【作者】 熊曾；

【导师】 胡成平；

【作者基本信息】 中南大学 ， 临床医学， 2012， 博士

【摘要】 目的：研究者发现厄洛替尼能引起裸鼠移植瘤血管构筑的变化,我们应用最新的容积灌注CT (volume perfusion computer tomography,VPCT)技术观察肺癌患者肿瘤全病灶血管灌注功能的变化,结果发现肺癌患者吉非替尼治疗前后VPCT灌注参数也会发生变化,这些证据表明直接作用于肺癌细胞的表皮生长因子受体酪氨酸激酶抑制剂(Epidermal growth factor receptor-tyrosine kinase inhibitor, EGFR-TKI)也可能同时影响肺癌的血管灌注水平。基于此,本课题分别在裸鼠肺癌移植瘤及肺癌患者中研究VPCT与血管构筑的关系,观察肺癌患者吉非替尼治疗前后VPCT灌注参数的变化,以此来判断EGFR-TKI的疗效,为寻求新的EGFR-TKI疗效评价指标提供技术支持与理论依据。方法：1)建立荷A549裸鼠移植瘤模型,随机分成贝伐单抗组和空白对照组,每组于d0,2,4,6,10随机选取6-8只小鼠,获得VPCT灌注参数[包括最高强化值(peak enhancement image, PEI)和血流量(blood flow,BF)]后立即处死,检测血管构筑指标,来评价移植瘤“血管正常化”和“血管稳态”水平的变化。评价“血管正常化”的指标包括周细胞覆盖率(microvessel pericyte coverage index,MPI)评价血管结构、电镜评价血管功能、乏氧标志物Pimonidazole检测评价血管效应。评价“血管稳态”的指标包括血管成熟指数(vessel maturity index,VMI)、血管生成调控因子平衡性、肿瘤细胞稳定性、电镜观察肿瘤与血管的相互关系。采用影像-病理对照研究方法在裸鼠移植瘤中观察CT灌注参数与血管构筑指标的关系。2)48例肺癌患者分别行容积灌注CT (VPCT)和多层螺旋CT灌注成像(multi-slice spiral comuted tomography perfusion, MS-CTP)后获取病理标本,检测血管构筑相关指标(微血管MPI和VMI、肿瘤增殖和凋亡),采用影像-病理对照研究方法在肺癌患者中探讨CT灌注参数与血管构筑的关系。3)18例行VPCT的肺癌患者进行吉非替尼单药靶向治疗及随访观察,吉非替尼治疗前及每治疗2周期(6周)后再次行VPCT获得治疗前后的灌注参数,同时用实体瘤治疗反应评价标准1.1(Response Evaluation Criteria in Solid Tumors,RECIST)评价疗效,探讨靶向治疗6周后VPCT灌注参数变化与RECIST评分的关系来评价近期疗效,探讨靶向治疗前后VPCT灌注参数的变化与中期疗效评价指标无疾病进展时间(progress free survival,PFS)的关系来评价中期疗效。结果：1)裸鼠肺癌移植瘤VPCT与微血管密度(microvessel density,MVD)及血管成熟度的关系：贝伐单抗处理4天后肿瘤血流量BF明显增高(P<0.01),微血管成熟度(周细胞覆盖率MPI和血管成熟指数VMI)明显增高(P<0.01),处理10天后肿瘤BF与微血管成熟度均下降到处理前水平(P>0.05),贝伐单抗处理后肿瘤最高强化值PEI与MVD均下降(P<0.01)。2)裸鼠肺癌移植瘤VPCT与“血管正常化”观察指标的关系：“血管正常化窗口期”发生在贝伐单抗处理后2-6天,“窗口期“时MPI增高,内皮细胞与周细胞连接紧密,基底膜完整性修复,肿瘤微环境乏氧改善,血流量BF增高。3)裸鼠肺癌移植瘤VPCT与“血管稳态”观察指标的关系：贝伐单抗处理后2-6天,肿瘤血管生成调控因子平衡性和肿瘤细胞稳定性增加,血流量BF增高。4)肺癌患者CT灌注参数与血管构筑的关系：血流量BF与微血管成熟度呈明显正相关(r为0.68,P为0.000),BF与CD31-MVD呈正相关(r=0.365,P=0.013),最高强化值PEI与微血管成熟度无明显相关(P均>0.05),PEI与CD31-MVD呈正相关(P<0.05)。CT灌注参数与肺癌增殖与凋亡均无明显关系(P均>0.05)。5) VPCT评价吉非替尼治疗EGFR突变的晚期肺腺癌患者近期疗效的作用：吉非替尼治疗6周后BF值上升的病例近期疗效欠佳。将高分化、中分化和低分化肺癌分别赋值1、2、3,治疗前BF与分化程度呈负相关(r=-0.904,P=-0.000),治疗6周后BF下降率与分化程度呈负相关(r=-0.525,P=0.025),BF下降的患者疗效更好(r=-0.636,P=0.005)。BF变化趋势与分化程度无相关(r=-0.353,P=0.151)。VPCT预测RECIST评价达部分缓解(partial response, PR)的敏感性、特异性、准确性、阳性预测值及阴性预测值分别为77.8%、66.7%、72.2%、70.0%及75.0%。6) VPCT评价吉非替尼治疗EGFR突变的晚期肺腺癌患者吉非替尼中期疗效的作用：按RECIST标准测量病灶体积变化获得的PFS(RECIST-PFS)与使用VPCT测量病灶血管灌注功能变化获得的PFS(VPCT-PFS)呈明显正相关(r=0.761,P=0.004)。VPCT-PFS短于RECIST-PFS (中位PFS12周VS30周),中位领先时间为12周。治疗前BF与中期疗效评价指标无明显关系,治疗2周期(6周)BF下降率与VPCT测得的PFS呈正相关(r=0.584,P=0.046),VPCT预测RECIST评价达疾病进展(progressive disease, PD)的敏感性、特异性、准确性、阳性预测值及阴性预测值分别为92.3%、60.0%、83.3%、85.7%及75.0%。结论：1) VPCT可以动态观察裸鼠肺癌移植瘤血管构筑的变化,并以此来观察裸鼠肺癌移植瘤的“血管正常化”现象和评价“血管稳态”水平；2)在裸鼠肺癌移植瘤及肺癌患者的影像病理对照试验中分别证实VPCT可以有效评价肺癌的血管构筑状态,灌注参数BF与PEI具有不同的病理基础；3)肺癌患者吉非替尼治疗后靶病灶血管灌注功能的变化早于体积的变化,VPCT灌注参数的变化是评价EGFR-TKI疗效的理想指标。更多还原

