Breast Tumor Tissues Research Articles

PET/CT with <sup>18</sup>F-Fluoroestradiol in First Diagnosed Breast Cancer. A Pilot Study

Purpose: To evaluate the informativity of PET/CT with 18F-FES in determining estrogen receptor (ER) positivity in breast tumor tissue, as well as in regional lymph nodes or distant metastatic foci. Material and methods: The data of 30 patients who underwent PET/CT with 18F-FES performed at the stage of primary diagnosis were retrospectively analyzed. The study included patients before the start of specific treatment or on the background of neoadjuvant hormone therapy, with previously determined expression of ER according to the results of IGC. 14 patients underwent 18F-FDG PET/CT with 2–7 days difference between both PET/CT. Results: In our study, the mean SUVmax value in the primary tumor focus was 4.8, the lowest SUVmax = 1.61, and the highest SUVmax = 18.86. 18F-FES accumulation considered suspicious for metastatic lesion in axillary lymph nodes on the affected side was detected in 16 patients (56 %), mean SUVmax = 5.2, lowest SUVmax = 0.99, highest SUVmax = 11.54. On subsequent histological examination, tumor changes in axillary lymph nodes were confirmed in 16/16 patients. Conclusion: PET/CT with 18F-FES is a diagnostic method for noninvasive evaluation of ER expression in tumor foci, which can complement the data of traditional diagnostic methods and has a significant clinical potential. In our study, the results of PET/CT with 18F-FES coincided with the data of immunohistochemical study in 100 % of cases.

Cross-linking breast tumor transcriptomic states and tissue histology

Identification of the gene expression state of a cancer patient from routine pathology imaging and characterization of its phenotypic effects have significant clinical and therapeutic implications. However, prediction of expression of individual genes from whole slide images (WSIs) is challenging due to co-dependent or correlated expression of multiple genes. Here, we use a purely data-driven approach to first identify groups of genes with co-dependent expression and then predict their status from WSIs using a bespoke graph neural network. These gene groups allow us to capture the gene expression state of a patient with a small number of binary variables that are biologically meaningful and carry histopathological insights for clinical and therapeutic use cases. Prediction of gene expression state based on these gene groups allows associating histological phenotypes (cellular composition, mitotic counts, grading, etc.) with underlying gene expression patterns and opens avenues for gaining biological insights from routine pathology imaging directly.

Abstract C044: Investigation of breast tumor biology and microenvironment in women of African descent using a single cell multiomic approach

Abstract Women of African descent are at an increased risk of developing and dying from aggressive subtypes of breast cancer. A connection between aggressive disease and Western Sub-Saharan African ancestry has been postulated, but it remains largely unknown to what extent breast cancer in Africa is reminiscent of breast cancer in U.S. African American (AA) women who experience disproportionately high mortality rates. We performed ATAC- and RNA-sequencing on 9 human triple-negative breast cancer cell lines of U.S. origin and discovered that African ancestry influences the chromatin landscape, leading to disparate transcription factor activity and downstream gene expression patterns indicative of an aggressive tumor biology. Here, we describe an ambitious study that employs single-nucleus ATAC- and RNA-sequencing (snMultiome) of frozen breast tumors to characterize chromatin accessibility and gene expression patterns with single-cell resolution in AA (n=32), Kenyan (n=18), and European American (EA, n=20) women in relation to ancestry and risk factor exposure. In an initial pilot in 10 Kenyan women, we successfully isolated intact, high-quality single nuclei from frozen breast tumor tissue through an optimized combination of enzymatic digestion and an automated tissue homogenizer. We performed multiome sequencing on a total of 36525 nuclei across these 10 pilot tumors using the 10x Genomics platform. Following filtering, normalization (SCT for snRNA; LSI for snATAC), peak calling (MACS2), and integration (Seurat-v4 for snRNA; Harmony for snATAC), we characterized tumor, stromal, and immune cell populations (totaling 10 distinct cell types). We observed striking intra- and inter-tumoral heterogeneity across Kenyan women, with increased abundance of immune subpopulations (myeloid, T-cells, B-cells) in triple-negative subtypes. After demonstrating technical feasibility and success in this sample subset, we are now performing integration, cell type annotation, and downstream analyses in our larger cohort to characterize differences in both tumor biology and the tumor microenvironment in European American, African American, and Kenyan women at the single cell level. To date, 70 tumors spanning breast cancer subtypes have been sequenced. We are currently performing downstream analyses in this cohort to characterize ancestry- and risk factor-related differences in the tumor epithelium and microenvironment at the single-cell level. This project holds the potential to yield crucial insights into how ancestry or other factors may influence the etiology of different breast cancer subtypes, as well as produce clinically actionable biomarkers and therapeutic targets to enhance precision medicine within patient populations at high risk for aggressive disease. Citation Format: Alexandra R. Harris, Huaitian Liu, Brittany Jenkins-Lord, Francis Makokha, Shahin Sayed, Gretchen Gierach, Stefan Ambs. Investigation of breast tumor biology and microenvironment in women of African descent using a single cell multiomic approach [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C044.

