Chemotherapy In Triple-negative Breast Cancer Research Articles

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 PO1-28-08: The Efficacy and Safety Results of Neoadjuvant Phase II study of Anlotinib plus Sintilimab Combined with Chemotherapy in Triple-negative Breast Cancer (NeoSACT)

Abstract Background: In triple-negative breast cancer (TNBC), the co-administration of programmed mortality 1 (PD-1) inhibitors and chemotherapy has demonstrated enhanced pathological complete response (pCR) rates and improved event-free survival (EFS). Previous research has suggested that antiangiogenic agents possess the ability to normalize aberrant tumor vasculature, thereby converting the immune-suppressive tumor microenvironment into an immune-supportive one. This study aims to assess the effectiveness and safety of combining the neoadjuvant small-molecule antiangiogenic drug Antilotinib with the PD-1 inhibitor Sintilimab and chemotherapy. Methods: In this study, a single-arm, open-label, phase II trial was conducted to evaluate the effectiveness of Anlotinib plus Sintilimab, in conjunction with nab-paclitaxel and carboplatin, followed by epirubicin and cyclophosphamide, in eligible patients with stage II-III TNBC. The treatment regimen consisted of four cycles of nab-paclitaxel (100 mg/m² on day 1, 8 and 15) plus carboplatin (AUC 5) every 21 days, followed by four cycles of epirubicin (90 mg/m2) plus cyclophosphamide (600 mg/m2) every 21 days. Additionally, patients received Sintilimab (200mg) every 21 days and Antilotinib (12mg, po on day 1-14, every 21 days) for eight cycles. Patient enrollment was conducted according to a Simon two-stage design. The primary endpoint was the rate of pCR based on the definition of ypT0/Tis ypN0. Secondary endpoints included residual cancer burden (RCB), EFS, overall survival (OS), adverse events (AE), and immune response biomarkers. Results: Between September 2021 and August 2023, a cohort of 31 patients was enrolled, with a median age of 48 years (range: 30-70 years). Among the participants, 21 out of 31 (67.8%) presented with clinical stage II disease, 23 out of 31 (74.2%) had T2 tumors, and 19 out of 31 (61.3%) exhibited clinically positive lymph nodes. In the initial phase of the study, a total of 11 patients were evaluated, out of which 7 patients achieved a pathological complete response (pCR) after surgery. Consequently, 20 patients were included in the subsequent stage, and among the 19 patients who were evaluable, 13 achieved pCR. One participant withdrew from the study prematurely following the third cycle due to grade 3 liver impairment, resulting in the discontinuation of study therapy as well as subsequent surgery. In the intention-to-treat (ITT) population, the overall pCR rate was determined to be 64.5% (20/31, 95% CI 45.4% - 80.8%). Additionally, 80.6% (25/31, 95%CI 62.5% - 92.5%) of the patients were assessed as having a RCB score of 0 or 1. A total of 31 patients were subjected to adverse event assessment. Among them, 12 patients (38.7%) experienced one or more grade 3/4 adverse events (AEs) according to the Common Terminology Criteria for Adverse Events (CTCAE) version 5.0. The grade 3/4 AEs encompassed rash (7 patients, 22.6%), anemia (4 patients, 12.9%), thrombocytopenia (3 patients, 9.7%), neutropenia (2 patients, 6.4%), leukopenia (2 patients, 6.4%), hypothyroidism (2 patients, 6.4%), and elevated AST/ALT (1 patient, 3.2%). Conclusions: The neoadjuvant regimen comprising the combination of Anlotinib and Sintilimab alongside chemotherapy demonstrated a pCR rate of 64.5%, with no observed emergence of novel toxicity signals. Ongoing investigations encompass biomarker assessments and survival analyses. Pertinent clinical trial details can be found under the identifier NCT04877821. Citation Format: Liulu Zhang, Mei Yang, Ciqiu Yang, Teng Zhu, Hong-Fei Gao, Kun Wang. The Efficacy and Safety Results of Neoadjuvant Phase II study of Anlotinib plus Sintilimab Combined with Chemotherapy in Triple-negative Breast Cancer (NeoSACT) [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 PO1-28-08.

