Breast Tissue Samples Research Articles

Abstract PS8-12: Interim analysis of a phase I dose escalation study of topical bexarotene in women at high risk for breast cancer

Abstract Background: Breast cancer prevention with anti-estrogens, including tamoxifen, raloxifene, and exemestane, has been shown to reduce the incidence of hormone receptor-positive breast cancer. However, agents that can reduce the incidence of hormone receptor negative breast cancer are currently lacking. Rexinoids such as bexarotene are vitamin A analogues that have been shown to be involved in cell differentiation, growth, and apoptosis. In preclinical mouse models that develop ER-negative breast cancers, bexarotene showed a significant reduction in mammary tumor development. Oral bexarotene has been evaluated in BRCA mutation carriers and significant decreases in cyclin D1 were noted in breast cells suggesting biological activity of bexarotene on breast tissue. Systemic side effects of hyperlipidemia and hypothyroidism were also found. Data from chemoprevention studies with topical 4-hydroxytamoxifen support the concept of topical agents penetrating into the breast tissue and exhibiting biological activity. We hypothesize that topical bexarotene can be applied to the breast as a prevention agent with penetration into the breast tissue and without subsequent systemic side effects as seen with oral bexarotene. Methods: Women at high risk for breast cancer were recruited and assigned to escalating doses of 1% topical bexarotene: 10mg (1ml) every other day, 10mg (1ml) daily and 20mg (2ml) daily for 4 weeks. Cohorts of 3-4 participants were enrolled and fully evaluated through 4 weeks prior to enrolling the next cohort. Each dose level enrolled a maximum of 10 participants. The primary endpoint of the study was to determine the recommended phase II dose of topical bexarotene 1% gel for evaluation in healthy at-risk women. Dose Limiting Toxicity (DLT) was defined as a grade 2 skin adverse event that persists for at least 6 days or any grade 3 or greater adverse event related to the study drug. A grade 2 skin adverse event that recurs and persists for at least 3 days is also a DLT. The Maximum Tolerated Dose (MTD) was defined as the highest dose level at which no more than 2 participants experience a DLT among 10 participants treated. Once the MTD was determined, interim biomarker analysis will be completed to assess bexarotene levels in serum and tissue samples. An expansion cohort of an additional 10 patients will be recruited at the MTD to further evaluate safety and toxicity. Secondary endpoints include serum bexarotene level, tissue bexarotene levels, and changes in thyroid function tests, lipid profile, and calcium. Results: Ten women were enrolled at the dose level of 10mg every other day and 9/10 participants experienced Grade 1 skin related events at the application site. Two participants reported Grade 2 skin related events at the application site but did not last long enough to be considered DLTs. Four women were enrolled at the second dose level of 10mg daily and 3/4 experience Grade 2 skin related events including 2 DLTs which stopped accrual to the study. Therefore, the MTD was determined to be 10mg every other day. No laboratory abnormalities were noted across either dose level and no grade 3 or 4 adverse events were reported. Conclusion: Maculopapular rash at treatment site was the most common adverse event related to study drug and resolved with discontinuation. Analysis is ongoing to assess bexarotene drug levels in serum and breast tissue samples. Citation Format: Parijatham S Thomas, Anisha B Patel, Diane Liu, J. Jack Lee, Seema Khan, Miguel Muzzio, Alejandro Contreras, Lana Vornik, Eileen P Dimond, Marjorie Perloff, Brandy Heckman-Stoddard, Powel H Brown. Interim analysis of a phase I dose escalation study of topical bexarotene in women at high risk for breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS8-12.

Abstract PS11-14: Advanced precision health resources in the Susan G Komen tissue bank at the IU simon comprehensive cancer center