Neutralizing tumor-related inflammation and reprogramming of cancer-associated fibroblasts by Curcumin in breast cancer therapy

Tumor-associated inflammation plays a vital role in cancer progression. Among the various stromal cells, cancer-associated fibroblasts are promising targets for cancer therapy. Several reports have indicated potent anti-inflammatory effects attributed to Curcumin. This study aimed to investigate whether inhibiting the inflammatory function of cancer-associated fibroblasts (CAFs) with Curcumin can restore anticancer immune responses. CAFs were isolated from breast cancer tissues, treated with Curcumin, and co-cultured with patients' PBMCs to evaluate gene expression and cytokine production alterations. Blood and breast tumor tissue samples were obtained from 12 breast cancer patients with stage II/III invasive ductal carcinoma. Fibroblast Activation Protein (FAP) + CAFs were extracted from tumor tissue, treated with 10 μM Curcumin, and co-cultured with corresponding PBMCs. The expression of smooth muscle actin-alpha (α-SMA), Cyclooxygenase-2(COX-2), production of PGE2, and immune cell cytokines were evaluated using Real-Time PCR and ELISA, respectively. Analyzes showed that treatment with Curcumin decreased the expression of genes α-SMA and COX-2 and the production of PGE2 in CAFs. In PBMCs co-cultured with Curcumin-treated CAFs, the expression of FoxP3 decreased along with the production of TGF-β, IL-10, and IL-4. An increase in IFN-γ production was observed that followed by increased T-bet expression. According to our results, Curcumin could reprogram the pro-tumor phenotype of CAFs and increase the anti-tumor phenotype in PBMCs. Thus, CAFs, as a component of the tumor microenvironment, are a suitable target for combination immunotherapies of breast cancer.

MDR1-expressing CD4+ T cells with Th1.17 features resist to neoadjuvant chemotherapy and are associated with breast cancer clinical response

BackgroundMultidrug resistance-1 (MDR1) transporter limits the intracellular accumulation of chemotherapies (paclitaxel, anthracyclines) used in breast cancer (BC) treatment. In addition to tumor cells, MDR1 is expressed on immune cell subsets...

Human Breast Tissue Microbiota Reveals Unique Microbial Signatures that Correlate with Prognostic Features in Adult Ethiopian Women with Breast Cancer.

Breast cancer (BC) is the leading cause of cancer mortality among women in Ethiopia. Overall, women of African ancestry have the highest death toll due to BC compared to other racial/ethnic groups. The cause of the disparity in mortality is unclear. Recently, studies conducted in the United States and other high-income countries highlighted the role of microbial dysbiosis in BC initiation, tumor growth, and treatment outcome. However, the extent to which inter-individual differences in the makeup of microbiota are associated with clinical and histopathological outcomes in Ethiopian women has not been studied. The goal of our study was to profile the microbiome in breast tumor and normal adjacent to tumor (NAT) tissues of the same donor and to identify associations between microbial composition and abundance and clinicopathological factors in Ethiopian women with BC. We identified 14 microbiota genera in breast tumor tissues that were distinct from NAT tissues, of which Sphingobium, Anaerococcus, Corynebacterium, Delftia, and Enhydrobacter were most significantly decreased in breast tumors compared to NAT tissues. Several microbial genera significantly differed by clinicopathological factors in Ethiopian women with BC. Specifically, the genus Burkholderia more strongly correlated with aggressive triple negative (TNBC) and basal-like breast tumors. The genera Alkanindiges, Anoxybacillus, Leifsonia, and Exiguobacterium most strongly correlated with HER2-E tumors. Luminal A and luminal B tumors also correlated with Anoxybacillus but not as strongly as HER2-E tumors. A relatively higher abundance of the genus Citrobacter most significantly correlated with advanced-stage breast tumors compared to early-stage tumors. This is the first study to report an association between breast microbial dysbiosis and clinicopathological factors in Ethiopian women.