Abstract PO5-13-08: Impact of systemic inflammation markers in response to neoadjuvant pembrolizumab and chemotherapy in triple-negative breast cancer - a retrospective analysis

Abstract Background: Immune checkpoint inhibitors (ICIs) have revolutionized the treatment landscape for triple-negative breast cancer (TNBC). Systemic inflammation has been linked to cancer development and overall poor outcomes. Routine blood parameters have been investigated as potential inflammatory biomarkers in patients (pts) with various types of cancer, such as the derived NLR (neutrophils/(leucocytes-neutrophils) ratio (dNLR). A high dNLR has been associated with worse survival in several types of cancer. Specifically, it has been linked to lower pathologic complete response (pCR) rates after neoadjuvant chemotherapy (CTx). We investigated the association between systemic inflammation markers (dNLR, LIPI score and platelet-to-lymphocyte ratio [PLR]) and pCR in early TNBC pts treated with CTx + pembrolizumab (Pemb). Methods: Retrospective analysis of pts with early TNBC treated with Pemb + CTx from April 2022 to March 2023. dNLR was estimated from analytical values of peripheral blood collected before treatment (baseline). dNLR was calculated as the ratio of the absolute neutrophil number to the difference between absolute total leukocyte and absolute neutrophil counts. Univariate and multivariate logistic regression analyses were used to explore the association of dNLR with main clinical characteristics and dNLR capability (distributed as a continuous variable, using median cut-off) to predict pCR. Results: The study included 86 TNBC pts with a median age of 51 years (IQR: 42.2 – 56-7). Most pts had stage II disease (64%) and presented grade 3 tumours (n=66; 78.5%). HER2 low tumours represented 27% (n=23) of the cohort. Germline mutations were present in 20 pts (23%) with the following distribution: BRCA1 (n=14), BRCA 2 (n=5) and PALB2 (n=1). The median stromal tumor infiltrating lymphocytes (TILs) was 20% (IQR: 10-30%). The median Ki67 was 70% (IQR:50%-80%). All pts received Pemb. The majority of pts received carboplatin plus paclitaxel, followed by EC90 (n=77 (90%), while 10% (n=9) did not receive anthracyclines. Data concerning surgical outcomes was available in 80 pts. Pathological complete response rate was 61.2% (n=49). Pts achieving pCR presented significantly higher TILs (p< 0.01). Residual cancer burden (RCB) class (n=80) was distributed as follows: 0: 61% (n=49); I: 13% (n=10); II: 20% (n=16); III: 1% (n=1) and non-assigned: 5% (n=4). In pts with germline mutations (n=20), the pCR rate was 85% (n=17). Median dNLR at baseline was 1.30 (interquartile range (IQR): 0.99 – 1.86). High baseline dNLR levels considered a continuous variable was associated with a higher likelihood of achieving a pCR in univariate (OR: 2.59; 95% CI: 1.01–6.6; p-value = 0.046) but not in multivariate (OR: 2.33; 95%; CI: 0.85–6.33; p-value = 0.097) logistic regression analysis (adjusted for stage and histologic grade). High baseline dNLR, defined by the median cut-off, was associated with an increased likelihood of achieving pCR in both univariate (OR: 3.85; 95% CI: 1.47–10.1; p-value = 0.006) and multivariate (OR: 3.72; 95% CI: 1.28–10.8; p-value = 0.016) logistic regression analysis (adjusted for stage and histologic grade). Conclusion: In this real-life cohort of early TNBC pts treated with Pemb and CTx we found similar pCR rates (61.2%) to the ones reported in the KN522 trial. No association between PLR or LIPI score with pCR was evidenced. High baseline dNLR (assessed as a continuous variable or by the median cut-off) was associated with a higher likelihood of pCR in the univariate analysis. In the multivariate analysis, only high baseline dNLR defined by the median cut-off retained an association with pCR after stage and histologic grade adjustment. Despite several limitations, these exploratory data provide initial evidence of a potential association between dNLR values and independent prediction of pCR. Further research to assess the role of dNLR as a predictive marker for pCR in early TNBC pts treated with CTx + immunotherapy is needed. Citation Format: Martina Spotti, Elie Rassy, Alessandro Viansone, Fiona Pham, Layal Rached, Barbara Pistilli, Charles-Antoine Dutertre, Joana Mourato Ribeiro. Impact of systemic inflammation markers in response to neoadjuvant pembrolizumab and chemotherapy in triple-negative breast cancer - a retrospective analysis [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 PO5-13-08.

Imaging of tumour microvasculature using high-resolution contrast-enhanced ultrasound with biological markers to characterise response to neoadjuvant chemotherapy in triple-negative breast cancer

Imaging of tumour microvasculature using high-resolution contrast-enhanced ultrasound with biological markers to characterise response to neoadjuvant chemotherapy in triple-negative breast cancer

Chlorin Conjugates in Photodynamic Chemotherapy for Triple-Negative Breast Cancer