Abstract Background: Understanding how hereditary, lifestyle and demographic risk factors influence the development of breast cancer is critical for its prevention and treatment. For this purpose, the Susan G. Komen Tissue Bank at the IU Simon Comprehensive Cancer Center (KTB) serves as the world’s primary repository for normal breast tissue and blood samples from healthy women (~5,500 tissue + blood donors and ~4,500 blood-only donors). In addition, KTB has received mammograms, H&E images and additional longitudinal health and wellness data from its donors via ongoing annual surveys. Whole genome sequencing has also recently been completed on a subset of 500 donors, including ~125 initially healthy donors who later went on to develop breast cancer. To make these disparate, multi-modal data accessible to the larger research community and add advanced precision health analytics capabilities to the KTB database, LifeOmic's Precision Health Cloud (PHC), a secure healthcare-compliant cloud platform, was chosen to host the KTB data moving forward. The PHC provides browsing and querying capabilities across all data including genomic, demographic, histology, donor surveys, and scanned images. Data from donors can be viewed individually as well as analyzed across cohorts of interest. Computation resources, such as informatics workflows and machine learning infrastructure, are available on the same secure environment to leverage the power of cloud computing without having to download large files. This is in addition to visual and custom analytics using Jupyter Notebooks accessible to collaborators. Methods: We loaded all available KTB data from ~10,000 donors into the PHC. Donor-reported data collected at the time of donation and subsequent follow-up questionnaire responses were loaded as FHIR Observations and Medications in the PHC. Additional imaging and genomic data loaded into the PHC include approximately 12,000 mammogram images, 5,000 H&E images, 5,000 ancestry-informative genotypes and 500 VCF and BAM files from whole genome sequencing. A timeline view of each donor shows data collected from each donation and follow-up surveys. All genetic variants were annotated upon ingestion into the PHC with functional effect on genes, population allele frequency, ClinVar clinical significance and in silico predictions for functional impact. Results: To demonstrate the utility of KTB data hosted in the PHC, we will show how the PHC can be used to directly analyze genomic and donor-reported data, specifically focusing on the recent whole genome sequencing data from 500 donors. Whole genome sequencing data will be used to calculate the genetic ancestry of each donor compared to their self-reported ancestry and previously predicted ancestry from a 41-SNP ancestry panel. Additionally, we will calculate genomewide breast cancer polygenic risk scores and compare these with the survey-based Tyrer-Cuzick and Gail score risk measures. We will assess how the polygenic risk score further stratifies the donors that have germline pathogenic variants in a known breast cancer gene. For the ~125 sequenced donors that went on to develop breast cancer we will also investigate correlations between family history of cancer, age of diagnosis, prior gynecological history and lifestyle differences. All generated results will remain in the PHC for access by future KTB collaborators to continue to build the utility and value of the KTB data. Conclusions: We have shown how hosting KTB data in the PHC opens new opportunities for advanced precision health research for breast cancer researchers worldwide. As more specimens and diverse data sets including genomic, longitudinal and imaging information are being tracked and deposited into KTB, this resource will continue to grow to enable diverse research needs. Citation Format: Steven M Bray, Swee Seong Wong, Baiju G Parikh, Kevin C Wood, Samuel T Rysdyk, Jill E Henry, Natascia Marino, Milan Radovich, Anna Maria V Storniolo. Advanced precision health resources in the Susan G Komen tissue bank at the IU simon comprehensive cancer center [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS11-14.

Abstract PS17-17: cGAS-STING pathway protein expression in human primary breast cancer

Abstract Background: The DNA-sensing nucleotidyl transferase enzyme cyclic GMP-AMP synthase (cGAS), its second-messenger product cyclic GMP-AMP (cGAMP), and the cGAMP sensor Stimulator of interferon Genes (STING) form a major cytoplasmic DNA-sensing mechanism in human cells [Barber GN. Nat Rev Immunol, 2015]. cGAMP-activated STING oligomers bind TANK-binding kinase 1 (TBK1), translocate from the endoplasmic reticulum to perinuclear vesicles, and phosphorylate transcription factor interferon (IFN) regulatory factor 3 (IRF3), resulting in IRF3 nuclear translocation to induce expression of type I IFNs such as IFNβ. This pathway is critical for controlling innate antiviral immune responses, and is implicated in anti-tumor adaptive immunity, yet little is known regarding its function in human primary breast cancer (BC). Methods: Immunohistochemistry (IHC) staining for cGAS (ab224144), STING (CST13647), TBK1 (HPA045797), IRF3 (ab25950) and IFNβ (ab238675) was performed on tissue microarrays constructed from 18 normal human breast tissue samples and 197 primary BC samples to characterize in-situ protein expression. Positive controls were normal colon, MCF10A cells, testis tissue, MCF-7 cells and normal colon for cGAS, STING, TBK1, IRF3 and IFNβ, respectively. Substitution of primary antibodies with 0.5% normal goat serum served as negative controls. Scoring of extent of the IHC-stained area was 0 for no IHC signal, 1 for <10%, 2 for 10% to 50%, and 3 for >50% of tumor cells. IHC intensity was scored as 0 for no IHC signal, 1 for weak, 2 for moderate, and 3 for strong. A final IHC score was the product of extent score and intensity score, as described [Xia T, et al. Cell reports, 2016]. Categorical classification of high versus low final IHC scores was determined using the median as an unbiased cutoff. Associations between cGAS-STING pathway protein constituents was interrogated by Pearson’s test, and associations between STING pathway IHC expression and breast tumor clinicopathological features were by Chi-square. Results: Among 197 primary BCs, 51.5% were estrogen receptor (ER) positive/ human epidermal growth factor receptor 2 (HER2) negative, 28.4% were HER2 positive and 19.8% were triple negative BC (TNBC). Distinct cGAS-STING pathway expression patterns were observed among three BC subtypes (Table 1). Frequent high expression of cGAS-STING pathway constituents was observed in ER+/HER2- BC. By contrast, high cGAS expression was not associated with high STING expression or effector protein IFNβ in either HER2+ BC, or in TNBC. IRF3 and IFNβ expression was significantly positively associated with hormone receptor status (both P< .001); IFNβ expression was significantly inversely associated with higher grade (P=0.008) and higher mean Ki67 index (18 vs 10, P=0.005). High expression of IFNβ was positively correlated with higher levels of TBK1 and IRF3 (R=0.262 P<.001; R=0.347, P=<.001, respectively). Conclusion: To our knowledge, this is the first study to report cGAS-STING pathway in situ protein expression in primary BC, and demonstrates distinct expression patterns amongst different primary BC clinical subtypes. A limitation of this study is that IHC methods were insufficient for detecting post-translational modifications or translocation of STING pathway proteins. These results provide foundation for understanding the mechanisms of innate immune response in BC, as well as for developing new therapeutic strategies aimed at facilitating adaptive immune anti-tumor responses. Citation Format: Yu Zong, Xiaofei Liu, Glen N Barber, Mark D Pegram. cGAS-STING pathway protein expression in human primary breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-17.