Identifying the effectiveness of 3D culture systems to recapitulate breast tumor tissue in situ.

Breast cancer heterogeneity contributes to chemotherapy resistance and decreased patient survival. To improve patient outcomes it is essential to develop a technology that is able to rapidly select the most efficacious therapy that targets the diverse phenotypes present within the tumor. Breast cancer organoid technologies are proposed as an attractive approach for evaluating drug responses prior to patient therapy. However, there remain challenges in evaluating the effectiveness of organoid cultures to recapitulate the heterogeneity present in the patient tumor in situ. Organoids were generated from seven normal breast and nineteen breast cancer tissues diagnosed as estrogen receptor positive or triple negative. The Jensen-Shannon divergence index, a measure of the similarity between distributions, was used to compare and evaluate heterogeneity in starting tissue and their resultant organoids. Heterogeneity was analyzed using cytokeratin 8 and cytokeratin 14, which provided an easily scored readout. In the in vitro culture system HER1 and FGFR were able to drive intra-tumor heterogeneity to generate divergent phenotypes that have different sensitivities to chemotherapies. Our methodology, which focuses on quantifiable cellular phenotypes, provides a tractable system that complements omics approaches to provide an unprecedented view of heterogeneity and will enhance the identification of novel therapies and facilitate personalized medicine.

Introduced the ITGB1-DT as a novel biomarker associated with five potential drugs using bioinformatics analysis of breast cancer proteomics data and RT-PCR

BackgroundBreast cancer (BC) has been identified as a significant contributor to the rising number of female cancer deaths. As, it has become clear that breast cancer development depends on the interplay of several biological factors against a single molecule. This research aimed to use proteomics to gain a regulatory and metabolic understanding of BC pathophysiology. MethodFor the study, a breast cancer proteomics dataset was downloaded from ProteomeXchange and then analyzed by employing MaxQuant and Perseus. Functional enrichment analysis through Metascape and Cytoscape software showed DEPs related biomedical phenomena with potential abruption. The expression of selected lncRNA in terms of the highest connectivity parameters was then quantitatively assessed through RT-PCR in 30 tumor tissues of breast cancer patients, as compared to the adjacent healthy ones. ResultThe results indicated that among the 3048 identified proteins, 1149 were differentially expressed, which could be mainly enriched in several key terms. Furthermore, the obtained findings revealed that ITGB1-DT was significantly overexpressed in tumor tissues. Moreover, we found five potential compounds that could be attributed to ITGB1-DT targets (ATN-161, Firategrast, SB-683698, dabigatran-etexilate, and tranexamic-acid). ConclusionThese analyses proposed that ITGB1-DT could be employed as a differentiated factor to identify breast tumor tissues in healthy samples. Besides this, Firategrast could be introduced as a potential remedial agent for breast cancer patients. Overall, from the analysis of a proteomics dataset, an integrative map was generated, and a novel biomarker that may have been implicated in the early detection of BC was introduced.

Chimeric RNAs reveal putative neoantigen peptides for developing tumor vaccines for breast cancer.

We present here a strategy to identify immunogenic neoantigen candidates from unique amino acid sequences at the junctions of fusion proteins which can serve as targets in the development of tumor vaccines for the treatment of breastcancer. We mined the sequence reads of breast tumor tissue that are usually discarded as discordant paired-end reads and discovered cancer specific fusion transcripts using tissue from cancer free controls as reference. Binding affinity predictions of novel peptide sequences crossing the fusion junction were analyzed by the MHC Class I binding predictor, MHCnuggets. CD8+ T cell responses against the 15 peptides were assessed through in vitro Enzyme Linked Immunospot (ELISpot). We uncovered 20 novel fusion transcripts from 75 breast tumors of 3 subtypes: TNBC, HER2+, and HR+. Of these, the NSFP1-LRRC37A2 fusion transcript was selected for further study. The 3833 bp chimeric RNA predicted by the consensus fusion junction sequence is consistent with a read-through transcription of the 5'-gene NSFP1-Pseudo gene NSFP1 (NSFtruncation at exon 12/13) followed by trans-splicing to connect withLRRC37A2 located immediately 3' through exon 1/2. A total of 15 different 8-mer neoantigen peptides discovered from the NSFP1 and LRRC37A2 truncations were predicted to bind to a total of 35 unique MHC class I alleles with a binding affinity of IC50<500nM.); 1 of which elicited a robust immune response. Our data provides a framework to identify immunogenic neoantigen candidates from fusion transcripts and suggests a potential vaccine strategy to target the immunogenic neopeptides in patients with tumors carrying the NSFP1-LRRC37A2 fusion.

