Samples Of Breast Cancer Patients Research Articles

Diagnostic utility of ESR1 mutation detection in liquid biopsy of metastatic breast cancer patients.

Molecular analysis of circulating cell-free DNA (cfDNA) extracted from peripheral blood plasma samples of metastatic breast cancer (BC) patients is of rising interest to find optimal therapeutic strategies. Detection of emerging resistance mutations against endocrine therapy is possible with this approach. Here we present the applicability of a laboratory-developed NGS assay in molecular pathology routine diagnostic, covering four genes with therapeutic (ESR1, PIK3CA, ERBB2) and prognostic (TP53) consequences in metastatic BC. We analyzed 162 liquid biopsy samples and 25 corresponding metastases from metastatic BC patients. In the liquid biopsies, we detected ESR1 mutations in 42 cases (25.9%) and ERBB2 mutations in six cases (3.7%), arguing for a change in therapy to fulvestrant, elacestrant, or neratinib. Furthermore, 17 cases had detectable TP53 mutations, associated with resistance against endocrine therapy. We conclude that liquid biopsy testing is a noninvasive, sensitive, and helpful method to optimize therapeutic decisions in metastatic BC.

Systematic screening of protein-coding gene expression identified VWF as a potential key regulator in anthracycline-based chemotherapy-exacerbated metastasis of breast cancer

BackgroundBreast cancer is the most commonly diagnosed cancer worldwide. Although major treatments represented by chemotherapy have shown effectiveness at the initial period, recurrence and metastasis still occur later after treatments. The alternation of the tumor microenvironment by chemotherapy is confirmed as a trigger of the elevated proliferation and migration of the remaining tumor cells.MethodsUsing bioinformatic methods, differential gene expression analysis was used to determine DEGs between post-chemotherapy and pre-chemotherapy samples of breast cancer patients, followed by survival analysis and ELISA analysis of the potential key genes. An in vitro model of 2 breast cancer cells lines was used to demonstrate the role of VWF in the evasion and migration of breast cancer cells, using cell migration, evasion and wound healing assays, PCR and molecular docking analysis.Results19 hub genes were further identified using GO and KEGG pathway analyses and WGCNA. The 5 secreted protein-coding genes with reported carcinogenesis effects (VWF, SVEP1, DPT, ADIPOQ, and LPL) were further analyzed in breast cancer patients and VWF was identified as a potential key regulator in the anthracycline-based chemotherapy-exacerbated metastasis. It was further confirmed that anthracycline-based chemotherapeutics doxorubicin exacerbated VWF upregulation and the evasion and migration of breast cancer cells. Based on molecular docking analysis and previous study, berberine was used as an inhibitor of VWF, and showed an effective inhibition of the doxorubicin-exacerbated VWF upregulation, migration and evasion in breast cancer.ConclusionsDoxorubicin-exacerbated evasion and migration through VWF upregulation. Berberine as an inhibitor of VWF was able to reversed the doxorubicin-exacerbated VWF upregulation and evasion and migration in breast cancer cells.

Double-negative T cells with a distinct transcriptomic profile are abundant in the peripheral blood of patients with breast cancer.

Double-negative T (DNT) cells comprise a distinct subset of T lymphocytes that have been implicated in immune responses. The aim of this study was to characterize the peripheral DNT population in breast cancer (BC) patients. DNT cells were isolated from the peripheral blood samples of BC patients and healthy controls by flow cytometry. The sorted DNT cells were analyzed by the Smart-seq2 for single-cell full-length transcriptome profiling. The differentially expressed genes (DEGs) between the BC and control groups were screened and functionally annotated by Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses using R. The protein-protein interaction (PPI) network of the DEGs was constructed using the CytoHubba and MCODE plug-in of Cytoscape software to identify the core genes. Survival status, DNA methylation level, immune infiltration and immune checkpoint expression were analyzed using Kaplan-Meier Plotter, UALCAN, MethSeuvr, TIMER, and TISIDB respectively. The sequencing results were verified by RT-qPCR. The percentage of DNT cells was higher in the BC patients compared to healthy controls. We identified 289 DEGs between the DNT populations of both groups. GO and KEGG pathway analyses revealed that the DEGs were mainly related to immunoglobulin mediated immune response, complement activation, and B cell receptor signaling. The PPI networks of the common DEGs were constructed using Cytoscape, and 10 core genes were identified, including TMEM176B, C1QB, C1QC, RASD2, and IFIT3. The expression levels of these genes correlated with the prognosis and immune infiltration in BC patients, and were validated by RT-qPCR (P < 0.05). DNT cells are abundant in patients with BC, and might exert anti-tumor immune responses by regulating genes such as TMEM176B and EGR1.

Identification of HOXC Gene Family as Prognostic and Immune-Related Biomarkers in Breast Cancer Through mRNA Transcriptional Profile and Experimental Validation.

