Biomarker For Triple-negative Breast Cancer Research Articles

Exploring a novel four-gene system as a diagnostic and prognostic biomarker for triple-negative breast cancer, using clinical variables

Triple-negative breast cancer (TNBC) is a subtype of breast cancer with a poor prognosis. This research aims to find real hub genes for prognostic biomarkers of TNBC therapy. The GEO datasets GSE27447 and GSE233242 were analyzed using R package limma to explore DEGs. The PPI was generated using the STRING database. Cytoscape software plug-ins were used to screen the hub genes. Using the DAVID database, GO functional enrichment and KEGG pathway enrichment analysis were performed. Different online expression databases were employed to investigate the functions of real hub genes in tumor driving, diagnosis, and prognosis in TNBC patients with various clinicopathologic characteristics. A total of one hundred DEGs were identified between both datasets. The seven hub genes were identified after the topological parameter analysis of the PPI network. The KEGG pathway and GO analysis suggest that four genes (PSMB1, PSMC1, PSMF1, and PSMD8) are highly enriched in proteasome and were finally considered as real hub genes. Additionally, the expression analysis demonstrated that hub genes were notably up-regulated in TNBC patients compared to controls. Furthermore, correlational analyses revealed the positive and negative correlations among the expression of the real hub genes and various ancillary data, including tumor purity, promoter methylation status, overall survival (OS), genetic alterations, infiltration of CD8+ T and CD4+ immune cells, and a few more, across TNBC samples. Finally, our analysis identified a couple of significant chemotherapeutic drugs, miRNAs and transcription factors (TFS) with intriguing curative potential. In conclusion, we identified four real hub genes as novel biomarkers to overcome heterogenetic-particular challenges in diagnosis, prognosis, and therapy for TNBC patients.

Radiologic imaging biomarkers in triple-negative breast cancer: a literature review about the role of artificial intelligence and the way forward

Abstract Breast cancer is one of the most common and deadly cancers in women. Triple-negative breast cancer (TNBC) accounts for approximately 10-15% of breast cancer diagnoses and is an aggressive molecular breast cancer subtype associated with important challenges in its diagnosis, treatment, and prognostication. This poses an urgent need for developing more effective and personalized imaging biomarkers for TNBC. Toward this direction, artificial intelligence (AI) for radiologic imaging holds a prominent role, leveraging unique advantages of radiologic breast images, being used routinely for TNBC diagnosis, staging, and treatment planning, and offering high-resolution whole-tumor visualization, combined with the immense potential of AI to elucidate anatomical and functional properties of tumors that may not be easily perceived by the human eye. In this review, we synthesize the current state-of-the-art radiologic imaging applications of AI in assisting TNBC diagnosis, treatment, and prognostication. Our goal is to provide a comprehensive overview of radiomic and deep-learning-based AI developments and their impact on advancing TNBC management over the last decade (2013-2024). For completeness of the review, we start with a brief introduction of AI, radiomics and deep learning. Next, we focus on clinically relevant AI-based diagnostic, predictive and prognostic models for radiologic breast images evaluated in TNBC. We conclude with opportunities and future directions for AI towards advancing diagnosis, treatment response predictions and prognostic evaluations for TNBC.

Abstract A053: Liquid biopsy-based detection of triple negative breast cancer using DNA methylation biomarkers

Abstract Triple-negative breast cancer (TNBC) presents significant clinical challenges due to its aggressive phenotype, rapid progression, and limited targeted treatment options. TNBC often mimics benign lesions on ultrasound and metastatic features can be missed in routine mammography screening leading to misdiagnosis and delayed treatment. These limitations led us to investigate whether a blood-based liquid biopsy could provide a non-invasive, sensitive method for early TNBC detection, potentially overcoming the constraints of conventional imaging techniques. To discover biomarkers for TNBC, we performed targeted bisulfite sequencing on a cohort of 46 breast cancer solid tissue biopsy samples, comprising 19 TNBC and 27 non-TNBC cases. We identified 150 differentially methylated regions (mean length 703bp) separating TNBC and non-TNBC samples. Upon linking each region to its nearest gene, we observed several genes previously associated with prognosis and survival (HOXB3, PAX9, SOX9) substantiating the clinical relevance of these biomarkers. Using public TCGA data (369 breast cancer samples), we integrated expression and methylation data and observed directionally consistent changes. For example, HOXB3 showed increased methylation at its associated differential regions and decreased expression in TNBC cases. To ascertain whether these biomarkers could be useful in a liquid biopsy approach, we performed targeted bisulfite- sequencing on cell-free DNA samples derived from prospectively collected patients with breast cancer. All samples had independent tissue-based assessment of ER, PR and HER2 using immunohistochemistry or in situ hybridization. We then built a feed-forward neural network classifier with classes TNBC and non-TNBC that used transfer-learning and the identified biomarkers to learn methylation signatures in the biopsy samples and map them to cell-free DNA. To emulate clinical workflow, where subtyping follows cancer diagnosis and tissue of origin identification, we evaluated cfDNA samples correctly identified as breast cancer using two independent machine learning models trained using 2,393 prospectively collected multi- indication cancer and non-cancer samples (NCT05435066). Subsequent TNBC subtyping of breast cancer-positive cases (N=56) accurately identified 77% (10/13) of TNBC samples and 91% (39/43) of non-TNBC cases, with overall accuracy of 84%. The estimated tumor content range for correctly predicted TNBC samples was 0.5-33% with the three mis-classified samples all showing very low tumor content (0.22%, 0.28%, 0.59%). Non-TNBC cases showed a tumor content range of 0.07-36% for correct and 2.3-14% for misclassified samples. In conclusion, we identified novel methylation biomarkers that distinguish TNBC in the cell-free DNA of patients without needing invasive tissue biopsies. The high sensitivity of our assay at low tumor fractions has the potential to improve outcomes through earlier intervention and may enable earlier screening in high-risk groups, monitoring of minimal residual disease and longitudinal monitoring during treatment. Citation Format: Jocelyn Charlton, Shiva Farashahi, Kade Pettie, Elie Massaad, Josh Hubbell, Feras Hantash, Kieran I Chacko. Liquid biopsy-based detection of triple negative breast cancer using DNA methylation biomarkers [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A053.

