Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Ductal Carcinoma Progression

ABSTRACT: To obtain insight into the molecular basis of ductal carcinoma in situ (DCIS) and its progression by infiltrating surrounding tissue, we have performed cellular-based gene expression analysis of pure DCIS and DCIS with co-existing invasive ductal carcinoma (IDC) and compared the histological and molecular aspects between these morphologically identical lesions seeking to find key genes involved in DCIS progression. For that, 30 samples were evaluated, 4 non-neoplastic (N), 5 pure DCIS, 11 DCIS with co-existing IDC (DCIS-IDC) and 10 IDC. All samples were laser capture microdissected and RNAs were amplified using T7-based methodology. Microarray technology was performed using a customized cDNA platform containing 4,608 human genes. To classify the 4 sample groups according to molecular similarity we performed comparisons of their general expression pattern using ANOVA test (pFDR<0,01) followed by Tukey´s test (fold>l2l). Among the 4 sample groups, as expected, non-neoplastic cells reported the most distinct gene expression pattern. However, among the 3 groups of neoplastic cells, in contrast to morphological aspects, pure DCIS had the most distinct expression profile when compared to the other lesions: DCIS-IDC and IDC. Additionally, by comparison among pure DCIS, DCIS-IDC and N, we identified 147 genes potentially involved in DCIS progression. Unsupervised hierarchical cluster based on expression profile of this gene-set could discriminate DCIS-IDC from 60% of pure DCIS samples. Keywords: disease state analysis Fresh-frozen human breast samples were retrieved from the Tumor Tissue Biobank of the Medical and Research Center - Hospital A. C. Camargo, São Paulo. This research was approved by Ethic Committee of the Medical and Research Center - Hospital A. C. Camargo under number 587/04 and a written informed consent signed by all participants. All patients had presented at least 5 years of follow-up. Thirty samples were evaluated, 4 non-neoplastic breast samples (MN), 5 pure DCIS (stage 0; DCIS-0), 11 DCIS with co-existing IDC (DCIS) and 10 IDC. The non-neoplastic samples were obtained from perilesional mammary specimens from patients submitted to resection of benign lesions. All cells types were laser capture microdissected using PixCell II LCM system (Arcturus Engineering, Mountain View, CA). The total RNA was extracted by using the PicoPureä RNA Isolation kit (Arcturus Engineering # KT0204). A two-round linear amplification procedure based on T7-driven amplification was carried out. Amplified RNA was then used in a transcriptase reverse reaction into cDNA in the presence of Cy3- or Cy5-labeled dCTP. HB4a normal luminal epithelial mammary cell line (O’Hare et al 1991) was amplified following the same protocol and used as reference for microarray hybridizations. Dye swap was performed for each sample analyzed and used as replicate samples. For the raw_data (lowess_data), see Web Link below.

ORGANISM(S): Homo sapiens

SUBMITTER: Nádia Castro

PROVIDER: E-GEOD-11042 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Evidence that molecular changes in cells occur before morphological alterations during the progression of breast ductal carcinoma.

Castro Nadia P NP Osório Cynthia A B T CA Torres César C Bastos Elen P EP Mourão-Neto Mário M Soares Fernando A FA Soares Fernando A FA Brentani Helena P HP Carraro Dirce M DM

Breast cancer research : BCR 20081017 5

<h4>Introduction</h4>Ductal carcinoma in situ (DCIS) of the breast includes a heterogeneous group of preinvasive tumors with uncertain evolution. Definition of the molecular factors necessary for progression to invasive disease is crucial to determining which lesions are likely to become invasive. To obtain insight into the molecular basis of DCIS, we compared the gene expression pattern of cells from the following samples: non-neoplastic, pure DCIS, in situ component of lesions with co-existing ...[more]

PMID: 18928525

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Project description: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. Breast cancer samples, 31 pure DCIS patients, 36 IDC patients, 42 mixed and 6 normal.

