Tissues Of Breast Cancer Patients Research Articles

The mutational landscape of Chinese breast cancer by liquid biopsy.

e13117 Background: Breast cancer is the most common malignancy worldwide. The novel technique of liquid biopsy provides further prospect of breast cancer management. However, the clinical application of liquid biopsy for breast cancer remains clearly defined. Methods: Blood samples of 145 Chinese patients diagnosed as breast cancer were collected. cfDNA were analyzed by next-generation sequencing technique using Haplox HapOnco panel. The mutations of cfDNA were analyzed and compared with genomic mutations detected within tumor tissues of breast cancer patients. Results: In total, 327 genetic mutations were detected in 145 patients using cfDNA. The top 5 high frequent alterations were TP53(50.3%), PIK3CA (42.8%), SYNE1 (16.6%), ESR1 (10.3%), CSMD3 (9.0%). Within TP53 mutations, 13.7% were frame shift insertions or deletions, 5.5% were in-frame insertions or deletions, 9.6% were splice site mutations. Within PIK3CA mutations, 1.6% were in-frame insertions or deletions, 1.6% were copy number variations. The average number of mutations for each patient was 7.2 (5.5 to 9.0, 95% CI). The mutated genes in cfDNA were compared with those in tumor tissues in publications. 208 mutated genes in tumor tissues were also detected in cfDNA. There was no significant difference in mutation rate for 83.6% genes in blood and tumor tissues. Conclusions: Most mutated genes in tumor tissues of breast cancer patients could be detected in cfDNA. The mutation rate for most genes had no significant difference in blood and tumor tissues, which indicated that liquid biopsy using cfDNA is a promising technique to improve breast cancer management.

Fusobacterium nucleatum-derived small extracellular vesicles facilitate tumor growth and metastasis via TLR4 in breast cancer

BackgroundThe contributive role of the microbiome in tumor progression has been reported in multiple studies, such as the Fusobacterium nucleatum (F. nucleatum) in breast cancer (BC). This study aimed to explore the role of F. nucleatum-derived small extracellular vesicles (Fn-EVs) in BC and preliminarily uncover the mechanism.MethodsTen normal and 20 cancerous breast tissues were harvested to investigate the gDNA expression of F. nucleatum and its relation with the clinical characteristics of BC patients. After isolating Fn-EVs by ultracentrifugation from F. nucleatum (ATCC 25,586), both MDA-MB-231 and MCF-7 cells were treated with PBS, Fn, or Fn-EVs, followed by being subjected to CCK-8, Edu staining, wound healing, and Transwell assays to detect their cell viability, proliferation, migration, and invasion. TLR4 expression in BC cells with diverse treatments was assessed by western blot. In vivo experiments were performed to verify its role in tumor growth and liver metastasis.ResultsThe F. nucleatum gDNA levels of breast tissues in BC patients were significantly higher than those in normal subjects, and positively associated with tumor size and metastasis. Fn-EVs administration significantly enhanced the cell viability, proliferation, migration, and invasion of BC cells, while knocking down TLR4 in BC cells could block these effects. Furthermore, in vivo study verified the contributive role of Fn-EVs in tumor growth and metastasis of BC, which might rely on its regulation of TLR4.ConclusionsCollectively, our results suggest that F. nucleatum plays an important role in BC tumor growth and metastasis by regulating TLR4 through Fn-EVs. Thus, a better understanding of this process may aid in the development of novel therapeutic agents.

Construction of Genetically Encoded Light-Up RNA Aptamers for Label-free and Ultrasensitive Detection of CircRNAs in Cancer Cells and Tissues.

Circular RNAs (circRNAs) as endogenous non-coding RNAs are characterized by covalently closed circular structures, and they widely exist in mammalian cells. The aberrant expression of circRNAs may result in various diseases. Herein, we demonstrate the construction of genetically encoded light-up RNA aptamers for ultrasensitive and label-free detection of circRNA mitochondrial tRNA translation optimization 1 (circMTO1) in cancer cells and tissues. The light-up RNA aptamers are generated by proximity ligation-activated recombinase polymerase amplification (RPA)-assisted transcription amplification. When circMTO1 is present, it initiates the proximity ligation reaction, activating RPA to produce numerous long double-stranded DNAs containing T7 promoters. Subsequently, the RPA products are identified by T7 RNA polymerase, initiating the transcription amplification reaction to generate abundant Spinach RNA aptamers. Spinach RNA aptamers can bind with DFHBI (3,5-difluoro-4-hydroxybenzylidene imidazolidinone) dye to produce a distinct fluorescence signal with near-zero background. This biosensor exhibits excellent selectivity and high sensitivity with a limit of detection of 2.54 aM. It can accurately monitor cellular circMTO1 at the single-cell level and discriminate the expression of circMTO1 between breast cancer patient tissues and healthy tissues. Notably, this biosensor can be employed to measure other nucleic acids by altering the corresponding target recognition sequences, providing a valuable platform for cancer diagnosis and biomedical study.

