Triple-negative Breast Cancer Disease Research Articles

Adjuvant nivolumab, capecitabine or the combination in patients with residual triple-negative breast cancer: the OXEL randomized phase II study

Chemotherapy and immune checkpoint inhibitors have a role in the post-neoadjuvant setting in patients with triple-negative breast cancer (TNBC). However, the effects of nivolumab, a checkpoint inhibitor, capecitabine, or the combination in changing peripheral immunoscore (PIS) remains unclear. This open-label randomized phase II OXEL study (NCT03487666) aimed to assess the immunologic effects of nivolumab, capecitabine, or the combination in terms of the change in PIS (primary endpoint). Secondary endpoints included the presence of ctDNA, toxicity, clinical outcomes at 2-years and association of ctDNA and PIS with clinical outcomes. Forty-five women with TNBC and residual invasive disease after standard neoadjuvant chemotherapy were randomized to nivolumab, capecitabine, or the combination. Here we show that a combination of nivolumab plus capecitabine leads to a greater increase in PIS from baseline to week 6 (91%) compared with nivolumab (47%) or capecitabine (53%) alone (log-rank p = 0.08), meeting the pre-specified primary endpoint. In addition, the presence of circulating tumor DNA (ctDNA) is associated with disease recurrence, with no new safety signals in the combination arm. Our results provide efficacy and safety data on this combination in TNBC and support further development of PIS and ctDNA analyses to identify patients at high risk of recurrence.

Pathologic and immunohistochemical prognostic markers in residual triple-negative breast cancer after neoadjuvant chemotherapy.

The persistence of residual tumour after neoadjuvant chemotherapy (NAC) in localised triple-negative breast cancer (TNBC) is known to have a negative prognostic value. However, different degrees of expression of some immunohistochemical markers may correlate with different prognoses. The expression of biomarkers with a known prognostic value, i.e., cytokeratin 5/6 (CK5/6), androgen receptor (AR), epidermal growth factor receptor (EGFR) proliferation-related nuclear antigen Ki-67, human epidermal growth factor receptor 2 (HER2), protein 53 (p53), forkhead box protein 3 (FOXP3), and cluster differentiation 8 (CD8), was analysed by immunohistochemistry in 111 samples after NAC in non-metastatic TNBC patients addressed to Georges-François Leclerc Cancer Centre Dijon, France. Clinical and pathological variables were retrospectively collected. Cox regression was used to identify immunohistochemical (IHC) and clinicopathological predictors of event-free survival (EFS) (relapse or death). Median age was 50.4 years (range 25.6-88.3), 55.9% (n = 62) were non-menopausal, 70 (63.1%) had stage IIA-IIB disease. NAC was mostly sequential anthracycline-taxanes (72.1%), and surgical intervention was principally conservative (51.3%). We found 65.7% ypT1, 47.2% lymph node involvement (ypN+), and 29.4% lymphovascular invasion (LVI). Most residual tumours were EGFR >110 (H-score) (60.5%, n = 66), AR ≥4% (53.2%, n = 58), p53-positive mutated (52.7%, n = 58), CD8 ≥26 (58.1%, n = 61), FOXP3 ≥7 (51.4%, n = 54), more than half in the stroma, and 52.3% (n = 58) HER2 score 0. After a median follow-up of 80.8 months, 48.6% had relapsed. Median EFS was 62.3 months (95% CI, 37.2-not reached (NR)). Factors independently associated with poor EFS were AR-low (p = 0.002), ypN+ (p < 0.001), and LVI (p = 0.001). Factors associated with lower overall survival (OS) were EGFR-low (p = 0.041), Ki-67 high (p = 0.024), and ypN+ (p < 0.001). Post-NAC residual disease in TNBC showed biomarkers specific to a basal-like subtype and markers of lymphocyte infiltration mostly present in the stroma. Prognostic markers for EFS were AR, LVI, and ypN and warrant further validation in a prognostic model.

Effects of tea catechin extracts from BB35 and purple (TRFK 306) tea clones on the gene expression of Egfr, App, Bcl2, Dnmt, Casp3, Hif1a, Gadd45b and Psmb5 genes involved in triple negative breast cancer diseases: In silico and in vitro study

Tea has been shown to contain metabolites that exhibit antioxidant, anti-inflammatory, anti-diabetic, anti-cancer and anti-cardio vascular diseases properties. This study aimed to carry out an in silico assessment of catechins on the genes previously shown to be involved in Triple Negative Breast Cancer (TNBC) disease and also to evaluate the effects of catechin extracts from purple (TRFK306) and BB35 tea clones on the expression patterns of genes in the 4T1 TNBC cell line as mentioned above. Identification and quantification of different catechin contents in these two tea clones were performed by using High-Performance Liquid Chromatography (HPLC). In silico assessment including Absorption, Distribution, Metabolism and Excretion (ADME) study for drug-likeness evaluation, Drug target prediction, Protein-protein interaction (PPI) network analysis and construction, Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) enrichment and analysis for targets and Molecular docking study was conducted. Simplified Molecular Input Line Entry System (SMILES) of 4 different tea catechins showed a target on 111 genes in TNBC and have high binding affinity to the receptor-binding pocket of chain A of Epidermal Growth Factor Receptor (EGFR) Extracellular Domains. Catechin extracts promoted the downregulation of expression of 6 genes (Egfr, App, Bcl2, Dnmt, Hif1a and Psmb5) and the upregulation of 2 genes (Casp3 and Gadd45b) in the 4T1 TNBC cell line. Catechin extracts from purple tea clones showed higher activity on gene expression levels of Egfr, Bcl2 and Casp3 than catechin extracts from BB35 tea clone.