【Abstract】 ObjectiveThe researcher found that erlotinib change the vasculature in nude mice transplanted tumor. We also found the volume perfusion CT (VPCT) parameters will be changed before and after the Gefitinib therapy in patients with lung cancer. These evidences suggest EGFR-TKI not only act directly on the tumor cell, may also has an effect on the vascular perfusion in lung cancer. Based on these, this paper discussed the relationships between the VPCT and the vascular architecture in nude mice transplantation tumors and patients with lung cancer respectively. Then the changes of VPCT parameters in lung cancer patients before and after treatment of Gefitinib were observed to determine the response of EGFR-TKI therapy. This paper tried to seek a new index to evaluate the effect of EGFR-TKI therapy, and aimed to provide technical support and theoretical basis of this new index.Methods(1) Nude mice transplanted tumors were randomly divided into bevacizumab group and blank control group.6-8mice were randomly selected in the day0,2,4,6and10in each group. After VPCT perfusion parameters (PEI and BF) were acquired, the mice were sacrificed immediately, then the indexes of vascular architecture were detected to evaluate the level of "vascular normalization" and "vascular homeostasis". The indexex of "vascular normalization" include vascular structure (MPI), vascular function (electron microscopic) and vascular effects (Pimonidazole expression). The indexes of "vascular homeostasis" include vascular maturation index (VMI), the balance of angiogenesis regulation factor, the stability of tumor cell and the relationship between tumor and vascular. Then we observed the relationships between CT perfusion parameters and pathological indexes by using image-pathologic correlation method in mice.(2)48patients with lung cancer received CT perfusion scan (including VPCT and MS-CTP) and pathological biopsy, the indexes of vascular architecture (MPI, VMI, tumor proliferation and apoptosis) were detected. The CT perfusion parameters were compared with pathological indexes by using image-pathologic correlation method in lung cancer patient.(3)18patients received VPCT scan, then followed by Gefitinib therapy and follow-up observation. VPCT was repeated and the therapy effect was evaluated with RECIST criteria. The VPCT parameters of18patients were compared with their short term effect and middle term effect.Resluts:(1) The relationship between VPCT and micro vessel density/maturation in nude mice:Tumor blood flow (BF) and tumor microvascular maturity (MPI and VMI) were increased significantly in day4(P<0.01), and down to the preoperative level in day10(P>0.05), however, PEI and microvessel density were decreased(P<0.01).(2) The relationship between VPCT and the indexes of "vascular normalization" in nude mice:The "vascular normalization window" occurred in2-6days after bevacizumab treatment. In the window, tumor MPI were increased, Endothelial cells and pericytes were closely connected, the integrity of basement membranes were repaired, hypoxia in tumor microenvironment was improved, tumor blood flow(BF) were increased.