Breast cancer (BC) is the most common malignancy in women worldwide, and more effective biomarkers are urgently needed for the prevention and treatment of BC. Our study aimed to investigate the role of the HOXC gene family (HOXCs) and its relationship with the immune response in BC. The differential expression of HOXCs and its clinical prognostic significance in BC were explored using bioinformatics analysis, and the cBioPortal database was used to evaluate the genetic mutation profile of the HOXCs in BC. The results indicated that the expression levels of HOXC4, 10, 11, 12, and 13 were significantly increased in BC tissues compared with the normal tissues, and expressions of these genes were closely associated with BC stage, among them, high expression levels of HOXC10 and HOXC13 predicted poor outcome in BC patients. In addition, to elucidate the essential role of HOXCs in the tumor microenvironment and immunotherapeutic response of BC, the impact of HOXCs on the regulation of immune infiltration in BC was comprehensively assessed. The result showed thatHOXC10 and HOXC13 expressions were significantly positively linked with the infiltration levels of CD8+T cell and M1 macrophage, while they werenegatively related to Mast and Natural killer cells, suggesting the important influence of HOXCs on regulating tumor immunity in BC patients. Lastly, the RT-qPCR assay was employed to validate HOXCs expression in samples of BC patients. In conclusion, HOXCs may be a promising prognostic indicator and couldregulate the immune infiltration in BC patients, thus being apromising targeted immunotherapy for BC.

Hydrogen Sulfide-Induced Activatable Photodynamic Therapy Adjunct to Disruption of Subcellular Glycolysis in Cancer Cells by a Fluorescence-SERS Bimodal Iridium Metal-Organic Hybrid.

The practical application of photodynamic therapy (PDT) demands targeted and activatable photosensitizers to mitigate off-target phototoxicity common in "always on" photosensitizers during light exposure. Herein, a cyclometalated iridium complex-based activatable photodynamic molecular hybrid, Cy-Ir-7-nitrobenzofurazan (NBD), is demonstrated as a biomedicine for molecular precision. This design integrates a hydrogen sulfide (H2S)-responsive NBD unit with a hydroxy-appended iridium complex, Cy-Ir-OH. In normal physiological conditions, the electron-rich Ir metal center exerts electron transfer to the NBD unit, quenches the excited state dynamics, and establishes a PDT-off state. Upon exposure to H2S, Cy-Ir-NBD activates into the potent photosensitizer Cy-Ir-OH through nucleophilic substitution. This mechanism ensures exceptional specificity, enabling targeted phototherapy in H2S-rich cancer cells. Additionally, we observed that Cy-Ir-NBD-induced H2S depletion disrupts S-sulfhydration of the glyceraldehyde-3-phosphate dehydrogenase enzyme, impairing glycolysis and ATP production in the cellular milieu. This sequential therapeutic process of Cy-Ir-NBD is governed by the positively charged central iridium ion that ensures mitochondria-mediated apoptosis in cancer cells. Dual-modality SERS and fluorescence imaging validate apoptotic events, highlighting Cy-Ir-NBD as an advanced theranostic molecular entity for activatable PDT. Finally, as a proof of concept, clinical assessment is evaluated with the blood samples of breast cancer patients and healthy volunteers, based on their H2S overexpression capability through SERS and fluorescence, revealing Cy-Ir-NBD to be a promising predictor for PDT activation in advanced cancer phototherapy.

Profuse color-evolution based aptasensor for mucin 1 detection utilizing urease-mediated color mixing of the mixed pH indicator

Mucin 1 is a significant tumor marker, and developing portable and cost-effective methods for its detection is crucial, especially in resource-limited areas. Herein, we developed an innovative approach for mucin 1 detection using a visible multicolor aptasensor. Urease-encapsulated DNA microspheres were used to mediate multicolor change facilitated by the color mixing of the mixed pH indicator, a mixed methyl red and bromocresol green solution. Distinct color changes were exhibited in response to varying mucin 1 concentrations. Notably, the color mixing of the mixed pH indicator was used to display various hues of colors, broadening the range of color variation. And color tonality is much easier to differentiate than color intensity, improving the resolution with naked-eyes. Besides, the variation of color from red to green (a pair of complementary colors) enhanced the color contrast, heightening sensitivity for visual detection. Importantly, the proposed method was successfully applied to detect mucin 1 in real samples, demonstrating a clear differentiation of colors between the samples of healthy individuals and breast cancer patients. The use of a mixed pH indicator as a multichromatic substrate offers the merits of low cost, fast response to pH variation, and plentiful color-evolution. And the incorporation of calcium carbonate microspheres to encapsulate urease ensures stable urease activity and avoids the need for extra urease decoration. The color-mixing dependent strategy opens a new way for multicolor detection of MUC1, characterized by vivid color changes.