Project description:Cutaneous melanomas are malignant radio and chemoresistant neoplasias presenting high morbidity and elevated mortality rates. Isolated Limb Perfusion (ILP) with Melphalan (Mel) is used in the treatment of non-resectable locally advanced melanomas of extremity sparing the limb from amputation in 40-50% of the cases. Adding the Tumor Necrosis Factor alpha (Tnfa) improves total complete response to 70-90%. The cellular and molecular mechanisms underlying the changes promoted by Mel and Tnfa are not completely understood. In this study we evaluated the impact of systemic Mel and Tnfa administration on tumor growth, analyzed the morphological changes promoted by each treatment, and searched for early molecular targets of Mel and Tnfa, alone or in combination, in a murine melanoma model. Morphological changes were analyzed by histological analysis, while microarray gene expression followed by quantitative RT-PCR were used to search for early molecular targets. We found that Mel systemic administration accounted for the impairment of tumor-growth (p<0.001) and improvement of survival. Tnfa co-administration augmented necrosis (p<0.024) and decreased mitotic rates (p=0.001). We identified a set of 118 potential molecular markers that might be correlated with the observed biologic response to treatment with Melphalan and Tnfa and which could represent potential therapeutic targets in melanoma. We used amplified RNA (aRNA) from 18 tumor samples (Control – 5 samples; Mel – 4 samples; Tnfa – 4 samples; and Mel+Tnfa – 5 samples). For replica hybridization with dye swap, aRNA from the tumors and from a pool composed of equal amounts of RNA extracted from the K1735M2, M2R, B16F10, and B16F1 melanoma cells were labeled with Alexa555 or Alexa647. Labeled aRNA samples were hybridized against a glass platform containing 16,128 immobilized sense oligonucleotides (Fox Chase Cancer Center, USA) corresponding to murine genes. Slides were pre-hybridized at 42ºC for approximately 6 hours in a solution containing Denhardt’s 5X (Ficoll 400 0.05g/mL, poli-vynil-pyrrolidone 0.05g/mL, bovine serum albumin 0.05g/mL), SSC (sodium saline citrate) 5X, 0.2% SDS (Sigma), and 1% BSA. For hybridization, we used a mix of 3.5µg of each labeled sample aRNA and reference aRNA. The hybridization solution contained Denhardt’s 5X, formamide 25% (Sigma), SSC 5X, SDS 0.1%, salmon sperm DNA 0.1mg/mL (GE Healthcare), Poly-A 0.1mg/mL (GE Healthcare), and Cot-1 0.1mg/mL (GE Healthcare), to a final volume of 100µL. Hybridizations were carried out on a Gene Tac hybridization station (Genomic Solutions) at 42ºC for about 16h. The slides were removed from the hybridization cassettes directly to a recipient containing a washing solution (SSC 2X, SDS 0.1%) at 42ºC, put in a slide rack, transferred to another recipient containing fresh solution, and washed under constant agitation at 42ºC for 5 min. After that, they were washed twice for 5min in a second wash solution (SSC 0.1X, SDS 0.1%) at room temperature and rinsed five times for 1min at room temperature with a SSC 0.1X solution. The slides were dried upside down in a centrifuge at 1500rpm for 5min. The slides were scanned with a confocal laser scanner (ScanArrayTM Express, Perkin-Elmer Life Sciences, USA), and the spot intensities processed with the help of the ScanArray Express program (Packard BioScience), with a 10µm resolution and a PMT of 60% for Alexa 555 and of 70% for Alexa 647. Each slide generated a data set for each channel corresponding to the dyes Alexa 555 and Alexa 647. The slides were scanned with a confocal laser scanner (ScanArrayTM Express, Perkin-Elmer Life Sciences, USA) with a 10µm resolution and a PMT of 60% for Alexa 555 and of 70% for Alexa 647, and data were extracted with ScanArray Express software (Packard BioScience) using the histogram method. Each slide generated a data set for each channel corresponding to the dyes Alexa 555 and Alexa 647. The Locally Weighted Scatter-plot Smoothing method (LOWESS), adjusted for linear and non-linear systematic variations, both of the intensity dependent type, mainly for low intensity spots, was employed for data normalization.

Dataset Information

Ductal Carcinoma Progression

Publications

Evidence that molecular changes in cells occur before morphological alterations during the progression of breast ductal carcinoma.

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