A new approach to the classification of mammographic masses and normal breast tissue

A new approach to mammographic mass detection is presented in this paper. Although different algorithms have been proposed for such a task, most of them are application dependent. In contrast, our approach makes use of a kindred topic in computer vision adapted to our particular problem. In this sense, we translate the eigenfaces approach for face detection/classification problems to a mass detection. Two different databases were used to show the robustness of the approach. The first one consisted on a set of 160 regions of interest (RoIs) extracted from the MIAS database, being 40 of them with confirmed masses and the rest normal tissue. The second set of RoIs was extracted from the DDSM database, and contained 196 RoIs containing masses and 392 with normal, but suspicious regions. Initial results demonstrate the feasibility of using such approach with performances comparable to other algorithms, with the advantage of being a more general, simple and cost-effective approach

A new approach to the classification of mammographic masses and normal breast tissue

A new approach to mammographic mass detection is presented in this paper. Although different algorithms have been proposed for such a task, most of them are application dependent. In contrast, our approach makes use of a kindred topic in computer vision adapted to our particular problem. In this sense, we translate the eigenfaces approach for face detection/classification problems to a mass detection. Two different databases were used to show the robustness of the approach. The first one consisted on a set of 160 regions of interest (RoIs) extracted from the MIAS database, being 40 of them with confirmed masses and the rest normal tissue. The second set of RoIs was extracted from the DDSM database, and contained 196 RoIs containing masses and 392 with normal, but suspicious regions. Initial results demonstrate the feasibility of using such approach with performances comparable to other algorithms, with the advantage of being a more general, simple and cost-effective approach

Fatty acid compositions of inositol and choline phospholipids of breast tumours and normal breast tissue

The fatty acid compositions of the -choline and -inositol phospholipids of breast tumours of women undergoing surgery for treatment of breast disease (malignant n = 12; benign n = 10) and normal breast tissue of women undergoing breast reduction surgery (n = 6) were determined. The fatty acid compositions of erythrocyte phospholipids were also determined in the same subjects and in an additional number of normal healthy volunteers (n = 16). Levels of oleic acid were lower in both phospholipid fractions of erythrocytes of women with breast disease and in the phosphatidylcholine fraction of breast tumours compared with normal breast tissue. Significantly higher levels of linoleic acid were found in erythrocytes of tumour-bearing subjects and a similar trend was evident in the phosphatidylcholine fraction of tumour compared with normal breast tissues. Conversely, lower levels of two of the products of linoleic acid chain elongation and desaturation, dihomogamma-linolenic and arachidonic acids, were found in the erythrocyte phospholipids of tumour-bearing subjects and in the choline phospholipids of breast tumour tissues. These data suggest that in women with breast disease, there may be inhibition of 6-desaturase, and enhanced activity of 9-desaturase, enzymes which play an important role in determining membrane phospholipid fatty acid composition. This pattern of altered fatty acid composition characteristic of erythrocyte phospholipids of tumour-bearing subjects and phosphatidylcholine of breast tumour tissue was less evident in the case of the breast tumour phosphatidylinositol in which differences other than those described were seen.

Correlation between carotenoid concentrations in serum and normal breast adipose tissue of women with benign breast tumor or breast cancer