Involvement of Total Antioxidant Activity and eNOS Gene rs1799983/ rs2070744 Polymorphisms in Breast Carcinogenesis

Globally, breast cancer is the common malignancy affecting women and understanding its associated molecular events could help in disease prevention and management strategies. The present study was set to investigate an association between total antioxidant capacity (TAC) and endothelial nitric oxide synthase (eNOS) polymorphisms with breast cancer. For this purpose, 100 subjects were participated in this work, including 50 female patients diagnosed with breast cancer recruited from Oncology hospital, Baghdad - Iraq and 50 healthy women as a control group. The concentration of antioxidants was measured in the serums collected from blood samples of breast cancer patients and healthy controls. While eNOS SNPs (rs1799983, G894T and rs2070744, T 786C) were assessed using TaqMan SNP genotyping and utilising genomic DNA extracted from the participants. The results showed that the antioxidants levels were significantly (P˂0.0001) reduced in blood samples of breast cancer patients in comparison to that of that healthy controls (0.144± 0.097 and 0.587±0.239 respectively). Additionally, the homozygous GG genotype G894T (rs1799983) could retain beneficial impact for the protection from breast cancer potential. While SNP genotyping results showed that both of the homozygous CC and heterozygous TC genotypes (rs2070744 T &gt;C SNP) seem to contribute to the susceptibility of breast cancer development in the investigated set of patients. Overall, the present study findings suggest an association between reduced antioxidant capacity and eNOS gene polymorphisms in breast carcinogenesis.

Abstract 3749: The evaluation of expression level of Cxcl12 gene in peripheral blood samples of breast cancer patients

Abstract Breast cancer, like many other cancer types, is linked to the activation and overexpression of CXCR4. The overexpression of CXCR4 is associated with factors such as tumor growth, angiogenesis, increased metastasis, and therapeutic resistance. Similarly, CXCL12, a specific ligand for CXCR4, plays a significant role in tumorigenesis processes, including survival, migration, and microenvironmental interactions. The CXCL12/CXCR4 signaling pathway has been found to impact the development and progression of breast cancer, regulating proliferation, motility, and metastasis. Hence, molecules within the CXCL12/CXCR4 signaling pathway hold promise for determining breast cancer prognosis, treatment monitoring, and serving as therapeutic agents. The study, conducted at the Istanbul University Institute of Oncology, obtained ethical approval from the Istanbul Faculty of Medicine Ethics Committee. Genetic materials from 300 breast cancer patients and 150 control subjects underwent standard testing, matched based on crucial parameters. Real-time PCR with the MIC qPCR Cycler was statistically analyzed using GraphPad Prism (p &amp;lt; 0.05). Descriptive statistics were presented for categorical and quantitative variables, while the Kolmogorov-Smirnov test assessed the normal distribution of quantitative variables. Statistical comparisons between groups were performed using the Mann-Whitney U test and the Kruskal-Wallis test. was used for calculations. Notably, among the three genes—LASP1, CXCL12, and CXCR4—LASP1 exhibited the highest mean expression level at 11.64 with a considerable standard deviation of 11.80. In contrast, CXCL12 had a much lower mean expression level at 2.96 and a tighter distribution, and CXCR4 displayed the lowest mean expression level at 0.37 with a standard deviation of 0.74. The analysis of diagnostic groups indicated no significant differences in mean LASP1 and CXCL12 levels, but a significant variance in mean CXCR4 levels (p&amp;lt;0.001). Specifically, CXCR4 expression in group Breast and ovarian cancer was notably lower compared to Breast cancer, Bilateral Breast cancer, and Breast and Other cancer, while group Breast cancer exhibited significantly lower CXCR4 expression compared to groups Bilateral Breast cancer and Breast and Other cancer. However, there was no significant difference in CXCR4 expression between groups of Bilateral Breast cancer and Breast and Other cancer. These findings reveal distinct expression patterns of CXCR4 across diagnostic groups, suggesting potential implications for diagnostic categorization and underlying molecular pathways in breast cancer. Understanding these expression disparities could be crucial in deciphering the molecular intricacies influencing disease progression and treatment responses. Citation Format: Arash Adamnejad Ghafour, Seref Bugra Tuncer. The evaluation of expression level of Cxcl12 gene in peripheral blood samples of breast cancer patients [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 3749.