Comprehensive analysis of the metabolomics and transcriptomics uncovers the dysregulated network and potential biomarkers of Triple Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is known for its aggressive nature, lack of effective diagnostic tools and treatments, and generally poor prognosis. The objective of this study was to investigate metabolic changes in TNBC using metabolomics approaches and explore the underlying mechanisms through integrated analysis with transcriptomics. In this study, serum untargeted metabolic profiles were first examined between 18 TNBC patients and 21 healthy control (HC) subjects using liquid chromatography-mass spectrometry (LC-MS), identifying a total of 22 significantly differential metabolites (DMs). Subsequently, receiver operating characteristic analysis revealed that 7-methylguanine could serve as a potential biomarker for TNBC in both the discovery and validation sets. Additionally, transcriptomic datasets were retrieved from the GEO database to identify differentially expressed genes (DEGs) between TNBC and normal tissues. An integrative analysis of the DMs and DEGs was conducted, uncovering potential molecular mechanisms underlying TNBC. Notably, three pathways-tyrosine metabolism, phenylalanine metabolism, and glycolysis/gluconeogenesis-were enriched, providing insight into the energy metabolism disorders in TNBC. Within these pathways, two DMs (4-hydroxyphenylacetaldehyde and oxaloacetic acid) and six DEGs (MAOA, ADH1B, ADH1C, AOC3, TAT, and PCK1) were identified as key components. In summary, this study highlights metabolic biomarkers that could potentially be used for the diagnosis and screening of TNBC. The comprehensive analysis of metabolomics and transcriptomics data offers a validated and in-depth understanding of TNBC metabolism.

Deciphering the landscape of triple negative breast cancer from microenvironment dynamics and molecular insights to biomarker analysis and therapeutic modalities.

Triple negative breast cancer (TNBC) displays a notable challenge in clinical oncology due to its invasive nature which is attributed to the absence of progesterone receptor (PR), estrogen receptor (ER), and human epidermal growth factor receptor (HER-2). The heterogenous tumor microenvironment (TME) of TNBC is composed of diverse constituents that intricately interact to evade immune response and facilitate cancer progression and metastasis. Based on molecular gene expression, TNBC is classified into four molecular subtypes: basal-like (BL1 and BL2), luminal androgen receptor (LAR), immunomodulatory (IM), and mesenchymal. TNBC is an aggressive histological variant with adverse prognosis and poor therapeutic response. The lack of response in most of the TNBC patients could be attributed to the heterogeneity of the disease, highlighting the need for more effective treatments and reliable prognostic biomarkers. Targeting certain signaling pathways and their components has emerged as a promising therapeutic strategy for improving patient outcomes. In this review, we have summarized the interactions among various components of the dynamic TME in TNBC and discussed the classification of its molecular subtypes. Moreover, the purpose of this review is to compile and provide an overview of the most recent data about recently discovered novel TNBC biomarkers and targeted therapeutics that have proven successful in treating metastatic TNBC. The emergence of novel therapeutic strategies such as chemoimmunotherapy, chimeric antigen receptor (CAR)-T cells-based immunotherapy, phytometabolites-mediated natural therapy, photodynamic and photothermal approaches have made a significant positive impact and have paved the way for more effective interventions.