Pipeline of Mass-spectrometry Data Processing for Diagnostic Molecular Marker Panel Obtaining Using the Example of Search Markers of Breast Cancer Metastasis

A pathology diagnostic using molecular marker is a perspective direction of clinical medicine. Mass-spectrometry (MS) is a one of methods, which are used for obtaining information about molecular profiles. Selection of species, essential for classification “case/control is an important task for data processing. Pipeline of data processing has been proposed using MS data, obtained during analysis of tumor breast tissue samples and health breast tissue samples, with the aim of metastasis marker selection. As a result, selection of lipid markers that belong to classes, related to metastasis and proliferation processes, makes it possible to create high sensitivity diagnostic model, based on logistic regression. The proposed method is applicable for data processing, obtained by MS analysis of other “omics”: metabolome, proteome, glycome.

Measurement of Breast Tissue Estrogens by Liquid Chromatography-tandem Mass Spectrometry

Although estrogen contribution estrogen to breast cancer development is not fully understood, an effective method of their measurement, in the mammary gland might provide additional insight. In this study, we have developed a LC-MS/MS method of simultaneous quantification of estrone and estradiol in breast tissue samples. Analytes were extracted with methyl tert-butyl ether by sonication and derivatized with dansyl chloride. Estrogens were analyzed by liquid chromatography-tandem mass spectrometry with an electrospray ionization source. Accuracy and precision were better than 20% for most concentrations. Although estrone and estradiol levels in normal and malignant breast tissue samples analyzed using our method insignificantly differed. The method developed may be used in further studies aimed at evaluating a role estrogens in breast cancer risk.

Assessment of Human Papillomavirus Infection and Risk Factors in Egyptian Women With Breast Cancer.

Numerous risk factors for breast cancer (BC) have been identified. High-risk human papilloma virus (HR-HPV) is the etiological agent of cervical cancer and in some cases of head and neck cancer, specifically oropharyngeal cancer, but the role of HR-HPV in evoking neoplasia in BC is still unclear. In this study, all women above the age of 18 visiting the oncology clinic at Al-Azhar university hospital and Ain Shams specialized hospital between the period of February 2017 and March 2018 were invited to participate. We determined the prevalence of HR-HPV genotypes 16, 18, and 31 in breast tissue samples from 72 women with treatment-naïve BC and 15 women with benign breast lesions (BBL) by quantitative real-time PCR (qRT-PCR) and primer sets targeting the E6 and E7 regions. High-risk human papilloma virus DNA was detected in 16 of 72 (22.2%) BC cases (viral load range = 0.3-237.8 copies/uL) and 0 of 15 women with BBL. High-risk human papilloma virus was detected in 14 of 16 (87.5%), 2 of 16 (12.5%), and 0 of 16 (0%) for genotypes 16, 18, and 31, respectively. Forty-three age-matched healthy Egyptian women were enrolled as controls for assessment of local risk factors that can be used to initiate a strategy of BC prevention in Egypt. Assessment of the risk factors demonstrated that low education level, passive smoking, lack of physical activity, family history of cancer, and use of oral contraception were significant risk factors for BC. In conclusion, our results lead us to postulate that HR-HPV infection may be implicated in the development of some types of BC in Egyptian women. In addition, identification of local risk factors can support practical prevention strategies for BC in Egypt.