Abstract C047: Multiomics approaches to identify African-ancestry specific phenotypes of triple negative breast cancer

Abstract Despite recent convergence of breast cancer (BC) incidence among African American (AA) and European American (EA) women, BC mortality remains 40% higher among AA women. This disparity is driven partly by BC heterogeneity, where distinct subtypes have differing prognosis and treatment modalities, defined by presence of estrogen and/or progesterone receptor (HR+), overexpression of HER2, or lack of all three biomarkers (triple negative BC (TNBC)). TNBC is the most aggressive subtype, diagnosed as late-stage disease and lacking targeted therapeutic options. TNBC represents ~10% of annual diagnoses, however prevalence is significantly higher among AA women. Our work in the International Center for the Study of Breast Cancer Subtypes (ICSBCS) revealed significant association of west African (AFR) ancestry and TNBC, where we report that Ghanaian and AA women have increased frequency of TNBC, compared to Ethiopian and EA women, and that TNBC patients have higher percentages of quantified AFR ancestry. To characterize AFR ancestry-specific TNBC phenotypes, we have employed transcriptomics and whole genome sequencing (WGS) methods. Our recent publication from an AFR ancestry enriched TNBC transcriptomics cohort (n = 26) leveraged genetic ancestry to identify 600+ AFR and 2000+ AFR sub-continental ancestry associated genes. Pathway analysis showed enrichment of immune cell trafficking and indicates distinct AFR ancestry phenotypes that can be leveraged for future therapeutic opportunities. Recently, we have completed WGS on 80+ AFR ancestry patients with TNBC disease (n = 59) and HR+ disease (n = 23); representing AA (n = 71), Ethiopian (n = 8) and Ghanaian patients (n = 3). TP53 was the most mutated gene, more commonly mutated among TNBC cases (83%) compared to HR+ cases (35%) (p &amp;lt; 0.0001). HR+ had significantly more GATA3 (26%) and PIK3CA (30%) mutations compared to TNBC (0% and 9%, p &amp;lt; 0.05). Prevalence of COSMIC mutational signatures shows higher Signature 1 among HR+ cases, and higher Signature 3 among TNBC cases (both p &amp;lt; 0.05). Signature 3 is associated with DNA double-strand break repair and germline BRCA1/2 mutation status, where 9 TNBC cases have elevated homologous repair deficiency status. Our genomic work utilizes the reference genome, which is primarily sequenced from a single individual. With the African Pan Genome (APG), a set of 125,000+ contigs distinct from the reference genome, we have aligned RNAseq reads to the APG that failed to map to the reference genome. Successful APG alignment of a subset of reads indicates that genes are expressed from these distinct AFR sequences. We also identified CpG sites in contigs with gene expression, showing AFR ancestry specific regulatory regions in the previously unmapped genome. We are working to determine the significance of these in the tumor microenvironment. This work represents a comprehensive, AFR ancestry enriched cohort with multiple genomic data approaches that will allow us to further characterize TNBC disease and define actionable biomarkers and targets for women of African ancestry. Citation Format: Rachel Martini, Max Chao, Timothy Chu, Brian Stonaker, Ishmael Kyei, Ernest Adjei, Mahteme Bekele, Kofi Gyan, Olivier Elemento, Nicolas Robine, John Carpten, Lisa Newman, Melissa Davis. Multiomics approaches to identify African-ancestry specific phenotypes of triple negative breast cancer [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C047.

National trends in neoadjuvant chemotherapy utilization in patients with early-stage node-negative triple-negative breast cancer: the impact of the CREATE-X trial.

Neoadjuvant chemotherapy (NAC) for triple-negative breast cancer (TNBC) allows for assessment of tumor pathological response and has survival implications. In 2017, the CREATE-X trial demonstrated survival benefit with adjuvant capecitabine in patients TNBC and residual disease after NAC. We aimed to assess national rates of NAC for cT1-2N0M0 TNBC before and after CREATE-X and examine factors associated with receiving NAC vs adjuvant chemotherapy (AC). A retrospective cohort study of women with cT1-2N0M0 TNBC diagnosed from 2014 to 2019 in the National Cancer Database (NCDB) was performed. Variables were analyzed via ANOVA, Chi-squared, Fisher Exact tests, and a multivariate linear regression model was created. 55,633 women were included: 26.9% received NAC, 52.4% AC, and 20.7% received no chemotherapy (median ages 53, 59, and 71years, p < 0.01). NAC utilization significantly increased over time: 19.5% in 2014-15 (n = 3,465 of 17,777), 27.1% in 2016-17 (n = 5,140 of 18,985), and 33.6% in 2018-19 (n = 6,337 of 18,871, p < 0.001). On multivariate analysis, increased NAC was associated with younger age (< 50), non-Hispanic white race/ethnicity, lack of comorbidities, cT2 tumors, care at an academic or integrated-network cancer program, and diagnosis post-2017 (p < 0.05 for all). Patients with government-provided insurance were less likely to receive NAC (p < 0.01). Women who traveled > 60 miles for treatment were more likely to receive NAC (p < 0.01). From 2014 to 2019, NAC utilization increased for patients with cT1-2N0M0 TNBC. Racial, socioeconomic, and access disparities were observed in who received NAC vs AC and warrants interventions to ensure equitable care.

