Abstract
SUMMARY The rearrangement of the gene encoding the transcription factor ETS-related gene (ERG) is thought to play a key role in the development of prostate cancer. However, the studies on the ERG mutations have been rarely reported in non-small cell lung carcinoma (NSCLC). Here, we reported genetic features regarding a case of a 68-year-old male patient who presented the primary synchronous multiple tumor lesions in the separated lungs. The patient was hospitalized due to the presence of tumor lesions at the right and left lungs revealed by a chest computerized tomography (CT) scan. After conducting lobectomies at the both lungs, the tumor nodules were all removed, and the histological analysis suggested adenocarcinoma at the both tumor lesions. The patient was diagnosed with synchronous multiple primary lung cancer (SMPLC) based on Martini-Melamed criteria and American College of Chest Physicians practice guidelines. An exome analysis of 315 genes in the two tumor lesions and a non-tumor lesion was conducted by using Illumina Nextseq500 platform from each tumor region to decipher a potential evolutional progress of SMPLC. Single or pair-end reads were first mapped to a human genome reference and filtered based on the mapping quality score. The read depth was ≥ 1 000× and the depth of coverage was 95%. The data revealed a discordant epidermal growth factor receptor (EGFR) from the separate lungs; additionally, a high frequency of point mutation on exon 9 H310P of the ERG gene was detected at the both sites of the tumor lesions. This case showed that a potential role of the molecular features analysis from each tumor lesion might contribute to the understanding of the evolutional development of SMPLC. This study suggests that the same environment may contribute certain gene(s) mutations in the same sites in the early stages of polyclonal tumor origins; meanwhile the extensive studies on these genes may help us understand the evolution and progress of tumor clones.
Keywords: Lung neoplasms; Neoplasms, multiple primary; ETS-related gene; Mutation; High-throughput nucleotide sequencing
E26转录因子(E26 transformation-specific，ETS)相关基因(ETS-related gene，ERG)是一种细胞生长调控基因，其与跨膜丝氨酸蛋白酶2基因(transmembrane serine protease 2，TMPRSS2)融合并重新排列，在前列腺癌发生中发挥关键作用[1-2]。最近的一项研究尝试采用大分子多肽靶向ERG，通过破坏其功能，用于治疗前列腺癌[3]，但较少见ERG在肺癌组织中表达和突变的报道。
本研究报道了1例术后确诊的同时性多原发肺癌(synchronous multiple primary lung cancer，SMPLC)患者，通过Illumina高通量测序平台对各癌组织及其癌旁组织的315个基因组合(panel)进行分析，发现在双侧肺癌组织中ERG相同位点存在高突变频率，并探讨其可能存在的临床价值。
1. 病例资料
1.1. 患者一般情况
患者，男性，68岁，因“体检发现双肺结节2年”于2016年3月就诊于浙江省嘉兴市第二医院肿瘤科，并收入院。2年前患者体检过程中通过检测胸部CT发现“双肺部结节”，后未行穿刺及随访，期间也一直未出现咳嗽、咳痰、胸痛等症状。患者有20余年吸烟史，10支/d。本次入院因查胸部CT提示:右肺上叶不规则条片状高密度影，大小约2.0 cm×2.0 cm，边缘见细长毛刺，其内见支气管通过(图 1A)；左肺下叶背段可见小片状毛玻璃样影，中央为实性成分，可见胸膜牵拉，大小约1.4 cm×3.3 cm(图 1B)。根据影像学不能排除双肺恶性病变，经完善各项检查，排除手术禁忌后行胸腔镜下左下肺癌根治术(左下肺背段切除+淋巴结清扫)+右上肺癌根治术(右上肺切除+淋巴结清扫)。术后病理提示左下肺浸润性中分化腺癌(2.0 cm×1.1 cm，部分贴壁生长，部分腺泡型)累及脏层胸膜，未见神经侵犯及脉管瘤栓，支气管切缘阴性，右上肺浸润性中-低分化腺癌(3.0 cm×1.8 cm，部分腺泡型，部分实性型)累及脏层胸膜，未见神经侵犯及脉管癌栓，支气管切缘阴性，淋巴结均未见转移，术后病理诊断为左下肺腺癌(T2N0M0，ⅠB)，右上肺癌(T2N0M0，ⅠB)。根据Martini-Melamed和美国胸科医师协会(American College of Chest Physicians，ACCP)2013年的诊断标准，患者为SMPLC [4-5]。
图1.
术前胸部CT扫描图像
The images of chest computed tomography (CT) before surgeries
A, the arrow showed a size of 2.0 cm×2.0 cm lesion with irregular high density shadow at the upper lobe of right lung; B, the arrow showed a size of 1.4 cm×3.3 cm with ground glass nodule in the bottom lobe of left lung.
