Research article
Establishment of the epithelial-specific transcriptome of normal and malignant human breast cells based on MPSS and array expression data
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* Corresponding author: A Munro Neville munroneville@f2s.com
1 Ludwig Institute for Cancer Research/University College London Breast Cancer Laboratory, 91 Riding House Street, London, W1W 7BS, UK
2 The Breakthrough Breast Cancer Research Centre, Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB, UK
3 Office of Information Technology, Ludwig Institute for Cancer Research and Swiss Institute of Bioinformatics, 1015 Lausanne, Switzerland
4 Ludwig Institute for Cancer Research, New York Branch at Memorial Sloan-Kettering Cancer Centre, New York, NY 10021, USA
5 The J. Craig Venter Institute, 9704 Medical Center Drive, Rockville, MD 20850, USA
6 Department of Neurodegenerative Disease, Institute of Neurology, London, WC1N 3BG, UK
Breast Cancer Research 2006, 8:R56 doi:10.1186/bcr1604
Published: 2 October 2006Additional files
Additional file 1:
A jpeg figure showing cell separation of normal and malignant breast epithelial cells. Purity of separated normal and malignant cells. (a) A short-term primary culture of breast epithelium stained with monoclonal antibodies specific for vimentin (green), CK 14 (red), CK 18 (blue) and CK 19 (purple), as visualised with appropriate class and sub-class specific fluorescence conjugated secondary antibodies (×150). The middle and right columns show the double immunomagneticallly separated luminal and myoepithelial preparations stained in the same manner, illustrating their homogeneity in respect of cells expressing luminal (CK 18/CK 19) and myoepithelial markers (CK 14/vimentin). (b) The irregular clusters of cohesive malignant epithelial cells obtained when a disaggregated tumour is subject to filtration, sedimentation and negative selection for fibroblast activation protein-positive reactive stromal cells and visualised by phase-contrast microscopy to identify samples with minimal microvessel and lymphocytic contamination (×400).
Format: JPEG Size: 2.8MB Download file
Additional file 2:
A Word document showing the pathology of primary breast tumours used for MPSS and microarray analysis. The pathological information of 15 primary breast tumours regarding grade, type, size of vascular invasion, lymph node status, estrogen (ER), progesteron (PR) and Her-2 status is provided.
Format: DOC Size: 51KB Download file
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Additional file 3:
A Word document providing a detailed description of the tissue microarray. Summary of clinicopathological features of patients included in the tissue microarrays. A cohort of 245 invasive breast carcinomas from 245 patients treated with surgery (wide local excision or mastectomy) and adjuvant anthracycline-based chemotherapy was retrieved from the Department of Histopathology files of the Royal Marsden Hospital with appropriate local Ethical Committee approval (Royal Marsden Hospital, London, UK). Representative blocks from 245 invasive breast carcinomas were reviewed by a pathologist (JSRF) and included in duplicate in two tissue microarray (TMA) blocks as previously described. In brief, 0.6 mm core tissue specimens were taken from selected areas of donor blocks (original tumour blocks) and precisely arrayed into two new recipient paraffin blocks (20 × 35 mm) with a custom-built precision instrument (Beecher Instruments, Silver Spring, MD, USA). The presence of tumour tissue in the arrayed sample was verified on a haematoxylin and eosin stained section. ER, PR, p53, vascular invasion, Ki67 (MIB-1) labelling index and nodal status were known for all samples. Follow up was available for 244 patients, ranging from 0.5 to 135.3 months (median = 67.3 months, mean = 67.3 months).
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Additional file 4:
An Excel file listing raw and annotated MPSS data for malignant and normal breast epithelial samples. Sequence signatures with their corresponding annotation and their expression in tpm are shown. Transcripts uniquely expressed in the malignant breast epithelium and in the normal luminal epithelium are highlighted in yellow and red, respectively.
Format: XLS Size: 3.4MB Download file
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Additional file 5:
An Excel file containing a table showing an overlay of the multiple microarray platforms based on the HTR database. Microarray featuers of Affymetrix U133 Plus 2.0 GeneChip and CodeLink™ Human Whole Genome Bioarray, Agilent Whole Human Genome Oligo Microarray 44 k cDNA array and 20 k brk cDNA microarray were mapped onto the HTR database.
Format: XLS Size: 5.3MB Download file
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Additional file 6:
An Excel file containing a table showing the semi-quantitative RT-PCR of transcripts belonging to the three groups (MPSS-only, MPSS-array confirmed and Array-only). Transcripts with their respective annotation, RT-PCR primer sequence and level of expression detected by RT-PCR are shown.
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Additional file 7:
An Excel file containing a table showing the differential tumour epithelial transcriptome. All 8,051 differentially expressed normal luminal versus tumour genes are listed with their HTR cluster_ID, microarray_ID and their respective fold change for each microarray platform, comprising the differentially expressed epithelial tumour transcriptome.
Format: XLS Size: 2.2MB Download file
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Additional file 8:
An Excel file containing a table showing the biological processes deregulated in the DTET. Biological processes of the up- and down-regulated transcripts are shown. Gene Ontology identifiers, description, total number of input genes, as well as P value are shown. The input genes for the most significant deregulated biological processes are provided by their gene names and their RefSeq accession numbers.
Format: XLS Size: 223KB Download file
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Additional file 9:
An Excel file containing a table showing the differential normal epithelial transcriptome. Luminal and myoepithelial transcriptomes based on multiple microarray analyses HTR cluster, microarray feature, fold change and P value are listed for each gene.
Format: XLS Size: 625KB Download file
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Additional file 10:
A Word document detailing the univariate analysis of POSTN on the tumour tissue microarray. Univariate analysis of clinicopathological and immunohistochemical data on the 245 tumour tissue microarray with respect to the epithelial expression of POSTN. P values were calculated by the log-rank test.
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Additional file 11:
Cumulative Kaplan-Meier curves for epithelial expression of POSTN. Probability of (a) overall survival (OS) and (b) disease free survival (DFS) for all patients; and probability of (c) OS and (d) DFS for only patients with ER positive tumours. P values were calculated by the log-rank test.
Format: EPS Size: 1.8MB Download file