Abstract 3788: EpCAM+ DCCs isolated from the bone marrow of breast cancer patients display high stemness and potency scores

Abstract Metastatic dissemination of cancer cells from primary to distant sites often occurs early and it has been shown that their detection is linked to poor outcomes. Eradication of disseminated cancer cells (DCC) has therefore become the primary goal of adjuvant therapies. Since the phenotype of DCC is largely unknown, we set out to search, isolate, and molecularly characterize these candidate metastasis founder cells from bone marrow of breast cancer patients, years before manifestation of metastasis. We screened more than 200 bone marrow samples of breast cancer patients with no evident metastasis (UICC stage M0) for EpCAM-positive cells. EpCAM positive cells in the bone marrow of healthy donors were used as a negative control. In addition, we isolated DCC from breast cancer patients with manifest metastasis (UICC stage M1). We then performed single cell RNA sequencing after whole transcriptome amplification of the collected DCC and the healthy donor (HD) control cells. To confirm the malignant origin of picked DCCs, we projected the transcriptome data into the bone marrow atlas, inferred copy number alterations, and searched for mutations shared with the matched primary tumor. EpCAM-positive cells from control patients mostly comprised plasma cells. In contrast, DCCs derived from M0-stage breast cancer patient formed a unique, non-overlapping cluster in the bone marrow atlas. UMAP-clustering placed M1-stage DCC clearly separate from M0 stage DCC. Among the collected M0-stage DCCs, we identified at least three separate subclusters. Strikingly, M0-stage DCC displayed much higher stemness and potency scores than M1-stage DCCs, reminiscent of embryonic cells. DCCs, identified by EpCAM, display very high transcriptional stemness scores when isolated before metastatic manifestation. Upon expansion and proliferation, stemness scores are reduced indicating epithelial re-differentiation. The embryonic phenotype of M0-stage DCCs may reflect an early adaptive mechanism that enables DCC to survive in an ectopic environment and may impact on the metastatic potential of the DCC. Citation Format: Elia Raya, Huiqin Koerkel-Qu, Laura Rudhart, Lisa-Marie Köhler, Anna Damboeck, Christoph Irlbeck, Catherine Botteron, Melanie Werner-Klein, Stephan Seitz, Christoph Klein. EpCAM+ DCCs isolated from the bone marrow of breast cancer patients display high stemness and potency scores [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 3788.

Abstract 5207: Hypermethylation of SOX17 is an important epigenetic mark in Afro-Brazilian breast cancers

Abstract Afro-Brazilian women suffer from higher excess breast cancer mortality after adjusting for age, level of education and stage at diagnosis. Limited studies have researched disease characteristics in this population, which can benefit from the identification of molecular markers that can provide an insight into who is at greater risk of mortality. Epigenetic biomarkers represent the combined effect of environmental and genetic factors. Particularly DNA methylation biomarkers can be valuable as biomarkers of breast cancer risk and prognosis. While studies in other populations have identified DNA methylation biomarkers associated with breast cancer and its clinicopathological outcomes, research on Brazilian cohorts has been limited in scope and focused on women of European descent. The goal of this work was to determine the epigenetic makeup of breast cancer in Brazilian women of African descent. To identify epigenetic biomarkers in Brazilian women who self-identified as Black or Brown, we conducted a case-series analysis investigating DNA methylation patterns in tumor (T) and adjacent non-tumor (NT) tissue. For this, we used the Illumina EPIC (850k) arrays to carry out DNA methylation profiling in 48 frozen tissue paired T and NT samples of breast cancer patients treated at the National Cancer Institute Breast Cancer Hospital in Rio de Janeiro, Brazil. We identified a total of 7,749 probes with Benjamini-Hochberg adjusted p-value &amp;lt; 0.0001 and differences between T and NT tissue above |0.3|. Of these, 5,976 or 77.12% had lower DNA methylation in T when compared to NT tissue; while 1,773 (22.88%) had higher DNA methylation. These were used to identify 21 differentially methylated regions (DMRs) that contained a minimum of seven cg sites, a mean difference larger than |0.2| and a maximum difference above |0.4|. DMRs were mostly (12) hypermethylated in T, and included PCDHGA, NKAPL, ZKSCAN4, SPAG6, CA3, DPP6, RGS22, NXPH1, ANK3, OTX2, SOX17, and CDO1. We used pyrosequencing to validate the levels of SOX17 in eighty-eight tumor and non-adjacent tumor tissue DNAs. We found SOX17 to be hypermethylated in T (44.5±19.1% vs 10.2±10.6, p&amp;lt;0.0001). Differences in DNA methylation in T and NT tissue we still observed by race/ethnicity categories, 43.9±18.6% vs 11.0.±13.2% (p&amp;lt;0.0001) for Brown, and 45.1±20.7% vs 8.8±3.0% for Black women (p&amp;lt;0.0001), respectively. Similarly to previously reported, SOX17 hypermethylation was found to be associated with Luminal A and B molecular subtypes (p&amp;lt;0.0001). To our knowledge, this is the first study to use a discovery approach to identify epigenetic biomarkers in Black and Brown Brazilian women. Our findings confirming previously reported data on SOX17 hypermethylation in breast cancer are encouraging. We expect to validate other epigenetic markers unique to this population that can ultimately aid in the molecular characterization of Brazilian breast cancers. Citation Format: Lissette Delgado-Cruzata, Diego J. Gomes de Paula, Jennifer G. Vieira, André L. Christianes, Tatiana Simao, Leonor B. Gusmao, Luis Felipe Ribeiro Pinto, Sheila Coelho Soares Lima. Hypermethylation of SOX17 is an important epigenetic mark in Afro-Brazilian breast cancers [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 5207.