Breast cancer (BC) is the most common type of cancer in women and the number of new cases in the US is still increasing each year. Triple-negative breast cancer (TNBC), which comprises 15–20% of all breast cancer, is a heterogeneous disease and is considered the most aggressive type of breast cancer due to the lack of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) expressions for treatments. Traditional chemotherapy is the standard protocol for the treatment of TNBC. Toxicity and multidrug resistance are major drawbacks to chemotherapy. The lack of molecular targets and poor prognosis for TNBC prompts an urgent need to discover novel therapeutic strategies to improve clinical outcomes and quality of life for patients. Photodynamic therapy (PDT) or light treatment is a binary anti-cancer procedure that uses a photosensitizer (PS) that, upon light activation, produces cytotoxic oxygen species, destroying tumor cells. PDT is minimally invasive and can be repeated a few times without accumulating significant toxicity in the surrounding tissues. The primary goal of this study was to investigate in vitro photodynamic chemotherapy as a ternary combination therapy using our synthesized photosensitizers (chlorin–vitamin conjugates and their corresponding indium complexes) co-treated with known chemotherapeutic agents (taxol, doxorubicin, cisplatin, fluorouracil, or methotrexate) in the presence of light and determine the optimum conditions as a pre-clinical study of an enhanced tumoricidal effect against TNBC. Our results indicated that the best combination for an effective chemophotodynamic effect involves a ternary treatment of the indium complex of the chlorin–lipoic acid conjugate (InCLA) co-treated with taxol, which exhibited strong synergism at the nanomolar concentration when combined in the presence of visible light irradiation. Other ternary combinations containing taxol with a synergistic anti-tumor effect against TNBC include chlorin–pantothenic acid (CPA) and chlorin–biotin (CBTN) conjugates. Several other ternary combinations containing InCLA, CBTN, and CPA with either cisplatin, fluorouracil, or methotrexate were identified to generate a synergistic or additive effect. The light dosage remained constant, but the dosages of photosensitizers and chemotherapy drugs were varied to obtain the lowest possible concentration for the desired effect. The synergistic, additive or antagonistic effects of the drug combinations were determined based on the Chou–Talalay method, with InCLA–taxol having the lowest combination index (CI) of 0.25. Fluorescence and transmission electron microscopy (TEM) images provided evidence of apoptosis as the preferred mode of cell death. Our study demonstrated the combination of PDT and chemotherapy as a potential treatment option for TNBC patients.

Impact of HER2 status on clinicopathological characteristics and pathological complete response to neoadjuvant chemotherapy in triple-negative breast cancer.

HER2-low triple-negative breast cancer (TNBC) accounted for up to 34%-39% of primary TNBC and 22.2%-32% of metastatic TNBC. Our study aims to explore the relationship between HER2 expression and clinicopathological characteristics, analyze the impact of HER2 expression on the pathological complete response (pCR) to neoadjuvant chemotherapy (NAC) in TNBC. This study involved 191 patients with TNBC who underwent operation after NAC from October 2021 to August 2022. Clinicopathological characteristics and the frequency of pCR were compared between HER2-low and HER2-0 TNBC. 42.2% (81/191) patients in our cohort were HER2-low. They exhibited differences in menopausal status, body mass index (BMI), androgen receptor (AR) expression, and histological grade (P < 0.05). Particularly, in HER2-low TNBC, AR was associated with tumor size, lymph node metastase, histological grade, and the incidence of multifocal disease (P < 0.05). The total pCR rate of entire cohor was 39.8%. Tumor size (P = 0.025), AR status (P = 0.033) and histological grade (P = 0.007) were significantly associated with the pCR rate of them, while the HER2 status did not exert a similar association. The multivariate analysis revealed that BMI (P = 0.004) and histological grade (P < 0.001) were associated with pCR of HER2-low TNBC, while tumor size (P = 0.034) and AR (P = 0.034) were associated with pCR of HER2-0 TNBC, respectively. In our cohort, HER2-low TNBC patients exhibits specific clinical characteristics and response features to NAC.

The PARTNER trial of neoadjuvant olaparib with chemotherapyin triple-negative breast cancer.

PARTNER is a prospective, phase II-III, randomized controlled clinical trialthat recruited patients with triple-negative breast cancer1,2, who were germlineBRCA1 and BRCA2 wild type3. Here we report the results of the trial.Patients (n = 559) were randomized on a 1:1 basis to receiveneoadjuvant carboplatin-paclitaxel with or without 150 mgolaparib twice daily, ondays 3 to 14, of each of four cycles (gap schedule olaparib, research arm) followed by three cycles of anthracycline-based chemotherapy before surgery. The primary end point was pathologic complete response (pCR)4, and secondary end points included event-free survival (EFS) and overall survival (OS)5. pCR was achieved in 51% of patients in the research arm and 52% in the control arm (P = 0.753). Estimated EFS at 36 months in the research and control arms was 80% and 79% (log-rank P > 0.9), respectively; OS was 90% and 87.2% (log-rank P = 0.8), respectively. In patients with pCR, estimated EFS at 36 months was 90%, and in those with non-pCR it was 70% (log-rank P < 0.001), and OS was 96% and 83% (log-rank P < 0.001), respectively. Neoadjuvant olaparib did not improve pCR rates, EFS or OS when added to carboplatin-paclitaxel and anthracycline-based chemotherapy in patients with triple-negative breast cancerwho were germline BRCA1 and BRCA2 wild type. ClinicalTrials.gov ID: NCT03150576 .

