原发性肝癌三维适形放疗的临床研究及大鼠肝脏照射后肝再生的实验研究

【作者】 任志刚；

【导师】 蒋国梁； 刘鲁明； 张小健； 欧周罗；

【作者基本信息】 复旦大学 ， 肿瘤学， 2009， 博士

【摘要】 第一部分CT/MRI图像融合技术应用于原发性肝癌三维适形和调强放疗中靶区的确定目的:探讨原发性肝癌（HCC）病人CT与MRI图像融合的精度,以及如何确定大体肿瘤体积（GTV）。材料与方法:13例HCC病人行深吸气末屏气模拟CT定位扫描及呼气末MRIT2相扫描,其中6例病人又加作深吸气末屏气快速MRIT2相（MRIT2F）扫描。互信息法进行图像融合,自动匹配后手动微调。图像融合精度的评价指标为CT和MRI图像上骨性标记点在融合图像上两者间的距离(D_CT-MRIT2,D_CT-MRIT2F)及肝脏交叠度P-LIVER_{CT-MRIT2（CT-MRIT2F）}。两位医师独立在CT及MRI图像上勾画GTV,分别计算依据CT及MRI勾画的GTV的体积、相互交叠的体积及融合后的总体积,并分别计算CT上GTV体积及MRI上GTV体积占融合后总体积的百分比。结果:D_CT-MRIT2及D_CT-MRIT2F分别为2.7mm±0.8mm及2.1mm±0.9mm。CT与MRIT2及MRIT2F的肝脏交叠度分别为85.0%±4.1%及92.7%±1.5%。两位医师在MRI上勾画的GTV差异非常小,但在CT上勾画GTV的差异有统计学意义。CT、MRIT2及MRIT2F上GTV的体积分别为387cm³±396cm³、488cm³±461cm³及597cm³±541cm³。CT及MRIT2上GTV的体积分别占融合后总体积的66.2%±13.5%及88.7%±10.2%,CT及MRIT2F上GTV的体积分别占融合后总体积的71.3%±12.7%及93.5%±4.8%。结论:用作CT和MRI融合的图像的采集尽可能在同一呼吸时相,保持同一体位,采用同样的扫描层厚和层间距。用自动配准软件,采用归一化的互信息法进行自动配准。用于放疗计划的GTV应该是CT和MRI图像上勾画GTV体积的总和。第二部分原发性肝癌三维适形放疗的Ⅰ期临床试验目的:确定三维适形及调强放疗（3DCRT/IMRT）应用于原发性肝癌（HCC）的最大耐受剂量（MTD）。材料及方法:入组标准为病理证实或临床诊断的HCC,单个病灶,直径大于5cm,不能行手术切除,无肝内播散和/或远处转移,肝硬化Child-Pugh A级。按肿瘤最大径分为2组:GroupⅠ（最大径＞5cm＜10cm）及GroupⅡ（最大径≥10cm）。GroupⅠ起始剂量46Gy,至最大剂量62Gy;GroupⅡ起始剂量40Gy,至最大剂量52Gy。常规分割,每次递增4Gy。剂量限制性毒性为≥3级急性肝脏、胃肠道毒性,任何5级的治疗相关性毒性或晚期放射性肝损伤（RILD）。结果:自2005年4月至2008年5月,共40例病人入组,其中GroupⅠ及GroupⅡ各20例。根据2006年AJCC分期,T2期20例,T3期17例,T4期3例。最后一随访时间为2009年1月,总的中位随访时间13个月,其中GroupⅠ为17.5个月,GroupⅡ为9个月。GroupⅠ中共发生11例（55.5%）急性治疗相关性毒副反应,其中1级9例,2级2例。GroupⅡ中共发生10例（50%）急性治疗相关性毒副反应,其中1级8例,2级2例。所有病人中,仅在GroupⅡ中的52Gy剂量水平在治疗后2个月发生1例非典型的RILD,经过2周对症治疗后死于肝衰竭。4例病人发生射野内复发,21例出现肝内播散,5例出现远处转移。2年局部控制率为90%,1、2年生存率分别为72%及62%。结论:ABC控制下的3DCRT/IMRT应用于局部进展期的HCC病人,肿瘤最大径＞5cm＜10cm的病人可以耐受62Gy,≥10cm的病人可以耐受52Gy,局部控制率令人满意。第三部分大鼠肝脏照射后肝再生的实验研究目的:对大鼠进行肝脏照射,检测受照射的肝脏是否出现再生现象,探讨再生的时间规律以及参与的细胞因子的作用。材料与方法:实验分为2部分:第1部分为将SD大鼠按处死时间不同随机分为5组,每组6只动物,分别为假照射组（0天）、照射后30天组、60天组、90天组和120天组。采用6MV-X射线单次照射,大鼠右半肝照射25Gy,左半肝照射5Gy。第2部分SD大鼠均为60天处死,随机分为5组,分别为假照射组（0Gy）,其余为右半肝照射25Gy,按左半肝照射剂量不同分为2.5Gy组、5Gy组、7.5Gy组及10Gy组,每组6只动物。分别在照射后相应时间点处死大鼠,取各组大鼠血清,ELISA法检测肝细胞生长因子（HGF）,酶法检测谷丙转氨酶（ALT）、谷草转氨酶（AST）及前白蛋白（PA）。HE染色检测左半肝核分裂相数,免疫组化测定PCNA及TGF-β1,实时荧光定量PCR测左右半肝HGFmRNA、TGF-β1mRNA及PCNAmRNA,流式细胞术检测左半肝增殖指数。结果:右半肝照射25Gy,左半肝照射5Gy大鼠在照射后60天,ALT和AST升高至峰值,PA降至最低,分别为正常大鼠的3.2倍、2.6倍及0.4倍,肝损伤最严重。ELISA示大鼠血清HGF在照射后30天升至峰值,与假照射组（0天）、照射后60天组、照射后90天组和照射后120天组比较有显著性差异（P＜0.05）。在5Gy照射区内,HGFmRNA及TGF-β1mRNA均在照射后30天升至峰值,PCNAmRNA及增殖指数表达峰值出现在照射后60天,有丝分裂相肝细胞数在照射后90天最多。在25Gy照射区内,HGFmRNA及TGF-β1mRNA均在照射后30天升至峰值,PCNAmRNA表达峰值出现在照射后90天。右半肝照射25Gy,左半肝照射10Gy组在1周至两周之间死亡4只。在左半肝照射2.5Gy至7.5Gy各组中,AST及ALT随剂量增加而增加,差异有统计学意义（P＜0.05）,但PA值差异无统计学意义（P＞0.05）。PCNAmRNA及TGF-β1mRNA随剂量增加表达增强,差异有统计学意义（P＜0.05）。有丝分裂相肝细胞数随剂量增加各组间差异无统计学意义。照射5Gy及7.5Gy肝细胞增殖指数差异无统计学意义（P＞0.05）,但均明显高于照射2.5Gy肝细胞（P＜0.05）。结论:照射25Gy的肝脏仍有再生的潜能;照射使再生的肝组织发生细胞周期阻滞,肝再生进程被延迟;大鼠无法耐受右半肝照射25Gy,左半肝10Gy,肝再生来不及代偿急性肝损伤。更多还原

