TF-Marker: a comprehensive manually curated database for transcription factors and related markers in specific cell and tissue types in human.

Transcription factors (TFs) play key roles in biological processes and are usually used as cell markers. The emerging importance of TFs and related markers in identifying specific cell types in human diseases increases the need for a comprehensive collection of human TFs and related markers sets. Here, we developed the TF-Marker database (TF-Marker, http://bio.liclab.net/TF-Marker/), aiming to provide cell/tissue-specific TFs and related markers for human. By manually curating thousands of published literature, 5905 entries including information about TFs and related markers were classified into five types according to their functions: (i) TF: TFs which regulate expression of the markers; (ii) T Marker: markers which are regulated by the TF; (iii) I Marker: markers which influence the activity of TFs; (iv) TFMarker: TFs which play roles as markers and (v) TF Pmarker: TFs which play roles as potential markers. The 5905 entries of TF-Marker include 1316 TFs, 1092 T Markers, 473 I Markers, 1600 TFMarkers and 1424 TF Pmarkers, involving 383 cell types and 95 tissue types in human. TF-Marker further provides a user-friendly interface to browse, query and visualize the detailed information about TFs and related markers. We believe TF-Marker will become a valuable resource to understand the regulation patterns of different tissues and cells.

Transcriptional regulation of normal human mammary cell heterogeneity and its perturbation in breast cancer.

The mammary gland in adult women consists of biologically distinct cell types that differ in their surface phenotypes. Isolation and molecular characterization of these subpopulations of mammary cells have provided extensive insights into their different transcriptional programs and regulation. This information is now serving as a baseline for interpreting the heterogeneous features of human breast cancers. Examination of breast cancer mutational profiles further indicates that most have undergone a complex evolutionary process even before being detected. The consequent intra-tumoral as well as inter-tumoral heterogeneity of these cancers thus poses major challenges to deriving information from early and hence likely pervasive changes in potential therapeutic interest. Recently described reproducible and efficient methods for generating human breast cancers de novo in immunodeficient mice transplanted with genetically altered primary cells now offer a promising alternative to investigate initial stages of human breast cancer development. In this review, we summarize current knowledge about key transcriptional regulatory processes operative in these partially characterized subpopulations of normal human mammary cells and effects of disrupting these processes in experimentally produced human breast cancers.

Of mice and women: a comparative tissue biology perspective of breast stem cells and differentiation.

Tissue based research requires a background in human and veterinary pathology, developmental biology, anatomy, as well as molecular and cellular biology. This type of comparative tissue biology (CTB) expertise is necessary to tackle some of the conceptual challenges in human breast stem cell research. It is our opinion that the scarcity of CTB expertise contributed to some erroneous interpretations in tissue based research, some of which are reviewed here in the context of breast stem cells. In this article we examine the dissimilarities between mouse and human mammary tissue and suggest how these may impact stem cell studies. In addition, we consider the differences between breast ducts vs. lobules and clarify how these affect the interpretation of results in stem cell research. Lastly, we introduce a new elaboration of normal epithelial cell types in human breast and discuss how this provides a clinically useful basis for breast cancer classification.

Distribution of Kaiso protein in mouse tissues.

The Kaiso protein was originally described as a BTB/POZ zinc-finger transcription factor and a p120-catenin-binding partner. It is a DNA methylation-dependent transcriptional repressor, but its biological role in mice is still unknown. Here, we characterized a Kaiso-specific antibody by examining Kaiso protein distribution by immunofluorescence microscopy in the following tissues and cell types of adult mice: skin, small intestine, mammary glands, urinary bladder, and others. This study is the first to demonstrate that Kaiso is expressed in most of the examined tissues. Kaiso was localized to the nucleus in almost all tissues. However, it was primarily cytoplasmic in photoreceptor cells in the eye (rods and cones). Furthermore, Kaiso is expressed in a specific subset of male germ cells that are characterized by partly positive PLZF and Bmi-1 staining. In this study, we present the first confirmation of the reliability of expression data using Kaiso knockout mice.

The fractional viscoelastic response of human breast tissue cells.