MEF2C and miR-194-5p: New Players in Triple Negative Breast Cancer Tumorigenesis.

Among breast cancer (BC) subtypes, the most aggressive is triple negative BC (TNBC), which is prone to metastasis. We previously found that microRNA (miR)-194-5p is downregulated at the early stages of TNBC brain metastasis development. Additionally, the transcription factor myocyte enhancer factor 2 (MEF2)C, a bioinformatically predicted miR-194-5p target, was increasingly expressed throughout TNBC brain metastasis formation and disease severity. However, the contributions of these two players to malignant cells' features remain undetermined. This study aimed at disclosing the role of miR-194-5p and MEF2C in TNBC tumorigenesis. The transfection of 4T1 cells with a silencer for MEF2C or with a pre-miRNA for miR-194-5p was employed to study TNBC cells' phenotypic alterations regarding epithelial and mesenchymal markers, as well as migratory capability alterations. MEF2C-silenced cells presented a decline in both vimentin and cytokeratin expression, whereas the overexpression of miR-194-5p promoted an increase in cytokeratin and a reduction in vimentin, reflecting the acquisition of an epithelial phenotype. Both treatments reduced TNBC cells' migration. These results suggest that MEF2C may determine TNBC cells' invasive properties by partially determining the occurrence of epithelial-mesenchymal transition, while the overexpression of miR-194-5p promotes a decline in TNBC cells' aggressive behavior and reinforces this miRNA's role as a tumor suppressor in TNBC.

A modified natural small molecule inhibits triple-negative breast cancer growth by interacting with Tubb3

BackgroundTriple-negative breast cancer (TNBC) is a malignant tumor without specific therapeutic targets and a poor prognosis. Chemotherapy is currently the first-line therapeutic option for TNBC. However, due to the heterogeneity of TNBC, not all of TNBC patients are responsive to chemotherapeutic agents. Therefore, the demand for new targeted agents is critical. β-tubulin isotype III (Tubb3) is a prognostic factor associated with cancer progression, including breast cancer, and targeting Tubb3 may lead to improve TNBC disease control. Shikonin, the active compound in the roots of Lithospermun erythrorhizon suppresses the growth of various types of tumors, and its efficacy can be improved by altering its chemical structure. PurposeIn this work, the anti-TNBC effect of a shikonin derivative (PMMB276) was investigated, and its mechanism was also investigated. Study Design/MethodsThis study combines flow cytometry, immunofluorescence staining, immunoblotting, immunoprecipitation, siRNA silencing, and the iTRAQ proteomics assay to analyze the inhibition potential of PMMB276 on TNBC. In vivo study was performed, Balb/c female murine models with or without the small molecule treatments. ResultsHerein, we screened 300 in-house synthesized analogs of shikonin against TNBC and identified a novel small molecule, PMMB276; it suppressed cell proliferation, induced apoptosis, and arrested the cell cycle at the G2/M phase, suggesting that it could have a tumor suppressive role in TNBC. Tubb3 was identified as the target of PMMB276 using proteomic and biological activity analyses. Meanwhile, PMMB276 regulated microtubule dynamics in vitro by inducing microtubule depolymerization and it could act as a tubulin stabilizer by a different process than that of paclitaxel. Moreover, suppressing or inhibiting Tubb3 with PMMB276 reduced the growth of breast cancer in an experimental mouse model, indicating that Tubb3 plays a significant role in TNBC progression. ConclusionThe findings support the therapeutic potential of PMMB276, a Tubb3 inhibitor, as a treatment for TNBC. Our findings might serve as a foundation for the utilization of shikonin and its derivatives in the development of anti-TNBC.

Beta-blockade enhances anthracycline control of metastasis in triple-negative breast cancer.

Beta-adrenergic blockade has been associated with improved cancer survival in patients with triple-negative breast cancer (TNBC), but the mechanisms of these effects remain unclear. In clinical epidemiological analyses, we identified a relationship between beta-blocker use and anthracycline chemotherapy in protecting against TNBC progression, disease recurrence, and mortality. We recapitulated the effect of beta-blockade on anthracycline efficacy in xenograft mouse models of TNBC. In metastatic 4T1.2 and MDA-MB-231 mouse models of TNBC, beta-blockade improved the efficacy of the anthracycline doxorubicin by reducing metastatic development. We found that anthracycline chemotherapy alone, in the absence of beta-blockade, increased sympathetic nerve fiber activity and norepinephrine concentration in mammary tumors through the induction of nerve growth factor (NGF) by tumor cells. Moreover, using preclinical models and clinical samples, we found that anthracycline chemotherapy up-regulated β2-adrenoceptor expression and amplified receptor signaling in tumor cells. Neurotoxin inhibition of sympathetic neural signaling in mammary tumors using 6-hydroxydopamine or genetic deletion of NGF or β2-adrenoceptor in tumor cells enhanced the therapeutic effect of anthracycline chemotherapy by reducing metastasis in xenograft mouse models. These findings reveal a neuromodulatory effect of anthracycline chemotherapy that undermines its potential therapeutic impact, which can be overcome by inhibiting β2-adrenergic signaling in the tumor microenvironment. Supplementing anthracycline chemotherapy with adjunctive β2-adrenergic antagonists represents a potential therapeutic strategy for enhancing the clinical management of TNBC.