O090 TRANSCRIPTOMICS OF BREAST CANCER TUMOUR AND NORMAL ADJACENT TUMOUR TISSUE REVEAL DIFFERENTIAL IMMUNE-ADIPOSE-CANCER GENE EXPRESSION

Abstract Introduction Surgical-related immunity dysfunction in normal adjacent tumour (NAT) tissue of breast cancer (BC) patients is associated with BC recurrence. Characterising inflammation and immunosuppression in NAT tissue at the time of surgical excision of the tumour, using transcriptomics, may reveal a tissue phenotype more vulnerable to surgical-related immunity dysfunction. Methods RNA-Seq read counts from matched BC patient tumour and NAT tissues from the cancer genome atlas (TCGA) (n=54) and from University College Cork (UCC) & Cork University Hospital (CUH) biobank (n=10) were used. Differential gene expression was analysed using EdgeR and pathway analysis by Gene Set Enrichment Analysis using Hallmark gene sets. Immune cell subsets were deconvoluted using CIBERSORTx using the LM22 signature matrix. Genes were significantly different with a False Discovery Rate (FDR)<0.1 and pathways enriched with an FDR<0.25. Results Adipogenesis and IL6 JAK STAT3 signalling pathways were enriched in NAT and not in tumour tissue (FDR<0.25). Adipogenesis genes ADIPOQ, FABP4, CD36, LPL and PPARG and IL6 were significantly upregulated in NAT relative to tumour (FDR<0.1). Patients stratified on NAT expression of these genes, with higher expression had worse overall survival (OS)(p<0.05). There was no difference in patient OS, stratified on tumour expression of ADIPOQ and IL6. M2 macrophages were significantly increased in NAT relative to tumour(p<0.05). Patients with more M2 macrophages in tumours had significantly worse OS(p<0.05). There was no difference in OS for NAT tissue with more M2 macrophages. Conclusion Surgical-related immunity dysfunction in NAT breast tissue, may worsen postoperative immune-adipose-residual breast cancer cell interactions, contributing to poorer patient outcome.

Evaluation of hematopoietic- and neurologic-expressed sequence 1-like (HN1L) protein levels in tissue and plasma of breast cancer patients

Objectives: Breast cancer is the second leading cause of cancer deaths among women. Therefore, there is a need for new approaches that increase the success of treatment in breast cancer. Cancer stem cells (CSCs) are associated with treatment resistance and metastasis, which are important problems in cancer treatment including breast tumors. In this study, the Hematopoietic- and neurologic-expressed sequence 1-like (HN1L), also known as Jupiter microtubule associated homolog 2 (JPT2) protein levels which is involved in the self-renewal of CSCs were evaluated in common and rare breast tumor types. Methods: In this context, HN1L protein levels were measured from plasma of 17 patients and from tumor and normal tissues of 9 patients by enzyme linked immunosorbent assay method. Results: Mean HN1L levels were measured as 1.63 ± 0.88 ng/mL in plasma samples, 2.18 ± 0.75 ng/mL in tumor tissue samples and 2.71 ± 0.88 ng/mL in normal tissue samples. A significant difference was observed between mean HN1L levels in plasma and normal tissue (p < 0.05). Correlation of HN1L protein levels with clinicopathological characteristics were analyzed. Accordingly, HN1L levels were positively correlated with tumor size and invasion status (r=0.425; p < 0.05 and r=0.449; p < 0.05, respectively). Conclusions: We believe that the importance of HN1L in management of breast cancers will be demonstrated more thoroughly when further studies are conducted with increased number of patients.

Expression of genes related to iron homeostasis in breast cancer

The dysfunctions in the metabolism of iron have an important role in many pathological conditions, ranging from disease with iron deposition to cancer. Studies on malignant diseases of the breast reported irregular expression in genes associated with iron metabolism. The variations are related to findings that have prognostic significance. This study evaluated the relationship of the expression levels of transferrin receptor 1 (TFRC), iron regulatory protein 1 (IRP1), hepcidin (HAMP), ferroportin 1 (FPN1), hemojuvelin (HFE2), matriptase 2 (TMPRSS6), and miR-122 genes in the normal and malignant tissues of breast cancer patients. The normal and malignant tissues from 75 women with breast malignancies were used in this study. The patients did not receive any treatment previously. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used in figuring the levels of gene expression associated with iron metabolism. When the malignant and normal tissues gene expression levels were analyzed, expression of TFRC increased (1.586-fold); IRP1 (0.594 fold) and miR-122 (0.320 fold) expression decreased; HAMP, FPN1, HFE2, and TMPRSS6 expressions did not change. FPN1 and IRP1 had a positive association, and this association was statistically significant (r = 0.266; p = 0.022). IRP1 and miR-122 had a positive association, and this association had statistical significance (r = 0.231; p = 0.048). Our study portrayed the important association between genes involved in iron hemostasis and breast malignancy. The results could be used to establish new diagnostic techniques in the management of breast malignancies. The alterations in the metabolism of malignant breast cells with normal breast cells could be utilized to achieve advantages in treatment.