【Abstract】 Part 1:CT and MRI image fusion in delineation of gross tumor volume for 3-D conformal and intensity modulated radiation therapy for hepatocellular carcinomaObjective:To investigate the accuracy of image registration with computed tomography（CT） and magnetic resonance imaging（MRI） and how to determine GTV for HCC.Material and Methods:Thirteen patients were enrolled in this study.The CT image was taken at inhalation phase,and MRIT2,at the time of peak exhalation. Additional image was taken by fast scanning of MRIT2（MRIT2F） at peak inhalation in six patients.After mutual information method to CT/MRI image registration, manual adjustment was made to optimize the accuracy of image fusion.The GTV and liver in each patient were independently contoured by two observers on CT,MRIT2 and MRIT2F images.The accuracy of image fusion was assessed by the distance between bone markers(D_CT-MRIT2,D_CT-MRIT2F) of CT and MRI on the fused image,and the ratio of liver overlap(P-LIVER_CT-MRIT2,P-LIVER_CT-MRIT2F).The measured were volumes of GTV contoured on CT(V-GTV_CT),or on MRIT2(V-GTV_MRIT2),or on MRIT2F(V-GTV_MRIT2F),their overlap(V-GTV_CT-MRIT2,V-GTV_CT-MRIT2F) and their composite volume(V-GTV_CT+MRIT2,V-GTV_CT+MRIT2F).The percentage of V-GTV_CT and V-GTV_MRIT2 on V-GTV_CT+MRIT2,V-GTV_CT and V-GTV_MRIT2F on V-GTV_CT+MRIT2F, V-GTV_CT-MRIT2 and V-GTV_CT-MRIT2F on V-GTV_CT was also calculated,respectively.Results:Accuracy of image fusion was the mean D_CT-MRIT2 of 2.7 mm±0.8mm and D_CT-MRIT2F of 2.1mm±0.9mm.The mean P-LIVER_CT-MRIT2 and P-LIVER_CT-MRIT2F were 85.9%±4.1%and 92.7%±1.5%.Interobserver difference was small for GTV defined by MRIT2,but there was significant difference for GTV defined by CT between two observers.V-GTV_CT,V-GTV_MRIT2 and V-GTV_MRIT2F were 387cm³±396cm³,488cm³±461cm³ and 597cm³±541cm³,respectively.The percentage of V-GTV_CT and V-GTV_MRIT2 on V-GTV_CT+MRIT2 was 66.2%±13.5%and 88.7%±10.2%,V-GTV_CT and V-GTV_MRIT2F on V-GTV_CT+MRIT2F was 71.3%±12.7% and 93.5%±4.8%,respectively.Conclusions:The fusion image of CT and MRI should be obtained at the same respiratory phase,in the same treatment position and with the same slice thickness and separation.Automatic registration was carried out by mutual information method using automatic registration software.When contouring GTV of HCC,CT and MRI image should be integrated.GTV used to be planned should be the sum of CT-defined GTV and MRI-defined GTV. Part 2:PhaseⅠtrial of radiation dose escalation in patients with locally advanced hepatocellular carcinomaPurpose:To determine the maximum tolerated dose（MTD） of 3-dimensional conformal radiation therapy（3DCRT）/intensity modulated radiation therapy（IMRT） for locally advanced hepatocellular carcinoma（HCC）.Materials and Methods:Inclusion criteria were as follows:pathologically confirmed or clinically diagnosed HCC,surgically unsectable or medically inoperable diseases,solitary intrahepatic lesion with diameter bigger than 5 