Abstract 5523: Impact of COVID-19 on longitudinal breast cancer research studies involving TNBC patients

Abstract Background Triple-negative breast cancer (TNBC) accounts for ~15% of breast cancer diagnoses but is linked to worse outcomes and comprises a disproportionate number of breast cancer deaths. The TNBC pilot study is a prospective longitudinal study to provide a critical resource for understanding TNBC disease. However, the pandemic impacted the collection of samples. Objective To highlight the impacts of COVID-19 on this longitudinal cancer translational research study including the patient's perspective and to develop recommendations to avoid future disruptions. Methods 389 participants were enrolled in the prospective longitudinal cohort, which collected serial blood samples for up to 5 years. Due to the pandemic, research was curtailed for 6 months due to concerns about patient safety, halting the collection of blood samples. Missed samples and data gaps were documented. To complement this, we initiated a survey capturing the patient perspective on their experience of the study disruption due to COVID. Results 217 enrolled participants missed a blood draw or had a collection outside the study window. 158 patients missed 1 time-point collection, and 59 patients missed ≥ 2 collections. Of the 217 participants who missed a collection, 6 disease recurrence diagnoses and 3 deaths occurred during research curtailment. The collection of survey responses from participants is ongoing and will be presented at the AACR Annual Meeting. Conclusion Missed samples resulted in irreplaceable data gaps critical to monitoring patient outcomes, and reduced cohort sampling during the pandemic. Our current knowledge of the risks suggests that with proper informed consent, collections could have continued. To mitigate disruption in future clinical studies, clear plans should be part of study design to provide continuity. The participants’ experience to be reported will also help researchers understand their issues and help develop policies. Missed collections and clinical events during COVID-19 research curtailment Number of patients Total with missed or late blood draws 217 Missed 1 collection 158 Missed ≥ 2 collections 59 Diagnosed with disease recurrence 6 Deaths 3 Citation Format: Cherie Bates, Esther Kong, Elena Zaikova, Samuel Aparicio, Karen Gelmon. Impact of COVID-19 on longitudinal breast cancer research studies involving TNBC patients. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5523.

Crosstalk between triple negative breast cancer and microenvironment.

Although many advances have been made in the treatment of breast cancer, for the triple negative breast cancer (TNBC) these therapies have not significantly increased overall survival. Tumor microenvironment (TME) plays an essential role to develop and control TNBC progression. Many preclinical and clinical studies are ongoing to treat patients with TNBC disease, but the effective therapies are currently not available. Here, we have reviewed recent progress in understanding of TNBC and advance in defining mechanisms of TNBC therapies and potential therapeutic strategies to overcome TNBC.

Detection of Increased Expression of Claudin-1 in Triple-Negative Breast Cancer: Analysis and Clinical-Pathological Correlation.

Background Triple-negative breast cancer (TNBC) is a highly aggressive disease that lacks therapeutic targets and prognostic biomarkers. Claudin-1 is a well-described tight junction protein with prognostic value in many human cancers. Aims The need for the discovery of biomarkers of TNBC disease was a major reason for this study. Claudin-1 is a tight junction protein that has shown promising results in the prognosis and management of cancer in general. In the breast, claudin-1 expression and significance have shown variable results, especially in TNBC patients. Our study assessed expression of claudin-1 in a group of TNBC patients, and correlated this expression with clinical-pathological parameters, and with the expression of β-catenin. Materials and methods Tissues from a group of 52 TNBC patients were retrieved from the archives of the community hospital. All related information including demographical, pathologicand clinical data were retrieved. Immunohistochemistry assays ofa rabbit polyclonal antibody anti-human claudin-1 were applied using theavidin-biotin peroxidase methodology. Results A statistically significant majority of TNBC cases positively expressed claudin-1 (81%, χ2=13.705; p<0.001).Most TNBC cases had grade 2 β-catenin expression (77.5%; p<0.001), and positive expression for claudin-1 correlated with that of β-catenin (χ2= 23.757; p<0.001). Claudin-1 and β-catenin expressions within tumour cells shared several features including absent or weakness of membranous expression, and redistribution of both proteins to the cytoplasm of tumour cells, and in some cases to the nuclei of these cells. Claudin-1 expression also correlates with adverse survival outcomes, where only four of 20 claudin-1-positive patients who received neo-adjuvant chemotherapy (NAC) achieved pathological complete response (pCR). Conclusions The above presents a complex role of claudin-1 in TNBC patients. In this study, claudin-1 expression was associated with poor prognostic features including invasion, metastases and adverse clinical outcomes. Claudin-1 expression in TNBC correlated with the expression of β-catenin, an important oncogene and a major contributor to the epithelial mesenchymal transition (EMT) phenomenon. Overall, the above results may serve as an impetus for further mechanistic studies to assess the exact role of claudin-1 in TNBC and its possible use in the management of this subset of breast cancer.

