二代测序在乳腺癌基因突变检测与分子标志物及治疗靶点筛选中的应用
谢菲，北京大学人民医院乳腺中心 副主任医师。专业方向为乳腺癌的临床与基础研究，家族遗传性乳腺癌。
陈彦丽，北京大学人民医院检验科硕士研究生，研究方向为二代测序在乳腺癌易感基因检测中的应用及新发突变的功能研究。
【摘要】乳腺癌是目前全球最常见的恶性肿瘤，也是导致女性癌症死亡的主要原因。二代测序（next-generation sequencing，NGS）技术的应用有助于探究乳腺癌的发病机制，改善患者的预后。在基因组水平，应用NGS技术检测乳腺癌患者的胚系基因突变情况，可识别新的风险基因或致病突变位点，以指导患者的靶向治疗和预后评估。在转录水平，应用转录组测序技术可筛选乳腺癌潜在的分子标志物和治疗靶点，为乳腺癌的筛查、诊断、治疗和预后评估提供新思路。
乳腺癌是目前全球最常见的恶性肿瘤[1]，发病率约占女性癌症的30%，死亡发病比（mortality-to-incidence ratio，MIR）为15%[2]。根据国家癌症中心的报道，2015年中国乳腺癌新发病例为30.4万，发病率位居女性癌症的首位，死亡病例为7.0万，死亡率位居女性癌症的第五位[3]。乳腺癌易感性与遗传因素相关，大约10%~15%的乳腺癌是由遗传基因突变引起的[4]，其中参与DNA损伤修复的基因突变会影响基因组的稳定性，导致乳腺癌的发生[5]。目前已确定了多个乳腺癌易感基因，应用NGS技术对患者进行胚系基因突变检测有助于指导治疗方案的选择和预后评估。此外，随着转录组测序技术的发展，通过对乳腺癌患者进行转录水平的分析，可以更好地了解乳腺癌的异质性和复杂性，从而筛选出新的分子标志物和治疗靶点，为乳腺癌的精准治疗提供理论依据。
一、DNA损伤修复缺陷与乳腺癌
1. DNA双链断裂修复通路：DNA损伤可分为两大类：DNA双链断裂（double-strand breaks，DSBs）和DNA单链断裂（single-strand breaks，SSBs）。DSBs的发生是由于各种各样的基因组损伤，包括环境应激（如电离辐射）或与复制相关的内部诱导损伤[6]。DSBs的修复有两条途径：同源重组修复（homologous recombination repair，HR）和非同源末端连接（non-homologous end joining，NHEJ）。HR利用同源姐妹染色单体上相同的遗传信息作为临时模板以替代受损DNA的核苷酸[7]，这种修复机制依赖于细胞周期，即在S期和G2期发挥作用[8]。HR可实现对DSBs的精准修复，因此是维持基因组完整性和细胞活性的有力机制。NHEJ通过DNA连接酶直接将DSBs末端进行连接，不依赖于同源DNA序列，这种方式虽然迅速和高效，但是在修复过程中可能造成一些序列缺失或者片段插入[9]。
2. 同源重组修复缺陷与乳腺癌：多个基因参与了HR通路，其中BRCA1、BRCA2、PALB2和BARD1对维持HR通路和细胞周期检查点的正常功能至关重要，BRIP1和RAD51C等也参与了HR通路和细胞周期检查点，在DNA修复机制中起着辅助作用[10, 11]。HR通路基因的突变失活会导致同源重组修复缺陷（homologous recombination repair deficiency，HRD），迫使细胞采用NHEJ进行DSBs修复，修复过程中错误的积累增加了乳腺癌的发生风险[12]。
