased cytotoxicity, a lower IC50 value, elevated reactive oxygen species, induced mitochondrial membrane depolarization, and a greater apoptosis index in TNBC cell lines compared to free LPT. This study highlights the considerable potential of LPT B-MM to induce apoptosis, showcasing its promise in enhancing payload capabilities and enabling SMVT specific targeting within the context of TNBC. LPT B-MM demonstrated the synergistic effects of LPT and α-TOS in inducing apoptosis in TNBC cells, leading to enhanced therapeutic efficacy. This novel combination therapy offers a promising strategy for improving treatment outcomes in TNBC patients and represents a significant advancement in the field of TNBC therapy.

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.

HISTOPATHOLOGICAL PARAMETERS AND INTRATUMORAL LYMPHOCYTES: CORRELATION WITH SURVIVAL OUTCOMES IN TRIPLE-NEGATIVE BREAST CANCER - A COMPREHENSIVE RETROSPECTIVE ANALYSIS

OBJECTIVE: Triple-negative breast cancer (TNBC) is a subtype characterized by aggressive tumor behavior and limited treatment options. This study aimed to investigate the relationship among age, pathological stage, proliferative index, presence of tumor infiltrating lymphocytes (TILs), and survival outcomes in TNBC. MATERIAL AND METHODS: Tumoral slides and blocks of 31 patients with triple negative breast cancer were retrieved from the pathology archive and retrospectively re-evaluated. The relationship among patient age, histopathological subtype of the tumor, tumor grade, lymph node grade, Ki-67 proliferation index and survival was evaluated. TILs were scored as mild, moderate and severe and the relationship with survival was evaluated. RESULTS: Regarding age and tumor stage, there was no significant correlation found (p=0,81 and p=0,89 respectively). However, when analyzing the N stage, a clear association was observed, with a higher proportion of patients aged 65 years or older displaying advanced N3 stage breast cancer (p=0.000013). A significant relationship was found between TILs and the Ki-67 proliferative index, with cases exhibiting high TILs also demonstrating a high proliferative index (p=0.003). Furthermore, increased TIL concentration was associated with a positive response to therapy and improved overall survival in TNBC patients (p=0.001). CONCLUSIONS: These findings emphasize the importance of considering age, pathological stage, proliferative index, and the presence of TILs in TNBC prognosis. Evaluation of TILs in routine histopathologic examination and inclusion in pathology reports, particularly in postmenopausal patients, could provide valuable information for future studies and guide treatment decisions. Additional research on immune-modulating therapies targeting TILs may hold promise for improving outcomes in TNBC patients.

Identification of a Gene Expression Signature to Predict the Risk of Early Recurrence and the Degree of Immune Cell Infiltration in Triple-negative Breast Cancer.

Patients with triple-negative breast cancer (TNBC) have a high rate of recurrence within 3 years of diagnosis and a high rate of death within 5 years compared to other subtypes. The number of clinical trials investigating various new agents and combination therapies has recently increased; however, current strategies benefit only a minority of patients. This study aimed to identify specific genes that predict patients at high risk of recurrence and the immune status of the tumor microenvironment at an early stage, thereby providing insight into potential therapeutic targets to improve clinical outcomes in TNBC patients. We evaluated the prognostic significance of microarray mRNA expression of 20,603 genes in 233 TNBC patients from the METABRIC dataset and further validated the results using RNA-seq mRNA expression data in 143 TNBC patients from the GSE96058 dataset. Eighteen differentially expressed genes (AKNA, ARHGAP30, CA9, CD3D, CD3G, CD6, CXCR6, CYSLTR1, DOCK10, ENO1, FLT3LG, IFNG, IL2RB, LPXN, PRKCB, PVRIG, RASSF5, and STAT4) identified in both datasets were found to be reliable biomarkers for predicting TNBC recurrence and progression. Notably, the genes whose low expression was associated with increased risk of recurrence and death were immune-related genes, with significant differences in levels of immune cell infiltration in the tumor microenvironment between high- and low- expression groups. Genes reported herein may be effective biomarkers to identify TNBC patients who will and will not benefit from immunotherapy and may be particularly important genes for developing future treatment strategies, including immunotherapy.