Methylation Analysis of Promoter Region of ETS-1 Gene in Human Breast Cancer Patients

This study aimed to compare the methylation status of ETS1 between 18 cancerous and cancer-side tissues in breast cancer patients using matrix-assisted laser ionization-time flight mass spectrometry.

RNA demethylation-driven functional supramolecular structure for label-free detection of m6A modification eraser FTO in human breast tissues

Fat mass and obesity-associated enzyme (FTO) can dynamically regulate N6-methyladenosine modification, and it is engaged in various cellular functions. Herein, we demonstrate the RNA demethylation-driven functional supramolecular structure for label-free detection of m6A modification eraser FTO in human breast tissues. The presence of FTO catalyzes the removal of methyl group in m6A, causing the cleavage of demethylated DNA by DpnII and the release of DNA primer. The resultant DNA primer hybridizes with circular template to initiate isothermal rolling circle amplification (RCA), producing abundant long ssDNA polymers with repeating sequences of G-quadruplex. Subsequently, N-methylmesoporphyrin IX (NMM) is selectively embedded into G-quadruplex DNAzyme to form a supramolecular NMM-G-quadruplex structure for the generation of an amplified fluorescence signal. Benefiting from high selectivity of DpnII toward demethylated DNA, high amplification efficiency of RCA, and high signal-to-noise ratio of G-quadruplex-NMM system, this assay can sensitively detect FTO with a limit of detection (LOD) of 3.10 × 10−16 M, screen RNA demethylase inhibitors, quantify FTO activity in cancer cells, and discriminate FTO activity between breast cancer patient tissues and healthy person tissues. Importantly, this assay can be homogeneously conducted in a label-free manner, with great potential in RNA demethylases-related pathogenesis research and clinical diagnostics.

Abstract 1331: A novel approach to treating hormonal breast cancer using clinical database and 3D ex vivo model

Abstract Type 2 diabetes (T2D) female patients have a higher risk of being diagnosed and have worse survival for breast cancer compared to non-diabetic females. Although the relationship between these two female diseases has been reported, specific functional studies on which subtypes of breast cancer are affected and which genes are regulated by T2D are little known. Here, I found that Forkhead Box A1 (FOXA1) and Metformin, anti-diabetic medication for T2D, affect hormonal positive (HR+) breast cancer tumor cell growth and metastasis. 14 diabetes-related genes highly expressed in three HR+ breast cancer cell lines but not other breast cancer subtypes using a 53,805 gene database obtained from NCBI GEO. Among the diabetes-related genes, FOXA1, MTA3, PAK4, FGFR3, and KIF22 were highly expressed in HR+ breast cancer from 4,032 breast cancer patient tissue samples using the Breast Cancer Gene Expression Omnibus. Specifically, the high expression of FOXA1 correlated to a worse ER+/PR+ breast cancer patient survival rate. Consistent with this, the loss of FOXA1 inhibited the tumor proliferation and invasion of MCF-7 and T47D HR+ breast cancer cell lines using in vitro. Metformin (1,1-dimethylbiguanide hydrochloride) conspicuously inhibits the tumor cell growth in MCF-7 human hormonal breast cancer cells. Metformin or FOXA1 deletion enhanced Tamoxifen-mediated tumor growth inhibition in HR+ breast cancer cell lines through the ex vivo three-dimensional (3D) organoid model. Therefore, HR+ breast cancer is closely related to T2D, and metformin and FOXA1 inhibition might be an optimal new treatment for patients with HR+ breast cancer when combined with tamoxifen, a hormone therapy. Citation Format: Chaeun (Christine) Song, SeungBaek Lee. A novel approach to treating hormonal breast cancer using clinical database and 3D ex vivo model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1331.