（1）BRCA1/2：BRCA1位于17号染色体上，由四个主要功能域组成[13]：N-端的RING（really interesting new gene）结构域、卷曲螺旋域（coiled-coil domain，CC）和两个BRCA1 C-末端（BRCA1 C-terminus，BRCT）结构域。在HR通路中，BRCA1是组成HR复合体的主干。BRCA1的RING结构域与BARD1结合，最终去除P53结合蛋白1（P53-Binding Protein-1，53BP1），使DNA修复通过HR途径进行。BRCA1的CC结构域与PALB2结合，协助HR复合体负载到DNA双链断裂部位。BRCA1还通过BRCT结构域与其他参与HR通路的蛋白相互作用[14]。大多数BRCA1的致病突变发生在RING结构域或BRCT结构域，使HR途径无法发挥作用[15]。
BRCA2位于13号染色体上，由一个包含8个重复基序（BRC repeats，BRCs）的主要功能域组成[16]。BRCA2通过BRCs与RAD51结合，负载RAD51到DNA损伤部位，催化HR途径的第一步[17]。
携带BRCA1致病突变的女性终生罹患乳腺癌的风险为72%，BRCA2相应的风险为69%[18]。男性乳腺癌总体上很少见，人群终生患病风险为0.1%，然而，携带BRCA1/2致病突变的男性终生罹患乳腺癌的风险明显增高，携带BRCA1的为1.2%，携带BRCA2的为6.8%[19]。大约50%的遗传性乳腺癌是由BRCA1/2胚系致病突变导致的，并多为早发性乳腺癌[18]。约15%~20%的三阴性乳腺癌（triple negative breast cancer，TNBC）患者与BRCA1/2致病突变有关，其中与BRCA1致病突变相关的乳腺癌中，有70%~85%是TNBC[20]。10%~15%的高危Luminal型乳腺癌患者携带BRCA1/2致病突变[21]。
（2）PALB2：PALB2位于16号染色体上，由四个主要功能域组成[22]：CC结构域、ETGE基序、染色质关联基序（chromatin association motif，ChAM）和WD40重复基序，其中WD40结构域包含一个核输出信号（nuclear export signal，NES）。当存在DNA损伤时，PALB2蛋白与DSBs位点的BRCA1结合，随后介导BRCA2和RAD51招募和负载到BRCA1复合体上[23]。当PALB2缺陷时，会导致BRCA1/BRCA2/RAD51复合体组装错误，尤其是影响BRCA1与BRCA2之间的相互作用[24, 25]。在无DNA损伤的情况下，在细胞核内约一半的PALB2与BRCA2结合，这表明PALB2介导BRCA2稳定的核内定位和积累，同时调控BRCA2的功能[26]。PALB2胚系致病突变的携带者，80岁时患乳腺癌的风险为53%[27]，家族性乳腺癌患者中PALB2的致病突变频率为0.6%~3.9%[21]。
（3）BARD1：BARD1位于2号染色体上，是一种泛素连接酶，由以下功能结构域组成：一个N-端的RING结构域、三个锚蛋白重复序列和两个C-端的BRCT结构域[28]。在DNA损伤或缺氧的情况下，通过依赖细胞周期的磷酸化介导BARD1的表达上调[29]。由于BARD1与BRCA1两端的结构域相似，两者结合形成异二聚体[30]，在HR的启动中发挥关键作用。BRCA1/BARD1通过发挥泛素连接酶作用，促进泛素的转移，从而导致53BP1的移除[31]，允许ATM识别DSBs部位并开始末端切除，随后BRCA1结合，HR通路被激活[32]。当BARD1发生缺陷时，53BP1无法移除，细胞将通过NHEJ途径对DSBs进行修复。
（4）BRIP1：BRIP1位于17号染色体上，包含7个解旋酶家族特异性结构域和一个C-端的BRCA1结合结构域[33]。BRIP1是一种DNA解旋酶，直接与BRCA1的BRCT结构域结合[34, 35]，BRCA1/BRIP1相互作用的发挥取决于BRIP1的磷酸化状态，这依赖于ATM诱导的DNA损伤检查点——G2/M检查点[35]。由于BRIP1的解旋酶活性与BRCA1相关，BRIP1的致病突变可能会影响HR对DSBs的修复。