MIR4435-2HG: A novel biomarker for triple-negative breast cancer diagnosis and prognosis, activating cancer-associated fibroblasts and driving tumor invasion through EMT associated with JNK/c-Jun and p38 MAPK signaling pathway activation

BackgroundBreast cancer has the highest incidence rate and causes the most fatalities among all female cancers worldwide. Triple-negative breast cancer (TNBC) is known for its strong invasiveness and higher rates of recurrence. In this research, we aimed to identify MIR4435-2HG as a promising long non-coding RNA (lncRNA) biomarker and therapeutic target for TNBC. MethodsUtilizing clinicopathological information and transcriptome data from The Cancer Genome Atlas (TCGA) database, we assessed the clinical relevance of MIR4435-2HG in breast cancer through univariate and multivariate COX regression, receiver operating characteristic (ROC) analysis, as well as Kaplan-Meier survival analysis. To investigate the biological role of MIR4435-2HG in TNBC, we conducted gene set enrichment analysis (GSEA), as well as Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Additionally, we constructed and validated a nomogram to predict disease-free survival (DFS). Both the R package “pRRophetic” and the Tumor Immune Dysfunction and Exclusion (TIDE) algorithm were employed to forecast the sensitivity to different therapeutics between the high- and low-MIR4435-2HG groups. We employed single-cell RNA sequencing analysis and tumor microenvironment infiltration analysis to investigate the potential involvement of MIR4435-2HG in the TNBC tumor microenvironment. Cellular biological behaviors were assessed utilizing CCK-8, transwell assays, and wound-healing assays. Furthermore, we performed RNA-seq, qRT-PCR, and western blotting analyses to elucidate and confirm the specific mechanisms underlying the role of MIR4435-2HG in TNBC. ResultsIn our study, we have identified MIR4435-2HG as a significant diagnostic and prognostic factor for TNBC. We observed that MIR4435-2HG is widely expressed and might have a significant impact on the reshaping of the TNBC tumor microenvironment. Patients with TNBC in the high-MIR4435-2HG group may show reduced sensitivity to cisplatin, doxorubicin, and gemcitabine and have an increased propensity for immune escape. Knockdown of MIR4435-2HG inhibits cancer-associated fibroblasts (CAFs) activation. Notably, MIR4435-2HG predominantly enhances the migratory and invasive capabilities of TNBC cells through the epithelial-mesenchymal transition (EMT) process. Mechanistically, we validated that MIR4435-2HG activates the JNK/c-Jun and p38 non-classical MAPK signaling pathway in TNBC cells. ConclusionsOur findings highlight the significant potential of MIR4435-2HG as a highly promising biomarker for TNBC. Targeting MIR4435-2HG could represent an appealing therapeutic approach for TNBC.

Major considerations on the relationship of triple negative breast cancer with microRNAs and nutrological triggers: a systematic review

Introduction: Triple-negative breast cancer (TNBC) accounts for 15% of all cases. Its incidence is usually higher in young women (under 40 years of age). The classification as triple-negative occurs because, in this case, there are no estrogen, progesterone, and HER2 receptors, which are responsible for controlling tumor growth. Higher levels of physical activity and better diet quality are associated with lower mortality from breast cancer in observational studies. Furthermore, physical activity and optimal nutrition can improve the relative dose intensity of chemotherapy, as they can activate miRNAs that regulate several biological processes. Objective: This was to conduct a systematic review to establish the main considerations of the relationship between triple-negative breast cancer and microRNAs as biomarkers and regulators of gene expression in cancer cells, as well as to show some nutritional triggers of microRNA activation desirable for breast cancer control. Methods: The PRISMA Platform systematic review rules were followed. The search was conducted from April to June 2024 in the Web of Science, Scopus, PubMed, Science Direct, Scielo, and Google Scholar databases. The quality of the studies was based on the GRADE and AMSTAR-2 instrument and the risk of bias was analyzed appropriately. According to the Cochrane instrument. Results and Conclusion: 112 articles were found, and 40 were evaluated in full and included in this article, 22 of which were included in the systematic review. Considering the Cochrane tool for risk of bias, the global assessment resulted in 15 studies with a high risk of bias and 25 studies that did not reach GRADE and AMSTAR-2. Most studies showed homogeneity in their results, with X2=81.5%>50%. It was concluded that an increasing number of studies have shown that non-coding RNA (ncRNA), including microRNA (miRNA) and long non-coding RNA (lncRNA), play a significant role in tumorigenesis. Although a diet and exercise intervention did not affect relative dose intensity, the intervention was associated with a higher pCR in patients with hormone receptor-positive/human epidermal growth factor receptor 2-negative and triple-negative breast cancer undergoing neoadjuvant chemotherapy. High expression levels of miR-27a/b, miR-210, and miR-454 were associated with shorter overall survival, whereas high expression levels of miR-454 and miR-374a/b were associated with disease-free survival. The miRNAs associated with triple-negative breast cancer may provide new avenues for early diagnosis and treatment of breast cancer. Furthermore, specific miRNAs may serve as potential prognostic biomarkers in triple-negative breast cancer.

