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Epigenetic reprogramming of Fallopian tube fimbriae in BRCA mutation carriers defines early ovarian cancer evolution

ABSTRACT: The development of non-invasive primary cancer preventive measures in humans require a thorough understanding of the initial cancer-driving molecular mechanisms. High grade serous extra-uterine M llerian cancers (HGSEMC; formerly classified as ovarian/tubal/peritoneal cancer) present at a very late stage and less than 40% of women survive 5 years. Although the recent TCGA initiatives revealed key molecular changes in established cancers, very little is known about the initial molecular alterations in cancer development. Analysis of normal tissue at extensively high risk prior to the development of any microscopic alterations is critical. BRCA1/2 mutation carriers have an up to 30 40 fold increased risk to develop ovarian cancer, preferentially HGSEMC. Despite a plethora of evidence linking mutations in BRCA1 or BRCA2 to cancer development the core components such as organ specificity (i.e. to breast and Fallopian Tube) are still missing. The Fallopian Tube of BRCA mutation carriers offers a unique opportunity to study carcinogenesis because these cancers originate only from the distal (i.e. fimbrial) end of the Fallopian Tube (which is in close proximity to the ovary), and not from the proximal end (which is close to the uterus). The ovary which is in extreme close proximity to the fimbriae provides an excellent soil for cancer cells which are likely shed from the fimbriae and once the cancer has been discovered the big bulk of tumour is usually present on the ovary and hence the majority of HGSEMC are also referred to as ovarian cancer . To study the earliest steps of human carcinogenesis we performed epigenome-wide DNA methylation (DNAme) analyses (using the Illumina 450k DNA methylation bead-array assay assessing DNAme at ~480 000 CpG sites) in 215 microscopic normal Fallopian Tube samples of BRCA1/2 mutation carriers (n=56) and controls (n=59) who had their tubes/ovaries removed for prophylactic or other reasons, respectively (for 52 and 49 individuals respectively we analysed both fimbrial and proximal Fallopian Tubes). In order to adjust for any epigenetic effects which are not of immediate importance to the carcinogenic process, for each volunteer we analysed both the fimbrial (at risk) and the proximal (non at risk) portion of the tubes separately.

ORGANISM(S): Homo sapiens

PROVIDER: GSE74845 | GEO | 2016/05/19

SECONDARY ACCESSION(S): PRJNA301709

REPOSITORIES: GEO

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Project description:The development of non-invasive primary cancer preventive measures in humans require a thorough understanding of the initial cancer-driving molecular mechanisms. High grade serous extra-uterine M llerian cancers (HGSEMC; formerly classified as ovarian/tubal/peritoneal cancer) present at a very late stage and less than 40% of women survive 5 years. Although the recent TCGA initiatives revealed key molecular changes in established cancers, very little is known about the initial molecular alterations in cancer development. Analysis of normal tissue at extensively high risk prior to the development of any microscopic alterations is critical. BRCA1/2 mutation carriers have an up to 30 40 fold increased risk to develop ovarian cancer, preferentially HGSEMC. Despite a plethora of evidence linking mutations in BRCA1 or BRCA2 to cancer development the core components such as organ specificity (i.e. to breast and Fallopian Tube) are still missing. The Fallopian Tube of BRCA mutation carriers offers a unique opportunity to study carcinogenesis because these cancers originate only from the distal (i.e. fimbrial) end of the Fallopian Tube (which is in close proximity to the ovary), and not from the proximal end (which is close to the uterus). The ovary which is in extreme close proximity to the fimbriae provides an excellent soil for cancer cells which are likely shed from the fimbriae and once the cancer has been discovered the big bulk of tumour is usually present on the ovary and hence the majority of HGSEMC are also referred to as ovarian cancer . To study the earliest steps of human carcinogenesis we performed epigenome-wide DNA methylation (DNAme) analyses (using the Illumina 450k DNA methylation bead-array assay assessing DNAme at ~480 000 CpG sites) in 215 microscopic normal Fallopian Tube samples of BRCA1/2 mutation carriers (n=56) and controls (n=59) who had their tubes/ovaries removed for prophylactic or other reasons, respectively (for 52 and 49 individuals respectively we analysed both fimbrial and proximal Fallopian Tubes). In order to adjust for any epigenetic effects which are not of immediate importance to the carcinogenic process, for each volunteer we analysed both the fimbrial (at risk) and the proximal (non at risk) portion of the tubes separately. Formalin fixed paraffin embedded (FFPE) tissue blocks were retrieved from UCL Biobank (NC09.13). Histopathological features of fimbrial and proximal section of Fallopian Tube from BRCA carriers and control cases were carefully examined. Cases negative for serous tubal intraepithelial carcinoma (STIC) lesions were chosen for DNA isolation to characterize pre-cancer epigenetic changes. For DNA isolation, a core of 3 x 0.6 mm was taken from each block representing fimbrial and proximal end of fallopian tubes from both BRCA carriers and matched control cases. The DNA was isolated using QIAamp(r) DNA FFPE Tissue Kit as per manufacturer's protocol with minor modifications (Dewaxing for 4 hours in xylene and proteinase digestion performed overnight, other procedures were as per the instructions). DNA was quantified using Nandrop and restored using the Infinium FFPE DNA Restore Kit and then 200 nanogram of DNA was bisulfite converted using the EZ DNA Methylation-Gold(tm) and subjected to methylation analysis on the Illumina Infinium Human Methylation450 BeadChip.

Project description:Mutations in BRCA1 and BRCA2 genes confer an increased lifetime risk for breast and ovarian cancer. Ovarian cancer risk can be decreased by risk-reducing salpingo-oophorectomy (RRSO). Studies on RRSO material have altered the paradigm of serous ovarian cancer pathogenesis. The purpose of this study was to identify candidate genes possibly involved in pathogenesis of serous ovarian cancer by carrying out a microarray analysis of differentially expressed genes in BRCA1/2- mutation positive ovarian and fallopian tube epithelium derived from RRSO surgery. Freshly frozen ovarian and fallopian tube samples from nine BRCA1/2 mutation carriers scheduled for RRSO were prospectively collected in comparison with five mutation-negative control patients undergoing salpingo-oophorectomy for benign indications. Microarray analysis of genome-wide gene expression was performed on ovarian and fallopian tube samples from BRCA1/2 and control patients. The validation of microarray data was performed by quantitative real-time polymerase chain reaction (qRT-PCR) in selected cases of RRSO samples, and also high grade serous carcinoma samples collected from patients with BRCA phenotype. From 22,733 genes, 454 transcripts were identified that were differentially expressed in BRCA1/2 mutation carriers when statistically compared to controls pooling all ovarian and fallopian tube samples together. Of these, 299 genes were statistically significantly downregulated and 155 genes were upregulated. Differentially expressed genes in BRCA1/2 samples reported here might be involved in serous ovarian carcinogenesis and provide interesting targets for further studies. Both fallopian tube and ovarian samples were collected from each BRCA1/2 mutation carrier resulting in eighteen mutation positive adnexal samples. Both fallopian tube and ovarian control samples were collected from one control patient while either ovarian or fallopian tube sample was available from four control patients, respectively, resulting in 6 adnexal control samples. High quality RNA was available from nine BRCA1/2-mutation positive ovarian and eight BRCA1/2-mutation positive fallopian tube samples and from three control ovarian and three control fallopian tube samples.

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Epigenetic reprogramming of Fallopian tube fimbriae in BRCA mutation carriers defines early ovarian cancer evolution

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