【作者】 王超；

【导师】 郭金和；

【作者基本信息】 东南大学 ， 肿瘤学， 2020， 博士

【摘要】 研究背景:世界范围内,食管癌在恶性肿瘤中的发病率排名第7位,肿瘤相关致死率排名第6位。在中国,95%以上的食管癌患者病理类型为食管鳞状细胞癌（ESCC）,多数患者就诊时已处于进展期,失去了手术根治的机会。吞咽困难是无法切除的食管癌患者的主要症状,这些患者需接受姑息治疗。自膨式金属支架（SEMS）置入术和近距离放射治疗是两种广泛认可的用于治疗恶性吞咽困难的姑息治疗方式。近年来,碘-125(¹²⁵I)粒子作为一种持续低剂量率近距离放疗模式,已被用于治疗多种不可切除或局部复发的恶性肿瘤。一系列临床研究显示,¹²⁵I粒子与部分覆膜SEMS的联合治疗可有效控制肿瘤,并延长进展期食管癌患者的生存期。在过去几年中,多种全覆膜SEMS被设计并应用于食管癌的治疗。全覆膜SEMS尤其适用于预计生存时间较长,且需要接受后续治疗和支架移除的患者。因此,与联合部分覆膜SEMS相比,¹²⁵I粒子联合全覆膜SEMS的治疗模式理论上将更适合于预计生存时间较长的患者。另一方面,尽管大量研究关注了粒子支架的临床疗效,然而¹²⁵I粒子在ESCC中的抗肿瘤机制仍有待研究。在本课题中,首先,我们通过体内外实验系统地研究¹²⁵I粒子在ESCC细胞中的作用和抗肿瘤机制。然后,我们通过联合¹²⁵I粒子和分节式全覆膜SEMS设计了一款新型全覆膜内照射支架,并通过临床研究评估该内照射支架的可行性、安全性和有效性。目的:研究¹²⁵I粒子对ESCC细胞株Eca-109和KYSE-150的作用和相关机制。方法:通过体外照射模型,给予细胞累积剂量为0、2、4、6和8 Gy的¹²⁵I粒子辐射。在特定的实验中,细胞接受si RNA、N-乙酰-L-半胱氨酸（NAC）或放线菌酮的处理。通过克隆形成试验和台盼蓝染色试验检测细胞的增殖和活力。通过流式细胞术检测细胞周期、凋亡、活性氧自由基（ROS）和细胞内Ca²⁺水平。通过Western blot和免疫荧光技术分析DNA损伤、caspase活化、自噬和内质网应激。通过光镜和透射电镜观察细胞形态和超微结构的改变。在动物实验中,构建Eca-109和KYSE-150的荷瘤鼠,并对其进行¹²⁵I粒子（0.8 m Ci）植入治疗。记录肿瘤体积和重量的变化情况,并对肿瘤组织进行HE染色、ROS荧光染色、TUNEL和免疫组化等组织病理学检测。结果:在两株细胞中,¹²⁵I粒子辐射显著抑制了细胞增殖,并诱导DNA损伤和G2/M细胞周期阻滞。¹²⁵I粒子辐射通过凋亡和类凋亡诱导细胞死亡。在照射后,Eca-109细胞主要通过诱导caspase依赖的凋亡而死亡,并在6 Gy时达到凋亡峰值。KYSE-150细胞通过诱导凋亡和类凋亡被杀死,伴有广泛的细胞内空泡形成。在两株细胞中,¹²⁵I粒子辐射诱导了自噬流,并且通过si ATG5抑制自噬增强了细胞的放射敏感性。¹²⁵I粒子辐射还诱导了细胞内的Ca²⁺过载和内质网应激。此外,¹²⁵I粒子辐射引起ROS过量产生,ROS清除剂NAC明显削弱了¹²⁵I粒子辐射对内质网应激、自噬、凋亡和类凋亡样空泡化的诱导作用。动物实验显示,¹²⁵I粒子辐射可引起组织内ROS产生,激活细胞凋亡和潜在的类凋亡,并抑制细胞增殖和肿瘤生长。结论:在ESCC细胞中,¹²⁵I粒子辐射在引起DNA损伤、G2/M细胞周期阻滞、胞内Ca2+过载和内质网应激之后,可通过凋亡和类凋亡诱导细胞死亡,同时,触发保护性的自噬。¹²⁵I粒子辐射诱导的细胞凋亡、类凋亡和自噬在很大程度上由ROS介导。目的:评估¹²⁵I粒子联合分节式全覆膜支架治疗食管癌伴吞咽困难患者的可行性、安全性和初步疗效。方法:通过将¹²⁵I粒子与分节式全覆膜SEMS结合,我们设计出了一种新型全覆盖内照射支架,并应用于临床。2017年6月至2019年1月,在同一家医院连续招募食管癌伴吞咽困难患者,进行该新型内照射支架的治疗。收集并分析相关临床数据,包括技术成功率、临床成功率、总生存期、支架通畅时间、支架再狭窄（组织/肿瘤增生）、支架移位和不良事件（CTCAE v4.0）。结果:共计39名患者（31名[79.5%]男性,平均年龄71.3±7.4岁）接受了该支架的置入。技术成功率为97.4%（38/39）,临床成功率为100.0%（39/39）。吞咽困难评分在术后1周内明显下降（P<0.001）,6个月内维持在相对较低的水平。患者中位生存期为201（95%CI 173²28）天,3月和6月的累积生存率分别为87.2%和56.4%。中位支架通畅时间为175（95%CI 128²22）天。5名（12.8%）患者发生了支架再狭窄。4名（10.3%）患者发生了支架移位,所有移位支架均成功移除。常见不良反应包括胸痛（59.0%）、食管出血（28.2%）和恶心呕吐（20.5%）。8名（20.5%）患者共发生了8例严重（等级≥3）不良事件。结论:¹²⁵I粒子联合分节式全覆膜支架置入术治疗食管癌安全、有效。该治疗模式有望延长患者的生存期和支架通畅时间。目的:比较¹²⁵I粒子联合分节式全覆膜支架与其联合部分覆膜支架在食管癌患者治疗中的疗效差异,并评估预后影响因素。方法:回顾性分析2012年1月至2019年1月行分节式全覆膜内照射支架（全覆膜支架组,77例）或部分覆膜内照射支架（部分覆膜支架组,69例）治疗的146例食管癌患者数据。根据技术成功率、临床成功率、总生存期、支架通畅时间、复发性吞咽困难和不良事件（CTCAE v4.0）对结果进行分析。使用log-rank检验和Cox比例风险模型评估总生存期和支架通畅时间。复发性吞咽困难可分为支架再狭窄和支架移位,通过计算原因别风险比（CSHR）的Cox比例风险回归模型和计算部分分布风险比（SHR）的FineGray回归模型进行分析。结果:全覆膜支架组的技术成功率为97.4%（75/77）,部分覆膜支架组的技术成功率为98.6%（68/69）（P>0.999）。两组的临床成功率均为100.0%。全覆膜支架组和部分覆膜支架组的中位生存时间相当（164天比152天;P=0.382）。术前吞咽困难评分4分（HR 1.624,95%CI 1.114².369,P=0.012）和远处转移（HR 5.752,95%CI 3.538⁹.351,P<0.001）是生存的独立危险因素。全覆膜支架具有延长支架通畅时间的趋势（143天比113天;P=0.057）。全覆膜支架组和部分覆膜支架组在吞咽困难复发率方面差异无统计学意义（24.7%比36.2%;SHR 0.676,95%CI 0.371¹.233,P=0.202）,Cox模型显示全覆膜支架组的累积发生风险更低（CSHR 0.529,95%CI 0.286⁰.977,P=0.042）。与部分覆膜支架相比,全覆膜支架能显著降低支架再狭窄率（14.3%比29.0%;CSHR 0.387,95%CI 0.185⁰.810,P=0.012;SHR 0.446,95%CI 0.215⁰.927,P=0.031）。在多因素分析中,支架类型（全覆膜支架比部分覆膜支架;CSHR 0.377,95%CI 0.179⁰.794,P=0.010;SHR 0.443,95%CI 0.212⁰.925,P=0.030）和支架直径（20mm比16 mm;CSHR3.920,95%CI 0.851¹8.063,P=0.080;SHR 4.479,95%CI 1.028¹9.515,P=0.046）是支架再狭窄的独立影响因素。全覆膜支架组和部分覆膜支架组在支架移位率方面差异无统计学意义（13.0%比7.2%;CSHR 1.674,95%CI 0.0.572⁴.897,P=0.347;SHR 1.852,95%CI 0.636⁵.398,P=0.259）。全覆膜支架组与部分覆膜支架组相比,胸痛较为少见（54.5%比71.0%;P=0.040）,两组在其他不良事件发生率方面无显著差异（P>0.05）。结论:在食管癌伴吞咽困难患者的治疗中,分节式全覆膜内照射支架在生存获益和安全性方面与部分覆膜内照射支架表现相当。相较于部分覆膜内照射支架,分节式全覆膜内照射支架能显著降低支架再狭窄率,具有延长支架通畅时间的潜能。更多还原