Racial disparities in outcomes of patients with stage I-III triple-negative breast cancer after adjuvant chemotherapy: a post-hoc analysis of the E5103 randomized trial.

Breast cancer mortality is higher in Black women than other racial groups. This difference has been partially attributed to a higher proportion of triple-negative breast cancer (TNBC). However, it is uncertain if survival disparities exist in racially diverse TNBC patients receiving similar treatments. Here, we examine racial differences in disease-related outcomes in TNBC patients treated on the E5103 clinical trial. From 2007 to 2011, 4,994 patients with stage I-III HER2-negative breast cancer were randomized to adjuvant chemotherapy with or without bevacizumab. This analysis was limited to the subset of 1,742 TNBC patients with known self-reported race. Unadjusted Kaplan-Meier curves and adjusted Cox-Proportional Hazards models were used to determine breast cancer events and survival outcomes. Of the analysis population, 51 (2.9%) were Asian, 269 (15.4%) Black, and 1422 (81.6%) White. Median age was 51 years. Patient characteristics, treatment arm, and local therapies were similar across racial groups. White women were more commonly node-negative (56% vs. 49% and 44% in Asian and Black women, respectively; p < 0.01). At a median follow-up of 46 months, unadjusted Kaplan-Meier locoregional and distant recurrence, and disease-free and overall survival, did not differ significantly by race. In Cox models adjusted for patient and tumor characteristics and treatment arm, race was not associated with any disease event. Larger tumor size and nodal involvement were consistently associated with breast cancer events. This clinical trial population of similarly treated TNBC patients showed no racial differences in breast cancer outcomes. Disease extent, rather than race, was associated with disease events.

HSA-nanobinders crafted from bioresponsive prodrugs for combined cancer chemoimmunotherapy-an in vitro exploration.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer still lacking effective treatment options. Chemotherapy in combination with immunotherapy can restrict tumor progression and repolarize the tumor microenvironment towards an anti-tumor milieu, improving clinical outcome in TNBC patients. The chemotherapeutic drug paclitaxel has been shown to induce immunogenic cell death (ICD), whereas inhibitors of the indoleamine 2,3- dioxygenase 1 (IDO1) enzyme, whose expression is shared in immune regulatory and tumor cells, have been revealed to enhance the anti-tumor immune response. However, poor bioavailability and pharmacokinetics, off-target effects and hurdles in achieving therapeutic drug concentrations at the target tissue often limit the effectiveness of combination therapies. This work describes the development of novel biomimetic and carrier-free nanobinders (NBs) loaded with both paclitaxel and the IDO1 inhibitor NLG919 in the form of bioresponsive and biomimetic prodrugs. A fine tuning of the preparation conditions allowed to identify NB@5 as the most suitable nanoformulation in terms of reproducibility, stability and in vitro effectiveness. Our data show that NB@5 effectively binds to HSA in cell-free experiments, demonstrating its protective role in the controlled release of drugs and suggesting the potential to exploit the protein as the endogenous vehicle for targeted delivery to the tumor site. Our study successfully proves that the drugs encapsulated within the NBs are preferentially released under the altered redox conditions commonly found in the tumor microenvironment, thereby inducing cell death, promoting ICD, and inhibiting IDO1.

Abstract 5215: Orphan noncoding RNA (oncRNA) liquid biopsy assay is prognostic for survival in patients with triple-negative breast cancer (TNBC) and residual disease