TBC1 domain-containing proteins are frequently involved in triple-negative breast cancers in connection with the induction of a glycolytic phenotype

Metabolic plasticity is a hallmark of cancer, and metabolic alterations represent a promising therapeutic target. Since cellular metabolism is controlled by membrane traffic at multiple levels, we investigated the involvement of TBC1 domain-containing proteins (TBC1Ds) in the regulation of cancer metabolism. These proteins are characterized by the presence of a RAB-GAP domain, the TBC1 domain, and typically function as attenuators of RABs, the master switches of membrane traffic. However, a number of TBC1Ds harbor mutations in their catalytic residues, predicting biological functions different from direct regulation of RAB activities. Herein, we report that several genes encoding for TBC1Ds are expressed at higher levels in triple-negative breast cancers (TNBC) vs. other subtypes of breast cancers (BC), and predict prognosis. Orthogonal transcriptomics/metabolomics analysis revealed that the expression of prognostic TBC1Ds correlates with elevated glycolytic metabolism in BC cell lines. In-depth investigations of the three top hits from the previous analyses (TBC1D31, TBC1D22B and TBC1D7) revealed that their elevated expression is causal in determining a glycolytic phenotype in TNBC cell lines. We further showed that the impact of TBC1D7 on glycolytic metabolism of BC cells is independent of its known participation in the TSC1/TSC2 complex and consequent downregulation of mTORC1 activity. Since TBC1D7 behaves as an independent prognostic biomarker in TNBC, it could be used to distinguish good prognosis patients who could be spared aggressive therapy from those with a poor prognosis who might benefit from anti-glycolytic targeted therapies. Together, our results highlight how TBC1Ds connect disease aggressiveness with metabolic alterations in TNBC. Given the high level of heterogeneity among this BC subtype, TBC1Ds could represent important tools in predicting prognosis and guiding therapy decision-making.

Identification of TAT as a Biomarker Involved in Cell Cycle and DNA Repair in Breast Cancer.

Breast cancer (BC) is the most frequently diagnosed cancer and the primary cause of cancer-related mortality in women. Treatment of triple-negative breast cancer (TNBC) remains particularly challenging due to its resistance to chemotherapy and poor prognosis. Extensive research efforts in BC screening and therapy have improved clinical outcomes for BC patients. Therefore, identifying reliable biomarkers for TNBC is of great clinical importance. Here, we found that tyrosine aminotransferase (TAT) expression was significantly reduced in BC and strongly correlated with the poor prognosis of BC patients, which distinguished BC patients from normal individuals, indicating that TAT is a valuable biomarker for early BC diagnosis. Mechanistically, we uncovered that methylation of the TAT promoter was significantly increased by DNA methyltransferase 3 (DNMT3A/3B). In addition, reduced TAT contributes to DNA replication and cell cycle activation by regulating homologous recombination repair and mismatch repair to ensure genomic stability, which may be one of the reasons for TNBC resistance to chemotherapy. Furthermore, we demonstrated that Diazinon increases TAT expression as an inhibitor of DNMT3A/3B and inhibits the growth of BC by blocking downstream pathways. Taken together, we revealed that TAT is silenced by DNMT3A/3B in BC, especially in TNBC, which promotes the proliferation of tumor cells by supporting DNA replication, activating cell cycle, and enhancing DNA damage repair. These results provide fresh insights and a theoretical foundation for the clinical diagnosis and treatment of BC.

Integrative Multiomic Profiling of Triple-Negative Breast Cancer for Identifying Suitable Therapies.

Triple-negative breast cancer (TNBC) is a heterogeneous disease that carries the poorest prognosis of all breast cancers. Although novel TNBC therapies in development are frequently targeted toward tumors carrying a specific genomic, transcriptomic, or protein biomarker, it is poorly understood how these biomarkers are correlated. To better understand the molecular features of TNBC and their correlation with one another, we performed multimodal profiling on a cohort of 95 TNBC. Our approach involved quantifying tumor-infiltrating lymphocytes through hematoxylin and eosin staining, assessing the abundance of retinoblastoma, androgen receptor, and PDL1 proteins through IHC, and carrying out transcriptomic profiling using the NanoString BC360 platform, targeted DNA sequencing on a subset of cases, as well as evaluating associations with overall survival. Levels of RB1 mRNA and RB proteins are better correlated with markers of retinoblastoma functionality than RB1 mutational status. Luminal androgen receptor tumors clustered into two groups with transcriptomes that cluster with either basal or mesenchymal tumors. Tumors classified as PDL1-positive by the presence of immune or tumor cells showed similar biological characteristics. HER2-low TNBC showed no distinct biological phenotype when compared with HER2-zero. The majority of TNBC were classified as basal or HER2-enriched by PAM50, the latter showing significantly improved overall survival. Our study contributes new insights into biomarker utility for identifying suitable TNBC therapies and the intercorrelations between genomic, transcriptomic, protein, and cellular biomarkers. Additionally, our rich data resource can be used by other researchers to explore the interplay between DNA, RNA, and protein biomarkers in TNBC.

High Caveolin-1 mRNA expression in triple-negative breast cancer is associated with an aggressive tumor microenvironment, chemoresistance, and poor clinical outcome.