二、乳腺癌患者HR通路基因突变检测现状
多年来，Sanger测序是检测BRCA1/2基因序列中单核苷酸改变、插入和缺失的金标准，多重连接探针扩增（multiplex ligation-dependent probe amplification，MLPA）技术用于检测大的基因组改变[36]。然而，Sanger测序和MLPA技术耗时长，成本高。应用NGS技术检测BRCA1/2突变可获得与Sanger测序一致的结果，且时间更短，成本更低[37, 38]。HR通路基因的致病突变会增加患乳腺癌的风险，然而单个基因的突变较为罕见，一次只检测一个基因既低效又昂贵。目前NGS技术使大规模平行测序成为可能[39]，可实现同时对多个HR通路基因进行突变检测。
NGS能检测个体DNA中的微小变异，根据美国医学遗传学与基因组学学会（American College of Medical Genetics and Genomics，ACMG）指南将变异分为致病、可能致病、意义不明确（variants of unknown significance，VUS）、可能良性和良性五类[40]。胚系DNA检测比体细胞DNA检测更敏感，侵入性更小，因此优先考虑胚系DNA检测[41]。如果胚系BRCA1/2突变检测结果为阴性，美国临床肿瘤学会（American Society of Clinical Oncology，ASCO）指南建议采集肿瘤样本检测体细胞突变[42-44]。
迄今为止，HR通路的多基因检测已在国外不同人种中广泛开展。Tung等人对1241例乳腺癌患者进行了乳腺癌相关基因的致病突变检测，其中BRCA1/2突变频率为13.3%，非BRCA1/2基因突变频率为6.4%，此外，该研究还对377例BRCA1/2阴性乳腺癌患者进行了检测，非BRCA1/2基因突变频率为3.7%[45]。Couch等人对来自多个中心的1824例三阴性乳腺癌患者进行了17基因检测组合的致病突变检测，其中HR通路基因详细突变情况如下：BRCA1的突变频率为8.5%，BRCA2为2.7%，PALB2为1.2%，BARD1为0.5%，BRIP1为0.44%，RAD51D为0.38%，RAD50为0.33%，RAD51C为0.33%[46]。
由于遗传背景的差异，国外不同人种的基因突变检测数据可能不适用于中国人群，因此部分国内研究探究了中国乳腺癌患者HR通路基因的突变情况。由于检测基因组合的不同和样本的差异，HR通路基因突变频率存在差异。Shao等人应用NGS技术对261例中国遗传性乳腺癌患者的HR通路基因进行了致病突变检测，其中BRCA1的突变频率为3.07%，BRCA2为4.21%。在纳入的其他HR通路基因中，突变频率由高到低分别为：CHEK2为1.92%，BRIP1为0.77%，MRE11为0.38%，PALB2为0.38%，RAD51C为0.38%[47]。Li等人对中国遗传性乳腺癌患者进行的多中心研究发现，BRCA1的致病突变频率为8.8%，BRCA2为8.6%，PALB2为1.2%，ATM为0.6%，CHEK2为0.6%，BARD1为0.5%，BRIP1为0.3%，RAD50为0.2%[48]。Sun等人对8085例中国乳腺癌患者进行了62基因检测组合的致病突变检测，发现基因的总突变率为24.1%，与遗传性乳腺癌患者类似[49]。Jian等人对乳腺癌患者和乳腺癌患者的健康亲属进行了27基因检测组合的致病突变检测，结果显示：乳腺癌患者的突变率为19.2%，健康亲属为12.5%[50]。除BRCA1/2外，HR通路其他基因致病突变较为罕见，有必要继续对乳腺癌患者进行检测，以补充中国乳腺癌相关基因突变数据库。
