Tumor Type

France
Breast Cancer - Subtype defined by an amplification of the HER2 gene
Project Profile
Funding Organizations
Research Organizations
Research Activities
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Clinic & Pathology

France: Centre Antoine-Lacassagne
Centre Georges François Leclerc (CGFL)
Centre Hospitalier Universitaire de Besançon
Centre Hospitalier Universitaire de Saint-Étienne
Centre Jean Perrin
Centre Léon-Bérard
Centre Val d'Aurelle
Clinique Mutualiste
Institut Bergonié
Institut Curie
Institut de Cancérologie de Lorraine (ICL) - Alexis Vautrin
Institut Gustave Roussy
Institut Jean-Godinot
Institut Paoli-Calmettes

The recruitment effort will be pursued and reinforced in order to collect biological material from 75 Her2 amplified breast cancer patients. A total of 14 collaborative centers have signed a collaborative agreement and are committed to provide samples to this project. This effort will be substantial as the centers, in order to provide tumor samples, have already looked into their retrospective series. Thus the recruitment will, in the future, be entirely prospective.

Sequencing & Analysis

France: Fondation Synergie Lyon Cancer
United Kingdom: Wellcome Trust Sanger Institute

Whole genome sequencing:
The normal/tumor DNA pairs of 75 cancer patients will be sequenced using HiSeq2000 with at least 30X coverage for the normal DNA and 45X for the tumor DNA.

Exome sequencing:
All pairs will be sequenced using an exon pull-down strategy.

RNA and miRNA sequencing:
All tumor RNAs will be sequenced to a coverage enabling the detection of messenger RNAs that are present at an average of one copy per cell or more.

Methylome sequencing:
Ten tumors will be selected based on the analysis of their DNA and RNA sequence, to be whole genome sequenced after treatment with bisulfite.

Analysis:
Raw sequence data need thorough analyses in order to deliver reliable information of biomedical interest. The Synergie-Lyon Cancer bioinformatics facility will perform a two-stage analytical procedure of the raw data. The first stage aims at the construction a clean sample BAM file. The second stage uses multiple specialized analytical pipelines. The most important ones are specialized in the detection of somatic mutations, altered copy number of chromosomal regions, balanced translocations. Complementary pipelines have been developed for cryptic intra-species human contamination; contamination of tumor DNA by normal DNA; and the characterisation of exogenous contamination by looking at unmapped high quality sequences. The transcriptomic data will be analyzed in relation with the genomic and epigenomic data in order to reveal correlations suggesting mechanisms for the deregulation of genetic expression in cancer cells. All identified mutations and chromosome rearrangements will be listed and provided to the biomedical community on a dedicated web server. They will be provided to the ICGC consortium.

Complementary Studies

France: Institut Curie
Institut Génétique Biologie Moléculaire Cellulaire (IBGMC)
United Kingdom: Wellcome Trust Sanger Institute

SNP arrays for presequencing analysis:
A presequencing characterization of the DNA to be sequenced is perfomed using SNP arrays (Illumina or Affymetrix). In addition to producing a copy number profile of the tumor genome, this technique also provides an allelic-ratio profile. Joint analysis of the two profiles yields an evaluation of the DNA index of the main clonal component of the tumor cells. It may also detect the presence of a secondary subclone associated with specific chromosome copy number changes. Most importantly, it evaluates the level of contamination of the tumor fragment with non-neoplastic cells, an information which is taken into account in the priority ranking of samples for sequencing and is required to correctly interpret the final results/conclusions.

SNP arrays for methylation:
All Tumor DNAs will be characterized using the Illumina Infinium 450 allowing assessment of the methylation status of more than 480,000 cytosines distributed over the whole genome.

Data Storage, Analysis & Management

France: Fondation Synergie Lyon Cancer

Synergie-Lyon Cancer bioinformatics operates a medium size data center devoted to this project. This data center is hosted by the Centre Leon Berard Computer facility which provides infrastructure, hardware maintenance and network security. All informatics solutions developed by the facility have been implemented on this equipment.

Project Summary

A total of 44 normal/tumor pairs have already been recruited and are currently being sequenced at the Sanger Institute.

The recruitment effort will be pursued and reinforced in order to collect biological material from an additional 31 Her2 amplified breast cancer patients within the SIGNAL2 project. A total of 14 collaborative centers have signed a collaborative agreement and are committed to provide samples to this project. This effort will be substantial as the centers, in order to provide tumor samples, have already looked into their retrospective series. Thus the recruitment will, in the future, be entirely prospective.

DNAs and RNAs will be purified according to previously defined procedures used for the first 44 patients. All DNAs and RNAs that are candidate to be sequenced will be thoroughly quality controlled using both a set of standard methods (spectrophotometry, fluorimetry, electrophoresis), but also advanced genomic techniques (SNP arrays, transcriptome profiling) in order to select the best material for sequencing. A special attention will be paid to ascertain that the matched samples (normal DNA, tumor DNA, tumor RNA) originate from the same individual and are not contaminated by other human DNAs.

The technology used to achieve whole genome sequencing will be close or identical to that used for the first 44 cases in order to keep a consistent set of data. The sequence coverage of the normal DNA will be at least 30X; that of the tumor DNA will be adapted to the proportion of non-cancer cells present in the tumor fragment used to isolate the DNA (as evaluated on a cryostat section). It will be over 45X.

The tumor RNA will be sequenced to a coverage enabling the detection of messenger RNAs that are present at an average of one copy per cell or more. Ten tumor DNAs will be selected based on the analysis of their DNA and RNA sequence, to be whole genome sequenced after treatment with bisulfite. This method allows the genome-wide identification of methylated cytosine and therefore provides a comprehensive methylome characterization of the tumor DNA.

Deep exon sequencing of all 75 tumors and matched normal DNAs will be achieved to characterize tumor heterogeneity.

All raw sequence data (unmapped BAM files or similarly compressed data files) will be stored for at least 4 years at the Synergie-Lyon Cancer bioinformatics facility. The sequence data will be thoroughly quality controlled using analytical pipelines established at the facility. Dedicated pipelines for the identification of somatic mutations, altered copy number of chromosomal regions, balanced translocations will be developed and applied. The transcriptomic data will be analyzed in relation with the genomic and epigenomic data in order to reveal correlations suggesting new mechanisms for the (de)regulation of genetic expression in cancer cells.

All identified mutations and chromosome rearrangements will be listed and provided to the biomedical community on a dedicated web server. They will be provided to the ICGC consortium.

Principal Investigators

• Alain Viari

Lead Jurisdiction