35 Novel statistical methods to unlock the clinical potential of liquid biopsy sampling

OBJECTIVES/GOALS: Decoding the origins of cell-free DNA (cfDNA) released from dying cells in a liquid biopsy sample (e.g. blood) can provide insight into the dynamic, organism-wide changes reflective of health and disease state. Making cfDNA an ideal target for genomic monitoring of disease-related changes. METHODS/STUDY POPULATION: Methylome-wide sequencing (WGBS) data present unique statistical challenges. To this end, we developed a novel statistical method using an Expectation-Maximization algorithm to decode the cellular origins of cfDNA fragments in liquid biopsies. Our flexible, probabilistic method leverages the co-regulation of neighboring CpG sites on the individual sequencing read to facilitate tissue of origin analysis, as opposed to prior methods that focus on the methylation rate of a single CpG site. We assess the performance of our model in various simulated settings and apply our model to an important clinical example in which we are able to detect early off-target tissue damage from radiation therapy via minimally invasive blood draws. RESULTS/ANTICIPATED RESULTS: We found our model more effective at capturing the range of biologically plausible methylation patterns on cfDNA read fragments compared to prior models that use single CpG sites. We also show our model is robust to high levels of noise inherent with WGBS data. We demonstrate the accuracy of cell-type proportion estimation on in-silico mixed cfDNA samples from real WGBS data. Finally, we use our model in a clinical application. We detect significant (p < 0.05) increases in cellular contributions from lung and cardiac tissue in breast cancer patients (n=15) undergoing radiation therapy compared to baseline. We also detect novel signals of radiation induced toxicity to the liver in right-sided breast cancer patients (n=8) receiving radiation treatment compared to matched left-sided breast cancer patients (n=7). DISCUSSION/SIGNIFICANCE: Here we address an unmet need in developing novel statistical methodologies that can handle the unique complexities of methylated cfDNA obtained from liquid biopsy samples. We also demonstrate the far-ranging clinical utility of serial liquid biopsy sampling to complement and advance standards of clinical care in oncology and other pathologies.

DNA methylation age in paired tumor and adjacent normal breast tissue in Chinese women with breast cancer

BackgroundFew studies have examined epigenetic age acceleration (AA), the difference between DNA methylation (DNAm) predicted age and chronological age, in relation to somatic genomic features in paired cancer and normal tissue, with less work done in non-European populations. In this study, we aimed to examine DNAm age and its associations with breast cancer risk factors, subtypes, somatic genomic profiles including mutation and copy number alterations and other aging markers in breast tissue of Chinese breast cancer (BC) patients from Hong Kong.MethodsWe performed genome-wide DNA methylation profiling of 196 tumor and 188 paired adjacent normal tissue collected from Chinese BC patients in Hong Kong (HKBC) using Illumina MethylationEPIC array. The DNAm age was calculated using Horvath’s pan-tissue clock model. Somatic genomic features were based on data from RNA sequencing (RNASeq), whole-exome sequencing (WES), and whole-genome sequencing (WGS). Pearson’s correlation (r), Kruskal–Wallis test, and regression models were used to estimate associations of DNAm AA with somatic features and breast cancer risk factors.ResultsDNAm age showed a stronger correlation with chronological age in normal (Pearson r = 0.78, P < 2.2e−16) than in tumor tissue (Pearson r = 0.31, P = 7.8e−06). Although overall DNAm age or AA did not vary significantly by tissue within the same individual, luminal A tumors exhibited increased DNAm AA (P = 0.004) while HER2-enriched/basal-like tumors exhibited markedly lower DNAm AA (P = < .0001) compared with paired normal tissue. Consistent with the subtype association, tumor DNAm AA was positively correlated with ESR1 (Pearson r = 0.39, P = 6.3e−06) and PGR (Pearson r = 0.36, P = 2.4e−05) gene expression. In line with this, we found that increasing DNAm AA was associated with higher body mass index (P = 0.039) and earlier age at menarche (P = 0.035), factors that are related to cumulative exposure to estrogen. In contrast, variables indicating extensive genomic instability, such as TP53 somatic mutations, high tumor mutation/copy number alteration burden, and homologous repair deficiency were associated with lower DNAm AA.ConclusionsOur findings provide additional insights into the complexity of breast tissue aging that is associated with the interaction of hormonal, genomic, and epigenetic mechanisms in an East Asian population.

Construction of a Single-Molecule Biosensor for Antibody-Free Detection of Locus-Specific N6-Methyladenosine in Cancer Cells and Tissues.