The mechanical response of a living cell is notoriously complicated. The complex, heterogeneous characteristics of cellular structure introduce difficulties that simple linear models of viscoelasticity cannot overcome, particularly at deep indentation depths. Herein, a nano-scale stress-relaxation analysis performed with an atomic force microscope reveals that isolated human breast cells do not exhibit simple exponential relaxation capable of being modeled by the standard linear solid (SLS) model. Therefore, this work proposes the application of the fractional Zener (FZ) model of viscoelasticity to extract mechanical parameters from the entire relaxation response, improving upon existing physical techniques to probe isolated cells. The FZ model introduces a new parameter that describes the fractional time-derivative dependence of the response. The results show an exceptional increase in conformance to the experimental data compared to that predicted by the SLS model, and the order of the fractional derivative (α) is remarkably homogeneous across the populations, with a median value of 0.48 ± 0.06 for the malignant population and 0.51 ± 0.07 for the benign. The cells' responses exhibit power-law behavior and complexity not associated with simple relaxation (SLS, α = 1) that supports the application of a fractional model. The distributions of some of the FZ parameters also preserve the distinction between the malignant and benign sample populations seen from the linear model and previous results while including the contribution of fast-relaxation behavior. The resulting viscosity, measured by a composite relaxation time, exhibits considerably less dispersion due to residual error than the distribution generated by the linear model and therefore serves as a more powerful marker for cell differentiation.

Abundance and distribution of polychlorinated biphenyls (PCBs) in breast tissue.

Many environmental chemicals accumulate in human tissues and may contribute to cancer risk. Polychlorinated biphenyls (PCBs) are associated with adverse health effects, but relationships between PCB exposure and breast cancer are unclear. In this study, we sought to determine whether bioaccumulation of PCBs differs within regions of the human breast and whether PCB levels are associated with clinical and pathological characteristics in breast cancer patients. Tissue sections (n=245) were collected from breast quadrants from 51 women with a diagnosis ranging from disease-free to metastatic breast cancer. Ninety-seven PCB congeners were assayed by high resolution gas chromatography. ANOVA was used to examine PCB distribution within the breast and relationships with clinical/pathological variables. Pearson product-moment correlations assessed relationships between age at mastectomy and PCB levels. PCBs were abundant in breast tissues with a median concentration of 293.4ng/g lipid (range 15.4-1636.3ng/g). PCB levels in breast tissue were significantly different (p<0.001) among functional groupings of congeners defined by structure-activity properties: Group I (28.2ng/g), Group II (96.6ng/g), Group III (166.0ng/g). Total PCB concentration was highly correlated with age at mastectomy, but the distribution of PCBs did not differ by breast quadrant. PCB levels were not associated with patient status or tumor characteristics. In conclusion, PCB congeners with carcinogenic potential were present at high levels in the human breast, but were not associated with clinical or pathological characteristics in breast cancer patients.

Identification of a novel percent mammographic density locus at 12q24.

Percent mammographic density adjusted for age and body mass index (BMI) is one of the strongest risk factors for breast cancer and has a heritable component that remains largely unidentified. We performed a three-stage genome-wide association study (GWAS) of percent mammographic density to identify novel genetic loci associated with this trait. In stage 1, we combined three GWASs of percent density comprised of 1241 women from studies at the Mayo Clinic and identified the top 48 loci (99 single nucleotide polymorphisms). We attempted replication of these loci in 7018 women from seven additional studies (stage 2). The meta-analysis of stage 1 and 2 data identified a novel locus, rs1265507 on 12q24, associated with percent density, adjusting for age and BMI (P = 4.43 × 10(-8)). We refined the 12q24 locus with 459 additional variants (stage 3) in a combined analysis of all three stages (n = 10 377) and confirmed that rs1265507 has the strongest association in the 12q24 region (P = 1.03 × 10(-8)). Rs1265507 is located between the genes TBX5 and TBX3, which are members of the phylogenetically conserved T-box gene family and encode transcription factors involved in developmental regulation. Understanding the mechanism underlying this association will provide insight into the genetics of breast tissue composition.

Human breast cancer and lymph node metastases express Gb3 and can be targeted by STxB-vectorized chemotherapeutic compounds.