Currently, there are few treatment-predictive and prognostic biomarkers in triple-negative breast cancer (TNBC). Caveolin-1 (CAV1) is linked to chemoresistance and several important processes involved in tumor progression and metastasis, such as epithelial-mesenchymal transition (EMT). Herein, we report that high CAV1 gene expression is an independent factor of poor prognosis in TNBC. CAV1 gene expression was compared across different molecular features (e.g., PAM50 subtypes). CAV1 expression was assessed in relation to clinical outcomes using Cox regression adjusted for clinicopathological predictors. Differential gene expression and gene set enrichment analyses were applied to compare high- and low-expressing CAV1 tumors. Tumor microenvironment composition of high- and low-expressing CAV1 tumors was estimated using ECOTYPER. Tumor tissue microarrays were used to evaluate CAV1 protein levels in stromal and malignant cells. In the SCAN-B (n = 525) and GSE31519 (n = 327) cohorts, patients with CAV1-high tumors had an increased incidence of early recurrence adjusted HR 1.78 (95% CI 1.12-2.81) and 2.20 (95% CI 1.39-3.47), respectively. In further analysis, high CAV1 gene expression was associated with a molecular profile indicating altered metabolism, neovascularization, chemoresistance, EMT, suppressed immune response, and active tumor microenvironment. Protein levels of CAV1 in malignant and stromal cells were not correlated with CAV1 gene expression. CAV1 gene expression in TNBC is a biomarker that merits further investigation in clinical trials and as a therapeutic target.

Global biomarker trends in triple-negative breast cancer research: a bibliometric analysis

Background: Triple-negative breast cancer (TNBC) is defined as breast cancer that is negative for estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER-2) in cancer tissue. The lack of specific biomarkers makes the diagnosis and prognosis of TNBC challenging. Method: A comprehensive literature review and bibliometric analysis was performed using CiteSpace, VOSviewer and Scimago Graphica. Results: TNBC biomarker research has been growing rapidly in recent years, reflecting the enormous academic interest in TNBC biomarker research. A total of 127 journals published relevant studies and 1749 authors were involved in the field, with developed countries such as the United States, France, and the United Kingdom contributing greatly to the field. Collaborative network analysis found that the research in this field has not yet formed good communication and interaction, and the partnership should be strengthened in the future in order to promote the in-depth development of TNBC biomarker research. Comprehensive analysis of keywords and co-cited literature, etc. found that TNBC biomarker research mainly focuses on immune checkpoint markers, microenvironment-related markers, circulating tumour DNA, metabolic markers, genomics markers and so on. These research hotspots will help to better understand the molecular characteristics and biological processes of TNBC, and provide more accurate biomarkers for its diagnosis, treatment and prognosis. Conclusions: The bibliometric analysis highlighted global trends and key directions in TNBC biomarker research. Future developments in TNBC biomarker research are likely to be in the direction of multi-omics integration, meticulous study of the microenvironment, targeted therapeutic biomarkers, application of liquid biopsy, application of machine learning and artificial intelligence, and individualised therapeutic strategies. Young scholars should learn and collaborate across disciplines, pay attention to new technologies and methods, improve their data analysis skills, and continue to follow up on the latest research trends in order to meet the challenges and opportunities in the field of TNBC biomarkers.

Identification of disulfidptosis-related clusters and construction of a disulfidptosis-related gene prognostic signature in triple-negative breast cancer

ObjectiveThis study aimed to explore disulfidptosis-related clusters of triple-negative breast cancer (TNBC) and build a reliable disulfidptosis-related gene signature for forecasting TNBC prognosis. MethodsThe disulfidptosis-related clusters of TNBC were identified based on public datasets, and a comparative analysis was conducted to assess their differences in the overall survival (OS) and immune cell infiltration. Morever, the differentially expressed genes (DEGs) between clusters were recognized. Then, the prognostic DEGs were then chosen. A prognostic signature was constructed by the prognostic DEGs, followed by nomogram construction, drug sensitivity, immune correlation, immunotherapy response prediction, and cluster association analyses. ResultsTwo disulfidptosis-related clusters of TNBC were identified, which had different OS and macrophage infiltration. Moreover, 235 DEGs were identified between two clusters. A prognostic signature was then constructed by five prognostic DEGs including HLA-DQA2, CCL13, GBP1, LAMP3, and SLC7A11. This signature was highly valuable in predicting prognosis. A nomogram was built by risk score and AJCC stage, which could forecast OS accurately. Moreover, patients with high-risk scores exhibited greater sensitivity to chemotherapy drugs such as lapatinib and had a lower immunotherapy response. ConclusionsTwo TNBC clusters linked to disulfidptosis were identified, with different OS and immune cell infiltration. Moreover, a five-disulfidptosis-related gene signature may be a powerful prognostic biomarker for TNBC.