N6-Methyladenosine (m6A) has emerged as a key post-transcriptional regulator in mRNA metabolism, and its dysregulation is associated with multiple human diseases. Herein, we construct a single-molecule fluorescent biosensor for antibody-free detection of locus-specific m6A in cancer cells and tissues. A 5'-biotinylated capture probe and a 3'-hydroxylated assistant probe are designed for the recognition of specific m6A-mRNA. The m6A-sensitive endoribonuclease MazF can identify and cleave the unmethylated mRNA, and the retained intact m6A-mRNA can hybridize with assistant probes and capture probes to achieve sandwich hybrids. The sandwich hybrids are immobilized on magnetic beads (MBs) to initiate the terminal deoxynucleotidyl transferase (TdT)-assisted polymerization, facilitating the continuous incorporation of Cy5-dATP to form long Cy5-polyA tails for the production of an on-bead amplified fluorescence signal. After magnetic separation and exonuclease digestion, numerous Cy5 fluorophores are released and subsequently measured by single-molecule detection. Especially, this biosensor is implemented simply and isothermally without the involvement of either radiolabeling or m6A-specific antibody. Moreover, this biosensor shows ultrahigh sensitivity with a detection limit of 2.24 × 10-17 M, and it can discriminate a 0.01% m6A level from a large pool of coexisting counterparts. Furthermore, this biosensor can be used for monitoring cellular m6A-mRNA expression and differentiating the m6A level in the breast cancer patient tissues from that in the healthy person tissues, providing a new avenue for clinical diagnosis and epitranscriptomic research.

Abstract P5-03-08: Joint associations of mammographic breast density and obesity on the presence of crown-like structures in the breast adipose tissue of breast cancer patients

Abstract Background: Obesity is an established risk factor for postmenopausal breast cancer and is associated with poor outcomes. Accumulating evidence suggests crown-like structures in the breast adipose tissue (CLS-B), a marker of local inflammation, play a role in explaining the obesity-breast cancer association. However, it is unknown whether breast tissue composition (i.e., the amount of fibroglandular tissue in the breast relative to fat) is related to CLS-B. Objective: We evaluated whether breast tissue composition, as reflected by mammographic breast density, is associated with breast adipose tissue inflammation, as indicated by the presence of CLS-B, and whether the combination of breast density and obesity increases the presence of CLS-B among newly diagnosed breast cancer patients. Methods: We examined the presence of CLS-B, detected by CD68 immunohistochemistry, in breast adipose tissue obtained via mastectomy from a quadrant uninvolved by tumor among 254 women with stage I–III breast cancer treated at Emory University Hospitals (2007–2012). Patient characteristics, including mammographic breast density (assessed on a mammogram up to 5 years before breast surgery) and body mass index (BMI) at diagnosis, were abstracted from electronic medical records. Mammographic density was assessed using the Breast Imaging Reporting and Data System (BI-RADS) density classification (1=almost entirely fat; 2=scattered fibroglandular densities; 3=heterogeneously dense; and 4=extremely dense); density was further categorized as fatty (BI-RADS 1-2) and dense (BI-RADS 3-4). Age and multivariable (MV)-adjusted logistic regression models were used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) for the independent and joint associations of breast density (dense vs. fatty) and obesity (BMI ≥30 kg/m2 vs. &amp;lt; 30 kg/m2) on the presence of CLS-B. Multivariable models adjusted for age (continuous, years) and parity (nulliparous, parous) with mutual adjustment of BMI (continuous, kg/m2) or mammographic breast density (dense, fatty) depending on the model. Results: Women with obesity were more likely to have fatty breasts than women without obesity (52% vs. 27%). Obesity was strongly associated with the presence of CLS-B in age-adjusted (OR=3.11, 95% CI: 1.79, 5.48) and multivariable models (MV-OR=3.70, 95% CI: 1.96, 7.15). There was no apparent association between dense breast tissue and presence of CLS-B in the age-adjusted model (OR=1.04, 95% CI: 0.59, 1.85). After additional adjustment for BMI and parity, we noted that women with dense breasts had higher odds of having CLS-B compared to those with fatty breasts (MV-OR=2.13, 95% CI: 1.07, 4.41). MV-ORs from the joint model (common referent: not obese, fatty breasts) were 1.54 (95% CI: 0.62, 4.24) for women without obesity but with dense breasts, 3.18 (95% CI: 1.15, 9.56) for women with obesity and fatty breasts, and 6.24 (95% CI: 2.23, 19.2) for women with obesity and dense breasts. Conclusions: Our findings suggest that dense rather than fatty breast tissue is associated with breast adipose tissue inflammation among women with breast cancer. Results of the joint analyses suggest obesity is more strongly predictive of CLS-B presence than breast density. However, density may be an important risk factor for CLS-B among women without obesity while patients with obesity and dense breast tissue are the most likely to have CLS-B present. Future studies may consider the mechanisms by which density leads to increased presence of CLS-B. Joint Associations Table Table 1. Joint associations between mammographic breast density (MD) and body mass index (BMI) on the presence of crown-like structures in breast adipose tissue (CLS-B) among 254 women diagnosed with invasive breast cancel (stage I-III) at Emory University between 2007 and 2012 Citation Format: Maret L. Maliniak, Rebecca L. Seidel, Kimberly Bertrand, Lauren E. McCullough. Joint associations of mammographic breast density and obesity on the presence of crown-like structures in the breast adipose tissue of breast cancer patients [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-03-08.

