Detection Of Triple Negative Breast Cancer Research Articles

Abstract PO3-16-05: Mitochondrial DNA mutation detection in tumors and circulating extracellular vesicles of triple negative breast cancer patients for biomarker development

Abstract Early detection of triple-negative breast cancer (TNBC) patients and their recurrence prediction are daunting tasks. We aim to develop mitochondrial DNA (mtDNA) based biomarkers to improve the management strategies of TNBC patients. We have undertaken mitochondrial genome (MG) enrichment and next-generation sequencing approaches to map the entire MG in 73 samples (64 tissues, 9 extracellular vesicle samples) from 32 highly-aggressive TNBC patients. Measurement of mtDNA and cardiolipin contents, in concert with NDUFB8 and SDHB proteins’ expression, was carried out in tumors and matched extracellular vesicles (EVs) of all the above TNBC patients. Overall, we identified 168 nonsynonymous mtDNA mutations, of which 73% (123/186) were coding and 27% (45/168) were non-coding. Twenty percent of mutations were nucleotide transversions. Respiratory complex I (RCI) appears to be the key target, which harbored 44% (74/168) of the overall mtDNA mutations. A panel of 11 hotspot mtDNA mutations was identified among 19-38% TNBCs, each of which was detectable in the matched EV samples with 82% specificity. Overall, 38% of the metastatic tumor-signature mtDNA mutations were detectable in matched EVs of the TNBC patients. An appreciable number of mtDNA mutations were homoplasmic (18%, 31/168), novel (14%, 23/168), and potentially pathogenic (9%, 15/168). The overall and RCI-specific mtDNA mutational load was recorded to be higher in African American (AA) compared to European American (EA) women with an exclusive abundance of respiratory complex (RC) protein NDUFB8 (RCI) and SDHB (RCII) therein. Increased mtDNA (p < 0.0001) content was measured in both tumors and matched EV samples along with an abundance of cardiolipin (p=0.0001) content in the EVs of the TNBC patients. Lethal tumor-signature mtDNA mutation detection and measurement of altered mtDNA and cardiolipin contents in the EVs at an early time point bear the potential to formulate noninvasive early detection and recurrence prediction strategies in TNBCs to improve the overall survival of the patients. Citation Format: Kunwar Vikramdeo, Shashi Anand, Sarabjeet Sudan, Paramahansa Pramanik, Seema Singh, Andrew Godwin, Ajay Singh, Santanu Dasgupta. Mitochondrial DNA mutation detection in tumors and circulating extracellular vesicles of triple negative breast cancer patients for biomarker development [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-16-05.

Dynamic light scattering microscopy sensing mitochondria dynamics for label-free detection of triple-negative breast cancer enhanced by deep learning.

We established a deep learning-based dynamic light scattering (DLS) microscopy sensing mitochondria dynamic for label-free identification of triple-negative breast cancer (TNBC) cells. The capacity of DLS microscopy to detect the intracellular motility of subcellular scatters was verified with the analysis of the autocorrelation function. We also conducted an in-depth examination of the impact of mitochondrial dynamics on DLS within TNBC cells, employing confocal fluorescent imaging to visualize the morphology of the mitochondria. Furthermore, we applied the DLS microscopy incorporating the two-stream deep learning method to differentiate the TNBC subtype and HER2 positive breast cancer subtype, with the classification accuracy achieving 0.89.

Anti-CD44 antibodies grafted immunoaffinity Fe3O4@MnO2 nanozymes with highly oxidase-like catalytic activity for specific detection of triple-negative breast cancer MDA-MB-231 cells

Cell-enzyme-linked immunosorbent assay (CELISA) is extensively applied for cancer diagnosis and screening because of its simple operation, high sensitivity, and intuitive color change. However, the unstable horseradish peroxidase (HRP), hydrogen peroxide (H2O2) and non-specificity have led to a high false negative rate, which limits its application. In this study, we have developed an innovative immunoaffinity nanozyme aided CELISA based on anti-CD44 monoclonal antibodies (mAbs) bioconjugated manganese dioxide-modified magnetite nanoparticles (Fe3O4@MnO2 NPs) for the specific detection of triple-negative breast cancer MDA-MB-231 cells. The CD44FM nanozymes were fabricated to replace unstable HRP and H2O2 to counteract possible negative effects in conventional CELISA. Results suggested that CD44FM nanozymes displayed remarkable oxidase-like activities over an extensive pH and temperature range. The bioconjugation of CD44 mAbs enabled CD44FM nanozymes to enter MDA-MB-231 cells selectively via over-expressed CD44 antigens on the membrane surface of these cells, and then catalyzed oxidation of the chromogenic substrate TMB, further achieving specific detection of these cells. Additionally, this study exhibited high sensitivity and low detection limit for MDA-MB-231 cells with a quantitation range of just 186 cells. To sum up, this report developed a simple, specific and sensitive assay platform based on CD44FM nanozymes, which could provide a promising strategy for targeted diagnosis and screening of breast cancer.