Results of the c-TRAK TN trial: a clinical trial utilising ctDNA mutation tracking to detect molecular residual disease and trigger intervention in patients with moderate- and high-risk early-stage triple-negative breast cancer

Post-treatment detection of circulating tumour DNA (ctDNA) in early-stage triple-negative breast cancer (TNBC) patients predicts high risk of relapse. c-TRAK TN assessed the utility of prospective ctDNA surveillance in TNBC and the activity of pembrolizumab in patients with ctDNA detected [ctDNA positive (ctDNA+)]. c-TRAK TN, a multicentre phase II trial, with integrated prospective ctDNA surveillance by digital PCR, enrolled patients with early-stage TNBC and residual disease following neoadjuvant chemotherapy, or stage II/III with adjuvant chemotherapy. ctDNA surveillance comprised three-monthly blood sampling to 12 months (18 months if samples were missed due to coronavirus disease), and ctDNA+ patients were randomised 2 : 1 to intervention : observation. ctDNA results were blinded unless patients were allocated to intervention, when staging scans were done and those free of recurrence were offered pembrolizumab. A protocol amendment (16 September 2020) closed the observation group; all subsequent ctDNA+ patients were allocated to intervention. Co-primary endpoints were (i) ctDNA detection rate and (ii) sustained ctDNA clearance rate on pembrolizumab (NCT03145961). Two hundred and eight patients registered between 30 January 2018 and 06 December 2019, 185 had tumour sequenced, 171 (92.4%) had trackable mutations, and 161 entered ctDNA surveillance. Rate of ctDNA detection by 12 months was 27.3% (44/161, 95% confidence interval 20.6% to 34.9%). Seven patients relapsed without prior ctDNA detection. Forty-five patients entered the therapeutic component (intervention n= 31; observation n= 14; one observation patient was re-allocated to intervention following protocol amendment). Of patients allocated to intervention, 72% (23/32) had metastases on staging at the time of ctDNA+, and 4 patients declined pembrolizumab. Of the five patients who commenced pembrolizumab, none achieved sustained ctDNA clearance. c-TRAK TN is the first prospective study to assess whether ctDNA assays have clinical utility in guiding therapy in TNBC. Patients had a high rate of metastatic disease on ctDNA detection. Findings have implications for future trial design, emphasising the importance of commencing ctDNA testing early, with more sensitive and/or frequent ctDNA testing regimes.

Programmed death-ligand 1 (PD-L1) expression predicts response to neoadjuvant chemotherapy in triple-negative breast cancer: a systematic review and meta-analysis

Background In patients with metastatic triple-negative breast cancer (TNBC), programmed death-ligand 1 (PD-L1) expression has been demonstrated to predict response to immunotherapy. It is unclear whether PD-L1 expression measured with currently available validated assays can predict chemotherapy response in patients with non-metastatic TNBC. Methods We conducted a systematic review and meta-analysis of clinical studies to assess the PD-L1 expression as a predictor of response to chemotherapy in non-metastatic TNBC using validated assays. The primary endpoint was pathological complete response (pCR) rate to neoadjuvant chemotherapy. Secondary endpoints included the prevalence of PD-L1 expression in non-metastatic TNBC and its impact on disease-free survival (DFS) and overall survival (OS). Moreover, RNA sequence data from the TCGA breast cancer cohort was used to define the relationship between PDCD1 expression and response to chemotherapy and prognosis. Results Nineteen studies were eligible for the meta-analysis with a total of 2403 patients with non-metastatic TNBC disease. The PD-L1-positive cohort had a significantly higher likelihood of achieving pCR with neoadjuvant chemotherapy (pooled odds ratio = 1.95; 95% CI = 1.39–2.73, p < 0.0001). In studies which reported long-term outcomes, PD-L1 positivity was associated with significantly better DFS and OS compared to PD-L1 negative patients (pooled hazard ratio = 0.51; 95% CI = 0.35–0.74, p < 0.0001 and 0.51; 95% CI = 0.27–0.94, p = 0.031, respectively). Transcriptomic data suggested that PD-L1 expression is a surrogate marker for the upregulation of key immune-related genes that mediate response to chemotherapy in TNBC. Conclusion This analysis clearly shows that patients with PD-L1 positive TNBC respond better to neoadjuvant chemotherapy and are associated with better survival outcomes compared to patients with PD-L1 negative tumours. The newly distinct quadruple negative breast cancer (QNBC) subtype should be defined as the BC subtype with the poorest outcome in the non-metastatic setting, highlighting the need for more aggressive therapy approaches.

Thymoquinone Alterations of the Apoptotic Gene Expressions and Cell Cycle Arrest in Genetically Distinct Triple-Negative Breast Cancer Cells.

Breast cancer (BC) is the most common cancer in women worldwide, and it is one of the leading causes of cancer death in women. triple-negative breast Cancer (TNBC), a subtype of BC, is typically associated with the highest pathogenic grade and incidence in premenopausal and young African American (AA) women. Chemotherapy, the most common treatment for TNBC today, can lead to acquired resistance and ineffective treatment. Therefore, novel therapeutic approaches are needed to combat medication resistance and ineffectiveness in TNBC patients. Thymoquinone (TQ) is shown to have a cytotoxic effect on human cancer cells in vitro. However, TQ’s mode of action and precise mechanism in TNBC disease in vitro have not been adequately investigated. Therefore, TQ’s effects on the genetically different MDA-MB-468 and MDA-MB-231 human breast cancer cell lines were assessed. The data obtained show that TQ displayed cytotoxic effects on MDA-MB-468 and MDA-MB-231 cells in a time- and concentration-dependent manner after 24 h, with IC50 values of 25.37 µM and 27.39 µM, respectively. Moreover, MDA-MB-231 and MDA-MB-468 cells in a scratched wound-healing assay displayed poor wound closure, inhibiting invasion and migration via cell cycle blocking after 24 h. TQ arrested the cell cycle phase in MDA-MB-231 and MDA-MB-468 cells. The three cell cycle stages in MDA-MB-468 cells were significantly affected at 15 and 20 µM for G0/G1 and S phases, as well as all TQ concentrations for G2/M phases. In MDA-MB-468 cells, there was a significant decrease in G0/G1 phases with a substantial increase in the S phase and G2/M phases. In contrast, MDA-MB-231 showed a significant effect only during the two cell cycle stages (S and G2/M), at concentrations of 15 and 20 µM for S phases and all TQ values for G2/M phases. The TQ effect on the apoptotic gene profiles indicated that TQ upregulated 15 apoptotic genes in MDA-MB-231 TNBC cells, including caspases, GADD45A, TP53, DFFA, DIABLO, BNIP3, TRAF2/3, and TNFRSF10A. In MDA-MB-468 cells, 16 apoptotic genes were upregulated, including TNFRSF10A, TNF, TNFRSF11B, FADD TNFRSF10B, CASP2, and TRAF2, all of which are important for the apoptotic pathway andsuppress the expression of one anti-apoptotic gene, BIRC5, in MDA-MB-231 cells. Compared to MDA-MB-231 cells, elevated levels of TNF and their receptor proteins may contribute to their increased sensitivity to TQ-induced apoptosis. It was concluded from this study that TQ targets the MDA-MB-231 and MDA-MB-468 cells differently. Additionally, due to the aggressive nature of TNBC and the lack of specific therapies in chemoresistant TNBC, our findings related to the identified apoptotic gene profile may point to TQ as a potential agent for TNBC therapy.

