Few studies have performed expression profiling of both miRNA and mRNA from the same primary breast carcinomas. In this study we present and analyze data derived from expression profiling of 799 ...miRNAs in 101 primary human breast tumors, along with genome-wide mRNA profiles and extensive clinical information.
We investigate the relationship between these molecular components, in terms of their correlation with each other and with clinical characteristics. We use a systems biology approach to examine the correlative relationship between miRNA and mRNAs using statistical enrichment methods.
We identify statistical significant differential expression of miRNAs between molecular intrinsic subtypes, and between samples with different levels of proliferation. Specifically, we point to miRNAs significantly associated with TP53 and ER status. We also show that several cellular processes, such as proliferation, cell adhesion and immune response, are strongly associated with certain miRNAs. We validate the role of miRNAs in regulating proliferation using high-throughput lysate-microarrays on cell lines and point to potential drivers of this process.
This study provides a comprehensive dataset as well as methods and system-level results that jointly form a basis for further work on understanding the role of miRNA in primary breast cancer.
Characteristic patterns of gene expression measured by DNA microarrays have been used to classify tumors into clinically relevant sub-groups. In this study, we have refined the previously defined ...sub-types of breast tumors that could be distinguished by their distinct patterns of gene expression. A total of 115 malignant breast tumors were analyzed by hierarchical clustering based on patterns of expression of 534 "intrinsic" genes and shown to subdivide into one basal-like, one ERBB2-overexpressing, two luminal-like, and one normal breast tissue-like subgroup. The genes used for classification were selected based on their similar expression levels between pairs of consecutive samples taken from the same tumor separated by 15 weeks of neoadjuvant treatment. Similar cluster analyses of two published, independent data sets representing different patient cohorts from different laboratories, uncovered some of the same breast cancer subtypes. In the one data set that included information on time to development of distant metastasis, subtypes were associated with significant differences in this clinical feature. By including a group of tumors from BRCA1 carriers in the analysis, we found that this genotype predisposes to the basal tumor subtype. Our results strongly support the idea that many of these breast tumor subtypes represent biologically distinct disease entities.
Gene expression profiling has been used to define molecular phenotypes of complex diseases such as breast cancer. The luminal A and basal-like subtypes have been repeatedly identified and validated ...as the two main subtypes out of a total of five molecular subtypes of breast cancer. These two are associated with distinctly different gene expression patterns and more importantly, a significant difference in clinical outcome. To further validate and more thoroughly characterize these two subtypes at the molecular level in tumors at an early stage, we report a gene expression profiling study using three different DNA microarray platforms.
Expression data from 20 tumor biopsies of early stage breast carcinomas were generated on three different DNA microarray platforms; Applied Biosystems Human Genome Survey Microarrays, Stanford cDNA Microarrays and Agilent's Whole Human Genome Oligo Microarrays, and the resulting gene expression patterns were analyzed. Both unsupervised and supervised analyses identified the different clinically relevant subtypes of breast tumours, and the results were consistent across all three platforms. Gene classification and biological pathway analyses of the genes differentially expressed between the two main subtypes revealed different molecular mechanisms descriptive of the two expression-based subtypes: Signature genes of the luminal A subtype were over-represented by genes involved in fatty acid metabolism and steroid hormone-mediated signaling pathways, in particular estrogen receptor signaling, while signature genes of the basal-like subtype were over-represented by genes involved in cell proliferation and differentiation, p21-mediated pathway, and G1-S checkpoint of cell cycle-signaling pathways. A minimal set of 54 genes that best discriminated the two subtypes was identified using the combined data sets generated from the three different array platforms. These predictor genes were further verified by TaqMan Gene Expression assays.
We have identified and validated the two main previously defined clinically relevant subtypes, luminal A and basal-like, in a small set of early stage breast carcinomas. Signature genes characterizing these two subtypes revealed that distinct molecular mechanisms might have been pre-programmed at an early stage in different subtypes of the disease. Our results provide further evidence that these breast tumor subtypes represent biologically distinct disease entities and may require different therapeutic strategies. Finally, validated by multiple gene expression platforms, including quantitative PCR, the set of 54 predictor genes identified in this study may define potential prognostic molecular markers for breast cancer.
