Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Genome-wide DNA methylation profiles in progression to in situ and invasive carcinoma of the breast with impact on gene transcription and prognosis

ABSTRACT: Ductal carcinoma in situ (DCIS) of the breast is a precursor of invasive breast carcinoma (IBC). DNA methylation alterations are thought to be an early event in progression of cancer, and may prove valuable as a tool in clinical decision making and for understanding neoplastic development. Genome-wide DNA methylation profiles of 285 breast tissue samples representing progression of cancer were generated using Illumina HumanMethylation450. Validation of methylation changes between normal and DCIS was performed in an independent dataset of 15 normal and 40 DCIS samples, and validation of a prognostic signature was performed on 583 breast cancer samples from The Cancer Genome Atlas. Using two independent datasets of normal breast tissue and DCIS revealed that DNA methylation profiles of DCIS were radically altered compared to normal breast tissue, involving almost 7000 genes (including CUL7 and ICAM2). Changes between DCIS and IBC involved around 1000 genes. In tumors, DNA methylation was associated with gene expression of almost 3000 genes (p<0.05, Bonferroni corrected) including both negative and positive correlations. A prognostic signature based on methylation level of 18 CpGs (representing genes such as IRF6, TBX5, ZNF259, KCTD21, EPN3, MACF1 and CSNK1G2) was associated to survival of breast cancer patients with invasive tumors, as well as to survival of patients with DCIS and mixed lesions of DCIS and IBC. This work demonstrates that changes in the epigenome occurs early in the neoplastic progression, provide evidence for the possible utilization of DNA methylation based markers of progression in the clinic, and highlights the importance of epigenetic changes in carcinogenesis. Bisulphite converted DNA from the 285 samples were hybridised to the Illumina Infinium 450k Human Methylation Beadchip

ORGANISM(S): Homo sapiens

SUBMITTER: Thomas Fleischer

PROVIDER: E-GEOD-60185 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:DCIS is a non-invasive precursor lesion to invasive breast carcinoma. We still have no understanding on why only some DCIS lesions evolve to invasive cancer while others appear not to do so during the life span of the patient. Here, we performed full exome (tumor vs. matching normal), transcriptome and methylome analysis of 30 pure high-grade DCIS (HG-DCIS) and 10 normal breast epithelial samples. Sixty two percent of HG-DCIS cases displayed mutations affecting cancer driver genes or potential drivers. Mutations were observed affecting PIK3CA (21% of cases), TP53 (17%), GATA3 (7%), MLL3 (7%) and single cases of mutations affecting CDH1, MAP2K4, TBX3, NF1, ATM and ARID1A. Significantly, 83% of lesions displayed numerous large chromosomal copy number alterations, suggesting they might precede selection of cancer driver mutations. Integrated pathway-based modeling analysis of RNA-seq data allowed us to identify two DCIS subgroups (DCIS-C1 and DCIS-C2) based on their tumor intrinsic subtypes, proliferative, immune scores and in the activity of specific signaling pathways. The more aggressive DCIS-C1 (highly proliferative, basal-like or ERBB2+) displayed signatures characteristic of activated Treg cells (CD4+/CD25+/FOXP3+) and CTLA4+/CD86+ complexes indicative of a tumor-associated immune suppressive phenotype. Strikingly, all lesions showed evidence of TP53 pathway inactivation. Similarly ncRNA and methylation profiles reproduce changes observed post-invasion. Among the most significant findings we observed upregulation of lncRNA HOTAIR in DCIS-C1 lesions and hypermethylation of HOXA5 and specific SOX genes. We conclude that most HG-DCIS lesions, in spite of representing a pre-invasive stage of tumor progression, displayed molecular profiles indistinguishable from invasive breast cancer. DNA from 24 out of 30 (80%) HG-DCIS samples and 5 normal breast organoids (total 29 samples) were subjected to reduced representation bisulfite sequencing analysis (RRBS) by using Illumina HiSeq2000 platform. Please note that description of samples employed for the NGS analyses including age, race, ER/PR immunohistochemistry results, ITIL/STIL scores and PAM50 classification is provided the 'Supplementary Data1_Samples data.xlsx' (available on Superseries record)

Project description: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.

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Genome-wide DNA methylation profiles in progression to in situ and invasive carcinoma of the breast with impact on gene transcription and prognosis

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