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.

Mammographic density changes after neoadjuvant chemotherapy in triple-negative breast cancer: Association with treatment and survival outcome

PurposeTo investigate the association of mammographic breast density with treatment and survival outcomes in patients with triple-negative breast cancer (TNBC) undergoing neoadjuvant chemotherapy (NAC). MethodsThis retrospective study evaluated 306 women with TNBC who underwent NAC followed by surgery between 2010 and 2019. The baseline density and the density changes after NAC were evaluated. Qualitative breast density (a-d) was evaluated using the Breast Imaging Reporting and Data System. Quantitative breast density (%) was evaluated using fully automated software (the Laboratory for Individualized Breast Radiodensity Assessment) in the contralateral breast. Multivariable logistic regression analysis was used to evaluate the association between breast density and pathologic complete response (pCR), stratified by menopausal status. Cox proportional hazard regression analysis was used to evaluate the association among breast density, the development of contralateral breast cancer, and the development of locoregional recurrence and/or distant metastasis. ResultsContralateral density reduction ≥10 % was independently associated with pCR in premenopausal women (odds ratio [OR], 2.5; p = 0.022) but not in postmenopausal women (OR, 0.9; p = 0.823). During a mean follow-up of 65 months, 10 (3 %) women developed contralateral breast cancer, and 68 (22 %) women developed locoregional recurrences and/or distant metastases. Contralateral density reduction ≥10 % showed no association with the occurrence of contralateral breast cancer (hazard ratio [HR], 3.1; p = 0.308) or with locoregional recurrence and/or distant metastasis (HR, 1.1; p = 0.794). ConclusionIn premenopausal women, a contralateral breast density reduction of ≥10 % after NAC was independently associated with pCR, although it did not translate into improved outcomes.

Sperm-Associated Antigen 5 Knockout Reduces Doxorubicin and Docetaxel Resistance in Triple-Negative Breast Cancer MDA-MB-231 and BT549 Cells.

Sperm-associated antigen 5 (SPAG5), also known as Astrin, was previously demonstrated as a biomarker for cellular resistance to major breast cancer therapies, including chemo-, endocrine- and targeted therapy. However, the contribution of SPAG5 to anthracycline- and taxane-based chemotherapy in triple-negative breast cancer (TNBC) remains controversial. In the present study, the SPAG5 knockout cell model was established by using clustered regularly interspaced palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system in MDA-MB-231 and BT549 TNBC cell lines. The knockout of SPAG5 was confirmed on both gene and protein levels using genomic PCR, DNA sequencing and western blotting. The functional loss of SPAG5 was determined by colony-formation assay. SPAG5-regulated doxorubicin- and docetaxel-resistance was assessed by MTT and apoptosis assays. The results indicated that all the SPAG5 knockout MDA-MB-231 and BT549 clones were biallelic, where one allele was replaced by the donor template, and the other allele had the same "T" insertion (indel) adjacent to the cutting sites of gRNAs at the exon 1 boundary, irrespective of the gRNAs and cell lines. The locus of indel interrupted the SPAG5 transcription by damaging the GT-AG mRNA processing rule. Deletion of SPAG5 decreased clonogenicity in both MDA-MB-231 and BT549 cells. SPAG5 was able to regulate the resistance and the drug-induced apoptosis of both doxorubicin and docetaxel. In conclusion, recombinant plasmid-based CRISPR-Cas9 technology can be used to delete the SPAG5 gene in the TNBC cell lines. SPAG5 has an important role in regulating cell proliferation and doxorubicin- and docetaxel-resistance in MDA-MB-231 and BT549 cells.