To evaluate the relationship between carotenoid concentrations in serum and breast tissue, we measured serum carotenoid concentrations and endogenous carotenoid levels in breast adipose tissue of women with benign breast tumor (n = 46) or breast cancer (n = 44). Before extraction, serum was digested with lipase and cholesterol esterase, and breast adipose tissue was saponified. Serum and tissue carotenoids were extracted with ether/hexane and measured by using HPLC with a C30 column. Serum retinoic acid was extracted with chloroform/methanol and measured using HPLC with a C18 column. There were no significant differences in serum carotenoids [lutein, zeaxanthin, cryptoxanthin (both α- and β-), α-carotene, all-trans β-carotene, 13-cis β-carotene and lycopene], retinoids (retinol, all-trans and 13-cis retinoic acids), and α- and -γ- tocopherol concentrations between benign breast tumor patients and breast cancer patients. A substantial amount of 9-cis β- carotene was present in adipose tissue and was the only carotenoid that had a significantly lower level in benign breast tumor patients than in breast cancer patients. Correlations between carotenoid concentrations in serum and in breast adipose tissue were determined by combining the data of the two groups. Concentrations of the major serum carotenoids except cryptoxanthin showed significant correlations with breast adipose tissue carotenoid levels. When the concentrations of serum carotenoids were adjusted for serum triglycerides or LDL, correlations between serum carotenoid concentrations and breast adipose tissue carotenoid levels markedly increased, including that of cryptoxanthin (P <0.001). The strong correlation between serum carotenoid concentrations and endogenous breast adipose tissue carotenoid levels indicate that dietary intake influences adipose tissue carotenoid levels as well as serum concentrations, and that adipose tissue is a dynamic reservoir of fat-soluble nutrients.

Establishment of the epithelial-specific transcriptome of normal and malignant human breast cells based on MPSS and array expression data.

INTRODUCTION: Diverse microarray and sequencing technologies have been widely used to characterise the molecular changes in malignant epithelial cells in breast cancers. Such gene expression studies to identify markers and targets in tumour cells are, however, compromised by the cellular heterogeneity of solid breast tumours and by the lack of appropriate counterparts representing normal breast epithelial cells. METHODS: Malignant neoplastic epithelial cells from primary breast cancers and luminal and myoepithelial cells isolated from normal human breast tissue were isolated by immunomagnetic separation methods. Pools of RNA from highly enriched preparations of these cell types were subjected to expression profiling using massively parallel signature sequencing (MPSS) and four different genome wide microarray platforms. Functional related transcripts of the differential tumour epithelial transcriptome were used for gene set enrichment analysis to identify enrichment of luminal and myoepithelial type genes. Clinical pathological validation of a small number of genes was performed on tissue microarrays. RESULTS: MPSS identified 6,553 differentially expressed genes between the pool of normal luminal cells and that of primary tumours substantially enriched for epithelial cells, of which 98% were represented and 60% were confirmed by microarray profiling. Significant expression level changes between these two samples detected only by microarray technology were shown by 4,149 transcripts, resulting in a combined differential tumour epithelial transcriptome of 8,051 genes. Microarray gene signatures identified a comprehensive list of 907 and 955 transcripts whose expression differed between luminal epithelial cells and myoepithelial cells, respectively. Functional annotation and gene set enrichment analysis highlighted a group of genes related to skeletal development that were associated with the myoepithelial/basal cells and upregulated in the tumour sample. One of the most highly overexpressed genes in this category, that encoding periostin, was analysed immunohistochemically on breast cancer tissue microarrays and its expression in neoplastic cells correlated with poor outcome in a cohort of poor prognosis estrogen receptor-positive tumours. CONCLUSION: Using highly enriched cell populations in combination with multiplatform gene expression profiling studies, a comprehensive analysis of molecular changes between the normal and malignant breast tissue was established. This study provides a basis for the identification of novel and potentially important targets for diagnosis, prognosis and therapy in breast cancer.

Tamoxifen down-regulates CaMKII messenger RNA levels in normal human breast tissue

