【作者】 李昕；

【导师】 陈军；

【作者基本信息】 天津医科大学 ， 外科学 胸心外科学（专业学位）， 2017， 博士

【摘要】 目的:本课题总结比较伴有恶性胸腔积液(Malignant Pleural Effusion,MPE)的晚期肺腺癌患接受以外科手术为主的综合治疗和单纯内科治疗后胸水治疗效果分析,总生存差异分析,以及接受手术治疗的外科组患者的无进展生存分析并对其临床因素进行分层比较分析。第二部分研究收集2014年-2015年在天津医科大学总医院肺部肿瘤外科接受手术治疗的9例伴有恶性胸腔积液的晚期肺腺癌患者的手术标本,对这些样本进行高通量测序,通过获得的基因突变数据进行分析寻找可能与该类患者发生胸膜转移相关的关键基因。方法:本研究收集了2007年1月-2015年3月在天津医科大学总医院接受过抗肿瘤治疗的195例初治时伴有恶性胸腔积液但不伴随远处器官转移的晚期肺腺癌患者的临床资料进行回顾分析,包括96例接受过以外科手术为主的综合治疗的手术组患者和99例仅接受过内科综合治疗的内科组患者。患者的临床特征包括年龄,性别,吸烟史,ECOG评分,是否接受手术治疗,手术方式,胸水治疗效果,化疗方案个数,是否接受过靶向药物治疗,具体病理分型,治疗过程中出现的器官转移部位,对以上这些因素进行分析,对于不同组间的疗效比较应用Χ2检验,对生存时间分析应用kaplan-meier法,long-rank对生存曲线的差异进行显著性检验,使用COX回归进行多因素分析。以p<0.05为具有显著性差异。所有数据应用SPSS 17.0统计软件处理。第二部分研究中收集了9例2014年-2015年在我科行外科手术治疗的伴有恶性胸腔积液的晚期肺腺癌患者的原发肿瘤组织标本,壁层胸膜转移组织标本及远癌的正常肺组织标本,运用Nextseq 500测序平台进行杂交捕获,对获得的基因文库进行数据分析,筛选掉1000 Genome database数据库已知的单核苷酸变异,同义突变,内含子及非编码区变异结果,同时过滤掉来自远癌正常肺组织的单核苷酸变异数据,得到1610个肿瘤相关的体细胞突变数据,根据COSMIC数据库及TCGA数据库中的热点单核苷酸变异以及肺癌核心突变数据集从中再筛选出80个热点突变数据,过滤掉低频等位基因(<10%),在原发肿瘤(C)与胸壁转移组织(P)中进行配对,最终对测序结果进行分析得出50个互相配对的可能与肿瘤发生胸膜转移相关的热点基因点突变数据。结果:本研究结果显示,外科组胸水控制效果CR 71例(74%),PR 20例(20.8%),RR为94.8%,内科组胸水控制效果CR 18例(18.2%),PR 52例(52.5%),RR为70.7%,运用CMH-Χ2检验进行分析发现,Location检验结果p=0.0037,外科组疗效优于内科组。全组195例患者自入院确诊为伴有恶性胸腔积液的原发肺腺癌开始至任何原因导致的死亡的全组中位生存时间(Median Survival Time,MST)为16个月(95%置信区间,95%Confidence Interval[95%CI]13.439-18.561),外科组患者MST 25个月(95%CI 22.373-27.627)显著长于内科组患者的MST 11个月(95%CI 9.978-12.022),两者间p<0.01;对全组患者进行COX多因素生存分析发现,接受外科治疗(风险比Hazard Ratio,HR 0.480 95%CI 0.301-0.765p=0.002),无吸烟史(HR 1.636 95%CI 1.163-2.301 p=0.005),ECOG评分0-1分(HR 2.735 95%CI 1.938-3.861 p<0.01),服用靶向药物(HR 0.430 95%CI0.276-0.671 p<0.01)是显著影响所有195例伴有恶性胸水的晚期肺腺癌患者总生存期的独立风险因素。外科治疗组患者的总生存在年龄,性别,吸烟史,1线接受过的化疗方案周期数,是否接受过靶向治疗,治疗过程中发生的远处器官转移:肺转移,骨转移,脑转移,肝转移等亚组中均较内科组患者有明显延长(p<0.01),伴有肾上腺转移的患者在外科组和内科组间未显示出明显的总生存期差异(p=0.220);对外科组患者进行单因素生存分析发现无吸烟史(p=0.001),ECOG评分0-1分(p<0.01),接受过靶向药物治疗(p<0.01),无肝转移(p=0.005),无脑转移(p=0.01),接受肺叶切除手术+胸膜固定术(p<0.01)或肺部分切除术+胸膜固定术(p=0.001)是改善外科组患者OS的因素,COX多因素生存分析显示,ECOG评分0-1分(p<0.01),服用靶向药物(p<0.01),接受肺叶切除手术+胸膜固定术或肺部分切除术+胸膜固定术(p<0.01),是显著影响外科组患者总生存的独立预后因素。而无吸烟史(p=0.016),ECOG评分0-1分(p<0.01),接受肺叶切除术+胸膜固定术(p<0.01)或接受肺部分切除术+胸膜固定术(p<0.01),病理类型为微乳头为主型腺癌(p=0.013),是改善外科组患者PFS的因素,差异有统计学意义,COX多因素生存分析发现,ECOG评分0-1分(p<0.01),接受肺叶切除术+胸膜固定术(p<0.01)或接受肺部分切除术+胸膜固定术(p<0.01),病理类型为微乳头为主型侵润性腺癌(p=0.002),是显著影响外科组患者PFS的独立风险因素,其差异具有统计学意义。第二部分实验中发现,9例患者中,有5例患者在原发肿瘤与转移组织中均捕获到了EGFR基因的高频突变(AF>10%)(55.6%,5/9),其中有3例患者表现为EGFR基因小片段插入缺失(INDEL),2例患者表现为EGFR基因单核苷酸变异(SNV)。有5例患者在原发肿瘤与转移组织中均捕获到了TP53基因的高频突变(AF>10%)(55.6%,5/9),其中有1例患者表现为TP53基因小片段插入缺失(INDEL),4例患者表现为TP53基因单核苷酸变异(SNV);有3例患者同时表达了TP53与EGFR基因的协同突变,且均为高频突变(AF>10%),其余检测到的配对基因突变包括FAT-3,MSH6,BARD1,PIK3CA,MAP3K1,NOTCH4,MET,PRKDC,ATM,KMT2D,FGF14,SMARCA4,AXL。结论:对于仅伴有恶性胸腔积液而不伴有远处器官转移的肺腺癌患者而言,接受外科手术可更有效的控制胸水的再生,且接受外科手术可以显著改善该类患者的总生存期。接受肺叶切除或肺部分切除术及胸膜部分切除固定术的患者不论PFS或是OS均优于仅接受胸膜部分切除固定术的患者。通过高通量测序分析发现EGFR基因与TP53基因的高频突变可能是伴有恶性胸腔积液的肺腺癌患者发生胸膜转移的重要相关基因,这将为伴有恶性胸腔积液的肺腺癌患者的未来多靶点协同靶向治疗发展提供新的方向。更多还原