BACKGROUND: The B-subunit of Shiga toxin (STxB) specifically binds to the glycosphingolipid Gb3 that is highly expressed on a number of human tumors and has been shown to target tumor cells in mouse models and ex vivo on primary colon carcinoma specimen. METHODS: Using a novel ex vivo STxB labeling (ESL) method we studied Gb3 expression in cytological specimens of primary human breast tumors from 107 patients, and in synchronous lymph node metastases from 20 patients. Fluorescent STxB was incubated with fine-needle aspiration (FNA) specimens, and Gb3 expression was evaluated by fluorescence microscopy. Furthermore, 11 patient-derived human breast cancer xenografts (HBCx) were evaluated for expression of Gb3 by ESL and FACS. In addition, the biodistribution of fluorescent STxB conjugate was studied after intravenous injection in a Gb3 positive HBCx model. RESULTS: Gb3 expression was detected in 62 of 107 patients (57.9%), mainly in epithelial tumor cells. Gb3 positivity correlated with estrogen receptor expression (p≤0.01), whereas absence of Gb3 expression in primary tumors was correlated with the presence of lymph node metastases (p≤0.03). 65% of lymph node metastases were Gb3 positive and in 40% of tested patients, we observed a statistically significant increase of metastatic Gb3 expression (p≤0.04). Using concordant ESL and flow cytometry analysis, 6 out of 11 HBCx samples were scored positive. Intravenous injections of fluorescent STxB into HBC xenografted mice showed preferential STxB accumulation in epithelial cells and cells with endothelial morphology of the tumor. CONCLUSION: The enhanced expression of Gb3 in primary breast carcinomas and its lymph node metastases indicate that the development of STxB-based therapeutic strategies is of interest in this pathology. Gb3 expressing HBCx can be used as a model for preclinical studies with STxB conjugates. Finally, the ESL technique on FNA represents a rapid and cost effective method for the stratification of patients in future clinical trials.

Lipidomic analysis of phospholipids from human mammary epithelial and breast cancer cell lines.

Alterations of phospholipid (PL) profiles have been associated to disease and specific lipids may be involved in the onset and evolution of cancer; yet, analysis of PL profiles using mass spectrometry (MS) in breast cancer cells is a novel approach. Previously, we reported a lipidomic analysis of PLs from mouse mammary epithelial and breast cancer cells using off-line thin layer chromatography (TLC)-MS, where several changes in PL profile were found to be associated with the degree of malignancy of cells. In the present study, lipidomic analysis has been extended to human mammary epithelial cells and breast cancer cell lines (MCF10A, T47-D, and MDA-MB-231), using TLC-MS, validated by hydrophilic interaction liquid chromatography-MS. Differences in phosphatidylethanolamine (PE) content relative to total amount of PLs was highest in non-malignant cells while phosphatidic acid was present with highest relative abundance in metastatic cells. In addition, the following differences in PL molecular species associated to cancer phenotype, metastatic potential, and cell morphology were found: higher levels of alkylacyl PCs and phosphatidylinositol (PI; 22:5/18:0) were detected in migratory cells, epithelial cells had less unsaturated fatty acyl chains and shorter aliphatic tails in PE and sphingomyelin classes, while PI (18:0/18:1) was lowest in non-malignant cells compared to cancer cells. To date, information about PL changes in cancer progression is scarce, therefore results presented in this work will be useful as a starting point to define possible PLs with prospective as biomarkers and disclose metabolic pathways with potential for cancer therapy.

Does mammographic density reflect the expression of breast cancer markers?

Mammographic density reflects variation in breast tissue composition as detected on mammogram. It is associated with a number of well-known breast cancer risk factors and itself is considered one of the strongest risk factors for breast cancer. If the expression of several proteins and genes within the breast tissue influences mammographic density in the same way as it influences breast cancer risk, then mammographic density might serve as an intermediate biomarker in future epidemiological studies on breast cancer. This has the potential to provide a quick means for predicting the effect of changes in the breast microenvironment on breast cancer risk without having to wait for an eventual development of breast cancer. In this review, the expression of several proteins and genes (growth factors, enzymes, proteoglycans and pro-inflammatory markers) within the breast tissue is shown to be associated with mammographic density. These proteins and genes are suspected to play a role in breast carcinogenesis. More studies assessing differential expression of proteins and genes in mammary epithelium and stroma and their association with mammographic density among premenopausal and postmenopausal women are required. Identification of proteins and genes influencing mammographic density may provide further insight on the molecular causes of breast cancer.

Pilot and feasibility study: prospective proteomic profiling of mammary epithelial cells from high-risk women provides evidence of activation of pro-survival pathways.