Abstract GS3-06: Primary results of the cTRAK TN trial: A clinical trial utilising ctDNA mutation tracking to detect minimal residual disease and trigger intervention in patients with moderate and high risk early stage triple negative breast cancer

Abstract Background Detection of circulating tumour DNA (ctDNA) in patients (pts) who have completed treatment for early-stage triple negative breast cancer (TNBC) is associated with a very high risk of future relapse. Identifiying those at high risk of subsequent relapse may allow tailoring of further therapy to delay or prevent recurrence. The c-TRAK TN trial assessed the utility of prospective ctDNA surveillance in pts treated for TNBC and the activity of pembrolizumab (P) in pts with ctDNA detected.. Methods c-TRAK TN, a multi-centre phase II trial with integrated prospective screening component, enrolled pts with early-stage TNBC and either residual disease following neoadjuvant chemotherapy, or tumour size &amp;gt;20mm and/or axillary lymph node involvement if adjuvant chemotherapy was given. Tumour tissue was sequenced to identify somatic mutations suitable for tracking using personalised digital PCR ctDNA assays (BioRad QX200). Pts had “active” ctDNA surveillance via blood sample testing every 3 months to 12 months (potential up to 18 months if samples missed due to COVID) during which time if ctDNA was detected (ctDNA+) pts could be randomised 2:1 to P (200mg i.v. q 3 weeks for 1 year) or observation (Obs). Pts and clinicians were blinded to ctDNA+ results unless they were allocated P, when staging scans were done and those free of clinical recurrence started treatment. Following advice from the Independent Data Monitoring Committee, the Obs arm closed on 16/06/2020 with all subsequent ctDNA+ pts allocated P. Following the completion of active ctDNA surveillance, 3-monthly visits continued to 24 months to be analysed retrospectively. The aim was to recruit 150 pts to ctDNA surveillance, assuming 30% would be ctDNA+ within 12 months, allowing ctDNA+ rate to be estimated with a 2-sided 95%CI of +/- 7.3%. Co-primary endpoints are i) rates of ctDNA detection by 12 and 24 months from start of ctDNA surveillance; ii) rates of sustained ctDNA clearance on P defined as absence of detectable ctDNA, or disease recurrence 6 months after starting P.. Results 208 pts were registered between 30/01/18 and 06/12/19, 185 had tumour sequenced, 171 (92.4%) had trackable mutations, and 161 entered ctDNA surveillance. The rate of ctDNA detection by 12 months after start of surveillance was 27.3% (44/161, 95% CI 20.6-34.9). ctDNA+ rates from baseline, 3, 6, 9 and 12 month ctDNA samples were 23/161 (14.3%), 6/115 (5.2%), 6/99 (5.1%), 7/84 (8.3%), and 2/84 (2.4%) respectively. An additional 2 pts were ctDNA+ on COVID extended active surveillance at 15 (1/51, 2%) or 18 months (1/11, 9%). 7 pts relapsed without prior ctDNA detection. 45 pts entered the therapeutic component of the trial (initially 31 to P and 14 to Obs). 1 Obs pt was re-allocated to P. Of pts allocated to P, 72% (23/32) had metastatic disease at time of ctDNA detection on staging scans (75% (12/16) who were ctDNA+ at baseline and 69% (11/16) at other timepoints). 4 pts declined to start P, largely due to COVID concerns. Of the 5 pts who commenced P, at the time of analysis none achieved sustained ctDNA clearance and 4 had recurred. In pts allocated to Obs, median time to recurrence was 4.1 months (95% CI: 3.2-not-defined).. Conclusion The c-TRAK TN trial is to our knowledge the first study to assess the proof-of-principle of whether ctDNA assays have clinical utility in guiding further therapy in TNBC. Relatively few pts commenced P treatment precluding assessment of potential activity. At enrollment, patients had a relatively high of rate of undiagnosed metastatic disease when imaged. Our findings have implications for future trial design, emphasizing the importance of early start of ctDNA testing, and more sensitive and/or more frequent ctDNA testing regimes. Citation Format: Nicholas Turner, Claire Swift, Ben Jenkins, Lucy Kilburn, Maria Coakley, Matthew Beaney, Lisa Fox, Katie Goddard, Isaac Garcia-Murillas, Peter Hall, Catherine Harper-Wynne, Tamas Hickish, Sarah Kernaghan, Iain Macpherson, Alicia Okines, Carlo Palmieri, Sophie Perry, Katrina Randle, Claire Snowdon, Hilary Stobart, Andrew Wardley, Duncan Wheatley, Simon Waters, Matthew Winter, Judith Bliss. Primary results of the cTRAK TN trial: A clinical trial utilising ctDNA mutation tracking to detect minimal residual disease and trigger intervention in patients with moderate and high risk early stage triple negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr GS3-06.