【Abstract】 Objective:This study compared the effect of pleural effusion control and overall survival(OS)of advanced lung adenocarcinoma with malignant pleural effusion(MPE)between surgical predominantly comprehensive treatment and internal medicine comprehensive treatment.The progression free survival(PFS)was also stratified analysised by clinical elements of surgical group.The second step we collected 9 surgical specimens of lung adenocarcinoma patients with malignant pleural effusion who accept surgical treatment in Tianjin Medical University General Hospital between 2014-2015.Using High-Throughput Sequencing technique discoverd the possibly key genes associated with pleural metastasis of this kind of patients.Methods: This study retrospective analysised the clinical data of 195 lung adenocarcinoma patients with only malignant pleural effusion but no distant organ metastasis who accept anti cancer therapy in Tianjin Medical University General Hospital between January 2007 to March 2015.Including 96 patients received surgical predominantly comprehensive treatment called surgical group and 99 cases received only internal medicine comprehensive treatment called internal medicine group.Clinical characteristics including age,gender,smoking history,ECOG score,whether to accept the surgical treatment,operation style,effect of pleural effusion control,number of chemotherapy cycle,whether to accept targeted therapy,pathological classification,organ metastasis,analysed of these factors.Using Χ2 test for curative effect comparison between different groups,survival curves were constructed using the kaplan-meier method and the differences were evaluated by log-rank test.Cox proportional hazard analysis was performed to evaluated multiple-factor analysis.Differences were considered significantly when p<0.05.All calculations were performed by SPSS17.0 software.The second step we collected the primary tumor tissue samples,pleural metastasis tissue specimens and normal tissues of 9 advanced lung adenocarcinoma cases with malignant pleural effusion who accept operation in our department between 2014-2015.Performed gene sequencing under Nextseq 500 sequencing platform,analysis the obtained gene library,filtered the 1000 Genome database known Single Nucleotide Polymorphisms(SNPs),Synonymous,intron,untranslated regions(UTR variants),single nucleotide mutations of normal tissue,get 1610 tumor related somatic mutations.We chose high allele fraction(>10%)and cancer-related hotspot single nucleotide variants(SNVs)(COSMIC/TCGA/8 lungcoregenes),finally got 50 gene mutations might be the key genes associated with pleural metastasis matched in primary tumor and metastasis tissue.Results: The results showed,pleural effusion treat effect of surgical group compared with internal medicine group were complete remission(CR)71 cases(74 %)vs 18 cases(18.2%),partial response(PR)20 cases(20.8%)vs 52 cases(52.5%),Response rate(RR)94.8% vs 70.7%;CMH-Χ2 test showed Location result p=0.0037,surgical group was better than internal medicine group.The median survival time(MST)of 195 patients was 16 months(95% Confidence Interval[95% CI],13.439-18.561).MST was significantly longer among the surgical group than internal medicine group(25 months vs 11 months,p<0.01).Cox analysis showed the independent prognostic factors to significantly