Deciphering Clonal Somatic Alterations associated with Homologous Recombination Deficiency and Synthetic Lethality in Mesothelioma

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HYPOTHESISA conserved subset of somatic gene alterations affecting HR lead to enrichment of mutational signature 3 in early mesothelioma evolution and confer PARP inhibitor induced synthetic lethalityEXPERIMENTAL DETAILSPhylogenetic analysis of Sig3+ and HRD-associated somatic alterations Single nucleotide variants (SNVs) from the MEDUSA50 cohort (ie. first 50 multi-regionally sequenced patients), will be called as clonal or subclonal using our variant calling phylogenetic analysis software pipeline. Mutation signature analysis of the partitioned SNV calls will be conducted to define clonal versus subclonal sig3 enrichment [6, 8, 9]. Using sig3+ or Sig3- as a binary classifier, clustering of HR regulating genomic aberrations will be conducted, incorporating both SNV and somatic copy number alterations (SCNAs). Germline heterozygous HRD associated mutations will be explored and correlated with clonal second hit (SNV or SCNA) events in clonal sig3+ patients. Using REVOLVER we will explore the association of sig3 with repeated evolutionary clusters to further define the mutational context of sig3. The prognostic significance of sig3+ mesothelioma will be explored with respect to patient's time to progress and overall survival following surgery.Sig3+ classification and Phenotyping. Primary cell lines will be established from resected primary mesotheliomas prospectively using methodology developed by our collaborator Imagen. A group of 14 MEDUSA cell lines (in addition to 6 already generated) will be sequenced using a cancer gene panel (novogene) and sig3 enrichment status (binary, present or absent) defined using the Sigma algorithm. These cell lines will be subjected to the Imagen high throughput drug panel testing platform, incorporating PARP inhibitors, to establish quantitative sensitivity indices. These will be correlated with Sig3+ status, DNA repair indices (gamma H2AX, COMET, and clonogenic assays). Using elastic net methodology, HRD gene correlations with drug sensitivity will be explored quantitatively with functional genetic (RNAi) validation of key drug-gene interactions.