Endocrine disrupting chemicals and breast cancer cells.

Many hundreds of endocrine disrupting chemicals (EDCs) have been measured as entering human breast tissue from a range of environmental sources, and this review focuses on discussion of mechanisms by which such EDCs may be contributing to the globally rising incidence of breast cancer. Many of the distinguishing features of breast cancer may be accounted for by EDC exposure, including, but not limited to, the fact that many EDCs possess estrogenic activity and exposure to estrogen is a main risk factor for breast cancer. Studies of the actions of EDCs in human breast cancer cells are aided by use of the conceptual framework of the hallmarks of cancer, and, acting by a variety of genomic and nongenomic mechanisms, EDCs have now been shown to enable all the hallmarks of cancer to develop in human breast cancer cells. Many studies report that hallmarks can develop at concentrations which are within the range of those measured in human breast tissues, especially when added as mixtures. The varied levels of different EDCs measured in individual breast tissue samples together with the overlapping and complementary mechanisms of action of the EDCs imply that thematic mechanisms will be driven inevitably by different chemical mixtures. Despite the complexity, EDCs do need to now be acknowledged as a risk factor for breast cancer in order for preventative strategies to include reduction in EDC exposure.

Kallikrein 11 Down-regulation in Breast Carcinoma: Correlation With Prognostic Parameters.

Expression of kallikrein-11 (KLK11) has been found to be related to the prognosis of various human cancer types but its physiological functions in the steps of breast cancer (BC) progression are still unknown. BC and adjacent normal breast tissue samples were collected from 28 patients. KLK11 expression levels were determined by real-time polymerase chain reaction for each sample and associations with known prognostic features were statistically analyzed. Although there was slight up-regulation in tumor tissues overall, significant down-regulation of KLK11 expression in tumor tissue was observed in the elderly and in patients with perineural invasion. Furthermore, tumor size, grade, mitotic score, necrosis, calcification, lymphatic invasion, hormone receptor status and Ki67 expression were associated with altered KLK11 level. Changes in expression levels of KLK11, associated with patient characteristics, might be used as complementary data in order to predict clinical outcome and prognosis in BC.

Altered expression level of ACSM5 in breast cancer: An integrative analysis of tissue biomarkers with diagnostic potential

BackgroundAbnormal gene expression levels of enzymes involved in fatty acid synthesis and oxidation pathways in breast cancer have been shown to associate with progression of breast cancer. However, little is known about the significance of alternations in fatty acid activation, as a key step in their metabolism. We aimed to recognize suitable biomarkers indicating the altered activation of fatty acids and examine their differential expression in breast cancer and adjacent normal breast tissue samples. Material and methodsTranscript levels of ACS enzymes were analyzed in TCGA data (RNAseq), breast cancer and normal breast samples by differential gene expression (DGE). Gene set enrichment analysis (GSEA) was performed to highlight the most enriched genes involved in the activation of fatty acids. Bioinformatic analysis was performed for the selection of LncRNAs. Gene expression analyses were performed using quantitative Real-time PCR (qRT-PCR) in breast cancer tissue samples and adjacent normal breast tissue samples. Correlation and survival analyses were performed using R program. ROC curves were created for TCGA and qPCR data to evaluate the prognostic potential of the biomarkers. ResultsGSEA analysis revealed that ACSM5 is highly enriched in normal breast tissue samples compared to breast cancer tissue samples. LINC00265 with highest co-efficient score with ACSM5 was selected for further analyses. The expression level of ACSM5 and LINC00265 positively correlated with each other and were significantly low in breast cancer tissues compared to normal breast tissues. Correlation analysis suggested a positive correlation between ACSM5 and LINC00265 genes. Survival analysis predicted prognostic potential for ACSM5. ROC curve analyses revealed that ACSM5 and LINC00265 can distinguish breast tumor samples from adjacent breast tissues. ConclusionOur study suggests ACSM5 gene as a potential biomarker with prognostic value in detection of altered activation of medium-chain fatty acids in breast cancer. In addition, the positive correlation between ACSM5 and LINC00265 suggests the presence of possible regulatory mechanism between them.

Identification of candidate biomarkers correlated with the pathogenesis and prognosis of breast cancer via integrated bioinformatics analysis.