Abstract PD3-10: Dual epigenetic/autophagy inhibition as a novel strategy to tackle triple negative breast cancer

Abstract Clinical Significance: Despite initial response to therapies, patients with triple negative breast cancer (TNBC) have the highest risk of metastatic relapse within 5 years of diagnosis. There is an urgent need for better treatments for this deadly disease. To this end, understanding the molecular drivers can reveal novel therapeutic targets. Background: The TNBC oncogenic program is known to be driven by MYC. MYC expression is regulated by bromodomain proteins and several bromodomain inhibitors such as JQ1 have proven effective at inhibiting MYC expression. However, resistance can occur rapidly upon activation of alternative cell survival mechanisms, such as autophagy. TNBC is noted for its elevated basal autophagy. Interestingly, publicly available datasets show that ULK3, an initial effector of the autophagy program, is upregulated in TNBC patients while the opposite is observed for the classical autophagy initiator ULK1. Moreover, TNBC patients with low ULK3 expression levels tend to achieve longer relapse free survival (RFS) than higher expression cohort, in opposition to ULK1. Importantly, ULK3 has not been investigated to date in regulating TNBC cell intrinsic autophagy and no drugs exist that can inhibit ULK3. Methods and Results: Given the importance of MYC and ULK3 in TNBC progression, our team developed a novel class of small molecules, namely dual BRD4/ULK3 inhibitors, that are superior to JQ1 alone in limiting TNBC viability. In vitro data demonstrate that TNBC cells are more sensitive to the cytotoxic activity of the dual inhibitors compared to other breast cancer subtypes. Further investigation through western blot analysis confirms the effectiveness of these novel dual inhibitors in abrogating MYC expression overtime, and completely blocking the autophagy program (measured by downstream ULK3, ULK1, LC3B, p62 protein levels), resulting in cell death. Interestingly, the human cell line SUM159R, that is resistant to BRD4 inhibitor JQ1 and cross-resistant to standard chemotherapeutics (doxorubicin) is also highly sensitive to our dual inhibitors, implying their potential use for the treatment of refractory TNBC disease. Excitingly, and for the first time, using a genetic silencing approach (shULK3 in SUM159), we demonstrated that ULK3 is critical for TNBC autophagy and cell survival. As a proof of concept, we reproduced the same genetic silencing experiment in a different cell line, namely WM1366-LC3BmCherry-GFP, an LC3B conveniently engineered system used to visualize proper autophagosome formation and material degradation through autophagy. We verified that both genetic ablation and pharmacologic inhibition of ULK3 are strategies that lead to cancer cell death. Conclusions: In conclusion, we 1) characterized a novel class of dual BRD4/kinase compounds, capable of inhibiting BRD4 activity and autophagy, and superior to the BRD4 inhibitor, JQ1. 2) showed that ULK3 is over-expressed in TNBC. 3) demonstrated that silencing ULK3 blocks autophagy in TNBC cell lines and results in significantly lower cell viability. 4) found that our dual inhibitors present a double bind for TNBC cells that results in increased cytotoxicity and potentially will be useful for the treatment of resistant TNBC. Clinically, we also posit that our dual inhibitors could be easily administered as a single agent, avoiding the potential complication of pharmacokinetics/pharmacodynamics associated with administering multiple therapies. Citation Format: Marilena Tauro, Tao Li, Conor C Lynch. Dual epigenetic/autophagy inhibition as a novel strategy to tackle triple negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD3-10.

The A20/TNFAIP3-CDC20-CASP1 Axis Promotes Inflammation-mediated Metastatic Disease in Triple-negative Breast Cancer.

The functions of the specific genes involved in the three types of breast cancer (BC) are unclear. A total of 53,805 genes were assessed from the RNA-sequencing database of BC cells and classified into those involved in hormonal positive (HR+) BC and triple-negative breast cancer (TNBC). Overall, distant metastasis-free, and relapse-free survival obtained from the Breast Cancer Gene-Expression Miner database containing 13,603 human breast cancer patient samples were assessed for gene associations using the RNA-sequencing database. To examine cell invasion and cytokine levels, inflammation-related genes were knocked down. The role of inflammation in cancer metastasis was confirmed using inflammatory inhibitors in a three-dimensional organoid ex vivo. Genes affecting inflammation and cancer metastasis were highly expressed in TNBC, unlike HR+ BC. The A20/TNFAIP3-CDC20-CASP1 axis, which includes inflammation-related genes found in TNBC, was associated with poor patient prognosis, cancer metastasis, and cytokine levels. Inflammation inhibitors prevented the metastasis of aggressive TNBC. The A20/TNFAIP3-CDC20-CASP1 axis is closely related to the metastatic potential of TNBC, and inflammation inhibitors might be a novel target therapy for TNBC.