DNA methylation biomarkers for noninvasive detection of triple-negative breast cancer using liquid biopsy.

Noninvasive detection of aberrant DNA methylation could provide invaluable biomarkers for earlier detection of triple-negative breast cancer (TNBC) which could help clinicians with easier and more efficient treatment options. We evaluated genome-wide DNA methylation data derived from TNBC and normal breast tissues, peripheral blood of TNBC cases and controls and reference samples of sorted blood and mammary cells. Differentially methylated regions (DMRs) between TNBC and normal breast tissues were stringently selected, verified and externally validated. A machine-learning algorithm was applied to select the top DMRs, which then were evaluated on plasma-derived circulating cell-free DNA (cfDNA) samples of TNBC patients and healthy controls. We identified 23 DMRs accounting for the methylation profile of blood cells and reference mammary cells and then selected six top DMRs for cfDNA analysis. We quantified un-/methylated copies of these DMRs by droplet digital PCR analysis in a plasma test set from TNBC patients and healthy controls and confirmed our findings obtained on tissues. Differential cfDNA methylation was confirmed in an independent validation set of plasma samples. A methylation score combining signatures of the top three DMRs overlapping with the SPAG6, LINC10606 and TBCD/ZNF750 genes had the best capability to discriminate TNBC patients from controls (AUC=0.78 in the test set and AUC=0.74 in validation set). Our findings demonstrate the usefulness of cfDNA-based methylation signatures as noninvasive liquid biopsy markers for the diagnosis of TNBC.

Actin cytoskeletal structure and the statistical variations of the mechanical properties of non-tumorigenic breast and triple-negative breast cancer cells

This paper presents the results of a study of the actin cytoskeletal structures and the statistical variations in the actin fluorescence intensities and viscoelastic properties of non-tumorigenic breast cells and triple-negative breast cancer cells at different stages of tumor progression. The variation in the actin content of the cell cytoskeletal structures is shown to be consistent with the viscoelastic properties of the cell as it progresses from non-tumorigenic to more metastatic states. The corresponding viscoelastic properties of the nuclei and the cytoplasm (Young's moduli, viscosities, and relaxation times) of the cells are also measured using Digital Image Correlation (DIC) and shear assay techniques. These properties are shown to exhibit statistical variations that are well characterized by normal distributions. The changes in the mean properties of individual cancer cells are tested using Fisher pairwise comparisons and the analysis of variance (ANOVA). The implications of the results are then discussed for the development of shear assay techniques and mechanical biomarkers for the detection of triple-negative breast cancer at different stages of tumor progression.

Triple Negative Breast Cancer Detection Using LC-MS/MS Lipidomic Profiling.

Breast cancer (BC) is a heterogeneous malignancy that is responsible for a great portion of female cancer cases and cancer-related deaths in the United States. In comparison to other major BC subtypes, triple negative breast cancer (TNBC) presents with a relatively low survival rate and a high rate of metastasis. This has led to a strong, though largely unmet, need for more sensitive and specific methods of early-stage TNBC (ES-TNBC) detection to combat its high-grade pathology and relatively low survival rate. The current study employs a liquid chromatography-tandem mass spectrometry assay capable of targeted, highly specific, and sensitive detection of lipids to propose two diagnostic biomarker panels for TNBC/ES-TNBC. Using this approach, 110 lipids were reliably detected in 166 human plasma samples, 45 controls, and 121 BC (96 non-TNBC and 25 TNBC) subjects. Univariate and multivariate analyses allowed the construction and application of a 19-lipid biomarker panel capable of distinguishing TNBC (and ES-TNBC) from controls, as well as a 5-lipid biomarker panel capable of differentiating TNBC from non-TNBC and ES-TNBC from ES-non-TNBC. Receiver operating characteristic curves with notable classification performances were generated from the biomarker panels according to their orthogonal partial least-squares discrimination analysis models. TNBC was distinguished from controls with an area under the receiving operating characteristic curve (AUROC) = 0.93, sensitivity = 0.96, and specificity = 0.76 and ES-TNBC from controls with an AUROC = 0.96, sensitivity = 0.95, and specificity = 0.89. TNBC was differentiated from non-TNBC with an AUROC = 0.88, sensitivity = 0.88, and specificity = 0.79 and ES-TNBC from ES-non-TNBC with an AUROC = 0.95, sensitivity = 0.95, and specificity = 0.87. A pathway enrichment analysis between TNBC and controls also revealed significant disturbances in choline metabolism, sphingolipid signaling, and glycerophospholipid metabolism. To the best of our knowledge, this is the first study to propose a diagnostic lipid biomarker panel for TNBC detection. All raw mass spectrometry data have been deposited to MassIVE (dataset identifier MSV000085324).