The potent potential of MFAP2 in prognosis and immunotherapy of triple-negative breast cancer

BackgroundsMicrofibril-associated protein 2 (MFAP2) is a protein presenting in the extracellular matrix that governs the activity of microfibrils through its interaction with fibrillin. While the involvement of MFAP2 in metabolic disorders has been documented, its expression and prognostic significance in triple-negative breast cancer (TNBC) remain unexplored.MethodsWe acquired datasets pertaining to breast cancer (BC) from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases. Next, a Venn diagram was used to identify the differentially expressed genes (DEGs). The DEGs were used to perform Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), protein–protein interaction (PPI), immune and survival analysis. The expressions of MFAP2, PD-1 and PD-L1 were examined by immunohistochemistry and western blot and their relationship with clinical pathological parameters were analyzed by clinical specimen samples from patients with TNBC. Tumor Immune Estimation Resource (TIMER, https://cistrome.shinyapps.io/timer/) was adopted to calculate the immune infiltration level of TNBC. The link between gene expression and tumor mutational burden (TMB) was described using Spearman’s correlation analysis.ResultsWe identified 66 differentially expressed genes (DEGs) that were up-regulated. Among these DEGs, MFAP2 was found to be overexpressed in TNBC and was associated with a lower probability of survival. This finding was confirmed through the use of immunohistochemistry and western blot techniques. Additionally, MFAP2 was found to be related to various pathological parameters in TNBC patients. Mechanistically, gene set enrichment analysis (GSEA) revealed that MFAP2 primarily influenced cellular biological behavior in terms of epithelial mesenchymal transition, glycolysis, and apical junction. Notably, MFAP2 expression was positively correlated with the abundance of macrophages, while a negative correlation was observed with the abundance of B cells, CD4 + T cells, CD8 + T cells, neutrophils and dendritic cells through immune analysis. Furthermore, it was observed that MFAP2 displayed a negative correlation not only with tumor mutational burden (TMB), a recognized biomarker for PD-1/PD-L1 immunotherapy, but also with PD-L1 in samples of TNBC.ConclusionMFAP2 may be an important prognostic biomarker for TNBC, as well as a viable target for immunotherapy in this disease.

Unveiling the complexity: Liquid biopsy-based proteomic exploration of triple-negative breast cancer in women.

e13138 Background: This groundbreaking study places a spotlight on breast cancer in women, with a specific emphasis on the challenging triple-negative breast cancer (TNBC) subtype. Delving into the intricate world of extracellular vesicles (EVs) from TNBC-diagnosed individuals, the research meticulously compares various conditions, including healthy controls, benign tumors, recurrent tumors, and those in remission. Employing cutting-edge techniques like liquid biopsy and LC-MS/MS, the study meticulously explores the proteomic profile of serum exosomes, aiming to pinpoint diagnostic and prognostic markers in breast cancer. Methods: In the initial phase, EVs were expertly isolated and characterized from blood plasma samples exclusively from women, utilizing state-of-the-art methods such as ultracentrifugation and nanoparticle tracking analysis (NTA). Despite applying rigorous statistical criteria, the study found no significant differences in EV concentrations and sizes among the diverse groups. However, the ensuing proteomic analysis uncovered 130 proteins, with Cystatin A and Histone H2A emerging as promising biomarkers for TNBC. To validate them, the study intricately tested tumor cell lines from distinct subtypes, representing luminal, triple-negative, and HER2 overexpressed. The examination of gene and protein expression through RT-qPCR and western blotting added a layer of depth to the research, unraveling the complexity of regulatory processes within cells and demonstrating a remarkable congruence between protein levels found in the proteomics from patients’ serum and in immortalized cell lines. Results: The proteomic analysis brought to light the absence of CSTA and HIST1H2A expression in benign tumor patients, varied levels in malignant tumor patients, and a substantial increase in TNBC patients pre-mastectomy. Validating these findings through western blotting further accentuated the potential of these proteins as distinguishing factors. Conclusions: In conclusion, this pioneering study not only underscores the immense potential of liquid biopsy-based proteomic analysis in identifying crucial diagnostic and prognostic markers in breast cancer but also sheds light on the intricate nuances of the challenging TNBC subtype. Our findings unveil valuable insights that pave the way for future personalized treatment strategies, marking a significant stride forward in breast cancer research.

Exploring the neutrophil-to-lymphocyte ratio as a prognostic biomarker in triple-negative breast cancer

Triple-negative breast cancer is a subtype characterized by the absence of estrogen receptor, progesterone Receptor, and Human Epidermal Growth Factor Receptor 2 receptors, limiting treatment options primarily to chemotherapy due to lack of targeted therapies typically used for other breast cancer types, hence necessitating research for improved therapeutic strategies and outcomes. The neutrophil and lymphocyte percentages, absolute counts, and the ratio of neutrophils to lymphocytes were evaluated in peripheral blood of 81 patients with triple-negative breast cancer (TNBC) and 25 healthy controls. This study observed trend of high neutrophil percentage was observed in patients with positive lymph node status as compared to negative lymph node status. Significant high absolute neutrophil count was observed in patients with positive LN status, High BR score and presence of metastasis. A trend of high neutrophil to lymphocyte ratio was observed in patients with presence of perineural invasion, presence of necrosis, and patients with presence of metastasis.

MIF as a potential diagnostic and prognostic biomarker for triple-negative breast cancer that correlates with the polarization of M2 macrophages.