Abstract P2-26-22: Elevated Expression of High Mannose Glycans in Tissue and Serum Samples of Breast Cancer Patients

Abstract Glycobiology has proved to be a new frontier in oncology research in recent years. One sector of glycobiology involves examining glycan expression of cancerous cells. Glycans are complex carbohydrates found on cell surfaces, and patterns of aberrant glycan expression, such as increased sialylation and fucosylation, have been proposed as potential novel biomarkers of cancer tumorigenesis and metastasis. However, to our knowledge, there is little literature examining the differences in expression of high mannose glycans, a subtype of N-linked glycans, which is the focus of this study. Thirty-nine samples of malignant and surrounding benign tissue from breast cancer patients were collected from the Prisma Health Cancer Institute Biorepository. Additionally, forty malignant serum samples were collected—ten of these samples were from the same patients included in the tissue samples. Serum samples from ten healthy volunteers were also obtained as controls. The malignant samples included specimens from breast cancer staged I through IV at the time of the patients’ diagnosis. Samples were analyzed using MALDI-TOF mass spectroscopy for the presence of glycans by Emory Glycomics and Molecular Interactions Core. The known masses of sixty-one unique glycans were then compared to the spectra of the samples analyzed to determine the type of glycans present, and the relative amount of each glycan present was determined using the area under the curve in the spectral data. Data analysis revealed the means of the area under the curve for high mannose glycans in the malignant and normal tissue samples to be 247.18 and 163.6 respectively; the t-test established the difference in these means to be statistically significant (p-value, 0.0138). This finding was consistent in serum samples; the mean area under the curve for malignant and normal serum was 1650.34 and 376.26 respectively (p-value, &amp;gt;.0001). Furthermore, there were differences in expression of high mannose glycans between stages of breast cancer, with expression in stage III being significantly greater than expression in stage II in both serum (p-value, .0097) and tissue (p-value, .0003) samples. The results of this analysis suggest a higher expression of high mannose glycans to be a promising biomarker for breast cancer. This could lead to important clinical applications such as being a helpful distinction in obtaining negative margins during tumor resection, as well as providing a non-invasive diagnostic method. Future research could include expanding the sample size and comparing the glycan composition of the samples to a library specific for high-mannose type glycans, with the goal of establishing a signature high-mannose expression pattern in each stage of cancer. Citation Format: Alex Kesic, Avery Funkhouser, Jonah Shealy, Julie Martin, W. Larry Gluck, William J. Edenfield, Anna Blenda. Elevated Expression of High Mannose Glycans in Tissue and Serum Samples of Breast Cancer Patients [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-26-22.

Abstract P2-22-01: Nuclear long non-coding RNA LINC00355 expression and treatment using antisense oligonucleotides in Estrogen Receptor positive late-stage relapse breast cancer

Abstract Late-stage relapse also known as late recurrence breast cancer is a metastatic disease that occurs more than five years after initial adjuvant endocrine therapy treatment. The 5-year relative survival rate is less than 30% and there are limited treatment options for patients with late-stage relapse because of poor performance status. We hypothesize that long non-coding RNA (lncRNA) protein interactions regulate genes to promote late-stage relapse and these interactions can be targeted using RNA therapeutics. To address this, we used RNA-sequencing to identify LINC00355 to be up-regulated in 24 late-stage relapse samples compared to a unique cohort of 72 primary breast cancer samples. Analysis of 480 primary breast cancer samples from The Cancer Genome Atlas (TCGA) also shows decrease expression of LINC00355 compared to late-stage relapse. To better assess LINC00355 cell type expression, we analyzed 26 publicly available primary breast cancer single cell sequencing data samples and detected 0.90% or less of cells expressing LINC00355, which was restricted to cancer epithelial cells. Due to cellular localization playing an important role in lncRNA function, we isolated nuclear and cytoplasmic lysates and orthogonally validated with RNAscope in situ hybridization (RNA-ISH) to show that LINC00355 is located in the nucleus of MCF7 long-term estrogen deprived (MCF7 LTED) cells. LTED cells are deprived of estrogen for longer than three years meant to recapitulate the acquired resistance to aromatase inhibitors. Next, we determined that LINC00355 binds to MENIN protein to regulate p27KIP expression to promote proliferation with UV crosslinking immunoprecipitation and chromatin immunoprecipitation qPCR of LINC00355 overexpression and mutant constructs. Furthermore, RNA-ISH assays validated high nuclear expression of LINC00355 and RNA-ISH protein co-detection assays showed co-localization of LINC00355-MENIN interaction in a panel of late-stage relapse breast cancer patient tissues compared to primary tissues. Lastly to determine clinical significance, we treated MCF7 LTED cells with two locked nucleic acid antisense oligos (ASOs) targeting LINC00355 to observe a significant decrease in proliferation, cell viability, and invasion. Overall, this is the first study to show the importance of lncRNA-protein interactions and therapeutically target lncRNAs with ASOs in late-stage relapse breast cancer. Moving forward, we intend to further understand how LINC00355 contributes to the progression of late-stage relapse with the intent of creating novel cancer diagnostics and therapies. Citation Format: DeAnna Wells, Kyla Gelev, Prasanth Thunuguntla, Reyka Jayasinghe, Li Ding, Jieya Shao, Cynthia Ma, Jessica Silva-Fisher. Nuclear long non-coding RNA LINC00355 expression and treatment using antisense oligonucleotides in Estrogen Receptor positive late-stage relapse breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-22-01.