Background:This study was carried out to identify potential key genes associated with the pathogenesis and prognosis of breast cancer (BC).Methods:Seven GEO datasets (GSE24124, GSE32641, GSE36295, GSE42568, GSE53752, GSE70947, GSE109169) were downloaded from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) between BC and normal breast tissue samples were screened by an integrated analysis of multiple gene expression profile datasets. Hub genes related to the pathogenesis and prognosis of BC were verified by employing protein–protein interaction (PPI) network.Results:Ten hub genes with high degree were identified, including CDK1, CDC20, CCNA2, CCNB1, CCNB2, BUB1, BUB1B, CDCA8, KIF11, and TOP2A. Lastly, the Kaplan–Meier plotter (KM plotter) online database demonstrated that higher expression levels of these genes were related to lower overall survival. Experimental validation showed that all 10 hub genes had the same expression trend as predicted.Conclusion:The findings of this research would provide some directive significance for further investigating the diagnostic and prognostic biomarkers to facilitate the molecular targeting therapy of BC, which could be used as a new biomarker for diagnosis and to guide the combination medicine of BC.

The expression of programmed death-ligand 1 in patients with invasive breast cancer.

The purpose of this study is to investigate the association between protein expression of programmed death-ligand 1 (PD-L1) and the clinicopathological features of patients with invasive breast cancer. Clinicopathological data of 651 patients with invasive breast carcinoma were collected over a 1-year period. Patients whose breast tissue samples did not express genes for the estrogen receptor (ER), progesterone receptor (PR), or human epidermal growth factor receptor-2 (HER2) were classified as triple-negative breast cancer (TNBC). The correlations of PD-L1 expression with clinicopathological features and overall survival were determined using Pearson's correlation coefficient and logistic binary regression analysis, respectively. Positive expression of PD-L1 was detected in 47% of patients with invasive breast carcinoma, compared with 69.3% of TNBC patients (P<0.05). Furthermore, expression of PD-L1 in patients with invasive breast carcinoma was significantly correlated with WHO grade, tumor size, vascular invasion, pathological stage, and the expression of ER, PR, nuclear associated antigen Ki67 (Ki67), p53 gene, cytokeratin 5/6 (CK5/6), and epidermal growth factor receptor (EGFR) (P<0.05). Logistic binary regression analysis showed that WHO grade, Ki67, p53, and EGFR were independent risk factors for the expression of PD-L1 in patients with invasive breast cancer. Moreover, PD-L1 expression in TNBC patients was significantly correlated with WHO grade, neuro-invasion, Ki67, CK5/6, and EGFR (P<0.05), but it was not correlated with age, tumor size, vascular invasion, number of lymph nodes, pathological stage, or the expression of ER, PR, p53, androgen receptor (AR), or vascular endothelial growth factor receptor (VEGFR) (P>0.05). The high expression rate of PD-L1 in invasive breast cancer is closely related to some clinicopathological features. Thus, immunotherapy with PD-L1 inhibitors could be a potential treatment strategy for patients with invasive breast cancer.

The potential of terahertz sensing for cancer diagnosis

The terahertz (THz) region lies between the microwave and infrared regions of the electromagnetic (EM) spectrum such that it is strongly attenuated by water and very sensitive to water content. Here, we numerically present what is to our knowledge the detecting system based on THz reflectance spectral responses data in the diagnosis of in vivo and ex vivo of some cancer's samples such as skin, breast and colon cancer tissue samples. The numerical analysis on the use of semiconductor metamaterial design/device as a complex refractive index (CRI) biosensor have been carried out. We demonstrate the application of terahertz pulse detecting (TPD) in reflection geometry for the study of normal and cancerous biological tissues. THz radiation has very low photon energy and thus it does not pose any ionization hazard for biological tissues. The sensitivity of THz radiation to polar molecules, such as water, makes TPD suitable to study the diseases in human body. By studying the THz pulse shape in the time domain, we have been able to differentiate between diseased and normal tissue for the study of basal cell carcinoma (BCC), breast and colon cancers. These results demonstrate the potential of TPD for the study of skin tissue and its related disorders, both in vivo and ex vivo. Findings of this study demonstrate the potential of TPD to depict breast and colon cancers and both in vivo and ex vivo of skin cancer and encourage further studies to determine the sensitivity and specificity of the technique.

Sequential Colocalization of ERa, PR, and AR Hormone Receptors Using Confocal Microscopy Enables New Insights into Normal Breast and Prostate Tissue and Cancers.