Normal mammary gland homeostasis requires the coordinated regulation of protein signaling networks. However, we have little prospective information on whether activation of protein signaling occurs in premalignant mammary epithelial cells, as represented by cells with cytological atypia from women who are at high risk for breast cancer. This information is critical for understanding the role of deregulated signaling pathways in the initiation of breast cancer and for developing targeted prevention and/or treatment strategies for breast cancer in the future. In this pilot and feasibility study, we examined the expression of 52 phosphorylated, total, and cleaved proteins in 31 microdissected Random Periareolar Fine Needle Aspiration (RPFNA) samples by high-throughput Reverse Phase Protein Microarray. Unsupervised hierarchical clustering analysis indicated the presence of four clusters of proteins that represent the following signaling pathways: (1) receptor tyrosine kinase/Akt/mammalian target of rapamycin (RTK/Akt/mTOR), (2) RTK/Akt/extracellular signal-regulated kinase (RTK/Akt/ERK), (3) mitochondrial apoptosis, and (4) indeterminate. Clusters 1 through 3 comprised moderately to highly expressed proteins, while Cluster 4 comprised proteins that are lowly expressed in a majority of RPFNA samples. Our exploratory study showed that the interlinked components of mitochondrial apoptosis pathway are highly expressed in all mammary epithelial cells obtained from high-risk women. In particular, the expression levels of anti-apoptotic Bcl-xL and pro-apoptotic Bad are positively correlated in both non-atypical and atypical samples (unadjusted P < 0.0001), suggesting a delicate balance between the pro-apoptotic and anti-apoptotic regulation of cell proliferation during the early steps of mammary carcinogenesis. Our feasibility study suggests that the activation of key proteins along the RTK/Akt pathway may tip this balance to cell survival. Taken together, our results demonstrate the feasibility of mapping proteomic signaling networks in limited RPFNA samples obtained from high-risk women and the promise of developing rational drug targets or preventative strategies for breast cancer in future proteomic studies with a larger cohort of high-risk women.

BRCA1 regulates human mammary stem/progenitor cell fate.

Although it is well established that women with germ-line mutations in the BRCA1 gene have a greatly increased lifetime incidence of breast and ovarian cancer, the molecular mechanisms responsible for this tissue-specific carcinogenesis remain undefined. The majority of these breast cancers are of the basal-like phenotype characterized by lack of expression of ER, PR, and ERBB2. Because this phenotype has been proposed to resemble that of normal breast stem cells, we examined the role of BRCA1 in human mammary stem cell fate. Using both in vitro systems and a humanized NOD/SCID mouse model, we demonstrate that BRCA1 expression is required for the differentiation of ER-negative stem/progenitor cells to ER-positive luminal cells. Knockdown of BRCA1 in primary breast epithelial cells leads to an increase in cells displaying the stem/progenitor cell marker ALDH1 and a decrease in cells expressing luminal epithelial markers and estrogen receptor. In breast tissues from women with germ-line BRCA1 mutations, but not normal controls, we detect entire lobules that, although histologically normal, are positive for ALDH1 expression but are negative for the expression of ER. Loss of heterozygosity for BRCA1 was documented in these ALDH1-positive lobules but not in adjacent ALDH1-negative lobules. Taken together, these studies demonstrate that BRCA1 plays a critical role in the differentiation of ER-negative stem/progenitor cells to ER-positive luminal cells. Because BRCA1 also plays a role in DNA repair, our work suggests that loss of BRCA1 may result in the accumulation of genetically unstable breast stem cells, providing prime targets for further carcinogenic events.

Proteomic analysis of rodent ribosomes revealed heterogeneity including ribosomal proteins L10-like, L22-like 1, and L39-like.

Heterogeneity of ribosome structure, due to variations in ribosomal protein composition, has been shown to be of physiological significance in plants and yeast. Mammalian genomics have demonstrated numerous genes that are paralogous to genes encoding ribosomal proteins. Although the vast majority are considered to be pseudogenes, mRNA expression of a few paralogues, such as human ribosomal protein L39-like/L39-2, has been reported. In the present study, ribosomes from the liver, mammary gland, and testis of rodents were analyzed using a combination of two-dimensional gel electrophoresis under radical-free and highly reducing conditions, and mass spectrometry. This system allowed identification of 78 ribosomal proteins and Rack1 from a single gel. The degree of heterogeneity was far less than that reported for plant and yeast ribosomes, and was in accord with published biochemical and genetic data for mammalian ribosomes. Nevertheless, an uncharacterized paralogue of ribosomal protein L22, ribosomal protein L22-like 1, was identified as a minor ribosomal component. Ribosomal proteins L10-like and L39-like, paralogues of ribosomal proteins L10 and L39, respectively, were found in ribosomes only from the testis. Reverse transcription-polymerase chain reaction yielded supportive evidence for specific expression of L10-like and L39-like in the testis. Newly synthesized L39-like is likely to be transported to the nucleolus, where ribosome biosynthesis occurs, and then incorporated into translating ribosomes in the cytoplasm. Heterogeneity of mammalian testicular ribosomes is structurally non-negligible, and may offer valuable insights into the function of the customized ribosome.

