HRD-IA signatures in pancreatic ductal adenocarcinoma

摘要缺乏同源重组的肿瘤显示高水平的拷贝数变异 (CNV)包括它们基因组中的扩增、缺失和断裂点。这种同源重组 基因缺陷(HRD)表型与多种癌症的致病BRCA突变有关 可以通过与药物的合成致命相互作用在临床上加以利用，例如聚ADP的抑制剂- 核糖聚合酶(PARP)和化疗方案，如FOLFIRINOX，可以提高DNA水平 肿瘤基因组的损伤。引人注目的是，BRCA1和BRCA2的野生型肿瘤亚群已经显示 对这些药物的临床反应相似。初步研究表明，反应性肿瘤已经升高 即使在BRCA基因或其他调节因子中没有变异的情况下，基因组畸变率也是如此 同源重组介导的DNA修复(如PALB2、ATM和BRIP1)。因此，其性质和范围 在肿瘤基因组中的这些损伤中，无论是哪种基因型，都为那些将 对DNA损伤和修复做出反应并进行靶向治疗。 据估计，2019年将有56,770名美国人被诊断出患有胰腺导管腺癌 (PDA)和45,750人将死于这种疾病，使PDA成为癌症死亡的第三大常见原因。 最近的临床试验在总体存活率方面取得了适度的改善。研究表明，类似于 在其他肿瘤中，增加的染色体畸变率定义了PDA子集中的HRD特征。 一个基本的假设是，这个基因组签名预测那些PDA患者可能对 PARP抑制剂和DNA损伤剂。然而，PDA活检很难定性，因为 复杂的基因组和异质细胞，因为癌细胞平均只占细胞的25% 在肿瘤内。此外，活组织检查经常包含多个肿瘤群体，这些肿瘤群体不能 通过基于形态的方法来区分。为了解决这些临床挑战，我们验证了DNA含量 基于流动分类的PDA组织。我们公布的方法产生高度纯化的(&gt；95%)适合于 从各种临床样本中进行全基因组分析。这些措施包括新鲜冷冻和固定福尔马林 低肿瘤细胞含量(10-20%)和高坏死量(90%)的石蜡包埋(FFPE)组织 和碎片。我们已经鉴定出PDA在其基因组中具有大量的间质畸变(IA) 与在HRD阳性的BRCAmut肿瘤中观察到的类似。我们的结果表明，IAS数量的增加 与PDA的临床疗效相关。在这项研究中，我们将建立分析和处理程序 然后，我们的HRD-IA检测建立了区分HRD+BRCAmut肿瘤的评分。然后我们将利用 用FOLFIRINOX处理样本以验证我们的HRD-IA方法在识别这些PDA上的应用 对DNA损伤靶向药物有反应的患者，BRCAmut和BRCAwt。这项工作的最后阶段将 验证我们的分选和基于CNV的HRD-IA方法在CLIA和临床应用中的应用。

ABSTRACT Tumors deficient in homologous recombination display high levels of copy number variants (CNVs) including amplifications, deletions, and breakpoints in their genomes. This homologous recombination deficiency (HRD) phenotype has been associated with pathogenic BRCA mutations in multiple cancers and can be exploited clinically through a synthetic lethal interaction with agents, such as inhibitors of poly ADP- ribose polymerase (PARP) and chemotherapy regimens such as FOLFIRINOX, that increase the level of DNA damage in tumor genomes. Strikingly, subsets of tumors that are wild type for BRCA1 and BRCA2 have shown similar clinical responses to these agents. Preliminary studies suggest that responder tumors have elevated numbers of genomic aberrations even in the absence of variants in the BRCA genes or in other mediators of homologous recombination mediated DNA repair (e.g. PALB2, ATM, and BRIP1). Thus the nature and extent of these lesions in tumor genomes, regardless of genotype, provides a biomarker for patients who will respond to DNA damage and repair targeting therapies. It is estimated that in 2019, 56,770 Americans will be diagnosed with pancreatic ductal adenocarcinoma (PDA) and 45,750 will die from the disease, making PDA the third most common cause of cancer death. Recent clinical trials have made modest improvements in overall survival. Studies suggest that similar to other tumors, elevated numbers of chromosomal aberrations define a HRD signature in a subset of PDAs. A fundamental hypothesis is that this genomic signature predicts those PDA patients likely to respond to PARP inhibitors and to DNA damaging agents. However PDA biopsies are difficult to characterize due to complex genomes and heterogeneous cellularity, as cancer cells represent on average only 25% of the cells within the tumor. Furthermore, biopsies frequently contain multiple neoplastic populations that cannot be distinguished by morphology based methods. To address these clinical challenges we validated DNA content based flow sorting of PDA tissues. Our published methods yield highly purified (>95%) samples suitable for whole genome analyses from a variety of clinical samples. These include both fresh frozen and formalin fixed paraffin embedded (FFPE) tissues with low tumor cell content (<10-20%) and high amounts (>90%) of necrosis and debris. We have identified PDAs with extensive numbers of interstitial aberrations (IAs) in their genomes similar to those observed in HRD-positive BRCAmut tumors. Our results suggest that elevated numbers of IAs correlate with clinical response in PDA. In this study we will establish analytical and processing procedures for our HRD-IA assay then establish a score that distinguishes HRD+ BRCAmut tumors. We will then exploit samples treated with FOLFIRINOX to validate the application of our HRD-IA assay to identify those PDA patients, BRCAmut and BRCAwt, who respond to DNA damage targeting agents. The final phase of this work will validate our sorting and CNV based HRD-IA assay for CLIA application and clinical use.