Simple SummaryAt present, platforms for multiplex immunohistochemistry (e.g., Opal) identify markers in distinct cell populations within a tissue section using multispectral fluorescence and optic microscopy. However, the optic resolution is not enough to colocalize markers at the subcellular level in the main epithelial or cancer population. We use confocal microscopy in multiplex detection of nuclear hormone receptors since they are an important part of the diagnosis and treatment of breast and prostate cancer. Moreover, we increased the quantitative dynamic range and resolution through increasing the signal/noise ration through reducing autofluorescence and increased longer antibody incubations. ColNu mIHCF identified distinct patterns of nuclear receptor colocalization in breast cancers. Furthermore, in prostate cancer all cancer epithelium was positive for ERa at the plasma membrane; and in normal prostate a small ERa+/p63+/AR− basal population suggest stem cell commitment to differentiation. ColNu mIHCF could be used for improving diagnosis and treatment in cancer.Multiplex immunohistochemistry (mIHC) use markers staining different cell populations applying widefield optical microscopy. Resolution is low not resolving subcellular co-localization. We sought to colocalize markers at subcellular level with antibodies validated for clinical diagnosis, including the single secondary antibody (combination of anti-rabbit/mouse-antibodies) used for diagnostic IHC with any primary antibody, and confocal microscopy. We explore colocalization in the nucleus (ColNu) of nuclear hormone receptors (ERa, PR, and AR) along with the baseline marker p63 in paired samples of breast and prostate tissues. We established ColNu mIHCF as a reliable technique easily implemented in a hospital setting. In ERa+ breast cancer, we identified different colocalization patterns (nuclear or cytoplasmatic) with PR and AR on the luminal epithelium. A triple-negative breast-cancer case expressed membrane-only ERa. A PR-only case was double positive PR/p63. In normal prostate, we identified an ERa+/p63+/AR-negative distinct population. All prostate cancer cases characteristically expressed ERa on the apical membrane of the AR+ epithelium. We confirmed this using ERa IHC and needle-core biopsies. ColNu mIHCF is feasible and already revealed a new marker for prostate cancer and identified sub-patterns in breast cancer. It could be useful for pathology as well as for functional studies in normal prostate and breast tissues.

Rapid assessment of breast tumor margins using deep ultraviolet fluorescence scanning microscopy.

.Significance: Re-excision rates for women with invasive breast cancer undergoing breast conserving surgery (or lumpectomy) have decreased in the past decade but remain substantial. This is mainly due to the inability to assess the entire surface of an excised lumpectomy specimen efficiently and accurately during surgery.Aim: The goal of this study was to develop a deep-ultraviolet scanning fluorescence microscope (DUV-FSM) that can be used to accurately and rapidly detect cancer cells on the surface of excised breast tissue.Approach: A DUV-FSM was used to image the surfaces of 47 (31 malignant and 16 normal/benign) fresh breast tissue samples stained in propidium iodide and eosin Y solutions. A set of fluorescence images were obtained from each sample using low magnification () and fully automated scanning. The images were stitched to form a color image. Three nonmedical evaluators were trained to interpret and assess the fluorescence images. Nuclear–cytoplasm ratio (N/C) was calculated and used for tissue classification.Results: DUV-FSM images a breast sample with subcellular resolution at a speed of . Fluorescence images show excellent visual contrast in color, tissue texture, cell density, and shape between invasive carcinomas and their normal counterparts. Visual interpretation of fluorescence images by nonmedical evaluators was able to distinguish invasive carcinoma from normal samples with high sensitivity (97.62%) and specificity (92.86%). Using N/C alone was able to differentiate patch-level invasive carcinoma from normal breast tissues with reasonable sensitivity (81.5%) and specificity (78.5%).Conclusions: DUV-FSM achieved a good balance between imaging speed and spatial resolution with excellent contrast, which allows either visual or quantitative detection of invasive cancer cells on the surfaces of a breast surgical specimen.

VIRUS-ASSOCIATED BREAST CANCER (REVIEW AND METAANALYSIS)

The assessment of the etiological role of viruses in the development of breast cancer remains the subject of intense study. This review examines the data on the presence/absence of viral infection in breast cancer tumors and its clinical significance. The reports on the association of breast cancer with three groups of viruses: HMTV (human Mammary Tumor Virus / MMTV-like), Epstein-Barr virus (EBV) and human papillomavirus (HPV) were analyzed. The authors carried out meta-analysis for each type of virus, which demonstrated the association of the viral infection with breast cancer tissue. A meta-analysis of 1389 breast cancer tissue samples and 750 normal breast tissue samples showed a high level of HMTV infection in the breast cancer tissue (30.7%). The relative risk of breast cancer associated with HMTV infection was 16.7 (95% CI: 7.0-39.7, p = 1.69x10-10). For EBV, the meta-analysis of 1131 breast tumor samples and 185 normal breast tissue samples (based on 9 primary studies) showed that the incidence of EBV infection was 30. .4% in tumor breast tissue and 4.3% in normal breast tissue. The relative risk of EBV-associated breast cancer was 3.3 (95% CI: 1.8-5.8, р=0.00006). The meta-analysis of HPV infection included 29 primary studies with 2,446 tumor tissue samples and 1,144 normal tissue samples. The prevalence of HPV in breast cancer samples and in normal tissue samples was 25% and 4.5%, respectively. The relative risk of breast cancer associated with HPV infection was 3.6 (95% CI: 2.3-5.6, p = 2.8x10-8). The data obtained indicate that the studies on the etiological role of HMTV, EBV and HPV in the development of breast cancer are promising.