cm,without extrahepatic and/or distant metastases,associated with cirrhosis of Child-Pugh A.The patients were divided into 2 subgroups:tumor diameter more than 5cm and less than 10cm（GroupⅠ） and larger than 10cm（GroupⅡ）.The starting radiation doses were 46Gy and 40Gy in 2Gy per fraction for GroupⅠand GroupⅡ,and up to a chosen maximum of 62 Gy and 52 Gy with 4.0 Gy increments of each cohort.Dose-limiting toxicity（DLT） was defined as acute≥Grade 3 liver,gastrointestinal toxicity,or any grade 5 treatment-related adverse event,or late complication of radiation-induced liver disease（RILD）.Results:From April 2005 to May 2008,a total of 40 HCC patients were enrolled in this study（20 in GroupⅠand 20 in GroupⅡ）.According to AJCC staging（2006）, there were 20 cases of T2（Ⅱstage）,17 cases of T3（ⅢA stage）and 3 cases of T4（ⅢB stage）,respectively.The last follow-up was performed in January 2009 with the median follow-up time of 17.5months for GroupⅠand 9 months for GroupⅡ.The total median follow-up time was 13 months for all patients.Total 11（55.5%） patients （9 Grade 1 and 2 Grade 2） occurred acute treatment-related toxicities in GroupⅠand 10（50%） patients（8 Grade 1 and 2 Grade 2） in GroupⅡ.For all patients,only one nonclassic RILD was observed in 52Gy dose level of GroupⅡ,which occurred two months after completion of radiation treatment.After two weeks of symptomatic treatments,the patients died of hepatic failure eventually.4 patients developed in-field recurrence;21 patients had intrahepatic spreading;5 patients had extrahepatic metastases.The 2-year local control rate for all the patient was 90%.The 1,2 year overall survival rates was 72%and 62%for all the patients.Conclusions:The radiation dose was safely escalated in locally advanced HCC patients by using 3D-CRT/IMRT and ABC to 62Gy for patients with tumor diameter less than 10cm and 52Gy for tumor diameter equal or more than 10 cm under strict normal liver DVH constraints.The local control is satisfied with acceptable toxicities. Part 3:An experimental study of liver regeneration after liver irradiation on rats.Objective:To examine whether irradiated liver has the ability to regenerate after liver irradiation,and to investigate the timing of liver regeneration and the roles of participated cytokines on irradiated liver of rats.Material and Methods:Two parts were included in this study（PartⅠand PartⅡ）.In PartⅠ,rats were randomly divided into 0-day-post radiation（sham-radiation）, 30-day-post-radiation,60-day-post-radiation,90-day-post radiation and 120-day-post-radiation group according to time of sacrifice on rats,and each group had six rats.The right half liver was irradiated with a single fraction dose of 25Gy and left half with 5Gy using 6MV-X ray.In PartⅡ,all rats were sacrificed at the time of 60 days post irradiation and randomly divided into 5 groups.Sham-radiation