Abstract 309: AHNAK nucleoprotein 2 tune oxidative stress and resistance to chemotherapy in triple negative breast cancer

Abstract Background: Resistance to chemotherapy is a major reason for the poorerprognosis in patients with triple negative breast cancer (TNBC). Growing evidence suggests that oxidative stress induced by reactive oxygen species (ROS) is an important factor in both tumourprogression and responses to chemotherapies, however, many signalling pathways and key molecular underlying the regulation of cancer cells to the ROS metabolism remains unclear. Aim: (i) To investigate the mechanistic role of AHNAK nucleoprotein 2 (AHNAK2) in TNBC and its links with breast cancer mortality, particularly caused by chemotherapeutic resistance; and (ii) to test a hypothesis that AHNAK2 facilitates the abnormal activation of ROSscavenging system, result in the resistance to chemotherapy in TNBC. Methods: The expression level of AHNAK2 was determined by immunohistochemical staining and analysed the mRNA expression profiles from The Cancer Genome Atlas of triple negative breast tumors. Roles of AHNAK2 in cancer cell growth, metastasis, chemotherapeutic resistanceand activation of oxygen scavenging systemwere determined by molecular and cell biology methods. A jetPEI nanocarrier was used as the vehicle for anti-AHNAK2 siRNAs in mouse models of cancer therapeutics. Results: AHNAK2 is a novel triple negative breast cancer target, which is overexpressed in TNBC tissues and significantly associated with clinical pathology of chemotherapeutic resistance and poor prognosis of tumour patients. Mechanistically, AHNAK2 antagonizes the ROS accumulation induced by epirubicinvia activating JAK-STAT signaling pathway and transportingphosphorylated STAT1 &amp; STAT4 to the nucleus to promote the expression of SOD2, a key gene in ROS metabolism.And epirubicin enhanced AHNAK2 expression by induced the accumulation of ROS,which hence the Sp1-driven transcription of AHNAK2,result inde novo resistance. Furthermore, AHNAK2-specific siRNAs encapsulated by jetPEI nanocarriers prominently inhibit tumor growth and epirubicin-induced resistance in vivo. Conclusions: AHNAK2 plays a key role in resistance to chemotherapyby regulating the oxidative stressand ROS metabolism,representing a future biomarker and therapeutic target of TNBC. Citation Format: Na Hao. AHNAK nucleoprotein 2 tune oxidative stress and resistance to chemotherapy in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 309.

DDX58 deficiency leads to triple negative breast cancer chemotherapy resistance by inhibiting Type I IFN-mediated signalling apoptosis.

Triple-negative breast cancer (TNBC) is characterized by its aggressive nature and absence of specific therapeutic targets, necessitating the reliance on chemotherapy as the primary treatment modality. However, the drug resistance poses a significant challenge in the management of TNBC. In this study, we investigated the role of DDX58 (DExD/H-box helicase 58), also known as RIG-I, in TNBC chemoresistance. The relationship between DDX58 expression and breast cancer prognosis was investigated by online clinical databases and confirmed by immunohistochemistry analysis. DDX58 was knockout by CRISPR-Cas9 system (DDX58-KO), knockdown by DDX58-siRNA (DDX58-KD), and stably over expressed (DDX58-OE) by lentivirus. Western blotting, immunofluorescence and qPCR were used for related molecules detection. Apoptosis was analyzed through flow cytometry (Annexin V/7AAD apoptosis assay) and Caspase 3/7 activity assay. Patients with lower expression of DDX58 led to lower rate of pathological complete response (pCR) and worse prognosis by online databases and hospital clinical data. DDX58-KD cells showed multiple chemo-drugs resistance (paclitaxel, doxorubicin, 5-fluorouracil) in TNBC cell lines. Similarly, DDX58-KO cells also showed multiple chemo-drugs resistance in a dosage-dependent manner. In the CDX model, tumours in the DDX58-KO group had a 25% reduction in the tumour growth inhibition rate (IR) compared to wild-type (WT) group after doxorubicin (Dox) treatment. The depletion of DDX58 inhibited proliferation and promoted the migration and invasion in MDA-MB-231 cells. The findings of our research indicated that DDX58-KO cells exhibit a reduction in Dox-induced apoptosis both in vivo and in vitro. Mechanistically, Dox treatment leads to a significant increase in the expression of double-stranded RNAs (dsRNAs) and activates the DDX58-Type I interferon (IFN) signaling pathway, ultimately promoting apoptosis in TNBC cells. In the process of TNBC chemotherapy, the deficiency of DDX58 can inhibit Dox-induced apoptosis, revealing a new pathway of chemotherapy resistance, and providing a possibility for developing personalized treatment strategies based on DDX58 expression levels.

Efficacy of Cold Atmospheric Plasma vs. Chemotherapy in Triple-Negative Breast Cancer: A Systematic Review.