尽管多基因检测已普遍开展，但其临床应用仍存在一些挑战：筛选能从检测中获益的患者、待检基因的选择和组合以及对突变结果的解读，尤其是低或中等外显率基因的突变和VUS。VUS结果的识别为临床医生和患者带来了挑战：基于VUS的额外筛查和检测可能导致过度治疗和管理不当，而携带VUS的患者可能会对突变的潜在影响产生额外的焦虑。目前还需要进一步的研究和指南来确定如何在发现VUS后对患者进行管理和治疗[51]。
三、PARP抑制剂在乳腺癌患者中的治疗进展
在存在HRD的细胞中，当其他参与DNA修复的基因或其他通路中维持细胞存活和增殖功能的关键基因缺陷时，会导致合成致死（synthetic lethality）效应。聚腺苷二磷酸核糖聚合酶（poly ADP ribose polymerase，PARP）由一组核蛋白组成，它们与受损DNA结合后被激活并参与DNA修复[52]。PARP能够识别SSBs，并通过碱基切除修复（base excision repair，BER）途径进行DNA损伤的修复[53]。当DNA单链断裂修复机制功能失调时，SSBs累积并转化为DSBs，此时可以通过HR途径修复DNA损伤并维持细胞活力[54]，若细胞同时存在HRD，则两者发挥合成致死效应，导致DNA损伤无法修复，细胞死亡。
PARP抑制剂结合在PARP蛋白催化中心的相同位置，阻止底物烟酰胺腺嘌呤二核苷酸（NAD+）的结合，从而阻碍聚腺苷二磷酸核糖的产生[55]。PARP1被捕获在DNA损伤处，形成一种毒性复合物，阻止复制叉的进程[56]。在携带胚系BRCA1/2致病突变的乳腺癌患者中，应用PARP抑制剂可抑制PARP的功能，使SSBs持续存在，并转化为DSBs，由于DSBs无法得到精确修复，导致DSBs积累，细胞死亡。
奥拉帕尼是首个被批准用于乳腺癌的PARP抑制剂，2018年，奥拉帕尼获得了美国食品药品监督管理局（Food and Drug Administration，FDA）的完全批准，用于治疗携带BRCA1/2致病突变的乳腺癌患者[57]。目前，基于两项Ⅲ期临床试验，奥拉帕尼和他拉唑帕尼已被FDA批准用于携带BRCA1/2致病突变的转移性乳腺癌患者的单药治疗[58-61]。多项研究发现，除了BRCA1/2致病突变相关的肿瘤对PARP抑制剂有反应，未携带BRCA1/2致病突变的乳腺癌、卵巢癌和前列腺癌患者也能从PARP抑制剂治疗中获益[62-64]。
尽管PARP抑制剂在BRCA1/2致病突变乳腺癌的治疗中取得了显著的成功，但其中超过40%的患者对PARP抑制剂的治疗无反应[65, 66]。此外，那些最初对治疗有反应的患者在长期使用后获得PARP抑制剂抗性，导致疾病进展。PARP抑制剂的耐药机制包括PARP抑制剂外排，回复突变恢复HR功能，通过上调NAD+合成来释放PARP，沉默BP53，PARP发生点突变和复制叉重新稳定后复制速率增加[67-69]。
此外，研究发现PARP抑制剂和细胞周期检查点抑制剂的联合应用可以增强疗效。激活的ATR/CHK1信号通路导致细胞周期阻滞，允许DNA进行修复[70, 71]。ATR/CHK1抑制剂可阻碍DNA损伤诱导的细胞周期阻滞，使具有染色体畸变的癌细胞进入有丝分裂，并最终诱导有丝分裂障碍和细胞死亡。据报道，CHK1抑制剂与PARP抑制剂发挥协同作用，杀死乳腺癌细胞[72-74]。与PARP类似，ATR/CHK1也被认为能与DNA修复缺陷联合发挥合成致死效应[75, 76]。对BRCA1/2缺陷的患者联合应用PARP抑制剂和ATR/CHK1抑制剂，可以增加DNA复制压力，进一步发挥合成致死效应。
四、应用转录组测序技术筛选乳腺癌患者潜在的治疗靶点
不仅对乳腺癌进行基因组水平的分析，多项研究还应用转录组测序技术对乳腺癌患者进行转录水平的分析，以探究乳腺癌的异质性和复杂性，从而筛选新的分子标志物和潜在的治疗靶点。
