WP1 – Molecular characterization and identification of clonal evolution
Rationale
Whole-genome, transcriptome and methylome profiling have revealed recurrent molecular alterations disrupting pathways involved in cellular growth, cell cycle, DNA repair and chromatin in brain tumours, many of which are attractive therapeutic targets.
Due to the high degree of intratumour heterogeneity, it is important to reconstruct the clonal evolution of the tumour, to identify therapeutic targets and mechanisms of resistance.
Objectives
- Deep molecular and neuropathology characterization
- Tumour phylogeny analysis
- Identification of somatic alterations associated with drug-sensitivity
Approach
Genome-wide profiling of cancer related alterations. Nucleic acids will be extracted from germline and tumour material and subjected to high-throughput sequencing at our sequencing facility at the Rigshospitalet (CLIA-certified Illumina NovaSeqs), ensuring cost-effective and rapid whole genome sequencing (WGS) (min. 40x coverage) of tumour and germline DNA (Figure 3).
We will perform sequencing analysis involving genomic alignment followed by single nucleotide variants (SNVs), short insertions and deletions (InDels) and larger structural variants (SVs) discovery using state-of-the-art pipelines developed in the context of the PanCancer Analysis of Whole Genomes (PCAWG), which are implemented in the Weischenfeldt group. Infinium EPIC array will be performed to identify differential methylation sites.
RNA will be subjected to strand-specific paired-end sequencing, aiming at minimum 50 million reads, to detect differential expression patterns associated with GBM subtypes and with e.g. stemness, as a marker for GSCs.
Tumour phylogeny reconstruction. We anticipate to obtain paired tumour material from primary and at least one relapse from at least 20 patients per year. Using sequencing data from these patients, we will reconstruct the tumour phylogeny using principles from evolution and phylogeny, by comparing the molecular alterations in the primary tumour to the matched sample at relapse, to identify clonal changes that have persisted treatment to cause relapse.
The method has successfully been applied in several projects including GBM patients [13–17]. Identification of somatic alterations associated with drug-sensitivity. We will use our existing database of FDA-approved oncology agents and FDA-approved repositioned drugs to identify potential drug targets from the catalogue of somatic alterations, including the N2M2 MASTER trial analysis protocol.
Endpoints
Inventory of genomic alterations, differential expression and methylation and drug-target candidates for every patient. Clonal somatic alterations associated with treatment resistance and relapse.
Expected impact
Identification of existing and novel mutational mechanisms in GBM, which will be utilized in the CFA2 and CFA3.