Breast cancer is a growing disease, with a high worldwide incidence and mortality rate among women. Among the various types, the treatment of triple-negative breast cancer (TNBC) remains a challenge. Considering the recent advances in cold atmospheric plasma (CAP) cancer research, our goal was to evaluate efficacy data from studies based on chemotherapy and CAP in TNBC cell lines and animal models. A search of the literature was carried out in the PubMed, Web of Science, Cochrane Library, and Embase databases. Of the 10,999 studies, there were fifty-four in vitro studies, three in vivo studies, and two in vitro and in vivo studies included. MDA-MB-231 cells were the most used. MTT, MTS, SRB, annexin-V/propidium iodide, trypan blue, and clonogenic assay were performed to assess efficacy in vitro, increasing the reliability and comprehensiveness of the data. There was found to be a decrease in cell proliferation after both chemotherapy and CAP; however, different protocol settings, including an extensive range of drug doses and CAP exposure times, were reported. For both therapies, a considerable reduction in tumor volume was observed in vivo compared with that of the untreated group. The treatment of TNBC cell lines with CAP proved successful, with apoptosis emerging as the predominant type of cellular death. This systematic review presents a comprehensive overview of the treatment landscape in chemotherapy and CAP regarding their efficacy in TNBC cell lines.

ACSL4-mediated membrane phospholipid remodeling induces integrin β1 activation to facilitate triple-negative breast cancer metastasis.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer and has a poor prognosis and a high propensity to metastasize. Lipid metabolism has emerged as a critical regulator of tumor progression and metastasis in other cancer types. Characterization of the lipid metabolic features of TNBC could provide important insights into the drivers of TNBC metastasis. Here, we showed that metastatic TNBC tumors harbor more unsaturated phospholipids, especially long-chain polyunsaturated fatty acids, at the sn-2 position of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) compared to primary tumors. Metastatic TNBC tumors upregulated ACSL4, a long-chain polyunsaturated acyl-CoA synthetase that drives the preferential incorporation of polyunsaturated fatty acids into phospholipids, resulting in the alteration of membrane phospholipid composition and properties. Moreover, ACSL4-mediated phospholipid remodeling of the cell membrane induced lipid-raft localization and activation of integrin β1 in a CD47-dependent manner, which led to downstream focal adhesion kinase (FAK) phosphorylation that promoted metastasis. Importantly, pharmacological inhibition of ACSL4 suppressed tumor growth and metastasis and increased chemosensitivity in TNBC models in vivo. These findings indicate that ACSL4-mediated phospholipid remodeling enables TNBC metastasis and can be inhibited as a potential strategy to improve the efficacy of chemotherapy in TNBC.

GSH-activable heterotrimeric nano-prodrug for precise synergistic therapy of TNBC

Combination chemotherapy is an effective approach for triple-negative breast cancer (TNBC) therapy, especially when drugs are administered at specific optimal ratios. However, at present, strategies involving precise and controllable ratios based on effective loading and release of drugs are unavailable. Herein, we designed and synthesized a glutathione (GSH)--responsive heterotrimeric prodrug and formulated it with an amphiphilic polymer to obtain nanoparticles (DSSC2 NPs) for precise synergistic chemotherapy of TNBC. The heterotrimeric prodrug was prepared using docetaxel (DTX) and curcumin (CUR) at the optimal synergistic ratio of 1: 2. DTX and CUR were covalently conjugated by disulfide linkers. Compared with control NPs, DSSC2 NPs had quantitative/ratiometric drug loading, high drug co-loading capacity, better colloidal stability, and less premature drug leakage. After systemic administration, DSSC2 NPs selectively accumulated in tumor tissues and released the encapsulated drugs triggered by high levels of GSH in cancer cells. In vitro and in vivo experiments validated that DSSC2 NPs released DTX and CUR at the predefined ratio and had a highly synergistic therapeutic effect on tumor suppression in TNBC, which can be attributed to ratiometric drug delivery and synchronous drug activation. Altogether, the heterotrimeric prodrug delivery system developed in this study represents an effective and novel approach for combination chemotherapy.

Assessment of the Efficacy of the Combination of RNAi of lncRNA DANCR with Chemotherapy to Treat Triple Negative Breast Cancer Using Magnetic Resonance Molecular Imaging.

