Weren Robbert D A, Mensenkamp Arjen R, Simons Michiel, Eijkelenboom Astrid, Sie Aisha S, Ouchene Hicham, van Asseldonk Monique, Gomez-Garcia Encarna B, Blok Marinus J, de Hullu Joanne A, Nelen Marcel R, Hoischen Alexander, Bulten Johan, Tops Bastiaan B J, Hoogerbrugge Nicoline, Ligtenberg Marjolijn J L
Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands.
Department of Pathology, Radboud University Medical Center, Nijmegen, The Netherlands.
Hum Mutat. 2017 Feb;38(2):226-235. doi: 10.1002/humu.23137. Epub 2016 Nov 9.
With the recent introduction of Poly(ADP-ribose) polymerase inhibitors, a promising novel therapy has become available for ovarian carcinoma (OC) patients with inactivating BRCA1 or BRCA2 mutations in their tumor. To select patients who may benefit from these treatments, assessment of the mutation status of BRCA1 and BRCA2 in the tumor is required. For reliable evaluation of germline and somatic mutations in these genes in DNA derived from formalin-fixed, paraffin-embedded (FFPE) tissue, we have developed a single-molecule molecular inversion probe (smMIP)-based targeted next-generation sequencing (NGS) approach. Our smMIP-based NGS approach provides analysis of both strands of the open reading frame of BRCA1 and BRCA2, enabling the discrimination between real variants and formalin-induced artefacts. The single molecule tag enables compilation of unique reads leading to a high analytical sensitivity and enabling assessment of the reliability of mutation-negative results. Multiplex ligation-dependent probe amplification (MLPA) and Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) were used to detect exon deletions of BRCA1 and methylation of the BRCA1 promoter, respectively. Here, we show that this combined approach allows the rapid and reliable detection of both germline and somatic aberrations affecting BRCA1 and BRCA2 in DNA derived from FFPE OCs, enabling improved hereditary cancer risk assessment and clinical treatment of ovarian cancer patients.
随着聚（ADP - 核糖）聚合酶抑制剂的近期问世，一种有前景的新型疗法已可用于肿瘤中携带BRCA1或BRCA2失活突变的卵巢癌（OC）患者。为了筛选可能从这些治疗中获益的患者，需要评估肿瘤中BRCA1和BRCA2的突变状态。为了可靠地评估来自福尔马林固定、石蜡包埋（FFPE）组织的DNA中这些基因的种系和体细胞突变，我们开发了一种基于单分子分子倒置探针（smMIP）的靶向新一代测序（NGS）方法。我们基于smMIP的NGS方法可对BRCA1和BRCA2开放阅读框的两条链进行分析，从而能够区分真实变异和福尔马林诱导的假象。单分子标签能够汇编独特的读数，从而实现高分析灵敏度，并能够评估突变阴性结果的可靠性。多重连接依赖性探针扩增（MLPA）和甲基化特异性多重连接依赖性探针扩增（MS - MLPA）分别用于检测BRCA1的外显子缺失和BRCA1启动子的甲基化。在此，我们表明这种联合方法能够快速、可靠地检测来自FFPE OC的DNA中影响BRCA1和BRCA2的种系和体细胞畸变，从而改善卵巢癌患者的遗传性癌症风险评估和临床治疗。