The expression of lncRNAs CASC2, NEAT1, LINC00299 in breast cancer tissues and their relationship with the XBP1 splicing rate in Iranian patients during 2014-2019: A cross-sectional study.

Breast cancer is a leading cause of incidence and mortality in women globally. Identifying new molecular markers can aid in cancer diagnosis, targeted therapy, and treatment monitoring. This study aimed to measure the expression of the X-box binding protein 1 (XBP1) gene, an index of the unfolded protein response (UPR), and long noncoding RNAs (lncRNAs), including Nuclear Enriched Abundant Transcript 1 (NEAT1), Cancer Susceptibility Candidate 2 (CASC2), and Long Intergenic Nonprotein Coding RNA 299 (LINC00299), as possible regulators of the UPR pathway. Total RNA was extracted from 40 samples of breast tumor tissues and their respective controls. The expression level of lncRNAs CASC2, NEAT1, and LINC00299 was quantified using reverse transcription-polymerase chain reaction (RT-PCR). The ratio of the spliced form of XBP1 to its unspliced form (XBP1u) was determined by PCR and electrophoresis. The results showed a 2.8-fold increase in the ratio of XBP1s/u in breast cancer tissues compared to adjacent nonmalignant samples (p < 0.05). Additionally, the level of lncRNAs NEAT1, CASC2, and LINC00299 in breast tumor tissues increased significantly by twofold, 1.5-fold, and 2.3-fold, respectively, compared to adjacent nonmalignant samples (p < 0.05). Based on the association between the expression of lncRNAs CASC2, LINC00299, and NEAT1 and the XBP1s/u ratio, these lncRNAs could be potential regulators of the UPR pathway. Also, CASC2 and NEAT1 genes could be suggested as suitable biomarkers to distinguish cancerous tissue from noncancerous breast tissue due to their significant increase in expression in cancerous samples compared to adjacent noncancerous.

Immunohistochemical Detection of the Expressed BRCA1 and BRCA2 Proteins in Microenvironment of Malignant Breast Cancerous Tissues Infected with Human Mammary Tumor Virus.

The objective of the study was to examine the prevalence of HMTV infection, its associations with breast malignant tissues, and the expression of BRCA1 and BRCA2 proteins. One hundred archival breast tissues, 40 biopsies from female patients with breast cancer (BC), and 20 healthy breast tissues from the control group were used in the study. Immunohistochemical analysis was conducted to detect the expressed BRCA1 and BRCA2 proteins. Digoxigenin-labeled HMTV probes were used in chromogenic in situ hybridization for the identification of HMTV in breast tumor tissues. The complementary sequence sites of the HMTV probe sequences were stained by NBT/BCIP as blue signals. There were 12 out of 40 (30%) benign breast tumorous tissues and 14 out of 40 (35%) BC tissues, while healthy control breast tissues were 10% (2 out of 20 tissues). Positive immunohistochemical (IHC) reactions for BRCA2 protein were observed in 12 out of 40 BC tissues (30.0%), 25% of benign breast tumorous tissues, and 5% of the control group. A significant (p < 0.05) statistical difference in the percentages of HMTV in the studied groups was found. HMTV might contribute to the development of subsets of benign and malignant breast tumors. The observed rates of defective or mutated BRCA1 and BRCA2 genes in healthy tissues indicate a role in the development of breast tumors.

Comparison of three 18F-labeled 2-nitroimidazoles for imaging hypoxia in breast cancer xenografts: [18F]FBNA, [18F]FAZA and [18F]FMISO