Long noncoding RNA (lncRNA) differentiation antagonizing noncoding RNA (DANCR) is overexpressed in human triple-negative breast cancer (TNBC) and promotes cell migration and proliferation. TNBC is limited in treatment options relative to hormone-receptor-positive breast cancer and is commonly treated with chemotherapy, which is often compromised by acquired resistance. DANCR has been implicated in the development of chemoresistance across multiple cancer types. Here, we applied magnetic resonance molecular imaging (MRMI) with a targeted contrast agent, MT218, specific to extradomain-B fibronectin (EDB-FN), a marker for epithelial-to-mesenchymal transition, to assess the therapeutic efficacy of the combination of paclitaxel and ZD2-PEG-ECO/siDANCR nanoparticles (ZD2-siDANCR-ELNP) to treat TNBC. The treatment of orthotopic MDA-MB-231 TNBC in mice with paclitaxel significantly suppressed tumor growth but with a significant increase of EDB-FN in the tumor, as revealed by MRMI and immunohistochemistry. Combining ZD2-siDANCR-ELNP with paclitaxel further reduced tumor sizes, along with reduced EDB-FN expression. Interestingly, MT218-MRMI revealed a lower reduction of tumor signal enhancement with the combination treatment than that with the siDANCR treatment alone, which was supported by higher cell density in the tumors treated with the combination therapy, as shown by histochemical analysis. MT218-MRMI clearly revealed the changes of the tumor microenvironment in response to various therapies and is effective to noninvasively assess the response of TNBC tumors to the therapies. Regulating oncogenic lncRNA DANCR is an effective strategy for improving the outcomes of chemotherapy in TNBC.

SIRT3 Expression Predicts Overall Survival and Neoadjuvant Chemosensitivity in Triple-Negative Breast Cancer.

The Sirtuin (SIRT) family consists of seven evolutionary conserved NAD-dependent deacetylases that play important roles in various cancers, including breast cancer (BC). SIRTs expression has been reported to have prognostic value in BC, but these studies used limited sample size and yielded inconsistent conclusions. This study evaluated the association of SIRT3 and other SIRT family members with survival and neoadjuvant chemotherapy outcomes. BC patients' data was obtained from the TCGA-BRCA, METABRIC and GEO databases, comprising 4336 samples. SIRTs expression and overall survival (OS) were analyzed using Kaplan-Meier analysis and Cox proportional hazards regression. SIRT3 expression levels were compared between pathologic complete response (pCR) and non-pCR groups after neoadjuvant chemotherapy in triple-negative breast cancer (TNBC). Protein-protein interaction networks were constructed using the STRING database. Gene set enrichment analysis (GSEA) was performed to explore potential functions of SIRT3. Through systematic analysis of SIRTs expression and OS of BC using three independent cohorts: TCGA-BRCA, METABRIC and GSE16446, we found that high SIRT3 expression was significantly associated with worse OS in TNBC in the TCGA-BRCA cohort, which was validated in the METABRIC and GSE16446 cohorts. SIRT3 expression was correlated with BC subtypes and American Joint Committee on Cancer (AJCC) T stage, but not with age-at-diagnosis, race, or tumor stage. Moreover, TNBC patients with higher SIRT3 expression had lower pCR rates after neoadjuvant chemotherapy (p = 6.40e-03) and SIRT3 expression was significantly lower in the pCR group than in the non-pCR group in TNBC (p = 4.2e-03). GSEA indicated that SIRT3 was involved in drug-related pathways such as oxidative phosphorylation, metabolism of xenobiotics by cytochrome P450, and drug metabolism. Our study suggests that SIRT3 is a potential biomarker for both OS and neoadjuvant chemosensitivity in TNBC. It may also assist in selecting suitable candidates and treatment options for TNBC patients.

Research and experimental verification on the mechanisms of cellular senescence in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with high heterogeneity, poor prognosis, and a low 10-year survival rate of less than 50%. Although cellular senescence displays extensive effects on cancer, the comprehensions of cellular senescence-related characteristics in TNBC patients remains obscure. Single-cell RNA sequencing (scRNA-seq) data were analyzed by Seurat package. Scores for cellular senescence-related pathways were computed by single-sample gene set enrichment analysis (ssGSEA). Subsequently, unsupervised consensus clustering was performed for molecular cluster identification. Immune scores of patients in The Cancer Genome Atlas (TCGA) dataset and associated immune cell scores were calculated using Estimation of STromal and Immune cells in MAlignantTumours using Expression data (ESTIMATE) and Microenvironment Cell Populations-counter (MCP-counter), Tumor Immune Estimation Resource (TIMER) and Estimating the Proportion of Immune and Cancer cells (EPIC) methods, respectively. Immunotherapy scores were assessed using TIDE. Furthermore, feature genes were identified by univariate Cox and Least Absolute Shrinkage and Selection Operator (LASSO) regression analyses; these were used to construct a risk model. Additionally, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and transwell assay were conducted for in vitro validation of hub genes. TNBC was classified into three subtypes based on cellular senescence-related pathways as clusters 1, 2, and 3. Specifically, cluster 1 showed the best prognosis, followed by cluster 2 and cluster 3. The levels of gene expression in cluster 2 were the lowest, whereas these were the highest in cluster 3. Moreover, clusters 1 and 3 showed a high degree of immune infiltration. TIDE scores were higher for cluster 3, suggesting that immune escape was more likely in patients with the cluster 3 subtype who were less likely to benefit from immunotherapy. Next, the TNBC risk model was constructed and validated. RT-qPCR revealed that prognostic risk genes (MMP28, ACP5 and KRT6A) were up-regulated while protective genes (CT83) were down-regulated in TNBC cell lines, validating the results of the bioinformatics analysis. Meanwhile, cellular experiments revealed that ACP5 could promote the migration and invasion abilities in two TNBC cell lines. Finally, we evaluated the validity of prognostic models for assessing TME characteristics and TNBC chemotherapy response. In conclusion, these findings help to assess the efficacy of targeted therapies in patients with different molecular subtypes, have practical applications for subtype-specific treatment of TNBC patients, and provide information on prognostic factors, as well as guidance for the revelation of the molecular mechanisms by which senescence-associated genes influence TNBC progression.