YB1 Is a Major Contributor to Health Disparities in Triple Negative Breast Cancer.

Simple SummaryTriple negative breast cancer (TNBC) is a devastating disease that affects many women, due to the lack of FDA-approved targeted therapy. In the absence of cell surface receptors ER, PR, and Her2 that can be targeted with hormonal and antibody treatments, cytotoxic chemotherapy remains the major course of treatment, with a dismal response and rapid recurrence due to the acquisition of resistance. TNBC is also twice as more prevalent in African American (AA) when compared to Caucasian American (CA) women. This study investigated the role of the YB1 gene in the disparities in TNBC between AA and CA women. We found that YB1 is highly expressed in TNBC tumors of AA origin when compared to CAs. Increased expression levels and activity of YB1 correlates with poor disease outcomes, resistance to chemotherapy, and the activation of the cancer stem cell (CSC) phenotype, with higher levels in AA than in CA TNBC tumors. More importantly, we found that the targeted inhibition of the expression and activity of YB1 significantly inhibited the oncogenic behavior of AA tumors through sensitization to chemotherapy and inhibition of CSCs. Our study is the first to show that YB1 activity may be a major biological contributor to the health disparities in TNBC, and that development of therapies that specifically target YB1 could reduce these disparities.Triple negative breast cancer (TNBC) is the most aggressive amongst all breast cancer (BC) subtypes. While TNBC tumors represent less than 20% of all BC subtypes, they are responsible for the most BC-related deaths. More significantly, when considering TNBC incidence across all racial/ethnic groups, TNBC accounts for less than 20% of all BCs. However, in non-Hispanic black women, the incidence rate of TNBC is more than 40%, which may be a contributing factor to the higher BC-related death rate in this population. These disparities remain strong even after accounting for differences in socioeconomic status, healthcare access, and lifestyle factors. Increased evidence now points to biological mechanisms that are intrinsic to the tumor that contribute to disparate TNBC disease burdens. Here, we show that YB1, a multifunction gene, plays a major role in the TNBC disparities between African American (AA) and Caucasian American (CA) women. We show in three independent TNBC tumors cohorts, that YB1 is significantly highly expressed in AA TNBC tumors when compared to CAs, and that increased levels of YB1 correlate with poor survival of AA patients with TNBC. We used a combination of genetic manipulation of YB1 and chemotherapy treatment, both in vitro and in animal models of TNBC to show that YB1 oncogenic activity is more enhanced in TNBC cell lines of AA origin, by increasing their tumorigenic and aggressive behaviors, trough the activation of cancer stem cell phenotype and resistance to chemotherapeutic treatments.

The Role of Breast Cancer Stem Cells in Chemoresistance and Metastasis in Triple-Negative Breast Cancer.

Simple SummaryTriple negative breast cancer lacks targeted therapies and has poor prognosis; chemotherapy is currently the main treatment modality. Growing evidence has shown that breast cancer stem cells are associated with tumor initiation and metastasis and may play a critical role in chemoresistance. Multiple targets against breast cancer stem cells are now under investigation. Recent advances in the role of breast cancer stem cells in triple negative breast cancer and the identification of cancer stem cell biomarkers have paved the way for the development of new targeted therapies. The discovery of potential molecular signaling pathways targeting breast cancer stem cells to overcome chemoresistance and prevent metastasis will improve the overall survival of patients with triple negative breast cancer.Triple negative breast cancer (TNBC) remains an aggressive disease due to the lack of targeted therapies and low rate of response to chemotherapy that is currently the main treatment modality for TNBC. Breast cancer stem cells (BCSCs) are a small subpopulation of breast tumors and recognized as drivers of tumorigenesis. TNBC tumors are characterized as being enriched for BCSCs. Studies have demonstrated the role of BCSCs as the source of metastatic disease and chemoresistance in TNBC. Multiple targets against BCSCs are now under investigation, with the considerations of either selectively targeting BCSCs or co-targeting BCSCs and non-BCSCs (majority of tumor cells). This review article provides a comprehensive overview of recent advances in the role of BCSCs in TNBC and the identification of cancer stem cell biomarkers, paving the way for the development of new targeted therapies. The review also highlights the resultant discovery of cancer stem cell targets in TNBC and the ongoing clinical trials treating chemoresistant breast cancer. We aim to provide insights into better understanding the mutational landscape of BCSCs and exploring potential molecular signaling pathways targeting BCSCs to overcome chemoresistance and prevent metastasis in TNBC, ultimately to improve the overall survival of patients with this devastating disease.

CDK4/6 inhibitors: A potential therapeutic approach for triple negative breast cancer.

Triple negative breast cancer (TNBC) cells lack expression of the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor‐2 (HER‐2). Thus, TNBC does not respond to hormone‐based therapy. TNBC is also an aggressive subtype associated with poorer prognoses compared to other breast cancers. Conventional chemotherapeutics are used to manage TNBC although systemic relapse is common with limited benefits being reported as well as adverse events being documented. Here, we discuss current therapies for TNBC in the neo‐ and adjuvant settings, as well as recent advancements in the targeting of PD‐L1‐positive tumors and inclusion of PARP inhibitors for TNBC patients with BRCA mutations. The recent development of cyclin‐dependent kinase (CDK) 4/6 inhibitors in ER‐positive breast cancers has demonstrated significant improvements in progression free survival in patients. Here, we review preclinical data of CDK 4/6 inhibitors and describe current clinical trials assessing these in TNBC disease.