improve the OS of 195 patients included accept operation(Hazard Ratio,HR 0.480 95%CI 0.301-0.765 p=0.002),no smoking history(HR 1.636 95%CI 1.163-2.301 p=0.005),ECOG 0-1 point(HR 2.735 95%CI 1.938-3.861 p<0.01),taking target therapy(HR 0.430 95%CI 0.276-0.671 p<0.01).Patients of surgical group showed significantly longer OS than of internal medicine group in age,sex,smoking history,whether accept target therapy,numbers of prior chemotherapy,sites of metastasis including lung,bone,brain,liver(p<0.01),except adrenal metastasis(p=0.220).Patients of surgical group showed significantly improved OS in subgroups of no smoking(p=0.001),ECOG 0-1 point(p<0.01),accept target therapy(p<0.01),no liver metastasis(p=0.005),no brain metastasis(p=0.01),accept pulmonary lobectomy with pleurodesis(p<0.01)or lung wedge resection with pleurodesis(p=0.001).Cox analysis showed the independent prognostic factors to significantly improve the OS of surgical group included ECOG 0-1 point(p<0.01),accept target therapy(p<0.01),accept pulmonary lobectomy with pleurodesis(p<0.01)or lung wedge resection with pleurodesis(p<0.01).And Patients of surgical group showed significantly improved PFS in subgroups of no smoking(p=0.016),ECOG 0-1 point(p<0.01),accept pulmonary lobectomy with pleurodesis(p<0.01)or lung wedge resection with pleurodesis(p < 0.01),micro nipple mainly adenocarcinoma(p=0.013),Cox analysis showed the independent prognostic factors to significantly improve the PFS of surgical group included ECOG 0-1 point(p<0.01),accept pulmonary lobectomy with pleurodesis(p<0.01)or lung wedge resection with pleurodesis(p<0.01),micro nipple mainly adenocarcinoma(p=0.002).In the second step,we found 5 of 9 patients(55.6%)with malignant pleural effusion harboured EGFR high frequency mutations(AF>10%)in both primary tumor and metastasis tissue,of which 3 cases showed deletion or insertion mutation(INDEL),2 cases showed single nucleotide variants(SNV).TP53 gene mutations were found in 5 of 9 cases(55.6%)in both primary tumor and metastasis tissue,of which 1 case showed deletion or insertion mutation(INDEL),4 cases showed single nucleotide variants(SNV).There were 3 cases(33.3%,3/9)carried EGFR and TP53 high frequency collaborative mutations at the same time(AF>10%).Other mutations were detected including FAT-3,MSH6,BARD1,PIK3 CA,MAP3K1,NOTCH4,MET,PRKDC,ATM,KMT2 D,FGF14,SMARCA4,AXL.Conclusion: For lung adenocarcinoma patients with malignant pleural effusion,accept operation can effectively control the effusion regeneration.And accepted srugery treatment can significantly improved the OS of this kind of patients.Accept pulmonary lobectomy with pleurodesis or lung wedge resection with pleurodesis were significantly improved the PFS and OS compared with patients accept only pleurodesis.High-throughput sequencing results showed,EGFR and TP53 gene mutations might be the key genes lead to parietal pleura metastasis of lung adenocarcinoma.These findings will provide a new direction of development for new collaborative target therapy methods for lung adenocarcinoma patients with malignant pleural effusion.更多还原