BackgroundTumour hypoxia is associated with increased metastasis, invasion, poor therapy response and prognosis. Most PET radiotracers developed and used for clinical hypoxia imaging belong to the 2-nitroimidazole family. Recently we have developed novel 2-nitroimidazole-derived PET radiotracer [18F]FBNA (N-(4-[18F]fluoro-benzyl)-2-(2-nitro-1H-imidazol-1-yl)-acet-amide), an 18F-labeled analogue of antiparasitic drug benznidazole. The present study aimed to analyze its radio-pharmacological properties and systematically compare its PET imaging profiles with [18F]FMISO and [18F]FAZA in preclinical triple-negative (MDA-MB231) and estrogen receptor-positive (MCF-7) breast cancer models. MethodsIn vitro cellular uptake experiments were carried out in MDA-MB321 and MCF-7 cells under normoxic and hypoxic conditions. Metabolic stability in vivo was determined in BALB/c mice using radio-TLC analysis. Dynamic PET experiments over 3 h post-injection were performed in MDA-MB231 and MCF-7 tumour-bearing mice. Those PET data were used for kinetic modelling analysis utilizing the reversible two-tissue-compartment model. Autoradiography was carried out in tumour tissue slices and compared to HIF-1α immunohistochemistry. Detailed ex vivo biodistribution was accomplished in BALB/c mice, and this biodistribution data were used for dosimetry calculation. ResultsUnder hypoxic conditions in vitro cellular uptake was elevated in both cell lines, MCF-7 and MDA-MB231, for all three radiotracers. After intravenous injection, [18F]FBNA formed two radiometabolites, resulting in a final fraction of 65 ± 9 % intact [18F]FBNA after 60 min p.i. After 3 h p.i., [18F]FBNA tumour uptake reached SUV values of 0.78 ± 0.01 in MCF-7 and 0.61 ± 0.04 in MDA-MB231 tumours (both n = 3), representing tumour-to-muscle ratios of 2.19 ± 0.04 and 1.98 ± 0.15, respectively. [18F]FMISO resulted in higher tumour uptakes (SUV 1.36 ± 0.04 in MCF-7 and 1.23 ± 0.08 in MDA-MB231 (both n = 4; p < 0.05) than [18F]FAZA (0.66 ± 0.11 in MCF-7 and 0.63 ± 0.14 in MDA-MB231 (both n = 4; n.s.)), representing tumour-to-muscle ratios of 3.24 ± 0.30 and 3.32 ± 0.50 for [18F]FMISO, and 2.92 ± 0.74 and 3.00 ± 0.42 for [18F]FAZA, respectively. While the fraction per time of radiotracer entering the second compartment (k3) was similar within uncertainties for all three radiotracers in MDA-MB231 tumours, it was different in MCF-7 tumours. The ratios k3/(k3 + k2) and K1*k3/(k3 + k2) in MCF-7 tumours were also significantly different, indicating dissimilar fractions of radiotracer bound and trapped intracellularly: K1*k3/(k2 + k3) [18F]FMISO (0.0088 ± 0.001)/min, n = 4; p < 0.001) > [18F]FAZA (0.0052 ± 0.002)/min, n = 4; p < 0.01) > [18F]FBNA (0.003 ± 0.001)/min, n = 3). In contrast, in MDA-MB231 tumours, only K1 was significantly elevated for [18F]FMISO. However, this did not result in significant differences for K1*k3/(k2 + k3) for all three 2-nitroimidazoles in MDA-MB231 tumours. ConclusionNovel 2-nitroimidazole PET radiotracer [18F]FBNA showed uptake into hypoxic breast cancer cells and tumour tissue presumably associated with elevated HIF1-α expression. Systematic comparison of PET imaging performance with [18F]FMISO and [18F]FAZA in different types of preclinical breast cancer models revealed a similar tumour uptake profile for [18F]FBNA with [18F]FAZA and, despite its higher lipophilicity, still a slightly higher muscle tissue clearance compared to [18F]FMISO.

Multivariate analysis of breast tissue using optical parameters extracted from a combined time-resolved fluorescence and diffuse reflectance system for tumor margin detection.

Breast conservation therapy is the preferred technique for treating primary breast cancers. However, breast tumor margins are hard to determine as tumor borders are often ill-defined. As such, there exists a need for a clinically compatible tumor margin detection system. A combined time-resolved fluorescence and diffuse reflectance (TRF-DR) system has been developed to determine the optical properties of breast tissue. This study aims to improve tissue classification to aid in surgical decision making. Normal and tumor breast tissue were collected from 80 patients with invasive ductal carcinoma and measured in the optical system. Optical parameters were extracted, and the tissue underwent histopathological examination. In total, 761 adipose, 77 fibroglandular, and 347 tumor spectra were analyzed. Principal component analysis and decision tree modeling were performed using only TRF optical parameters, only DR optical parameters, and using the combined datasets. The classification modeling using TRF data alone resulted in a tumor margin detection sensitivity of 72.3% and specificity of 88.3%. Prediction modeling using DR data alone resulted in greater sensitivity and specificity of 80.4% and 94.0%, respectively. Combining both datasets resulted in the improved sensitivity and specificity of 85.6% and 95.3%, respectively. While both sensitivity and specificity improved with the combined modeling, further study of fibroglandular tissue could result in improved classification. The combined TRF-DR system showed greater tissue classification capability than either technique alone. Further work studying more fibroglandular tissue and tissue of mixed composition would develop this system for intraoperative use for tumor margin detection.