1.2. 二代测序分析双侧癌灶315个基因突变情况
通过高通量测序检测手术病理石蜡组织标本(左、右肺癌组织及癌旁组织)315个基因，了解多个肺癌灶(克隆)形成及各自特有的基因特征。采用美国Illumina公司Nextseq500/550测序仪平台，标本包括左、右肺癌组织及其癌旁组织，下机数据量≥2.5 Gb，Q30≥90%，组织样本平均测序深度≥1 000×，覆盖度≥95%，比对人类肿瘤基因组数据库，通过对315个基因组合的外显子点突变、插入-缺失(insertion-deletion，InDel)、融合(fusion)等基因变异的分析，去除胚系突变后，选取体细胞突变且突变频率≥5%的基因，突变频率的计算是基因中检测到的位点突变数除以该位点检测的总次数。去除胚系突变后发现，两侧肺癌组织存在不同基因突变谱系，在左侧癌灶发现驱动基因表皮生长因子受体(epidermal growth factor receptor，EGFR)21号外显子L858R位点突变，突变频率16%，但在右侧癌灶未发现EGFR突变。除EGFR 21号外显子L858R突变外，两侧癌灶存在不同基因突变频率≥5%的其他体细胞突变见表 1。通过二代测序分析，还发现左侧和右侧癌灶存在ERG相同位点(9号外显子H310P突变)较高频率突变，分别为9%和8%(表 1)。
表1.
315个基因组合中突变频率≥5%的体细胞突变在两侧癌灶间的对比
Comparison of somatic cell gene mutation with mutaton frequency ≥5% between the two separate tumor lesions
| Items | Tumor nodule | Gene | Chromosome location | Gene mutation | Density | Mutation frequency |
| Discordant (≥5%) | Left side | EGFR | 7p11.2 | exon21, c.2573T>G, p.L858R | 1 844 | 15%(290/1 884) |
| ACVR1B | 12q13.13 | exon2, c.106T>A, p.C36S | 554 | 9%(48/554) | ||
| GRIN2A | 16p13.2 | exon11, c.2326G>A, p.D776N | 647 | 9% (56/647) | ||
| ATR | 3q23 | exon47, c.7912C>T, p.L2638F | 483 | 7% (32/483) | ||
| EGFR | 7p11.2 | exon19, c.2240T>C, p.L747S | 2 906 | 7% (207/2 906) | ||
| CBFR | 16q22.1 | exon5, c.481C>T, p.R161W | 951 | 5% (48/951) | ||
| NF1 | 17q11.2 | exon33, c.4462C>T, p.R1488C | 775 | 5% (35/775) | ||
| NOTCH2 | 1p12 | exon34, c.7153A>C, p.T2385P | 914 | 5% (44/914) | ||
| Right side | ARID2 | 12q12 | exon5, c.539G>A, p.C180Y | 588 | 6%(34/588) | |
| ARID2 | 12q12 | exon15, c.1960C>G, p.P654A | 1 058 | 5% (48/1 058) | ||
| Concordant(≥5%) | Left side | ERG | 21q22.2 | exon9, c.929A>C, p.H310P | 637 | 9% (60/637) |
| Right side | ERG | 21q22.2 | exon9, c.929A>C, p.H310P | 929 | 8% (74/929) |
1.3. ERG在癌症基因组图谱中的突变及其预后分析
利用cBioportal数据分析平台(http://cbioportal.org)，对癌症基因组图谱(the cancer genome atlas，TCGA)非小细胞肺癌mRNA表达数据集进行研究，发现在1 144肺腺癌、鳞癌患者中存在27例突变(2.4%，27/1 144)，突变的类型包括扩增、深度缺失等(图 2)。存在ERG突变的肺癌患者(共21例，死亡5例)平均中位生存时间为88.16个月，ERG未突变肺癌患者(共933例，死亡267例)平均中位生存时间为43.26个月，两者间总生存时间差异无统计学意义(P=0.443，Log-rank检验, 图 3)。
图2.
利用cBioportal数据分析平台研究TCGA非小细胞肺癌ERG突变情况
The mutation of ERG was analyzed from the TCGA non-small-cell lung carcinoma samples by using the cBioportal data analysis platform
图3.