Tamoxifen was proven to reduce the incidence of breast cancer by 49% in women at increased risk of the disease in the Breast Cancer Prevention Trial. In order to identify potential candidates to explain the preventive effect induced by tamoxifen on breast cancer, normal breast tissue obtained from 42 fibroadenoma patients, randomly assigned to receive placebo or tamoxifen, was analyzed by the reverse Northern blot and RT-PCR techniques. The cDNA fragments used on Northern blot membranes were generated by the Human Cancer Genome Project funded by the Ludwig Institute for Cancer Research and FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo, Brazil). Total RNA was obtained from normal breast tissue from patients with clinical, cytological and ultrasound diagnosis of fibroadenoma. After a 50-day treatment with tamoxifen (10 or 20 mg/day) or placebo, normal breast tissue adjacent to the tumor was collected during lumpectomy with local anesthesia. One differentially expressed gene, Calcium/calmodulin-dependent protein kinase II (CaMKII), was found to be down-regulated during TAM treatment. CaMKII is an ubiquitous serine/threonine protein kinase that has been implicated in the diverse effects of hormones utilizing Ca2+ as a second messenger as well as in c-fos activation. These results indicate that the down-regulation of CaMKII induced by TAM might represent alternative or additional mechanisms of the action of this drug on cell cycle control and response to hormones in normal human breast tissue.

Mapping loss of heterozygosity in normal human breast cells from BRCA1/2 carriers

We have studied loss of heterozygosity at the BRCA1 and BRCA2 loci in 992 normal cell clones derived from topographically defined areas of normal tissue in four samples from BRCA1/BRCA2 mutation carriers. The frequency of loss of heterozygosity in the clones was low ( 1.01%), but it was found in all four samples, whether or not a tumour was present. Topographical mapping revealed that the genetic changes were clustered in some breast samples. Our study confirms the previous finding that a field of genetic instability can exist around a tumour, suggesting that sufficient tissue must be removed at surgery to avoid local recurrence. We also demonstrate that such a field of genetic change can exist in morphologically normal tissue before a tumour develops and, for the first time, we demonstrate that the field is of a size greater than one terminal duct-lobular unit. The genetic changes are not identical, however, which suggests that genetic instability in these regions may play an early role in tumour development. We also confirm and extend our original observation of loss of the wild-type BRCA1 allele in some clones, and loss of the mutant allele in others, demonstrating that loss of either allele is a stochastic event.

Decreased Expression Of Stem Cell Markers By Simvastatin In 7,12-dimethylbenz(a)anthracene (dmba)-induced Breast Cancer.

Simvastatin, a competitive inhibitor of HMG-CoA reductase widely used in the treatment and prevention of hyperlipidemia-related diseases, has recently been associated to in vitro anticancer stem cell (CSC) actions. However, these effects have not been confirmed in vivo. To assess in vivo anti-CSC effects of simvastatin, female Sprague-Dawley rats with 7,12-dimethyl-benz(a)anthracene (DMBA)-induced mammary cancer and control animals were treated for 14 days with either simvastatin (20 or 40 mg/kg/day) or soybean oil (N = 60). Tumors and normal breast tissues were removed for pathologic examination and immunodetection of CSC markers. At 40 mg/kg/day, simvastatin significantly reduced tumor growth and the expression of most CSC markers. The reduction in tumor growth (80%) could not be explained solely by the decrease in CSCs, since the latter accounted for less than 10% of the neoplasia (differentiated cancer cells were also affected). Stem cells in normal, nonneoplastic breast tissues were not affected by simvastatin. Simvastatin was also associated with a significant decrease in proliferative activity but no increase in cell death. In conclusion, this is the first study to confirm simvastatin anti-CSC actions in vivo, further demonstrating that this effect is specific for neoplastic cells, but not restricted to CSCs, and most likely due to inhibition of cell proliferation.

Analysis of breast cancer by small angle X-ray scattering (SAXS)

Small angle X-ray scattering (SAXS) images of normal breast tissue and benign and malignant breast tumour tissues, fixed in formalin, were measured at the momentum transfer range of 0.063 nm(-1) <= q (=4 pi sin(theta/2)/lambda) <= 2.720 nm(-1). Four intrinsic parameters were extracted from the scattering profiles (1D SAXS image reduced) and, from the combination of these parameters, another three parameters were also created. All parameters, intrinsic and derived, were subject to discriminant analysis, and it was verified that parameters such as the area of diffuse scatter at the momentum transfer range 0.50 <= q <= 0.56 nm(-1), the ratio between areas of fifth-order axial and third-order lateral peaks and third-order axial spacing provide the most significant information for diagnosis (p < 0.001). Thus, in this work it was verified that by combining these three parameters it was possible to classify human breast tissues as normal, benign lesion or malignant lesion with a sensitivity of 83% and a specificity of 100%.