group（0Gy） was also included.Other rats irradiated to right half liver with dose of 25Gy were randomly divided into 2.SGy,5Gy,7.5Gy,10Gy group according to the dose irradiated to left half liver,and each group also had six rats.All the rats were sacrificed at the scheduled time in each group of the two parts.Blood serum,right and left half liver tissue were collected.Hepatocyte growth factor（HGF）,Alanine aminotranferease（ALT）,aspartate aminotransferase（AST） and Prealbumin（PA） in blood serum were measured in each group.Mitotic index in liver tissue was assessed by H.E.staining.The expression of proliferation cell nuclear antigen（PCNA） and transforming growth factor-β1（TGF-β1） were confirmed by immunohistochemical staining.HGFmRNA,PCNAmRNA and TGF-β1mRNA were analysed quantificationally by real-time PCR.Proliferation index was determined by flow cytometry（FCM）.Results:ALT and AST in blood serum of rats irradiated 25Gy to right half liver and 5Gy to the left escalated to their peak values at the time of 60 days after irradiation,and were 3.2 and 2.6 folds of normal level,respectively.PA in blood serum dropped to its peak value at the time of 60 days after irradiation,and was 0.4 folds of normal level.HGF in blood serum reached its peak at the time of 30 days after irradiation.Comparing to sham-radiation（0 day）,60-day-post radiation, 90-day-post radiation and 120-day-post-radiation group,the differences were all significant（P＜0.05）.In the liver tissue irradiated 5Gy,HGFmRNA and TGF-β1mRNA increased to their peak values at the time of 30 days after irradiation, PCNAmRNA and Proliferation index at the time of 60 days after irradiation and mitotic index at the time of 90 days after irradiation.In the liver tissue irradiated 25Gy,HGFmRNA and TGF-β1mRNA augmented to their peak values at the time of 30 days after irradiation and PCNAmRNA at the time of 90 days after irradiation. Four rats in the group of right half liver irradiated 25Gy and left half 10Gy died between the first and the second week.In groups irradiating left half liver form 2.5Gy to 7.5Gy,the expression of AST,ALT,PCNAmRNA and TGF-β1mRNA increased significantly with the dose escalation（P＜0.05）.But there were no significant difference of PA and Mitotic index between groups mentioned above.Proliferation index in groups irradiating left half liver 5Gy and 7.5Gy showed no significant difference（P＞0.05）,but both were higher than that in group irradiating 2.5Gy （P＜0.05）.Conclusions:The liver tissue irradiated 25Gy still has regenerative potential; Irradiation can block the cell cycle of regenerative liver tissue and delay the course of liver regeneration;Rats can not tolerate the dose of 25Gy irradiated to right half liver and 10Gy to left half liver,and liver regeneration can not compensate for the acute injury of liver function in time.更多还原