Abstract Introduction: Residual disease (RD) after neoadjuvant systemic therapy is associated with high recurrence risk in patients with TNBC. oncRNAs are a novel category of small RNAs that are largely absent in healthy tissue but enriched in tumors and can be detected using a blood-based assay. This study investigated impact of an oncRNA recurrence risk model on outcomes in TNBC patients with RD. Methods: Study population included stage I-III TNBC patients with RD and available end-of-treatment (EOT) serum samples who were enrolled in a multisite prospective cohort study. EOT samples were collected after completion of all curative treatment (local/systemic). Small RNAs were isolated from EOT serum, sequenced at average depth of 76.5 ± 12.5 million 50bp single end reads, and annotated using a bespoke bioinformatics pipeline to identify oncRNAs. Cancer risk scores were generated using an oncRNA based tumor detection artificial intelligence model trained on 451 treatment naive breast cancer samples and 470 samples from individuals without known cancer diagnosis. Score cutpoint for high vs low recurrence risk was determined through ROC analysis. Impact of EOT oncRNA risk category on event free survival (EFS) and overall survival (OS) was estimated by Kaplan Meier method and compared by log rank test followed by Cox regression. Residual cancer burden (RCB) was determined according to classification by Symmans et al. Results: oncRNA isolation/score generation was successful for 79 out of 80 TNBC patients with RD and available EOT serum sample. Median age was 48 years and 39% had node positive disease. RCB class distribution was as follows: RCB I=27%, RCB II=49%, RCB III=18%. Training set (n=39) was used to define oncRNA risk score cutpoint. In the testing set (n=40), 38% were classified as oncRNA high-risk and 62% as oncRNA low-risk. oncRNA risk category was not associated with baseline T stage, nodal status, or RCB class. oncRNA high-risk status was associated with lower EFS and OS; 3y EFS was 47% and 73% (HR 2.76, 95% CI 0.92-9.24, p=0.058) and OS 53% and 76% (HR 3.78, 95% CI 1.19-12.00, p=0.016) in high and low risk groups, respectively. In multivariable analysis including oncRNA risk status, T stage, nodal status, and RCB class, oncRNA high risk status retained significant association with lower EFS (HR 7.70, 95% CI 1.33-44.64, p=0.023) and OS (HR 7.99, 95% CI 1.36-47.00, p=0.022). Conclusion: EOT oncRNA liquid biopsy assay was independently prognostic for outcomes in TNBC patients with RD. More than half of patients in the oncRNA high-risk group suffered an EFS event by 3 years. oncRNA risk score has potential to provide prognostic utility complementary to clinicopathologic characteristics in patients with TNBC. These findings should be confirmed in other TNBC studies and may provide insights for patient stratification/selection in RD adjuvant therapy intensification trials. Citation Format: Rachel Yoder, Jennifer Yen, Mehran Karimzadeh, Joshua M. Staley, India Fernandez, Adam C. Heinrich, Fereydoun Hormozdiari, Jeffrey Gregg, Andrew K. Godwin, Irene Acerbi, Raaj Trivedi, Babak Alipanahi, Shane R. Stecklein, Priyanka Sharma. Orphan noncoding RNA (oncRNA) liquid biopsy assay is prognostic for survival in patients with triple-negative breast cancer (TNBC) and residual disease [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 5215.

Abstract 1162: Tumor-immune microenvironment associated with obesity in triple-negative breast cancer in the Mayo Clinic cohort