NuMA influences higher order chromatin organization in human mammary epithelium.

The coiled-coil protein NuMA is an important contributor to mitotic spindle formation and stabilization. A potential role for NuMA in nuclear organization or gene regulation is suggested by the observations that its pattern of nuclear distribution depends upon cell phenotype and that it interacts and/or colocalizes with transcription factors. To date, the precise contribution of NuMA to nuclear function remains unclear. Previously, we observed that antibody-induced alteration of NuMA distribution in growth-arrested and differentiated mammary epithelial structures (acini) in three-dimensional culture triggers the loss of acinar differentiation. Here, we show that in mammary epithelial cells, NuMA is present in both the nuclear matrix and chromatin compartments. Expression of a portion of the C terminus of NuMA that shares sequence similarity with the chromatin regulator HPC2 is sufficient to inhibit acinar differentiation and results in the redistribution of NuMA, chromatin markers acetyl-H4 and H4K20m, and regions of deoxyribonuclease I-sensitive chromatin compared with control cells. Short-term alteration of NuMA distribution with anti-NuMA C-terminus antibodies in live acinar cells indicates that changes in NuMA and chromatin organization precede loss of acinar differentiation. These findings suggest that NuMA has a role in mammary epithelial differentiation by influencing the organization of chromatin.

Stromal vapors for real-time molecular guidance of breast-conserving surgery.

Achieving radical tumor resection while preserving disease-free tissue during breast-conserving surgery (BCS) remains a challenge. Here, mass spectrometry technologies were used to discriminate stromal tissues reported to be altered surrounding breast tumors, and build tissue classifiers ex vivo. Additionally, we employed the approach for in vivo and real-time classification of breast pathology based on electrosurgical vapors. Breast-resected samples were obtained from patients undergoing surgery at MUMC+. The specimens were subsequently sampled ex vivo to generate electrosurgical vapors analyzed by rapid evaporative ionization mass spectrometry (REIMS). Tissues were processed for histopathology to assign tissue components to the mass spectral profiles. We collected a total of 689 ex vivo REIMS profiles from 72 patients which were analyzed using multivariate statistical analysis (principal component analysis-linear discriminant analysis). These profiles were classified as adipose, stromal and tumor tissues with 92.3% accuracy with a leave-one patient-out cross-validation. Tissue recognition using this ex vivo-built REIMS classification model was subsequently tested in vivo on electrosurgical vapors. Stromal and adipose tissues were classified during one BCS. Complementary ex vivo analyses were performed by REIMS and by desorption electrospray ionization mass spectrometry (DESI-MS) to study the potential of breast stroma to guide BCS. Tumor border stroma (TBS) and remote tumor stroma (RTS) were classified by REIMS and DESI-MS with 86.4% and 87.8% accuracy, respectively. We demonstrate the potential of stromal molecular alterations surrounding breast tumors to guide BCS in real-time using REIMS analysis of electrosurgical vapors.

Organoid cultures from normal and cancer-prone human breast tissues preserve complex epithelial lineages.

Recently, organoid technology has been used to generate a large repository of breast cancer organoids. Here we present an extensive evaluation of the ability of organoid culture technology to preserve complex stem/progenitor and differentiated cell types via long-term propagation of normal human mammary tissues. Basal/stem and luminal progenitor cells can differentiate in culture to generate mature basal and luminal cell types, including ER+ cells that have been challenging to maintain in culture. Cells associated with increased cancer risk can also be propagated. Single-cell analyses of matched organoid cultures and native tissues by mass cytometry for 38 markers provide a higher resolution representation of the multiple mammary epithelial cell types in the organoids, and demonstrate that protein expression patterns of the tissue of origin can be preserved in culture. These studies indicate that organoid cultures provide a valuable platform for studies of mammary differentiation, transformation, and breast cancer risk.