Abstract 756: Differences in composition, diversity, and function of the fecal microbiota of post-menopausal breast cancer patients compared to healthy, age-matched controls

Abstract Gut microbiota are a community of organisms that colonize the intestinal tract with essential roles in immune function, gut epithelial integrity, and metabolite production. Dysbiosis occurs when the gut microbiota environment is negatively altered causing detrimental changes to these functions, which may predispose an individual to breast cancer development. To understand the relationship between dysbiosis and breast cancer development, fecal microbiota samples were collected from 27 post-menopausal breast cancer patients of varying hormone receptor statuses (ER+HER2-, ER+Her2+, ER-HER2+, or ER-Her2-) prior to starting chemotherapeutic treatment. Samples were compared to 25 healthy controls, and all samples underwent 16srRNA sequencing. Samples from breast cancer patients and control groups were compared for significant differences (P&amp;lt;.05) in alpha (within sample) and beta (between sample) diversity. Using the Shannon Index and Generalized Least Squares Model, significant differences in alpha diversity were identified between all breast cancer types vs. controls (P=.0104), and between ER+HER2- breast cancer vs. controls (P=.00153). Healthy controls exhibited a higher mean Shannon Index than breast cancer patients, thus higher within sample diversity. Using principal coordinate analyses, significant differences in beta diversity were identified between all breast cancer types vs. controls (P = .01199), and ER+HER2- breast cancer vs. control (P = .008991). Further analyses identified significant taxa of microbiota with functions related to breast cancer development and progression. Of note, Lachnoclostridium, a genus of bacteria that can produce β-glucuronidase, which can deconjugate estrogen, was increased in all breast cancer types and ER+HER2- breast cancer when compared to controls. Deconjugation of estrogen can cause elevated blood estrogen levels, potentially increasing Estrogen Receptor binding in hormone-receptor-positive breast cancer. Additionally, Akkermansia, a genus implicated in gut barrier dysfunction, was decreased in ER+HER2- patients, while Parvimonas, a tumorigenic cell-cycle regulator, was increased. Elucidating if these effects are causative or reactionary may have important implications for understanding breast cancer etiology. Samples are currently undergoing additional metagenomic analyses using long-read sequencing to identify specific bacteria, viruses, fungi, parasites, and phages that may be contributing to dysbiosis. Overall, this study is the first, to our knowledge, to employ short and long-read sequencing to the fecal samples of untreated postmenopausal breast cancer patients. We provide mechanistic insight into the relationship between microbiota and breast cancer development, with the intent of informing future patient screening and prevention. Citation Format: Nadia Kabbej, Frederick Ashby, Erika Atencio, Erinn Rosenkrantz, Dominique Day, Khloe Dang, Josee Gauthier, Rachel Newsome, John Sommerville, Taqwa Naas, Raad Gharaibeh, Christian Jobin, Amira Quevedo, Coy D. Heldermon. Differences in composition, diversity, and function of the fecal microbiota of post-menopausal breast cancer patients compared to healthy, age-matched controls [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 756.

RANK and RANKL Expression in Tumors of Patients with Early Breast Cancer

AbstractThe receptor activator of nuclear factor-κB (RANK) pathway was associated with the pathogenesis of breast cancer. Several studies attempted to link the RANK/RANKL pathway to prognosis; however, with inconsistent outcomes. We aimed to further contribute to the knowledge about RANK/RANKL as prognostic factors in breast cancer. Within this study, protein expression of RANK and its ligand, RANKL, in the tumor tissue was analyzed in association with disease-free survival (DFS) and overall survival (OS) in a study cohort of patients with early breast cancer.607 samples of female primary and early breast cancer patients from the Bavarian Breast Cancer Cases and Controls Study were analyzed to correlate the RANK and RANKL expression with DFS and OS. Therefore, expression was quantified using immunohistochemical staining of a tissue microarray. H-scores were determined with the cut-off value of 8.5 for RANK and 0 for RANKL expression, respectively.RANK and RANKL immunohistochemistry were assessed by H-score. Both biomarkers did not correlate (ρ = −0.04). According to molecular subtypes, triple-negative tumors and HER2-positive tumors showed a higher number of RANK-positive tumors (H-score ≥ 8.5), however, no subtype-specific expression of RANKL could be detected. Higher RANKL expression tended to correlate with a better prognosis. However, RANK and RANKL expression could not be identified as statistically significant prognostic factors within the study cohort.Tumor-specific RANK and RANKL expressions are not applicable as prognostic factors for DFS and OS, but might be associated with subtype-specific breast cancer progression.