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that plays a crucial role in antitumor immunity. However, the role of MIF in influencing the tumor microenvironment (TME) and prognosis of triple-negative breast cancer (TNBC) remains to be elucidated. Using R, we analyzed single-cell RNA sequencing (scRNA-seq) data of 41 567 cells from 10 TNBC tumor samples and spatial transcriptomic data from two patients. Relationships between MIF expression and immune cell infiltration, clinicopathological stage, and survival prognosis were determined using samples from The Cancer Genome Atlas (TCGA) and validated in a clinical cohort using immunohistochemistry. Analysis of scRNA-seq data revealed that MIF secreted by epithelial cells in TNBC patients could regulate the polarization of macrophages into the M2 phenotype, which plays a key role in modulating the TME. Spatial transcriptomic data also showed that epithelial cells (tumor cells) and MIF were proximally located. Analysis of TCGA samples confirmed that tumor tissues of patients with high MIF expression were enriched with M2 macrophages and showed a higher T stage. High MIF expression was significantly associated with poor patient prognosis. Immunohistochemical staining showed high MIF expression was associated with younger patients and worse clinicopathological staging. MIF secreted by epithelial cells may represent a potential biomarker for the diagnosis and prognosis of TNBC and may promote TNBC invasion by remodeling the tumor immune microenvironment.

Lactylproteome analysis indicates histone H4K12 lactylation as a novel biomarker in triple-negative breast cancer.

The established link between posttranslational modifications of histone and non-histone lysine (K) residues in cell metabolism, and their role in cancer progression, is well-documented. However, the lactylation expression signature in triple-negative breast cancer (TNBC) remains underexplored. We conducted a comprehensive lactylproteome profiling of eight pairs of TNBC samples and their matched adjacent tissues. This was achieved through 4-Dimensional label-free quantitative proteomics combined with lactylation analysis (4D-LFQP-LA). The expression of identified lactylated proteins in TNBC was detected using immunoblotting and immunohistochemistry (IHC) with specific primary antibodies, and their clinicopathological and prognostic significance was evaluated. Our analysis identified 58 lactylation sites on 48 proteins, delineating the protein lactylation alteration signature in TNBC. Bioinformatic and functional analyses indicated that these lactylated proteins play crucial roles in regulating key biological processes in TNBC. Notably, lactylation of lysine at position 12 (H4K12lac) in the histone H4 domain was found to be upregulated in TNBC. Further investigations showed a high prevalence of H4K12lac upregulation in TNBC, with positive rates of 93.19% (137/147) and 92.93% (92/99) in TNBC tissue chip and validation cohorts, respectively. H4K12lac expression correlated positively with Ki-67 and inversely with overall survival (OS) in TNBC (HR [hazard ratio] =2.813, 95%CI [credibility interval]: 1.242-6.371, P=0.0164), suggesting its potential as an independent prognostic marker (HR=3.477, 95%CI: 1.324-9.130, P=0.011). Lactylation is a significant post-translational modification in TNBC proteins. H4K12lac emerges as a promising biomarker for TNBC, offering insights into the lactylation profiles of TNBC proteins and linking histone modifications to clinical implications in TNBC.

Abstract PO5-03-10: The prognostic role of androgen receptor status in patients with triple negative breast cancer with an associated ductal carcinoma in situ