【Abstract】 Background: Esophageal cancer is the seventh most common cancer and the sixth leading cause of cancer-related mortality worldwide.In China,over 95% of patients with esophageal cancer have esophageal squamous cell carcinoma（ESCC）,and most of them are diagnosed with an advanced stage,so not eligible for surgery.Dysphagia is the major symptom of patients with unresectable esophageal cancer,and these patients should be considered for palliative treatment.Placement of a self-expandable metallic stent（SEMS）and intraluminal brachytherapy are wellestablished procedures for the palliative treatment of malignant dysphagia.In recent years,brachytherapy via radioactive iodine-125(¹²⁵I)seeds,a kind of continuous low-dose-rate irradiation,has been applied to the treatment of various unresectable or locally recurrent cancers.A series of studies reported that the combination therapy of ¹²⁵Iseed brachytherapy and partially covered SEMS placement shows good tumor control and survival benefits in patients with advanced esophageal cancer.In the past few years,various fully covered SEMSs have been designed and used in patients with advanced esophageal cancer,especially if patients have a longer life expectancy,and they are receiving additional palliative therapy and require stent removal.Theoretically,for patients with longer life expectancy,¹²⁵I seeds in combination with a fully covered stent would be more suitable than those with a partially covered stent.Besides,although many studies focused on the efficacy of the stent loaded with ¹²⁵Iseeds,the anti-cancer mechanisms of ¹²⁵Iseed radiation in ESCC have yet not been studied.In this study,firstly,we systematically investigated the effects and anti-cancer mechanisms of ¹²⁵I seed radiation in ESCC cells in vitro and in vivo.Next,we designed a novel fully covered irradiation stent through combining ¹²⁵I seeds and segmented fully covered SEMS,and investigated the feasibility,safety,and preliminary efficacy of this irradiation stent through the clinical trial.Objective: To investigate the effects and mechanisms of ¹²⁵I seed radiation on human ESCC cells（Eca-109 and KYSE-150）.Methods: Cells were exposed to the cumulative radiation dose of 0,2,4,6,and 8 Gy in the in vitro irradiation model.In certain experiments,the cells were treated with si RNA,N-Acetyl-Lcysteine（NAC）,or cycloheximide.Colony formation and trypan blue assays were used to assess cell proliferation and viability.Flow cytometry was used to assess cell cycle,apoptosis,reactive oxygen species（ROS）,and intracellular Ca²⁺ levels.Western blot and immunofluorescence were used to analyze DNA damage,caspase activation,autophagy,and endoplasmic reticulum（ER）stress.Changes of morphology and ultrastructure were investigated by light and transmission electron microscopy.In animal experiments,mice bearing Eca-109 and KYSE-150 ESCC xenograft were treated with ¹²⁵I seed implantation（0.8 m Ci）.Changes in tumor volume and weight were recorded.Histology assays,including H&E staining,ROS fluorescence staining,TUNEL,and immunohistochemistry,were performed.Results: ¹²⁵I seed radiation significantly inhibited cell proliferation,and induced DNA damage and G2/M cell cycle arrest in both cell lines.