Surface-enhanced Raman spectroscopy of the filtrate portions of the blood serum samples of breast cancer patients obtained by using 30 kDa filtration device

Raman spectroscopy is reliable tool for analyzing and exploring early disease diagnosis related to body fluids, such as blood serum, which contain low molecular weight fraction (LMWF) and high molecular weight fraction (HMWF) proteins. The disease biomarkers consist of LMWF which are dominated by HMWF hence their analysis is difficult. In this study, in order to overcome this issue, centrifugal filter devices of 30 kDa were used to obtain filtrate and residue portions obtained from whole blood serum samples of control and breast cancer diagnosed patients. The filtrate portions obtained in this way are expected to contain the marker proteins of breast cancer of the size below this filter size. These may include prolactin, Microphage migration inhabitation factor (MIF), γ-Synuclein, BCSG1, Leptin, MUC1, RS/DJ-1 present in the centrifuged blood serum (filtrate portions) which are then analyzed by the SERS technique to recognize the SERS spectral characteristics associated with the progression of breast cancer in the samples of different stages as compared to the healthy ones. The key intention of this study is to achieve early-stage breast cancer diagnosis through the utilization of Surface Enhanced Raman Spectroscopy (SERS) after the centrifugation of healthy and breast cancer serum samples with Amicon ultra-filter devices of 30 kDa. The silver nanoparticles with high plasmon resonance are used as a substrate for SERS analysis. Principal Component Analysis (PCA) and Partial Least Discriminant Analysis (PLS-DA) models are utilized as spectral classification tools to assess and predict rapid, reliable, and non-destructive SERS-based analysis. Notably, they were particularly effective in distinguishing between different SERS spectral groups of the cancerous and non-cancerous samples. By comparing all these spectral data sets to each other PLSDA shows the 79 % accuracy, 76 % specificity, and 81 % sensitivity in samples with AUC value of AUC = 0.774 SERS has proven to be a valuable technique for the rapid identification of the SERS spectral features of blood serum and its filtrate fractions from both healthy individuals and those with breast cancer, aiding in disease diagnosis.

Advancing canine mammary tumor diagnostics: Unraveling the diagnostic potential of Cytokeratin 19 through droplet digital PCR analysis

Cytokeratin 19 (CK19) is a complex intracytoplasmic cytoskeletal protein primarily localized in the ducts of the mammary gland and skin epithelial cells. In humans, the expression of CK19 gene within circulating tumor cells (CTCs) extracted from blood samples of breast cancer patients reflects tumor cell activity, offering valuable insights for predicting early metastatic relapse or monitoring treatment effectiveness. However, knowledge of serum tumor markers is limited in veterinary oncology. Recently, droplet digital PCR (ddPCR), has been employed to explore rare target genes due to its heightened sensitivity and accuracy as a novel molecular diagnostic tool. The objectives of this study were to investigate the expression of the CK19 mRNA in CTCs, non-neoplastic mammary tissues, and both benign and malignant canine mammary tumors (CMTs) through ddPCR analysis. In Study I, we optimized the discard volume for blood samples to reduce CK19 contamination from skin epithelial cells post-venipuncture. The results revealed that discarding the initial 3 mL of blood was adequate and effective in eliminating CK19 mRNA contamination. In Study II, after the removal of the initial 3 mL of blood, we investigated CK19 mRNA-positive CTCs in the peripheral blood of normal healthy dogs, including those with benign and malignant CMTs. Intriguingly, CK19 mRNA was undetectable in all blood samples. The expression of CK19 mRNA in mammary tissues was investigated in Study III. The copy number (CN) ratios of the CK19 gene in non-neoplastic mammary tissues (14.77 ± 14.65) were significantly higher (P < 0.05) than those in benign (4.23 ± 3.35) and malignant groups (6.56 ± 5.64). Notably, no difference was observed between the benign and malignant groups. In conclusion, CK19 mRNA appeared unlikely to be a suitable candidate as a biomarker in the peripheral blood of CMTs, while the CN ratio in mammary tissues could serve as a potential discriminator between non-neoplastic and CMT groups, complementing the gold standard of histopathological examination.

A Bio-Inspired Free-Standing Film with Versatility: From Heterogeneous CTCs Programed Isolating to Breast Cancer Molecular Typing.