Abstract Abstract Background Triple negative breast cancer (TNBC) is defined by the lack of expression of the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. Prognostic immunohistochemical biomarkers in TNBC have been studied in recent years such as the androgen receptor (AR) which is expressed in 10-40% of TNBC. However the prognostic value of AR expression is not clear. Here, we studied the prognostic significance of AR expression in combination with the presence of a ductal carcinoma in situ (DCIS). Considering DCIS is a precursor of invasive ductal carcinoma, we hypothesize that TNBC with co-existing DCIS and presence of AR expression is less aggressive and patients are older at diagnosis. Methods We analyzed data retrospectively from all patients with stage 1-3 TNBC who underwent primary surgery and adjuvant chemotherapy in the University hospitals Leuven, between 01-01-2000 and 31-12-2017. Patient and tumor related characteristics were compared between two subgroups, one with co-existing DCIS and one with pure invasive carcinoma, without co-existing DCIS. AR expression was assessed by immunohistochemistry (IHC). We used AR expression in ≥1% and ≥10% of cells as cut-off scores. The prognostic role of the expression of AR in combination with a co-existing DCIS was analyzed, using the distant metastasis rate as primary endpoint. Results are presented as hazard ratios (HR) with 95% confidence intervals (CI). Secondary endpoints were associations of AR expression with clinical-pathological characteristics, time between diagnosis and metastasis, and disease specific mortality. Results In the 426 included patients with TNBC, co-existing DCIS was present in 66.7%; AR expression was expressed ≥1% in 29.3% and ≥10% in 21.4% of cases. Median age at diagnosis was 51 years (range: 22-85y). Age at diagnosis was independent of DCIS, dependent of AR expression; in DCIS positive cases, median age was 49 years if AR negative (IHC < 1%), 53 years if AR positive (IHC ≥1%), and 56 years if AR positive (IHC ≥10%) (p=0.006). In contrast in DCIS negative cases, median age was 51 years if AR negative (IHC < 1%), 51 if AR positive (IHC ≥1%), and 51 years if AR positive (IHC ≥10%) (p=0.895). AR expression was DCIS dependent and was ≥1% in 34.9% and ≥10% in 25.0% of patients in the DCIS group compared to 18.3% and 14.1% in the non-DCIS group (p=0.001 and p< 0.001 respectively). In both subgroups there was no significant difference for AR positive versus AR negative cases in lymph node involvement, tumor grade, tumor size and Nottingham Prognostic Index. Patients with a coexisting DCIS and AR expression did not have a different incidence of distant relapse compared to AR negative cases (AR ≥1%: p=0.2803 and AR ≥10%: p=0.5527). Of patients with coexisting DCIS, 12.0% (95% CI: 7.8; 17.1) in the AR negative group, 8.2% (95% CI: 3.8; 14.6) in the AR ≥ 1% group and 7.1% (95% CI: 2.6; 14.7) in the AR ≥ 10% group, had distant relapse within 2 years. Conclusion In patients with TNBC, AR expression is associated with older age in case of co-existing DCIS and patients with co-existing DCIS are more frequently AR positive. In patients with co-existing DCIS, there was no significant difference in distant relapse between AR negative and AR positive cases. Citation Format: Micaëlle Merckx. The prognostic role of androgen receptor status in patients with triple negative breast cancer with an associated ductal carcinoma in situ [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 PO5-03-10.

Abstract PO2-15-10: Identifying new immune-related biomarkers in TNBC with a look at PD-L1 cell-autonomous role

Abstract Background The programmed death ligand 1 (PD-L1) has recently emerged as a target immunotherapy in Triple-negative breast cancer (TNBC). However, the tumor-intrinsic role of PD-L1 and its pathway still needs to be fully clarified. Recently, a close association between CD73, PD-L1, cancer cell steaminess and epithelial-to-mesenchymal transition phenotypes is emerging. In addition, we have previously demonstrated that miR-320a and miR-145 target PD-L1, whereas miR-30 family is known to target CD73. In this study, we aimed at investigating the role of these miRNAs as prognostic and predictive dynamic biomarkers for immunotherapy in TNBC. Additionally, we investigated whether PD-L1 exerts cellular autonomic functions in TNBC beyond its role in the immune checkpoint. Results CD73 and PD-L1 expression was assessed in a panel of breast cancer lines and the non-malignant breast cells MCF-10. We showed a significantly higher CD73 and PD-L1 expression in MDA-MB-231 (TNBC, Basal B) than in MCF-10. Conversely, in MCF-7 cells (HR +, Luminal A), CD73 and PD-L1 expression is lower than in MCF-10. Furthermore, we showed that low PDL1 expression is correlated with high levels of miR-320a and miR-145, and CD73 expression is inversely related to miR-30 expression. These putative biomarkers were also validated in two patients with different PD-L1 status. From the analysis of the data obtained by real-time qRT-PCR, we found high plasma level of CD73 and low plasma level of miR-320a, miR-145 and miR-30 in the patient with PD-L1 positive BC. On contrary, in the patient with PD-L1-negative BC, we found low plasma level of CD73 and high plasma level of miR-320a, miR-145 and miR-30. Furthermore, to explore whether PD-L1 could also have an intrinsic role in tumor development and invasiveness, we used stably MDA–MB-231 PD-L1-silenced cells. We found that cell growth, colony formation, migration rate and the ability to form spheres are consistently reduced upon shRNA-mediated PDL1 silencing. In particular, we observed a reduction in the number, diameter and volume of the 3D spheres compared to the control cells. To further demonstrate the role of PD-L1, we treated the PDL1-high expression cells MDA-MB-231, PD-L1 silenced MDA-MB-231 clones, and PD-L1 low expression cells MCF-7 with Durvalumab, an anti-PD-L1. Interestingly, we observed that in PD-L1 silenced clones and MCF-7 cells, the sphere-forming ability was increased in presence of Durvalumab treatment. On the contrary, in MDA-MB-231, Durvalumab inhibited the sphere-forming ability. Conclusion Our data confirm that PD-L1 and CD73 are targets of miR-320a/miR-145 and miR-30 respectively. These could be potential predictive dynamic biomarkers for chemotherapy, ICIs and anti-CD73 therapeutic approach. Future validations of these biomarkers in an extensive series of TNBC patients are needed to support their use in clinical practice. Here, we further characterized the cellular autonomic role of PD-L1 in breast cancer and showed a differential role of basal PD-L1 expression in PD-L1 checkpoint inhibitors treatment efficacy. This suggests a potential role in monitoring PD-L1 expression indirect biomarkers (i.e. miR-320a, miR-145 and CD73) during ICIs treatment.