Identification of Neoplasias of Breast Tissues Using a Powder Diffractometer

An investigation was carried out to study the potential use of the angular distribution of scattered photons by human breast samples for a rapid identification of neoplasias of breast tissues. This technique has possible applications as diagnostic aid for breast cancer. In this work, a commercial powder diffractometer was used to obtain the scattering profiles from breast tissues histopathologically classified as normal breast tissues, fibroadenomas (benign breast diseases) and carcinomas (malignant breast diseases), in the interval 0.02 angstrom(-1) < x < 0.62 angstrom(-1). The experimental methods and data corrections are discussed in detail, and they included background subtraction, polarization, self-attenuation and geometric effects. The validation of the experimental procedure was achieved through an analysis of water sample. The results showed that the scattering profile is a unique impression of each type of tissue, being correlated with their microscopic morphological features. Multivariate analysis was applied to these profiles in order to verify if the information carried by these scattering profiles allow the differentiation between normal, benign and malignant breast tissues. The statistical analysis results showed that a correct identification of 75% of the analyzed samples is accomplished. The values of sensibility and specificity of this method in correctly differentiating between normal and neoplastic samples were 95.6% and 82.3%, respectively, while the values for differentiation between benign and malignant neoplasias were 78.6% and 62.5%. These initial results indicate the feasible use of commercial powder diffractometer to provide a rapid diagnostic with a high sensitivity.

Expression of the estrogen receptor-associated immunophilins, cyclophilin 40 and FKBP52, in breast cancer

The estrogen receptor alpha (ER alpha) is implicated in the development of breast cancer. The immunophilins, cyclophilin 40 (CyP40) and FKBP52, are associated with ER alpha and other steroid receptors in mutually exclusive heterocomplexes and may differentially modulate receptor activity. Since previous studies have not assessed the levels of these immunophilins in breast cancer, we examined 10 breast cancer cell lines for mRNA and protein expression of CyP40 and FKBP52 and for amplification of the CyP40 gene. In addition, 26 breast carcinomas, including seven with matched normal breast tissue, were examined for mRNA expression of both immunophilins. CyP40 and FKBP52 were ubiquitously expressed in breast cancer cell lines, but there were significant differences in their pattern of expression. FKBP52 protein levels were generally an order of magnitude greater than those for CyP40. FKBP52 mRNA expression correlated strongly with protein expression and was significantly higher in ER alpha-positive compared with ER alpha-negative cell lines. However, CyP40 mRNA expression did not correlate with protein expression, nor did expression of this immunophilin correlate with ER alpha status. Relatively high expression of CyP40 in one cell line (BT-20) could be attributed to amplification of the CyP40 gene. Both immunophilins were also ubiquitously expressed in breast carcinomas, and we demonstrate for the first time that both CyP40 and FKBP52 mRNA are overexpressed in breast tumors compared to matched normal breast controls. The overexpression of CyP40 and FKBP52, coupled with relative differences in their expression in tumors, may have important functional implications for ER alpha and other steroid receptors in breast cancer.