Abstract B038: A preliminary study on predicting pathological complete response to neoadjuvant chemotherapy in triple-negative breast cancer

Abstract Purpose In this preliminary study, we are aiming to develop a deep learning model to predict pathological complete response (pCR) to neoadjuvant chemotherapy (NAC) for triple-negative breast cancer (TNBC) patient based on dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). Background Neoadjuvant chemotherapy (NAC) has become a grown treatment method for patients with triple-negative breast cancer (TNBC). However, not every patient can achieve pathological complete response (pCR) to NAC. NAC also has side effects such as nausea, hair loss, appetite loss, mouth sores, and nerve damage. Additionally, delaying surgery to remove the tumor can allow it to spread, making treatment more difficult. Therefore, it is necessary to predict pCR to NAC reliably in a non-invasive way. Deep learning methods, especially convolutional neural network (CNN), have been shown great success in image-based treatment response prediction. Methods In this preliminary study, totally 78 TNBC patients on DCE-MRI collected from public dataset (Duke University) were used, among which are 23 patients with pCR and 55 patients without pCR. For each patient, the median slice from the tumor area of the fat-saturated gradient echo T1-weighted pre-contrast sequence MRI was extracted. A convolutional neural network model was built to predict whether the patients receive pCR or not after NAC. In the constructed prediction model, three convolutional layers were used to capture the features from DCE-MRI. Each convolutional layer was followed by a max-pooling layer to reduce the dimensions of the feature maps. Two fully connected layers were added into the model so that the model has a binary outcome which provides the prediction of pCR. Additionally, “ReLU” was used as the activation function in all the layers except for the output layer which used “softmax” instead. By using augmentation technique, we solved the problems of small sample size and unbalance data. Results In this study, 75% dataset were used for training and the remaining 25% were used for testing. The model we developed has a 75% accuracy, with a sensitivity of 0.67 and a specificity of 0.79. These results indicate that the model performs well in predicting patients who do not achieve pathological complete response (pCR). Additionally, the area under the receiver operating characteristic (AUC) curve is calculated as 0.74, indicating the outstanding performance. Citation Format: Xi Chen, Zhiguo Zhou. A preliminary study on predicting pathological complete response to neoadjuvant chemotherapy in triple-negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr B038.

Local dose-dense chemotherapy for triple-negative breast cancer via minimally invasive implantation of 3D printed devices

Dose-dense chemotherapy is the preferred first-line therapy for triple-negative breast cancer (TNBC), a highly aggressive disease with a poor prognosis. This treatment uses the same drug doses as conventional chemotherapy but with shorter dosing intervals, allowing for promising clinical outcomes with intensive treatment. However, the frequent systemic administration used for this treatment results in systemic toxicity and low patient compliance, limiting therapeutic efficacy and clinical benefit. Here, we report local dose-dense chemotherapy to treat TNBC by implanting 3D printed devices with time-programmed pulsatile release profiles. The implantable device can control the time between drug releases based on its internal microstructure design, which can be used to control dose density. The device is made of biodegradable materials for clinical convenience and designed for minimally invasive implantation via a trocar. Dose density variation of local chemotherapy using programmable release enhances anti-cancer effects in vitro and in vivo. Under the same dose density conditions, device-based chemotherapy shows a higher anti-cancer effect and less toxic response than intratumoral injection. We demonstrate local chemotherapy utilizing the implantable device that simulates the drug dose, number of releases, and treatment duration of the dose-dense AC (doxorubicin and cyclophosphamide) regimen preferred for TNBC treatment. Dose density modulation inhibits tumor growth, metastasis, and the expression of drug resistance-related proteins, including p-glycoprotein and breast cancer resistance protein. To the best of our knowledge, local dose-dense chemotherapy has not been reported, and our strategy can be expected to be utilized as a novel alternative to conventional therapies and improve anti-cancer efficiency.