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

HYPOTHESISA保守的影响HR的体细胞基因改变的亚集导致突变信号3在早期间皮瘤进化中的丰富，并提供了PARP抑制剂诱导的合成癫痫的经验检测来自MEDUSA50队列的Sig3+和HRD相关体细胞改变单核苷酸变体(SNV)的遗传学分析(即。前50个多区域测序的患者)，将被称为克隆或亚克隆，使用我们的变异调用系统发育分析软件流水线。将对分割的SNV调用进行突变特征分析，以确定克隆性与亚克隆性sig3浓缩[6，8，9]。使用Sig3+或Sig3-作为二进制分类器，将结合SNV和体细胞拷贝数改变(SCNAs)对HR调节基因组畸变进行聚类。在克隆性sig3+患者中，将探索种系杂合子HRD相关突变，并与克隆性二次命中(SNV或SCNA)事件相关。利用左轮手枪，我们将探索sig3与重复进化簇的关联，以进一步定义sig3的突变背景。Sig3+间皮瘤的预后意义将从患者进展时间和手术后总存活率方面进行探讨。我们将使用我们的合作者Imagen开发的方法，从切除的初级间皮瘤中前瞻性地建立原代细胞系。一组14个Medusa细胞株(除了已经产生的6个)将使用癌症基因小组(Novogene)和使用Sigma算法定义的sig3浓缩状态(二元、存在或不存在)进行测序。这些细胞系将接受Imagen高通量药物面板测试平台，其中包含PARP抑制剂，以建立定量敏感性指标。这将与Sig3+状态、DNA修复指数(伽马H_2AX、彗星和克隆形成试验)相关。使用弹性网络方法，将通过关键药物-基因相互作用的功能遗传(RNAi)验证来定量探索HRD基因与药物敏感性的相关性。

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.