Allelic loss of cyclophilin 40, an estrogen receptor-associated immunophilin, in breast carcinomas

Purpose: Cyclophilin 40 (CyP40) is an estrogen receptor-associated protein which appears to modify receptor function. The aim of this study was to determine the extent of allelic loss at the CyP40 locus in a panel of breast carcinomas using a newly characterized microsatellite marker located upstream of the CyP40 gene and then to correlate this with losses at chromosomal sites for cancer-associated genes. Methods: Allelic loss at CyP40 was determined from patients' matched tumor and normal breast tissue using Genescan 672 software analysis of fluorescently labeled, PAGE-separated PCR products incorporating the marker. For each patient, allelic loss at CyP40 was then assessed and compared with losses at markers for various cancer-associated genes. Results: Allelic loss was detected in 30% of breast carcinomas from patients heterozygous for the CyP40 marker. All carcinomas demonstrating allelic loss were grade II or III invasive ductal carcinomas and generally showed multiple losses at other sites near known cancer-associated genes. Conclusions: The polymorphic marker which we characterized was useful in determining allelic loss at the CyP40 locus in breast cancer patients and when applied in these studies in conjunction with various cancer-associated gene markers, suggests that deletions in the region of the CyP40 gene might be a late event in breast tumor progression.

Estradiol-regulated expression of the immunophilins cyclophilin 40 and FKBP52 in MCF-7 breast cancer cells

The immunophilins, cyclophilin 40 (CyP40) and FKBP52, are associated with the unactivated estrogen receptor in mutually exclusive heterocomplexes and may differentially modulate receptor activity, We have recently shown that CyP40 and FKBP52 mRNA's are differentially elevated in breast carcinomas compared with normal breast tissue. Other studies suggest that such alterations ill the ratio of immunophilins might potentially influence steroid receptor function. Studies were therefore initiated to investigate the influence of estradiol on CyP40 and FKBP52 expression in MCF-7 breast cancer cells. Over a 24-h-treatment period with estradiol, CyP40 and FKBP52 mRNA expression was increased approximately five- and 14-fold, respectively. The corresponding protein levels were also elevated in comparison to controls. The antiestrogen, ICI 182,780, was an antagonist for CyP40 and FKBP52 mRNA induction. Cycloheximide treatment did not inhibit this increased immunophilin expression, suggesting that estradiol-mediated activation is independent off de novo protein synthesis. Treatment of MCF-7 cells with estradiol resulted in an increased half-life of both CyP40 and FKBP52 mRNA, as determined by actinomycin D studies. These results suggest that estradiol regulates CyP40 and FKBP52 mRNA expression through both transcriptional and posttranscriptional mechanisms. (C) 2001 Academic Press.

Reciprocal changes in gene expression profiles of cocultured breast epithelial cells and primary fibroblasts

The importance of epithelial-stroma interaction in normal breast development and tumor progression has been recognized. To identify genes that were regulated by these reciprocal interactions, we cocultured a nonmalignant (MCF10A) and a breast cancer derived (MDA-MB231) basal cell lines, with fibroblasts isolated from breast benign-disease adjacent tissues (NAF) or with carcinoma-associated fibroblasts (CAF), in a transwell system. Gene expression profiles of each coculture pair were compared with the correspondent monocultures, using a customized microarray. Contrariwise to large alterations in epithelial cells genomic profiles, fibroblasts were less affected. In MDA-MB231 highly represented genes downregulated by CAF derived factors coded for proteins important for the specificity of vectorial transport between ER and golgi, possibly affecting cell polarity whereas the response of MCF10A comprised an induction of genes coding for stress responsive proteins, representing a prosurvival effect. While NAF downregulated genes encoding proteins associated to glycolipid and fatty acid biosynthesis in MDA-MB231, potentially affecting membrane biogenesis, in MCF10A, genes critical for growth control and adhesion were altered. NAFs responded to coculture with MDA-MB231 by a decrease in the expression of genes induced by TGF beta 1 and associated to motility. However, there was little change in NAFs gene expression profile influenced by MCF10A. CAFs responded to the presence of both epithelial cells inducing genes implicated in cell proliferation. Our data indicate that interactions between breast fibroblasts and basal epithelial cells resulted in alterations in the genomic profiles of both cell types which may help to clarify some aspects of this heterotypic signaling. (C) 2009 UICC