Regulation of the stem‑like properties of estrogen receptor‑positive breast cancer cells through NR2E3/NR2C2 signaling.

Cancer stem cells (CSCs) are major drivers of metastasis, drug resistance and recurrence in numerous cancers. However, critical factors that can modulate CSC stemness have not been clearly identified. Nuclear receptor subfamily 2 group E member 3 (nr2e3) expression has been previously reported to be positively associated with drug sensitivity and favorable clinical outcomes in patients with estrogen receptor (ER)+ breast cancer. This suggests that nr2e3 expression may be inversely associated with CSC stemness in this type of tumor cells. The present study aimed to investigate the regulatory roles of NR2E3 in the stem-like properties of ER+ breast cancer cells and to identify the underlying mechanisms. Bioinformatics analysis was performed using the data derived from the Cancer Genome Atlas database. Nr2e3-specific shRNA and nuclear receptor subfamily 2 group C member 2 (nr2c2) overexpressed plasmids were constructed to silence and enhance the expression of nr2e3 and nr2c2, respectively. Transwell and wound healing experiments were conducted to evaluate the migration and invasion ability of MCF7 cells, while colony formation tests were used to evaluate the clonality. Flow cytometry was used to detect the percentage of CD44+CD24-/low cells. Reverse transcription-quantitative PCR and western blotting were performed to detect expression at the mRNA and protein levels. The results showed that compared with normal breast tissues and MCF10A cells, the expression of nr2e3 was increased in ER+ breast tumor tissues and cell lines. Nr2e3 silencing promoted the migration, invasion and colony-forming ability of the ER+ MCF7 cells. It also increased the expression of epithelial-mesenchymal transition markers and stem cell-related transcription factors, in addition to the percentage of CD44+CD24-/low cells. The expression of nr2e3 and nr2c2 was found to be positively correlated. Nr2e3 knockdown decreased the mRNA and protein expression levels of nr2c2, whereas nr2c2 overexpression reversed the elevated CD44+CD24-/low cell ratio and the increased migratory activity caused by nr2e3 silencing. The results of the present study suggest that NR2E3 may serve an important role in modulating the stem-like properties of ER+ breast cancer cells, where NR2E3/NR2C2 signaling may be a therapeutic target in ER+ breast cancer.

Evaluation of miR-146 and miR-196 as potential biomarkers in a sample of Iraqi breast cancer patients

Breast cancer is a heterogeneous disease defined by molecular types and subtypes. It constitutes the most commonly-diagnosed cancer and the leading cause of cancer death in women worldwide, according to the International Agency for Research on Cancer (IARC) World Cancer Reports in 2020. The study aimed to evaluate the miR-146 and miR-196 expression level and their association with the ca15-3 serum level of the participants diagnosed with breast cancer. There were 105 samples, three groups of 35 fresh blood samples and FFPE Tissue samples, which were collected as malignant, benign and healthy control. CA15-3 concentration was elevated in a malignant group with a mean equal to (36.14 Units/ml) in comparison to (27.07 Units/ml) for the benign group and (14.34 Units/ml) for the healthy control group (p&lt;0.01). The results revealed that the expression of miR-146 in Malignant breast tumor tissue was (2.378 ±0.76) times more, while in benign breast tissue, with the fold of expression (1.197 ±0.38) in comparison with apparently healthy tissue. At the same time, the expression of miR-196 in Malignant breast tumor tissue was (8.11 ±2.15) times more, while in benign breast tissue, with a fold of expression (2.584 ±0.84) compared with apparently healthy tissue with highly significant differences. Keyword: Breast Cancer, miR-146, miR-196, ca15-3, FFPE

Redlining-associated methylation in breast tumors: the impact of contemporary structural racism on the tumor epigenome.