(3) The relationship between VPCT and the indexes of "vascular homeostasis" in nude mice:the balance of angiogenesis regulation factor and the stability of tumor cell were improved in2-6days after bevacizumab treatment, tumor blood flow(BF) were increased.(4) The relationship between CT perfusion parameters and vascular architecture in patients with lung cancer:There was a positive correlation between BF and microvascular maturity (r=0.680, P=0.000), and there was a weakly positive correlation between BF and CD31-MVD(r=0.365, P=0.013). There was no correlation between PEI and microvascular maturity (P>0.05), and there was a weakly positive correlation between PEI and CD31-MVD (P<0.05). There was no correlation between CT perfusion parameters and the stability of tumor cell (P>0.05)(5) Evaluating the short term effect of Gefitinib in patients with advanced lung adenocarcinoma by VPCT:Short term effect was poor in the case which the BF increase after6weeks of targeted therapy. BF before therapy was a negative correlated with differentiation grade (r=-0.904, P=0.000).There was a negative correlation between rate of BF decline and the differentiation grade (r=-0.525, P=0.025); There was a negative correlation between the trend of BF and RECIST criteria (r=-0.636, P=0.005), but there was no significant correlation with the differentiation grade(P=0.151).The VPCT can predict the effect of RECIST criteria, the sensitivity, specificity, accuracy, positive predictive value and negative predictive value of VPCT is77.8%、66.7%、72.2%、70.0%and75.0%.(6) Evaluating the middle term effect of Gefitinib in advanced lung adenocarcinoma by VPCT:There was a positive correlation between RECIST-PFS and VPCT-PFS (r=0.761, P=0.004); The VPCT-PFS was shorter than RECIST-PFS(the median PFS12weeks vs30weeks), the median lead time was12weeks. There was no relationship between BF before therapy and the middle term effect. There was a positive correlation between rate of BF decline after6weeks of targeted therapy and VPCT-PFS (r=0.584, P=0.046).The VPCT can predict the progress of RECIST criteria, the sensitivity, specificity, accuracy, positive predictive value and negative predictive value of VPCT is92.3%、60.0%、83.3%、85.7%and75.0%.Conclusion:(1) VPCT can be used to evaluate dynamically the changes of vascular architecture, then to evulate the "vascular normalization" and "vascular homeostasis" in nude mice.(2) VPCT is a good technology to evaluate dynamically the changes of vascular architecture by using image-pathologic correlation method in nude-mice transplanted tumor model and patients with lung cancer. VPCT parameters, such as BF and PEI, have different pathological basis.(3) The changes of VPCT parameters is earlier than the change of tumor volume, and the changes of VPCT parameters are ideal indexes to evaluate the effect of EGFR-TKI therapy in lung cancer.更多还原