Method for Determining Gelatinolytic Activity in Tissue: In Situ Gelatin Zymography.

To explore the physiological or pathological roles of proteases, it is important to be able to detect and precisely localize them in a tissue, to differentiate between inactive and active forms, as well as to quantify and determine the nature of the enzyme that degrades a given substrate. Here we present an in situ gelatin zymography method that allows for a precise localization of active gelatin-degrading enzymes in a tissue section. In this method, dye-quenched gelatin is put on top of a tissue section. During an incubation period, active gelatinolytic enzymes will degrade the substrate and fluorescent signals are emitted from the locations of these enzymes.

Morphologic and molecular evolutionary pathways of low nuclear grade invasive breast cancers and their putative precursor lesions: further evidence to support the concept of low nuclear grade breast neoplasia family.

We have previously provided evidence showing an association between some precursor lesions with low nuclear grade breast carcinomas (LNGBCs). In this study, further immunophenotypic support to our proposed route of pathogenesis of LNGBC and their precursor lesions was provided. Precursor lesions including columnar cell lesions, atypical ductal hyperplasia, ductal carcinoma in situ, usual epithelial hyperplasia, and lobular neoplasia were compared with matching "morphologically normal" terminal lobular duct units and matching invasive carcinoma. The epithelial cells in the putative precursor flat epithelial atypia, atypical ductal hyperplasia, lobular neoplasia, ductal carcinoma in situ lesions, and their coexisting LNGBC were negative for basal and myoepithelial markers, but positive for CK19/18/8, estrogen receptor (ER)-alpha, Bcl-2, and cyclin D1. The ER-alpha/ER-beta expression ratio increased during carcinogenesis, as did expression of cyclin D1 and Bcl-2. p53 immunopositivity was found 3% in LNGBC versus 43% in high nuclear grade breast carcinoma (HNGBC), whereas ataxia telangiectasia mutated expression was absent or reduced in 22% of LNGBC versus 53% of HNGBC cases. In summary, our findings support the concept that flat epithelial atypia is the earliest morphologically identifiable nonobligate precursor lesion of LNGBC. These may represent a family of precursor, in situ and invasive neoplastic lesions belonging to the luminal "A" subclass of breast cancer. The balance between ER-alpha and ER-beta expression may be important in driving cyclin D-1 and Bcl-2 expression. Ataxia telangiectasia mutated may be one of the alternative regulatory mechanisms to TP53 mutation or dysfunction in low-grade and high-grade breast carcinoma. Our findings support the concept that progression of LNGBC to HNGBC (basal-like or HER2+) phenotype is an unlikely biologic phenomenon.

Comparative evaluation of trace metal distribution and correlation in human malignant and benign breast tissues.

Selected trace metals were analyzed in human malignant and nonmalignant (benign) breast tissue samples by the flame atomic absorption spectrophotometric method. In malignant tissues, dominant mean concentrations were revealed by Na, K, Ca, Mg, Fe, Zn, and Al at 927, 552, 231, 61.7, 36.5, 18.3, and 8.94 microg/g, respectively, while the mean metal levels in benign tissues were 903, 435, 183, 63.3, 24.7, 14.5, and 10.1 microg/g, respectively. Average concentrations of Cd, Co, Cr, Cu, Fe, Mn, K, Ca, and Zn were noted to be significantly higher in the malignant tissues compared with the benign tissues. Significantly strong correlations (r > 0.50) in malignant tissues were observed between Mn and Co, Mn and Cd, Cd and Cr, Fe and Mn, Cd and Co, Fe and Co, Mg and Pb, Cd and Fe, Mg and Ni, Pb and Ni, Ni and Sr, and Fe and Pb, whereas, Cd and Co, Cd and Mn, Co and Mg, Co and Mn, Cu and Mn, Co and Ni, Mg and Ni, Cd and Cu, Cd and Ni, Ca and Mg, Mn and Pb, Cu and Ni, Fe and Ni, Cd and Mg, Co and Cu, Cr and Na, and Cd and Cr revealed strong and significant relationships in benign tissues at p < 0.001. Principal component analysis of the metals data yielded six principal components for malignant tissues and five principal components for benign tissues, with considerably different loadings, duly supported by cluster analysis. The study revealed a considerably different pattern of distribution and mutual correlations of trace metals in the breast tissues of benign and cancerous patients.