Place-based measures of structural racism have been associated with breast cancer mortality, which may be driven, in part, by epigenetic perturbations. We examined the association between contemporary redlining, a measure of structural racism at the neighborhood level, and DNA methylation in breast tumor tissue. We identified 80 Black and White women diagnosed and treated for a first-primary breast cancer at Emory University Hospitals (2008-2017). Contemporary redlining was derived for census tracts using the Home Mortgage Disclosure Act database. Linear regression models were used to calculate the association between contemporary redlining and methylation in breast tumor tissue. We also examined epigenetic age acceleration for two different metrics, regressing β values for each cytosine-phosphate-guanine dinucleotide (CpG) site on redlining while adjusting for covariates. We employed multivariable Cox-proportional hazards models and 95% confidence intervals (CI) to estimate the association between aberrant methylation and mortality. Contemporary redlining was associated with 5 CpG sites after adjustment for multiple comparisons (FDR<0.10). All genes were implicated in breast carcinogenesis, including genes related to inflammation, immune function and stress response (ANGPT1, PRG4 and PRG4). Further exploration of the top 25 CpG sites, identified interaction of 2 sites (MRPS28 and cg11092048) by ER status and 1 site (GDP1) was associated with all-cause mortality. Contemporary redlining was associated with epigenetic age acceleration by the Hannum metric (β=5.35; CI 95%=0.30,10.4) and showed positive but non-significant correlation with the other clock. We identified novel associations between neighborhood contemporary redlining and the breast tumor DNA methylome, suggesting that racist policies leading to inequitable social and environmental exposures, may impact the breast tumor epigenome. Additional research on the potential implications for prognosis is needed.

A STUDY OF MMP7 EXPRESSION IN TRIPLE NEGATIVE BREAST CANCER PATIENTS

MMP7 has a part in preventing apoptosis and promoting angiogenesis. According to both in vivo and in vitro evidence, MMP7 is mostly seen during tumour invasion and metastasis. Controlling MMP7 expression may be a method for treating this cancer because its overexpression encourages the occurrence, progression, and invasiveness of breast cancer. The aim is to study the expression of MMP7 in triple negative breast cancer patients. Formalin fixed paraffin embedded breast tumor tissues from 50 triple negative breast cancer patients were collected for this study. immunohistochemistry method was used to study MMP7 expression and correlated with clinical and pathological parameters as well as disease status. MMP7 positivity was observed in 48% triple negative breast cancer patients. No significant correlation of MMP7 expression with age, menopausal status, tumour size, histopathology, stage and lymph node status were observed. A significant higher incidence of MMP7 expression was noted in patients with high grade tumor (63%) as compared to patients with low grade tumor (00%). Further, a significant higher incidence of MMP7 was observed in patients with high BR score (65%) as compared to the patients with low BR score (00%). Also, a significant higher incidence was observed in patients with relapse (63%) than patients in remission (35%). A significant higher incidence of MMP7 expression was observed with respect to disease free survival in univariate survival analysis. In multivariate survival analysis, for disease free survival MMP7 entered at step 1 and lymph node status and MMP7 entered at step 2. The findings imply that MMP7 is important for tumour invasion and metastasis, and that elevated MMP7 expression is associated with tumour development and recurrence.

Assessment of resection margins during breast-conserving surgery using multimodal optical coherence tomography

Background. Breast cancer (BC) has been ranked first in morbidity and mortality among the female population in Russian Federation for several years. Rapid and accurate intraoperative examination of the cleanliness of resection margins is the main condition for breast-conserving surgery (BCS) to reduce the risks of local recurrence.Aim. To examine the tumor margins and assess the cleanliness of the resection margins in BCS in patients diagnosed with BC using multimodal optical coherence tomography (MM OCT) with comparative planned histological and additional molecular genetic analysis.Materials and methods. The study was carried out on postoperative samples of tumor and non-tumor breast tissue obtained from 115 patients with BC T1–2N0M0G2–3 stage IA–IIA, after BCS in the volume of radical resection or lumpectomy with histological control of the resection margins. A spectral domain MM OCT device (OCT 1300-E, BioMedTech LLC, Russia) was used, which provides structural OCT images to study the backscattering properties of tissue and allows performing a quantitative assessment of the elastic properties of the tissue by compression optical coherence elastography (OCE).Results. The MM OCT method was able to determine the exact margins between the tumor and non-tumor breast tissue. The main signs of the tumor were a decrease in the level and depth of penetration of the OCT signal in the structural OCT images, as well as an increase in the stiffness values (&gt;200 kPa) in the OCE images with the highest stiffness values (&gt;600 kPa) in the area of tumor cell accumulations. The use of compression OCE made it possible to detect clusters of tumor cells in the margins of resection at a distance of 5 mm from the visible border of the tumor. Among the 132 studied resection margins in 6 cases, the positive resection margin was determined by the OCE method, which was subsequently confirmed histologically. The negative resection margins were characterized by the lowest stiffness values (&lt;200 kPa) in the OCE images and were confirmed by both histological analysis and molecular genetic analysis. ROC-analysis established a threshold stiffness value of 159.8 kPa, which indicates the presence of tumor tissue at the resection margins in the presence of a high stiffness area larger than 500 µm. The high diagnostic accuracy of the OCE method (98.5 %) was determined to distinguish between the negative and positive resection margins during BCS.Conclusion. The use of MM OCT with OCE modality makes it possible to clearly distinguish different types of breast tissue in BC (adipose tissue, connective tissue, accumulation of tumor cells). MM OCT can be used as an additional intraoperative tool for visualizing tumor cells boundaries and assessing the cleanliness of resection margins in real time during BCS.