Developing a robust strategy for profiling heterogeneous circular tumor cells specifically, distinguishing the phenotypes of which in blood sample of cancer patient precisely, and releasing them sequentially, is significant for cancer management by liquid biopsy. Herein, a bio-inspired free-standing and flexible film composed of TiO2 nanotube and silk fibroin, fabricated with multiply dynamic bioactive surface (TSF/MDBS) by a simple and eco-friendly way including using polydopamine chemistry and dual dynamic covalent chemistry, is reported. The as-prepared TSF/MDBS binds specific peptides toward cells with epithelial biomarker and human epithelial growth factor receptor 2 (HER2) biomarker, and antifouling agents bovine serum albumin for obviating platelets and proteins adhering of blood, can capture heterogeneous CTCs with enhanced capability due to the cytocompatible soft film and exquisite surface design, and further release the captured cells as program, by specifically breaking down the covalent bonds in sequence via the action of adding biocompatible molecules fructose and glutathione. By applying the TSF/MDBS, it can be tailored into desired pieces for identifying CTCs with different phenotypes (HER2-high and HER2-low) from the unprocessed blood samples of breast cancer patients, and finally profiling these heterogeneous CTCs, to discriminate HER2 positive or negative of breast cancer patients in clinical applications.

Construction of a ferroptosis-based prognostic model for breast cancer helps to discriminate high/low risk groups and treatment priority.

Breast cancer is a common malignant tumor associated with high morbidity and mortality. The role of ferroptosis, a regulated form of cell death, in breast cancer development and prognosis remains unclear. This study aims to investigate the relationship between ferroptosis-related genes and breast cancer and develop a prognostic model. RNA-seq expression datasets and clinical samples of breast cancer patients were obtained from public databases. Immunity- and drug resistance-related data were integrated. A preliminary screening was performed, resulting in the identification of 73 candidate ferroptosis factors. Univariate Cox regression analysis was conducted to select 12 genes, followed by LASSO Cox regression analysis to construct a prognostic risk prediction model consisting of 10 ferroptosis-related genes. The model was further characterized by immune cell infiltration. The expression levels of ferroptosis-related genes were validated in human breast cancer cell lines, and immunohistochemical (IHC) analysis was conducted on cancer specimens to assess ferroptosis-related protein expression. The study identified 10 ferroptosis-related genes that were significantly associated with breast cancer prognosis. The constructed prognostic risk prediction model showed potential for predicting the prognostic value of these genes. In addition, the infiltration of immune cells was observed to be a characteristic of the model. The expression levels of ferroptosis-related genes were confirmed in human breast cancer cell lines, and IHC analysis provided evidence of ferroptosis-related protein expression in cancer specimens. This study provides a novel prognostic model for breast cancer, incorporating 10 ferroptosis-related genes. The model demonstrates the potential for predicting breast cancer prognosis and highlights the involvement of immune cell infiltration. The expression levels of ferroptosis-related genes and proteins further support the association between ferroptosis and breast cancer development.

Blood Plasma Circulating DNA-Protein Complexes: Involvement in Carcinogenesis and Prospects for Liquid Biopsy of Breast Cancer.

Circulating DNA (cirDNA) is a promising tool in translational medicine. However, studies of cirDNA have neglected its association with proteins, despite ample evidence that this interaction may affect the fate of DNA in the bloodstream and its molecular functions. The goal of the current study is to shed light on the differences between the proteomic cargos of histone-containing nucleoprotein complexes (NPCs) from healthy female (HFs) and breast cancer patients (BCPs), and to reveal the proteins involved in carcinogenesis. NPCs were isolated from the blood samples of HFs and BCPs using affinity chromatography. A total of 177 and 169 proteins were identified in NPCs from HFs and BCPs using MALDI-TOF mass spectrometry. A bioinformatics analysis revealed that catalytically active proteins, as well as proteins that bind nucleic acids and regulate the activity of receptors, are the most represented among the unique proteins of blood NPCs from HFs and BCPs. In addition, the proportion of proteins participating in ion channels and proteins binding proteins increases in the NPCs from BCP blood. However, the involvement in transport and signal transduction was greater in BCP NPCs compared to those from HFs. Gene ontology term (GO) analysis revealed that the NPC protein cargo from HF blood was enriched with proteins involved in the negative regulation of cell proliferation, and in BCP blood, proteins involved in EMT, invasion, and cell migration were observed. The combination of SPG7, ADRB1, SMCO4, PHF1, and PSMG1 NPC proteins differentiates BCPs from HFs with a sensitivity of 100% and a specificity of 80%. The obtained results indirectly indicate that, in tandem with proteins, blood cirDNA is an important part of intercellular communication, playing a regulatory and integrating role in the physiology of the body.