¹²⁵I seed radiation induced cell death through both apoptosis and paraptosis.After irradiation,Eca-109 cells were primarily killed by inducing caspase-dependent apoptosis,with a peak value at 6 Gy.KYSE-150 cells were eliminated by inducing both apoptosis and paraptosis,which is characterized by extensive cytoplasmic vacuolation.¹²⁵I seed radiation induced autophagic flux in both cell lines,and autophagy inhibition by si ATG5 enhanced radiosensitivity.Moreover,¹²⁵I seed radiation induced intracellular Ca²⁺ overload and ER stress in both cell lines.Furthermore,in both cell lines,¹²⁵I seed radiation induced ROS overproduction,and ROS scavenger,NAC,significantly attenuated the effect of ¹²⁵I seed radiation on ER stress,autophagy,apoptosis,and paraptotic vacuoles.Animal experiments showed that ¹²⁵I seeds radiation induced ROS generation,triggered cell apoptosis and potential paraptosis,and inhibited cell proliferation and tumor growth.Conclusions: In ESCC cells,¹²⁵I seed radiation induces cell death through both apoptosis and paraptosis,following induction of DNA damage,G2/M cell cycle arrest,intracellular Ca²⁺ overload,and ER stress.Meanwhile,it triggers protective autophagy.¹²⁵I seed radiationinduced apoptosis,paraptosis,and autophagy are mediated considerably by ROS.Objective: To assess the feasibility,safety,and preliminary efficacy of ¹²⁵I seeds in combination with segmented fully covered stents in patients with dysphagia caused by esophageal cancer.Methods: Through reasonably combining ¹²⁵I seeds and segmented fully covered SEMS,a novel fully covered irradiation stent was designed and used in the clinic.Between June 2017 and January 2019,consecutive patients with dysphagia caused by esophageal cancer were recruited for treatment with this novel irradiation stent at a single hospital.Data on technical success,clinical success,overall survival,stent patency,tissue/tumor growth,stent migration,and adverse events（CTCAE v4.0）were collected and analyzed.Results: A total of 39 patients（31 [79.5%] men,mean age of 71.3±7.4 years）received treatment with this irradiation stent.The technical success rate was 97.4%（38/39）,and the clinical success rate was 100.0%（39/39）.Dysphagia scores decreased significantly within the first week（P<0.001）,and remained at a relatively low level thereafter.The median overall survival was 201 days（95% CI 173-228）,and the 3-and 6-month cumulative survival rates were 87.2% and 56.4%.The median stent patency period was 175 days（95% CI 128-222）.Tissue/tumor growth was observed in 5（12.8%）patients.Stent migration was observed in 4 patients（10.3%）,and all migrated stents were removed successfully.The common adverse events were chest pain（59.0%）,hemorrhage（28.2%）,and