Mutations of BRCA1, BRCA2, and PALB2 Genes in Breast Tumor Tissue: Relationship with the Effectiveness of Neoadjuvant Chemotherapy and Disease Prognosis.

The study included 66 patients with breast cancer. DNA copy number aberrations (CNA) were assessed by high-density CytoScanHD™ Array micro matrix analysis. Gene mutations were assessed by sequencing on the MiSeq™ Sequencing System using the Accel-Amplicon BRCA1, BRCA2, and PALB2 Panel. It has been established that the presence of a normal copy number of PALB2 is associated with a lack of response to chemotherapy in Taxotere-containing treatment regimens (p = 0.05). In addition, the presence of a PALB2 deletion is associated with 100% metastatic survival rates (log-rank test p = 0.04). As a result of sequencing, 25 mutations were found in the BRCA1 gene, 42 mutations in BRCA2, and 27 mutations in the PALB2 gene. The effect of mutations on the effectiveness of treatment is controversial, but an effect on the survival of patients with breast cancer has been shown. So, in the presence of pathogenic mutations in the BRCA2 gene, 100% metastatic survival is observed (log-rank test p = 0.05), as well as in the elimination of PALB2 mutations during treatment (log-rank test p = 0.07). Currently, there is little data on the effect of chromosomal aberrations and mutations in the BRCA1/2 and PALB2 genes on the effectiveness of treatment and prognosis of the disease. At the same time, the study of these genes has great potential for testing focused on a personalized approach to the treatment of patients with breast cancer.

The prognostic significance of KLRB1 and its further association with immune cells in breast cancer.

Killer cell lectin-like receptor B1 (KLRB1) is an important member of the natural killer cell gene family. This study explored the potential value of KLRB1 as a breast cancer (BC) biomarker and its close association with the tumor immune microenvironment during the development of BC. We examined the differential expression of KLRB1 in pan-cancer. Clinical and RNA-Seq data from BC samples were evaluated in The Cancer Genome Atlas (TCGA) and validated in Gene Expression Omnibus (GEO) datasets and by immunohistochemistry (IHC) staining. The relationship between KLRB1 and clinical parameters was explored through Chi-square tests. The diagnostic value of KLRB1 was evaluated using a receiver operating characteristic (ROC) curve. Survival analysis was tested by Kaplan-Meier curves to demonstrate the relationship between KLRB1 and survival. Univariable and multivariate cox regression analyses were carried out as well. The analysis of immune infiltration level and gene set enrichment analysis (GSEA) were conducted to examine KLRB1's mechanism during the progression of BC. We used the Tumor Immune Estimation Resource (TIMER), the Cancer Single-cell Expression Map (CancerSCEM) database, the Tumor Immune Single-cell Hub (TISCH) database, and the Cell-type Identification by Estimating Relative Subsets of RNA Transcripts (CIBERSORT) method to explore KLRB1's association with immune infiltration level and different quantitative distribution of immune cells. The relevant signaling pathways in BC associated with KLRB1 were identified using GSEA. The expression of KLRB1 was downregulated across the majority of cancers including BC. The lower KLRB1 expression group exhibited shorter relapse free survival (RFS) and overall survival (OS). IHC staining showed that KLRB1 staining was weaker in breast tumor tissues than in paratumors. Additionally, GSEA identified several pathway items distinctly enriched in BC. KLRB1 expression level was also positively related to the infiltrating number of immune cells in BC. Moreover, the CancerSCEM and TISCH databases as well as the CIBERSORT method demonstrated the close relationship between KLRB1 and immune cells, particularly macrophages. Low KLRB1 expression was considered an independent prognostic biomarker and played an important role in the tumor immune microenvironment of BC patients.