Local aromatase activity alterations in breast cancer tissues: A potential way of decision support for clinicians

Background and aimsIt is becoming evident that local estrogen exposure is important in postmenopausal breast cancer patients. The microenvironment is established by breast stromal cells based on communication with tumor cells that is essential to cancer development, invasion, and metastasis. Here we investigated aromatase activity levels in both tumor and matched stromal tissues by showing their impact on the manufacturing of local estrogen and tumor progression in cases of invasive ductal carcinoma (IDC). MethodsTumor (T) and tumor-associated stroma (TAS) neighboring tissues were acquired from each postmenopausal patient, diagnosed with IDC, and categorized as luminal A (n = 20). The control group was formed from tumor-free breast tissue samples (N, n = 12). A microsomal-based technique was created to compare breast tissue aromatase activities using liquid chromatography – mass spectrometry. FindingsWe observed that the TAS tissues have the highest aromatase activities (p < 0.05). High progesterone receptor (PR) intensity levels were found to be decreasing the activity level in these tissues significantly (p < 0.05). Tumor tissue specific aromatase activity levels of postmenopausal patients' were tend to be lower compared to healthy premenopausal subjects' (3 fold, p < 0.001). In addition low activity in tumor tissues were associated with low grade and late stage cancers. ConclusionsEarly detection and personalized therapy is essential for postmenopausal breast cancer patients. Together, our in-house tandem mass spectrometry technique has the potential for further development and standardization for the measurement of aromatase activity and may assist clinicians decide on therapy policies for postmenopausal IDC patients which could be an invaluable asset for precise and specific evaluation.

Metals and breast cancer risk: a prospective study using toenail metal biomarkers

Background: Certain metals are known or suspected carcinogens and have been found in breast tissue samples. Toenails are a stable matrix that reflect exposure 6-12 months before collection and measurements correlate well over time. We prospectively examined a large panel of toenail metals in relation to breast cancer risk and were the first to consider whether multiple metal biomarkers jointly influence risk. Methods: The Sister Study is prospective cohort of 50,884 women who enrolled between 2003-2009 with follow-up for breast cancer through September 2016. We measured 15 metals in toenails collected at enrollment using a case-cohort design of 1,495 cases and a randomly-selected sub-cohort of 1,605 women. For individual metals, multivariable-adjusted hazard ratios (HRs) and 95% confidence intervals (CIs) were estimated using Cox regression with robust variance. We examined associations overall and stratified by race, estrogen receptor (ER), and menopausal status. Quantile g-computation was used to examine joint associations between multiple metals and breast cancer risk. Results: In individual metal models, arsenic was associated with a non-linear increase in breast cancer risk (2nd vs. 1st tertile, HR=1.22; 95% CI: 1.01-1.49). This association was stronger for ER+ breast cancer (HR=1.31; 95% CI: 1.06-1.62). In non-Hispanic Blacks, zinc was associated with an elevated risk (3rd vs. 1st tertile, HR=1.40; 95% CI: 0.97-2.02). Molybdenum was inversely associated with breast cancer overall (3rd vs. 1st tertile, HR=0.83; 95% CI: 0.68-1.00) and particularly for ER- breast cancer (3rd vs. 1st tertile, HR=0.57; 95% CI: 0.37-0.87). A simultaneous increase in multiple metals was not associated with breast cancer risk. Conclusions: In this prospective study considering multiple toenail metals in relation to breast cancer, we found that individual metals and metal mixtures were not consistently associated with a higher risk. However, a few metals appeared to be related to breast cancer risk in certain subgroups.