Metagenomics Analysis of Breast Microbiome Highlights the Abundance of Rothia Genus in Tumor Tissues

Breast cancer is one of the main global priorities in terms of public health. It remains the most frequent cancer in women and is the leading cause of their death. The human microbiome plays various roles in maintaining health by ensuring a dynamic balance with the host or in the appearance of various pathologies including breast cancer. In this study, we performed an analysis of bacterial signature differences between tumor and adjacent tissues of breast cancer patients in Morocco. Using 16S rRNA gene sequencing, we observed that adjacent tissue contained a much higher percentage of the Gammaproteobacteria class (35.7%) while tumor tissue was characterized by a higher percentage of Bacilli and Actinobacteria classes, with about 18.8% and 17.2% average abundance, respectively. Analysis of tumor subtype revealed enrichment of genus Sphingomonodas in TNBC while Sphingomonodas was predominant in HER2. The LEfSe and the genus level heatmap analysis revealed a higher abundance of the Rothia genus in tumor tissues. The identified microbial communities can therefore serve as potential biomarkers for prognosis and diagnosis, while also helping to develop new strategies for the treatment of breast cancer patients.

Carbonic Anhydrase IX in Tumor Tissue and Plasma of Breast Cancer Patients: Reliable Biomarker of Hypoxia and Prognosis

Carbonic anhydrase IX (CA IX) is recognized as an excellent marker of hypoxia and an adverse prognostic factor in solid tumors, including breast cancer (BC). Clinical studies confirm that soluble CA IX (sCA IX), shed into body fluids, predicts the response to some therapeutics. However, CA IX is not included in clinical practice guidelines, possibly due to a lack of validated diagnostic tools. Here, we present two novel diagnostic tools-a monoclonal antibody for CA IX detection by immunohistochemistry and an ELISA kit for the detection of sCA IX in the plasma-validated on a cohort of 100 patients with early BC. We confirm that tissue CA IX positivity (24%) correlates with tumor grading, necrosis, negative hormone receptor status, and the TNBC molecular subtype. We show that antibody IV/18 can specifically detect all subcellular forms of CA IX. Our ELISA test provides 70% sensitivity and 90% specificity. Although we showed that this test could detect exosomes in addition to shed CA IX ectodomain, we could not demonstrate a clear association of sCA IX with prognosis. Our results indicate that the amount of sCA IX depends on subcellular CA IX localization, but more strictly on the molecular composition of individual molecular subtypes of BC, particularly on metalloproteinases inhibitor expression.

An electrostatic force-independent dephosphorylation-driven chemiluminescent biosensor for sensitive and rapid detection of poly(ADP-ribose) polymerase-1 in human breast tissues