nausea（20.5%）.A total of 8 serious events（grade ≥3）occurred in 8（20.5%）patients.Conclusions: The combination therapy of ¹²⁵Iseeds and placement of a segmented fully covered stent is safe and effective for esophageal cancer.It may prolong the overall survival and stent patency period.Objective: To compare differences in efficacy between ¹²⁵I seeds in combination with a fully covered stent and those with a partially covered stent in patients with esophageal cancer,and assess prognostic factors.Methods: Data of 146 patients who underwent fully covered irradiation stent（FCIS;n=77）or partially covered irradiation stent（PCIS;n=69）placement for esophageal cancer from January 2012 to January 2019 were retrospectively analyzed.Outcomes were measured in terms of technical success,clinical success,overall survival,stent patency,recurrent dysphagia,and adverse events（CTCAE v4.0）.Overall survival and stent patency were evaluated using the logrank test and the Cox proportional hazards model.Recurrent dysphagia,subdivided into tissue/tumor growth and stent migration,was analyzed by both Cox proportional hazards regression on the cause-specific hazard ratio（CSHR）and Fine-Gray regression on subdistributional hazard ratio（SHR）.Results: The technical success rate was 97.4%（75/77）in the FCIS group and 98.6%（68/69）in the PCIS group（P>0.999）.The clinical success rate was 100.0% in both groups.The median overall survivals were comparable between the FCIS and PCIS groups（164 days vs.152 days;P=0.382）.A dysphagia score of 4（HR 1.624,95% CI 1.114-2.369,P=0.012）and metastasis（HR 5.752,95% CI 3.538-9.351,P<0.001）were independent risk factors for survival.A tendency towards a longer stent patency period was seen in the FCIS group（143 days vs.113 days;P=0.057）.There was no statistically significant difference in the recurrent dysphagia rate between the FCIS and PCIS groups（24.7% vs.36.2%;SHR 0.676,95% CI 0.371-1.233,P=0.202）,but lower cumulative hazard was found in the FCIS group（CSHR 0.529,95% CI 0.286-0.977,P=0.042）.Compared with PCISs,FCISs were associated with a decrease in tissue/tumor growth rate（14.3% vs.29.0%;CSHR 0.387,95% CI 0.185-0.810,P=0.012;SHR 0.446,95% CI 0.215-0.927,P=0.031）.In the multivariate analysis,stent types（FCIS vs.PCIS;CSHR 0.377,95% CI 0.179-0.794,P=0.010;SHR 0.443,95% CI 0.212-0.925,P=0.030）and stent diameter（20 mm vs.16 mm;CSHR 3.920,95% CI 0.851¹8.063,P=0.080;SHR 4.479,95% CI 1.028¹9.515,P=0.046）were independent factors for tissue/tumor growth.Stent migration rates were statistically comparable between the FCIS and PCIS groups（13.0% vs.7.2%;CSHR 1.674,95% CI 0.0.572-4.897,P=0.347;SHR 1.852,95% CI 0.636-5.398,P=0.259）.Chest pain was less common in the FCIS group than that in the PCIS group（54.5% vs.71.0%;P=0.040）.No significant differences were observed in the rates of other adverse events（P>0.05）.Conclusions: For patients with dysphagia caused by esophageal cancer,FCIS can provide survival benefits and safety comparable to those of a PCIS.Compared with the PCIS,FCIS is more successful in preventing tissue/tumor growth,and it could prolong the stent patency period.更多还原