Identification of key genes unique to the luminal a and basal-like breast cancer subtypes via bioinformatic analysis

BackgroundBreast cancer subtypes are statistically associated with prognosis. The search for markers of breast tumor heterogeneity and the development of precision medicine for patients are the current focuses of the field.MethodsWe used a bioinformatic approach to identify key disease-causing genes unique to the luminal A and basal-like subtypes of breast cancer. First, we retrieved gene expression data for luminal A breast cancer, basal-like breast cancer, and normal breast tissue samples from The Cancer Genome Atlas database. The differentially expressed genes unique to the 2 breast cancer subtypes were identified and subjected to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses. We constructed protein–protein interaction networks of the differentially expressed genes. Finally, we analyzed the key modules of the networks, which we combined with survival data to identify the unique cancer genes associated with each breast cancer subtype.ResultsWe identified 1114 differentially expressed genes in luminal A breast cancer and 1042 differentially expressed genes in basal-like breast cancer, of which the subtypes shared 500. We observed 614 and 542 differentially expressed genes unique to luminal A and basal-like breast cancer, respectively. Through enrichment analyses, protein–protein interaction network analysis, and module mining, we identified 8 key differentially expressed genes unique to each subtype. Analysis of the gene expression data in the context of the survival data revealed that high expression of NMUR1 and NCAM1 in luminal A breast cancer statistically correlated with poor prognosis, whereas the low expression levels of CDC7, KIF18A, STIL, and CKS2 in basal-like breast cancer statistically correlated with poor prognosis.ConclusionsNMUR1 and NCAM1 are novel key disease-causing genes for luminal A breast cancer, and STIL is a novel key disease-causing gene for basal-like breast cancer. These genes are potential targets for clinical treatment.

Genome-wide DNA methylation profiling in human breast tissue by Illumina TruSeq methyl capture EPIC sequencing and infinium methylationEPIC beadchip microarray

ABSTRACT A newly-developed platform, the Illumina TruSeq Methyl Capture EPIC library prep (TruSeq EPIC), builds on the content of the Infinium MethylationEPIC Beadchip Microarray (EPIC-array) and leverages the power of next-generation sequencing for targeted bisulphite sequencing. We empirically examined the performance of TruSeq EPIC and EPIC-array in assessing genome-wide DNA methylation in breast tissue samples. TruSeq EPIC provided data with a much higher density in the regions when compared to EPIC-array (~2.74 million CpGs with at least 10X coverage vs ~752 K CpGs, respectively). Approximately 398 K CpGs were common and measured across the two platforms in every sample. Overall, there was high concordance in methylation levels between the two platforms (Pearson correlation r = 0.98, P < 0.0001). However, we observed that TruSeq EPIC measurements provided a wider dynamic range and likely a higher quantitative sensitivity for CpGs that were either hypo- or hyper-methylated (β close to 0 or 1, respectively). In addition, when comparing different breast tissue types TruSeq EPIC identified more differentially methylated CpGs than EPIC-array, not only out of additional sites interrogated by TruSeq EPIC alone, but also out of common sites interrogated by both platforms. Our results suggest that both platforms show high reproducibility and reliability in genome-wide DNA methylation profiling, while TruSeq EPIC had a significant improvement over EPIC-array regarding genomic resolution and coverage. The wider dynamic range and likely higher precision of the estimates by the TruSeq EPIC may lead to the identification of novel differentially methylated markers that are associated with disease risk.

High positivity values for bovine leukemia virus in human breast cancer cases from Minas Gerais, Brazil.

Bovine leukemia virus (BLV) is a retrovirus that causes lymphoma in cattle worldwide and has also been associated with breast cancer in humans. The mechanism of BLV infection in humans and its implication as a primary cause of cancer in women are not known yet. BLV infection in humans may be caused by the consumption of milk and milk-products or meat from infected animals. Breast cancer incidence rates in Brazil are high, corresponding to 29.5% a year of cancer cases among women. In 2020, an estimated 66,280 new cases of breast cancer are expected, whereas in 2018 breast cancer has led to 17,572 deaths, the highest incidence and lethality among cancers in women in this country that year. BLV infection occurrence ranges from 60 to 95% in dairy herds. In addition, there are some regions, such as the Minas Gerais State, southeastern Brazil, where the population traditionally consume unpasteurized dairy products. Taken together, this study aimed to verify if there is a higher association between breast cancer and the presence of BLV genome in breast tissue samples within this population that consumes raw milk from animals with high rates of BLV infection. A molecular study of two BLV genes was carried out in 88 breast parenchyma samples, between tumors and controls. The amplified fragment was subjected to BLV proviral sequencing and its identity was confirmed using GenBank. BLV proviral genes were amplified from tumor breast parenchyma samples and healthy tissue control samples from women, revealing a 95.9% (47/49) and 59% (23/39) positivity, respectively. Our results show the highest correlation of BLV and human breast cancer found in the world to date within the population of Minas Gerais, Brazil.