Abstract Background: Previous studies demonstrated that obesity is associated with poor outcomes and significantly worse pathological complete response (pCR) in triple-negative breast cancer (TNBC) treated with conventional chemotherapy. Emerging studies described a phenomenon termed the obesity paradox, which referred to improved outcomes observed in obese patients treated with immune checkpoint inhibitors (ICIs). Our study aimed to determine distinct immune features in TNBC across body mass index (BMI). Methods: Characteristics and outcomes of TNBC patients treated with neoadjuvant pembrolizumab-based chemotherapy at Mayo Clinic were collected. Normal BMI was defined as 18-25 kg/m2, overweight 25-30, and obese BMI ≥ 30. NanoString CosMx, a spatial multi-omics single-cell imaging (SMI), was performed in 75 samples from the Mayo Clinic TNBC cohort (Leon-Ferre BCRT 2018). Differential expression as log 2-fold change (FC) was estimated from the linear mixed model with significance defined as two-sided p&amp;lt; 0.05. Results: Between 2021-2023, there were 108 TNBC patients treated with neoadjuvant pembrolizumab. 25.9% were obese, 32.4% were overweight, and 41.7% had normal BMI. In contrast to previous studies with conventional chemotherapy, obese patients numerically had a higher pCR rate of 71.4% with pembrolizumab-based chemotherapy compared to 55.6% in normal and 62.9% in overweight patients. However, this difference did not reach statistical significance (obese vs. normal weight, OR 2.26, 95%CI 0.89-6.06, p 0.094). We further evaluated spatially-resolved immune features associated with obesity using CosMx SMI in 75 samples from the MC TNBC cohort. Obese patients had significantly higher intratumoral B cells (log2FC 1.88, p 0.017), stromal B cells (log2FC 2.79, p 0.001), stromal plasmablasts (log2FC 1.94, p 0.047), and stromal macrophages (log2FC 1.80, p 0.017) compared to patients with normal BMI. Obese patients also had significantly lower intratumoral myeloid dendritic cells (mDC) (log2FC -1.27, p 0.037). Similar trends were observed after standardizing cell counts per 100 tumor or stromal cells, including higher intratumoral B cells (log2FC 3.00, p 0.001), intratumoral plasmablasts (log2FC 2.00, p 0.006), intratumoral natural killer (NK) cells (log2FC 1.66, p 0.044), and stromal B cells (log2FC 1.50, p 0.008). Differential gene analyses for intratumoral B cells revealed that genes (CD74, HLA-C, HLA-DRB1, HLA-DRA, HLA-DRB) associated with the positive regulation of type 2 immune or chemokine response were upregulated. Conclusions: Using in-depth analysis with spatially defined context, our study showed that obese TNBC patients had significantly higher B cell infiltration, which is associated with improved outcomes with ICIs. Future research on how obesity alters the immune microenvironment is needed to identify potential pathways to improve outcomes with ICIs in TNBC. Citation Format: Songhan Pang, Yi Liu, Yaohua Ma, Zhuo Li, Roberto A. Leon-Ferre, David Zahrieh, David W. Hillman, Judy C. Boughey, James N. Ingle, Krishna R. Kalari, Fergus J. Couch, Matthew P. Goetz, Keith L. Knutson, Saranya Chumsri, E. Aubrey Thompson. Tumor-immune microenvironment associated with obesity in triple-negative breast cancer in the Mayo Clinic cohort [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 1162.

Integrative analysis identifies cancer cell-intrinsic RARRES1 as a predictor of prognosis and immune response in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer with poor prognosis and limited treatment options. Although immune checkpoint inhibitors (ICIs) have been proven to improve outcomes in TNBC patients, the potential mechanisms and markers that determine the therapeutic response to ICIs remains uncertain. Revealing the relationship and interaction between cancer cells and tumor microenvironment (TME) could be helpful in predicting treatment efficacy and developing novel therapeutic agents. By analyzing single-cell RNA sequencing dataset, we comprehensively profiled cell types and subpopulations as well as identified their signatures in the TME of TNBC. We also proposed a method for quantitatively assessment of the TME immune profile and provided a framework for identifying cancer cell-intrinsic features associated with TME through integrated analysis. Using integrative analyses, RARRES1 was identified as a TME-associated gene, whose expression was positively correlated with prognosis and response to ICIs in TNBC. In conclusion, this study characterized the heterogeneity of cellular components in TME of TNBC patients, and brought new insights into the relationship between cancer cells and TME. In addition, RARRES1 was identified as a potential predictor of prognosis and response to ICIs in TNBC.

Increased ACSL6 Expression Predicts a Favorable Prognosis in Triple-negative Breast Cancer.

