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Study on methylation as risk and prognostic factor for breast cancer

Final Report Summary - METHYLBRECA (Study on methylation as risk and prognostic factor for breast cancer.)

Project number: 622853
Project title: MethylBRECA: Study on methylation as risk and prognostic factor for breast cancer.
Call identifier: FP7-PEOPLE-2013-IIF
Funding scheme: Marie Curie Actions— International Incoming Fellowships (IIF)
Researcher: Dr Laura Baglietto
Scientist in charge: Dr Françoise Clavel-Chapelon
Period: from 1/11/2014 to 31/10/2016

The main aim of the MethylBRECA project was to study the role of DNA methylation as diagnostic and prognostic factor of breast cancer. The project was conducted within the team Generation and Health, CESP, U1018, Inserm, France.

DNA methylation refers to the covalent addition of a methyl group to the 5-carbon of the cytosine ring. Methylation occurs in cytosine followed by a guanine (CpG dinucleotyde) and especially in CpGs Islands, that is DNA regions rich in CpGs dinucleotydes. Methylation of CpGs located in the promoter of genes is involved in the regulation of gene expression. More than 90% of all genomic methylated cytosine are not involved in gene expression, but lie on CpG dinucleotides located in transposable repetitive elements called transposons, including the long interspersed elements (LINE-1) and the short interspersed elements (SINE). Global hypomethylation, as well as hypomethylation of transposable repetitive elements, have been associated with reduced chromosomal stability and altered genome function (1).The epigenetic state of a genomic region may undergo alterations as a consequence of environmental stimuli. Such alterations may eventually increase the risk of certain diseases, like cancer including breast cancer (2-4).

During the two years of the fellowship, we have built the foundations to conduct a nested breast cancer case-control study within the French E3N prospective cohort, aimed at studying:
(1) the role of DNA methylation in pre-diagnostic blood in relation to breast cancer risk;
(2) the role of DNA methylation in tumour tissue of the cases in relation to prognosis.

In the past two years, Dr Baglietto has conceived and submitted requests for funding to obtain the necessary resources to conduct the project; only two of these requests were approved recently. The French Fondation ARC pour la Recherche Contre le Cancer funded the four-year project Xenair that started in June 2016 with the objective of investigating the effect of air pollution on breast cancer risk; within Xenair we will quantify methylation in DNA extracted from pre-diagnostic blood from 200 cases and 200 controls. In 2016, the French Ligue Contre le Cancer funded a three-year project (2017-2019) to identify breast cancer-related epigenetic and mutational signatures within which we will quantify methylation in the DNA extracted from the tumour tissue of 100 cases.

At the time the MethylBRECA project started, the most cost-effective array to quantify genome-wide DNA methylation was the Infinium HumanMethylation450 Beadchip by Illumina (HM450K). The application of the HM450K to DNA extracted from formalin-fixed paraffin-embedded (FFPE) tumour material has been more challenging than application to high quality DNA extracted from blood, because DNA extracted form FFPE samples is often of low quality and highly degraded. A protocol was developed for the DNA methylation analysis with the HM450K array of DNA extracted from FFPE tumour enriched samples (5). The protocol, applied to breast cancer FFPE samples from the Melbourne Collaborative Cohort Study (MCCS), led to the identification of a differential methylation profile between tumour samples and the adjacent normal breast tissues (6). Recently, the HM450K array has been replaced by the Infinium MethylationEPIC array, suitable also for FFPE samples, according to the manufacturer. In France, in the nineties when many of the E3N cases had been diagnosed, Bouin fixation of the tumour samples was common practice; only a minority of the samples were FFPE. We conducted a pilot study to test whether the quality of the DNA extracted from Bouin samples was suitable for the methylation array, but even DNA extraction by sonication did not guarantee the adequate DNA quality (pilot study conducted in collaboration with the pathology laboratory of Centre Leon Bérard in Lyon, France and the Genetic and Molecular Epidemiology Unit of HuGeF Foundation in Turin, Italy). In the Centre de Resources Biologiques (CRB) of Gustave Roussy in Villejuif, we have identified fresh frozen breast tumours for 130 cases diagnosed during the follow-up of the E3N cohort; these cases will represent the core sample of our breast tumour study. For a subset of samples with FFPE material available, we will process also DNA from FFPE tissue samples and compare the results with those obtained from DNA from frozen specimens.

During the fellowship, Dr Baglietto extended the concepts and ideas developed for breast cancer and applied them to other cancer types, in particular lung cancer. In a study conducted within the NOWAC cohort and replicated in another three cohorts, Dr Baglietto and colleagues identified two loci in the AHRR and F2RL3 genes whose methylation levels are strongly associated with lung cancer risk and appear to partially mediate the effect of smoking on risk (7). In a second study using the MCCS and EPIC-Italy cohorts and replicated in another three cohorts, Dr Baglietto and colleagues found that methylation levels at the two loci in AHRR and F2RL3 and methylation at another four different genomic locations were inversely associated with lung cancer risk (8). Methylation of five of the identified CpGs was lowest in current smokers and increased in former smokers with time since quitting, supporting the growing evidence that smoking may lead to DNA methylation changes measurable in peripheral blood and useful as predictive markers for lung cancer risk, especially in former smokers.

1. Esteller M. Epigenetics in cancer. N Engl J Med. 2008;358(11):1148-1159. doi:10.1056/NEJMra072067.
2. Jovanovic J, Rønneberg JA, Tost J, Kristensen V. The epigenetics of breast cancer. Mol Oncol. 2010;4(3):242-254. doi:10.1016/j.molonc.2010.04.002.
3. Severi G, Southey MC, English DR, et al. Epigenome-wide methylation in DNA from peripheral blood as a marker of risk for breast cancer. Breast Cancer Res Treat. 2014;148(3):665-673. doi:10.1007/s10549-014-3209-y.
4. van Veldhoven K, Polidoro S, Baglietto L, et al. Epigenome-wide association study reveals decreased average methylation levels years before breast cancer diagnosis. Clin Epigenetics. 2015;7(1):67. doi:10.1186/s13148-015-0104-2.
5. Wong EM, Joo JE, McLean CA, et al. Tools for translational epigenetic studies involving formalin-fixed paraffin-embedded human tissue: applying the Infinium HumanMethyation450 Beadchip assay to large population-based studies. BMC Res Notes. 2015;8(1):543. doi:10.1186/s13104-015-1487-z.
6. Wong EM, Joo JE, McLean CA, et al. Analysis of the breast cancer methylome using formalin-fixed paraffin-embedded tumour. Breast Cancer Res Treat. 2016;160(1):173-180. doi:10.1007/s10549-016-3971-0.
7. Fasanelli F, Baglietto L, Ponzi E, et al. Hypomethylation of smoking-related genes is associated with future lung cancer in four prospective cohorts. Nat Commun. 2015;6:10192. doi:10.1038/ncomms10192.
8. Baglietto L, Ponzi E, Haycock P, et al. DNA methylation changes measured in pre-diagnostic peripheral blood samples are associated with smoking and lung cancer risk. Int J Cancer. 2017;140(1):50-61. doi:10.1002/ijc.30431.