乳腺癌是一类异质性疾病，根据雌激素受体（estrogen receptor，ER）、孕激素受体（progesterone receptor，PR）和人类表皮生长因子受体2（human epidermal growth factor receptor 2，HER2）的表达情况，主要分为四个亚型[77]：Luminal A型（ER+，PR≥20%，HER2-）、Luminal B型（ER+，PR＜20%，和/或HER2+）、HER2过表达型（ER-，PR-，HER2+）和TNBC（ER-，PR-，HER2-）[78]。TNBC约占15%~20%的乳腺癌病例，患者的临床病理特征表现为发病年龄早、肿瘤体积大、易复发和转移[79]。与其他三种乳腺癌亚型相比，TNBC患者的早期转移率较高，预后较差[80]。由于缺乏有效的分子靶点，TNBC患者的治疗选择有限，临床治疗仍以化疗为主，一旦发生转移和扩散，患者的5年生存率则不足30%[81]，亟需寻找潜在的治疗靶点。
应用转录组测序技术可实现对mRNAs和非编码RNAs（包括lncRNAs和microRNAs）的全面分析，以筛选肿瘤相关的新靶点。lncRNAs参与基因调控，在癌症的发展和预后评估中扮演着重要角色[82-84]。特定的lncRNAs表达谱与不同亚型的乳腺癌相关，DSCAM-AS1在ER阳性的乳腺癌中特定存在，并增强肿瘤的侵袭性和耐药性[85]，AFAP1-AS1在HER2过表达型和TNBC中表达失调[86, 87]。UCA1，GAS5和XIST被证实是乳腺癌相关的肿瘤抑制因子，而HOTAIR，TINCR和DSCAM-AS1则被称为致癌的lncRNAs[88, 89]。目前基于转录组测序技术，已经能够描述乳腺癌的miRNA表达特征，从而揭示许多miRNA的异常表达[90]。Toda等人探究了TNBC肿瘤组织的miRNA表达谱，发现了104个差异表达的miRNA，其中表达显著下调的miR-204-5p发挥肿瘤抑制作用，调控多个乳腺癌相关的原癌基因[91]。
Yuan等人应用转录组测序技术对Luminal B型乳腺癌肿瘤组织和癌旁组织进行了分析，以鉴定差异表达的lncRNA和mRNA，并构建lncRNA-mRNA共表达网络，结果显示：MALAT1-S100A7，MIAT-CCL5和WT1-AS-WT1在Luminal B型乳腺癌中发挥重要作用，为进一步了解Luminal B型乳腺癌的发病机制提供新的思路[92]。Tian等人对公共数据库里的基因表达数据进行生信分析，发现和验证了4个关键基因（FAM171A1，NDFIP1，SKP1和REEP5），这些基因可能作为乳腺癌潜在的治疗靶点和预后标志物[93]。Chen等人应用公共数据库比较TNBC与其他亚型乳腺癌的转录水平差异，结果显示：转录激活状态是TNBC与其他亚型乳腺癌的重要区别；HORMAD1是与TNBC预后不良相关的关键差异基因；表观遗传学治疗和针对HORMAD1的靶向治疗可能有助于改善TNBC患者的预后[94]。
因此，基于转录组测序技术和公共数据平台，能够深入探究乳腺癌的发病机制，为乳腺癌的筛查、诊断、治疗和预后评估提供新的线索。目前TNBC患者的预后较差，治疗选择有限，亟需寻找更特异的分子标志物和治疗靶点，以改善患者的预后。此外，通过转录组测序技术筛选到的分子标志物，需要进一步评估其作为乳腺癌患者治疗靶点的可行性，从而真正实现其临床应用价值。
五、结语与展望
本文分析了二代测序技术在乳腺癌研究中的应用。在基因组水平，应用二代测序技术对HR通路基因进行突变检测，有助于指导乳腺癌患者的治疗和预后评估，高危人群的风险评估。在转录水平，应用转录组测序技术对乳腺癌肿瘤组织的基因表达情况进行全面分析，有利于筛选潜在的分子标志物和治疗靶点，为患者提供更多的治疗选择，以改善患者的预后。
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