Long-chain acyl-coenzyme A synthases (ACSLs) are responsible for the catalysis of fatty acids into their corresponding fatty acyl-CoAs. The dysregulation of ACSLs has been increasingly recognized in cancer patients. However, the function of ACSL6 in triple-negative breast cancer (TNBC) is still completely unknown. In this study, immunohistochemistry was applied to detect ACSL6 protein expression using a TNBC tissue microarray. Additionally, the mRNA levels of ACSL6 in human normal tissues and pancancer tissues were analyzed using Genotype Tissue Expression (GTEx) datasets and The Cancer Genome Atlas (TCGA) database. The correlations between the levels of ACSL6 expression and clinical characteristics were analyzed. The survival analysis of ACSL6 in TNBC was carried out using the Kaplan‒Meier Plotter online tool. Associations of ACSL6 with immune infiltration analyses were conducted using the ESTIMATE, CIBERSORT, and TISIDB databases. The relationship between ACSL6 and sensitivity to drugs was analyzed from Genomics of Drug Sensitivity in Cancer (GDSC). The results indicated a significant increase in ACSL6 expression in TNBC tissues compared to adjacent normal tissues. However, high ACSL6 expression was significantly associated with favorable survival outcomes in TNBC patients. Enrichment analysis revealed that coexpressed genes of ACSL6 were significantly enriched in various immunity processes. ACSL6 was positively correlated with the infiltration of memory CD4 T cells, while a negative correlation was found between ACSL6 and M2 macrophages and resting dendritic cells. Further analysis revealed that high levels of ACSL6 correlated with increased survival outcomes in cancer patients who received immunotherapy. Altogether, the current findings highlight the potential value of ACSL6 as a diagnostic and prognostic marker in the treatment of TNBC.

Emerging treatment approaches for triple-negative breast cancer.

Approximately, 15% of global breast cancer cases are diagnosed as triple-negative breast cancer (TNBC), identified as the most aggressive subtype due to the simultaneous absence of estrogen receptor, progesterone receptor, and HER2. This characteristic renders TNBC highly aggressive and challenging to treat, as it excludes the use of effective drugs such as hormone therapy and anti-HER2 agents. In this review, we explore standard therapies and recent emerging approaches for TNBC, including PARP inhibitors, immune checkpoint inhibitors, PI3K/AKT pathway inhibitors, and cytotoxin-conjugated antibodies. The mechanism of action of these drugs and their utilization in clinical practice is explained in a pragmatic and prospective manner, contextualized within the current landscape of standard therapies for this pathology. These advancements present a promising frontier for tailored interventions with the potential to significantly improve outcomes for TNBC patients. Interestingly, while TNBC poses a complex challenge, it also serves as a paradigm and an opportunity for translational research and innovative therapies in the field of oncology.

Unraveling Connective Tissue Growth Factor as a Therapeutic Target and Assessing Kahweol as a Potential Drug Candidate in Triple-Negative Breast Cancer Treatment.

Triple-negative breast cancer (TNBC) is characterized by aggressive behavior and limited treatment options, necessitating the identification of novel therapeutic targets. In this study, we investigated the clinical significance of connective tissue growth factor (CTGF) as a prognostic marker and explored the potential therapeutic effects of kahweol, a coffee diterpene molecule, in TNBC treatment. Initially, through a survival analysis on breast cancer patients from The Cancer Genome Atlas (TCGA) database, we found that CTGF exhibited significant prognostic effects exclusively in TNBC patients. To gain mechanistic insights, we performed the functional annotation and gene set enrichment analyses, revealing the involvement of CTGF in migratory pathways relevant to TNBC treatment. Subsequently, in vitro experiments using MDA-MB 231 cells, a representative TNBC cell line, demonstrated that recombinant CTGF (rCTGF) administration enhanced cell motility, whereas CTGF knockdown using CTGF siRNA resulted in reduced motility. Notably, rCTGF restored kahweol-reduced cell motility, providing compelling evidence for the role of CTGF in mediating kahweol's effects. At the molecular level, kahweol downregulated the protein expression of CTGF as well as critical signaling molecules, such as p-ERK, p-P38, p-PI3K/AKT, and p-FAK, associated with cell motility. In summary, our findings propose CTGF as a potential prognostic marker for guiding TNBC treatment and suggest kahweol as a promising antitumor compound capable of regulating CTGF expression to suppress cell motility in TNBC. These insights hold promise for the development of targeted therapies and improved clinical outcomes for TNBC patients.