Molecular subtypes of serous ovarian cancer have been recently described. Using data from independent datasets including over 900 primary tumour samples, we show that deregulation of the Let-7 ...pathway is specifically associated with the C5 molecular subtype of serous ovarian cancer. DNA copy number and gene expression of HMGA2, alleles of Let-7, LIN28, LIN28B, MYC, MYCN, DICER1, and RNASEN were measured using microarray and quantitative reverse transcriptase PCR. Immunohistochemistry was performed on 127 samples using tissue microarrays and anti-HMGA2 antibodies. Fluorescence in situ hybridisation of bacterial artificial chromosomes hybridized to 239 ovarian tumours was used to measure translocation at the LIN28B locus. Short interfering RNA knockdown in ovarian cell lines was used to test the functionality of associations observed. Four molecular subtypes (C1, C2, C4, C5) of high-grade serous ovarian cancers were robustly represented in each dataset and showed similar pattern of patient survival. We found highly specific activation of a pathway involving MYCN, LIN28B, Let-7 and HMGA2 in the C5 molecular subtype defined by MYCN amplification and over-expression, over-expression of MYCN targets including the Let-7 repressor LIN28B, loss of Let-7 expression and HMGA2 amplification and over-expression. DICER1, a known Let-7 target, and RNASEN were over-expressed in C5 tumours. We saw no evidence of translocation at the LIN28B locus in C5 tumours. The reported interaction between LIN28B and Let-7 was recapitulated by siRNA knockdown in ovarian cancer cell lines. Our results associate deregulation of MYCN and downstream targets, including Let-7 and oncofetal genes, with serous ovarian cancer. We define for the first time how elements of an oncogenic pathway, involving multiple genes that contribute to stem cell renewal, is specifically altered in a molecular subtype of serous ovarian cancer. By defining the drivers of a molecular subtype of serous ovarian cancers we provide a novel strategy for targeted therapeutic intervention.
Combining gene expression microarrays and high resolution magic angle spinning magnetic resonance spectroscopy (HR MAS MRS) of the same tissue samples enables comparison of the transcriptional and ...metabolic profiles of breast cancer. The aim of this study was to explore the potential of combining these two different types of information.
Breast cancer tissue from 46 patients was analyzed by HR MAS MRS followed by gene expression microarrays. Two strategies were used to combine the gene expression and metabolic data; first using multivariate analyses to identify different groups based on gene expression and metabolic data; second correlating levels of specific metabolites to transcripts to suggest new hypotheses of connections between metabolite levels and the underlying biological processes. A parallel study was designed to address experimental issues of combining microarrays and HR MAS MRS.
In the first strategy, using the microarray data and previously reported molecular classification methods, the majority of samples were classified as luminal A. Three subgroups of luminal A tumors were identified based on hierarchical clustering of the HR MAS MR spectra. The samples in one of the subgroups, designated A2, showed significantly lower glucose and higher alanine levels than the other luminal A samples, suggesting a higher glycolytic activity in these tumors. This group was also enriched for genes annotated with Gene Ontology (GO) terms related to cell cycle and DNA repair. In the second strategy, the correlations between concentrations of myo-inositol, glycine, taurine, glycerophosphocholine, phosphocholine, choline and creatine and all transcripts in the filtered microarray data were investigated. GO-terms related to the extracellular matrix were enriched among the genes that correlated the most to myo-inositol and taurine, while cell cycle related GO-terms were enriched for the genes that correlated the most to choline. Additionally, a subset of transcripts was identified to have slightly altered expression after HR MAS MRS and was therefore removed from all other analyses.
Combining transcriptional and metabolic data from the same breast carcinoma sample is feasible and may contribute to a more refined subclassification of breast cancers as well as reveal relations between metabolic and transcriptional levels. See Commentary: http://www.biomedcentral.com/1741-7015/8/73.
Ductal carcinoma
in situ (DCIS) is a non-invasive form of breast cancer where cells restricted to the ducts exhibit an atypical phenotype. Some DCIS lesions are believed to rapidly transit to ...invasive ductal carcinomas (IDCs), while others remain unchanged. Existing classification systems for DCIS fail to identify those lesions that transit to IDC. We studied gene expression patterns of 31 pure DCIS, 36 pure invasive cancers and 42 cases of mixed diagnosis (invasive cancer with an
in situ component) using Agilent Whole Human Genome Oligo Microarrays 44k. Six normal breast tissue samples were also included as controls. qRT-PCR was used for validation. All DCIS and invasive samples could be classified into the “intrinsic” molecular subtypes defined for invasive breast cancer. Hierarchical clustering establishes that samples group by intrinsic subtype, and not by diagnosis. We observed heterogeneity in the transcriptomes among DCIS of high histological grade and identified a distinct subgroup containing seven of the 31 DCIS samples with gene expression characteristics more similar to advanced tumours. A set of genes independent of grade, ER-status and HER2-status was identified by logistic regression that univariately classified a sample as belonging to this distinct DCIS subgroup. qRT-PCR of single markers clearly separated this DCIS subgroup from the other DCIS, and contains samples from several histopathological and intrinsic molecular subtypes. The genes that differentiate between these two types of DCIS suggest several processes related to the re-organisation of the microenvironment. This raises interesting possibilities for identification of DCIS lesions both with and without invasive characteristics, which potentially could be used in clinical assessment of a woman’s risk of progression, and lead to improved management that would avoid the current over- and under-treatment of patients.