Poly (ADP-ribose) polymerase-1 (PARP-1) is a multi-domain nuclear enzyme that mediates gene transcription and DNA repair. Accurate detection of PARP-1 is essential to clinical diagnostic. However, conventional PARP-1 assays rely on electrostatic force with the involvement of lengthy and complex construction processes and false positive. Herein, we construct an ultrasensitive chemiluminescent biosensor for rapid detection of PARP-1 in human breast tissues by integrating PARP-1-directed hyperbranched poly(ADP-ribose) (PAR) formation with alkaline phosphatase (ALP)-3-(2′-spiroadamantyl)-4-methoxy-4- (3′’-phosphoryloxyphenyl)-1,2-dioxetane (AMPDD)-mediated chemiluminescence system. Upon activation by dsDNA, PARP-1 cleaves nicotinamide adenine dinucleotide (NAD+), initiating the repeated polymerization of biotinylated ADP-ribose to form the dsDNA- PAR-biotin complex. The subsequent assembly of dsDNA-PAR-biotin complexes onto AuNPs through Au-S covalent bond results in the construction of the AuNPs-dsDNA-biotin nanostructure. AuNPs-dsDNA-biotin nanostructure subsequently captures SA-ALP to form the AuNPs-dsDNA-ALP nanostructure. After centrifugation and separation, the AuNPs-dsDNA-ALP nanostructure initiates the dephosphorylation of AMPPD to produce a high chemiluminescent signal. In this assay, only PARP-1 activated by dsDNA can cleave NAD+, efficiently eliminating background signal. The introduction of frozen method greatly shortens the assay time. Benefiting from high precision of the PARP-1-catalyzed NAD+ cleavage, high efficiency of biotinylated ADP-ribose polymerization reaction, and high signal-to-noise ratio of ALP-AMPPD chemiluminescence system, this biosensor can rapidly and sensitively detect PARP-1 with a detection limit of 2.94 × 10−7 U/μL, accurately screen PARP-1 inhibitors, and quantify PARP-1 at single-cell level. Most importantly, it can differentiate PARP-1 expression between breast cancer patient tissues and healthy person tissues, with promising applications in clinical diagnosis and biomedical research.

Mix-and-Detection Assay with Multiple Cyclic Enzymatic Repairing Amplification for Rapid and Ultrasensitive Detection of Long Noncoding RNAs in Breast Tissues.

Long noncoding RNAs (lncRNAs) are valuable biomarkers and therapeutic targets, and they play essential roles in various pathological and biological processes. So far, the reported lncRNA assays usually suffer from unsatisfactory sensitivity and time-consuming procedures. Herein, we develop a mix-and-read assay based on multiple cyclic enzymatic repairing amplification (ERA) for sensitive and rapid detection of mammalian metastasis-associated lung adenocarcinoma transcript 1 (lncRNA MALAT1). In this assay, we design two three-way junction (3WJ) probes including a 3WJ template and a 3WJ primer to specifically recognize lncRNA MALAT1, and the formation of a stable 3WJ structure induces cyclic ERA to generate triggers. The resulting triggers subsequently hybridize with a free 3WJ template and act as primers to initiate new rounds of cyclic ERA, generating abundant triggers. The hybridization of triggers with signal probes forms stable double-stranded DNA duplexes that can be specifically cleaved by apurinic/apyrimidinic endonuclease 1 to produce a high fluorescence signal. This assay can be carried out in a mix-and-read manner within 10 min under an isothermal condition (50 °C), which is the rapidest and simplest method reported so far for the lncRNA MALAT1 assay. This method can sensitively detect lncRNA MALAT1 with a limit of detection of 0.87 aM, and it can accurately measure endogenous lncRNA MALAT1 at the single-cell level. Moreover, this method can distinguish lncRNA MALAT1 expression in breast cancer patient tissues and their corresponding healthy adjacent tissues. Importantly, the extension of this assay to different RNAs detection can be achieved by simply replacing the corresponding target recognition sequences.

Proteome profiling of ductal carcinoma in situ.

DCIS is the most common type of non-invasive breast cancer, accounting for about 15 to 30%. Proteome profile is used to detect biomarkers in the tissues of breast cancer patients by mass spectrometry. This study aimed to obtain the expression profile of DCIS proteome, and the expression profile of invasive biomarkers, and finally to introduce a dedicated biomarker panel to facilitate the prognosis and early detection for in situ breast cancer patients. In this study, 10 patients with breast cancer (DCIS) were studied. Benign (marginal) and cancerous tissue samples were obtained from patients for proteomics experiments. Initially, all tissue proteins were extracted using standard methods, and the proteins were separated using two-dimensional electrophoresis. Then, the expression amount of the extracted proteins was determined by ITRAQ. The data were analysed by R software, and gene ontology was utilised for describing the protein in detail. 30 spots on gel electrophoresis were found in the tumor tissue group (sample), and 15 spots in the margin group (control) with P <0.05. Healthy and cancerous tissue gels showed that 5 spots had different expression. VWF, MMP9, ITGAM, MPO and PLG protein spots were identified using the site www.ebi.ac.uk/IPI. Finally, protein biomarkers for breast tumor tissue with margin were introduced with the names of P04406, P49915, P05323, P06733, and P02768. There are 5 critical proteins in inducing cancer pathways especially complement and coagulation cascades. The hall markers of a healthy cell to be cancerous are proliferation, invasion, angiogenesis, and changes in the immune system. Hence, regulation of protein plays a key role in developing recurrence to breast cancer in margins.