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

假设影响HR的体细胞基因改变的保守子集导致早期间皮瘤演变中突变标记3的富集，并赋予PARP抑制剂诱导的合成致死性。前50个多区域测序的患者），将使用我们的变体调用系统发育分析软件管道被称为克隆或亚克隆。将对划分的SNV调用进行突变特征分析，以确定克隆与亚克隆sig 3富集[6，8，9]。使用Sig 3+或Sig 3-作为二元分类器，将进行HR调节基因组畸变的聚类，合并SNV和体细胞拷贝数改变（SCNA）。将探索生殖系杂合HRD相关突变，并将其与克隆sig 3+患者中的克隆二次命中（SNV或SCNA）事件相关。使用REVOLVER，我们将探索sig 3与重复进化簇的关联，以进一步定义sig 3的突变背景。sig 3+间皮瘤的预后意义将在患者的进展时间和手术后的总生存率方面进行探讨。Sig 3+分类和表型。原代细胞系将建立从切除的原发性间皮瘤前瞻性使用我们的合作者Imagen开发的方法。将使用癌症基因组（novogene）和sig 3富集状态（二元、存在或不存在）对一组14个MEDUSA细胞系（除已生成的6个细胞系外）进行测序，sig 3富集状态使用Sigma算法定义。这些细胞系将接受Imagen高通量药物组检测平台（包含PARP抑制剂），以建立定量敏感性指数。这些将与Sig 3+状态、DNA修复指数（gamma H2 AX、COMET和克隆形成测定）相关。使用弹性网络方法，HRD基因与药物敏感性的相关性将通过关键药物-基因相互作用的功能遗传学（RNAi）验证进行定量研究。

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.