Recent studies have found an association between certain TP53 mutations and resistance to anthracycline-based primary medical therapy in breast cancer. The purpose of this study was to investigate ...whether TP53 mutational status also might influence the response to a non-anthracycline-containing regimen in primary breast cancer.
Thirty-five patients with locally advanced breast cancer were investigated for TP53 mutations before receiving combination chemotherapy with 5-fluorouracil (1000 mg/m(2) on days 1 and 2) and mitomycin (6 mg/m(2) on day 2), administered every 3 weeks for 2-10 cycles in the neoadjuvant setting.
Mutations in the TP53 gene, in particular those affecting loop domains L2 or L3 of the p53 protein, were associated with lack of response to chemotherapy (i.e., increase in the diameter product of tumor lesion by >/=25%; P = 0.177 for all mutations and P = 0.006 for those affecting L2/L3 domains, respectively). No statistically significant correlation between TP53 LOH and response to therapy was seen.
This study revealed a significant association between lack of response to 5-fluorouracil and mitomycin and mutations affecting the L2/L3 domains of the p53 protein. Together with our previous finding that such mutations predict resistance to weekly doxorubicin, our data suggest that mutations affecting this particular domain of the p53 protein may cause resistance to several different cytotoxic compounds applied in breast cancer treatment.
•Copper open-cell foams were washcoated with a commercial Cu/ZnO/Al2O3 catalyst.•Foams and powders were tested in the methanol synthesis at relevant conditions.•Foams and powders were also tested in ...the RWGS reaction at atmospheric pressure.•In both the reactions foams showed lower activity than powdered catalyst.•RWGS is a simple probe reaction for ranking the activity of Cu/ZnO/Al2O3 catalysts.
We herein present the preparation of washcoated copper open-cell foam prototypes by dip-blowing a ball-milled aqueous slurry made of commercial powdered Cu/ZnO/Al2O3 methanol catalyst, deionized water and methylhydroxyethylcellulose. We then show the results of the chemical testing of such prototypes in the low temperature-low pressure methanol synthesis at relevant industrial conditions (i.e. T=505K, P=50 bara, H2/CO/CO2/CH4/N2 feed molar ratio=73.2/8.3/2.6/5.1/11.2). Three levels of temperature (485, 505 and 525K) were investigated, keeping constant all the other operating conditions. The performances were compared to those of the original powder.
We found that washcoated copper foams coated with a 75μm thick catalytic layer were active in the methanol synthesis, but showed lower COx conversion and MeOH productivity than the original powder. Slurried powders (i.e. slurry dried and calcined) showed the same chemical activity as the washcoated foams, therefore ruling out any effect related to the deposition step and singling out the slurry preparation and/or calcination procedure as responsible for a change in the catalytic performances.
We also tested the same catalytic systems in the reverse-water–gas-shift (RWGS) reaction at P=1 bara, T=505K and with H2/CO2=20. The campaign of low pressure tests confirmed the lower activity of slurried powders with respect to the original catalytic powder and leads us to the conclusion that the RWGS reaction can be considered as a representative probe reaction for ranking the intrinsic activity of Cu/ZnO/Al2O3 methanol catalysts without requiring testing under the high pressures typical of the commercial methanol synthesis process.
Storey and co-workers recently presented results indicating that the allele encoding arginine in the codon-72 polymorphism of the p53 gene represents a significant risk factor in the development of ...cancers associated with human papilloma virus (HPV). The form of the p53 protein carrying an arginine residue at this position was found to be significantly more susceptible to degradation by the HPV E6 protein than by the proline form. Genotype analysis of 30 cervical tumours and 12 skin carcinomas revealed that the homozygous Arg/Arg genotype was overrepresented compared with 41 controls. We have now analysed this polymorphism in leukocyte DNA from a larger sample of cancer patients and controls but have found no significant overrepresentation of this genotype.
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