通过cBioportal数据分析平台研究TCGA非小细胞肺癌ERG突变与总生存率的关系
The correlation of ERG mutation and overall survival was analyzed from the TCGA non-small-cell lung carcinoma samples by using the cBioportal data analysis platform
2. 讨论
目前依据Martini-Melamed以及ACCP 2013年的诊断标准，结合手术观察到的现象(如各癌灶间彼此孤立，包括位于不同的肺段或肺叶、各癌灶共同引流区域无肿瘤细胞累计、无纵隔淋巴结及远处转移)，综合评估各个肿块的病理学特征，最终得出SMPLC的诊断[5]。通过高通量二代测序技术分析各个癌灶的基因特征，将对同时性或异时性多原发肿瘤的诊断以及肿瘤多克隆灶的起源提供非常关键的生物学信息。本例患者通过影像学、术后病理等检查可以较为明确地诊断为SMPLC，双侧肺部肿瘤分期均为ⅠB期，另外, 通过分子病理检测，发现驱动基因EGFR突变在不同癌灶存在差异，进一步证实患者存在多原发肺癌灶。通过对每个癌灶克隆进行二代测序全外显子检测，特别是对较早期SMPLC的不同癌组织进行全外显子测序检查，可能会对SMPLC的发生提供更为精准的个体化信息。
对SMPLC的病因学进行研究发现，肺鳞癌及小细胞肺癌患者吸烟影响支气管树的不同敏感细胞后形成肺的浸润性癌，可以同时或序贯发生[6]，异时性多原发肺鳞癌的发生和发展可能是多处肺组织因不断暴露在吸烟等致癌性刺激下的结果。目前肺腺癌的病因并不是很明确，肺腺癌高发于从未吸烟的女性患者，有研究证据表明肺腺癌患者往往存在驱动基因的突变，其中包括了Kirsten大鼠肉瘤病毒癌基因(Kirsten rat sarcoma viral oncogene homolog, KRAS)、EGFR、磷脂酰肌醇-3激酶(phosphatidylinositol 3-kinase，PI3KCA)及间变性淋巴瘤激酶(anaplastic large cell lymphoma，ALK)等[7]。2017年的一项大样本非小细胞肺癌(可切除的ⅠA至ⅡA期患者)研究，通过对327个区域进行全外显子组测序发现，EGFR、间质上皮转移因子(mesenchymal to epithelial transition factor，MET)和鼠类肉瘤滤过性毒致癌同源体B1(v-Raf murine sarcoma viral oncogene homolog B1，BRAF)突变大部分属于早期突变和克隆性突变，而参与DNA损伤修复等维持基因组完整性的基因，如PIK3CA、神经纤维瘤病1型(neurofibromatosis 1，NF1)、TP53和Notch尽管在75%以上的肿瘤发生突变，但在时间轴上发生较晚[8]。
本病例为术后病理证实的同时性多原发肺腺癌，通过对该病例不同癌灶的高通量分析，我们发现两个病灶间存在EGFR不一致突变，在左侧癌灶发现了EGFR 21号外显子高频率的L858R突变，而在右侧癌灶未发现EGFR突变。我们还发现在左侧和右侧癌灶中均有较高突变率的ERG 9号外显子H310P同位点突变。ERG是ETS家族的转录因子，ERG融合基因激活是引起前列腺癌发生和发展最为普遍的基因改变[9]，最近一项在前列腺癌中的研究表明，针对ERG为靶点的多肽，可以显著降低肿瘤细胞的增殖、浸润和生长[3]。ERG和尤文氏肉瘤(Ewing’s sarcoma，EWS)基因融合会导致尤文氏肉瘤发生[10]。最近的一项研究也提示，ERG在内皮细胞的表达下调参与了内皮细胞向间质细胞转化(endothelial-to-mesenchymal transition，EndMT)，而EndMT被认为与肿瘤进展有关[11]。本例患者两侧肺癌病理分型均为ⅠB期，属于病程早期，不同癌结节同时存在ERG基因改变，提示对于该患者，在疾病发生的早期相同环境中致癌因素可能导致了ERG 9号外显子相同位点突变，考虑此位点的突变存在于癌灶转化的早期阶段，各癌克隆“进化”发展过程中逐步产生差异。目前，关于ERG蛋白表达及其突变在非小细胞肺癌中的研究较少，通过对TCGA非小细胞肺癌数据库进行分析发现，ERG的突变率只有2.4%(21/933)，且ERG在肺癌中的融合突变也尚未见相关报道。虽然ERG突变的肺癌患者平均中位生存时间(88.16个月)长于ERG野生型患者(43.26个月)，但是总生存时间差异并无统计学意义，这可能是ERG突变患者样本数量较少的原因。
综上所述，本病例提示，对分期较早的SMPLC手术切除的各癌灶标本进行高通量测序分析，除可以辅助传统病理诊断外，还可能对各癌灶起源的研究提供有价值的线索。
Funding Statement
嘉兴市科技计划(2016AY23054)、浙江省医药卫生科技计划(2016KYB292)
Supported by Science and Technology Bureau of Jiaxing (2016AY23054), Health Bureau of Zhejiang Province (2016KYB292)
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