RANK and RANKL Expression in Tumors of Patients with Early Breast Cancer

AbstractThe receptor activator of nuclear factor-κB (RANK) pathway was associated with the pathogenesis of breast cancer. Several studies attempted to link the RANK/RANKL pathway to prognosis; however, with inconsistent outcomes. We aimed to further contribute to the knowledge about RANK/RANKL as prognostic factors in breast cancer. Within this study, protein expression of RANK and its ligand, RANKL, in the tumor tissue was analyzed in association with disease-free survival (DFS) and overall survival (OS) in a study cohort of patients with early breast cancer.607 samples of female primary and early breast cancer patients from the Bavarian Breast Cancer Cases and Controls Study were analyzed to correlate the RANK and RANKL expression with DFS and OS. Therefore, expression was quantified using immunohistochemical staining of a tissue microarray. H-scores were determined with the cut-off value of 8.5 for RANK and 0 for RANKL expression, respectively.RANK and RANKL immunohistochemistry were assessed by H-score. Both biomarkers did not correlate (ρ = −0.04). According to molecular subtypes, triple-negative tumors and HER2-positive tumors showed a higher number of RANK-positive tumors (H-score ≥ 8.5), however, no subtype-specific expression of RANKL could be detected. Higher RANKL expression tended to correlate with a better prognosis. However, RANK and RANKL expression could not be identified as statistically significant prognostic factors within the study cohort.Tumor-specific RANK and RANKL expressions are not applicable as prognostic factors for DFS and OS, but might be associated with subtype-specific breast cancer progression.

Exosomal circRNA RHOT1 promotes breast cancer progression by targeting miR-204-5p/ PRMT5 axis

BackgroundCircular RNA RHOT1 (circRHOT1) plays crucial roles in tumorigenesis by competing with microRNAs. It is largely abundant in tumor cell-derived exosomes. Meanwhile, cancer-derived exosomes participate in diverse biological processes. However, the expression patterns and functions of exosomal circRHOT1 in breast cancer remain unknown. This study is aimed to investigate and elucidate the exosomal circRHOT1/miR-204-5p/PRMT5 axis in breast cancer.MethodsThe exosomes derived from serum samples of breast cancer patients and breast cancer cell lines were characterized using transmission electron microscopy and Western blot. MTT, colony formation, wound healing, and transwell assays were utilized to analyze cell proliferation, migration, and invasion of breast cancer cells. Flow cytometry was used for apoptosis analysis. The bioinformatics method was employed to screen differentially expressed novel circRNAs and predict the microRNA targets of circRHOT1. Dual-luciferase reporter gene assays were performed to verify their direct interaction. Finally, Xenograft experiments were used to investigate the effect of exosomal circRHOT1 on tumor growth in vivo.ResultsCircRHOT1 exhibited significantly high expression in exosomes derived from the serum of breast cancer patients and breast cancer cell lines, which suggested its potential diagnostic value. Breast cancer-derived exosomes promoted the cell proliferation, migration, invasion, and epithelial-mesenchymal transition of breast cancer cells while inhibiting apoptosis. However, exosomes with downregulated circRHOT1 inhibited the growth of co-cultured cells. Mechanistically, circRHOT1 acted as a sponge of miR-204-5p and promoted protein arginine methyltransferase 5 (PRMT5) expression. Moreover, miR-204-5p inhibitor and pcPRMT5 could reverse the tumor suppressive effects mediated by circRHOT1-knockdown. Furthermore, treatment with exosomes derived from breast cancer cells with circRHOT1 knockdown attenuated tumor growth in tumor-bearing nude mice, which was accompanied by a reduction in PRMT5 expression and an enhancement of miR-204-5p expression.ConclusionThe exosomal circRHOT1 may promote breast cancer progression by regulating the miR-204-5p/PRMT5 axis. The current study strengthens the role of circRHOT1, miR-204-5p, and PRMT5 in breast cancer development and provides a potential treatment strategy for breast cancer.

Case Report: The Relationship of CCND1 RS614367 Polymorphism with Clinicopathological Features

Recent studies have shown that the CCND1 rs614367 polymorphism increases the risk of breast cancer and its invasive nature. However, studies evaluating the relationship of the CCND1 rs614367 polymorphism based on the clinicopathology of breast cancer patients in Indonesia were still limited. This study is aimed to determine the CCND1 rs614367 polymorphism in breast cancer and its relationship with the patient's clinicopathology. Methods: This study was a cross-sectional study on 45 samples of breast cancer patients. After collecting demographic and clinical data, PCR and sequencing will be performed on all blood samples to determine the CCND1 rs614367 polymorphism. All variables that have been collected will be analyzed using SPSS version 25.0 to determine the relationship between the CCND1 rs614367 polymorphism and the clinicopathology of breast cancer patients. The CCND1 rs614367 gene polymorphism in breast cancer subjects showed that 25 (55.5%) and 20 (44.5%) subjects had C and T alleles. Subjects aged ≥ 50 years old had a significant 4.45 risk of having the T allele type (p=0.037). In addition, subjects with metastases (M1) were also at a significant 4.89 times risk of having the T allele type (p=0.015). Subjects with histological grade III also had a significantly 4.77 times risk of having the T allele type (p=0.013). In conclusion, there was a significant relationship between CCND1 rs614367 polymorphism and breast cancer subjects' clinopathology features (age, metastasis, and grade). More than half of the subjects with this polymorphism had the C allele.