奥希替尼联合抗血管生成靶向药对人肺腺癌细胞抑制作用的实验研究

目的:通过体外细胞学实验初步探究奥希替尼联合两种不同机制的抗血管靶向治疗药物（贝伐珠单抗或阿帕替尼）治疗表皮生长因子受体（EGFR）敏感突变和T790M耐药突变肺腺癌细胞的抗肿瘤活性及其作用机制。方法:培养人肺腺癌细胞PC-9（E19 del）和H1975（E21 L858R/E20 T790M）,CCK-8法检测奥希替尼及抗血管靶向治疗药物（贝伐珠单抗或阿帕替尼）单药或联合处理肺腺癌细胞48 h后的抑瘤率;蛋白质印迹法检测EGFR及其下游AKT和ERK信号通路蛋白表达情况。结果:PC-9和H1975肺腺癌细胞对奥希替尼敏感且呈剂量依赖性。奥希替尼联合抗血管生成靶向药物（贝伐珠单抗,阿帕替尼）较同等浓度的单药奥希替尼可增加对PC-9和H1975细胞株的抑瘤率（P<0.05）。低浓度奥希替尼联合高浓度阿帕替尼（1 000 nmol/L）的抑制作用与高浓度奥希替尼相当（P>0.05）。随着联合的阿帕替尼浓度的升高,对PC-9和H1975细胞抑瘤率也有一定程度的提高（P<0.05）。不同处理因素对PC-9细胞的抑制率均高于对H1975细胞（P<0.01）。随着奥希替尼浓度的上升,p-EGFR、p-AKT、p-ERK磷酸化蛋白表达逐渐降低。结论:奥希替尼联合贝伐珠单抗或阿帕替尼会进一步增强对EGFR敏感突变或T790M突变肺腺癌细胞的杀伤作用。奥希替尼与阿帕替尼联合使用具有很强的抑瘤活性,具有很好的应用前景。奥希替尼单药或与抗血管形成药联合作用可能是进一步下调EGFR及其下游AKT和ERK信号通路的活化。     

奥希替尼联合贝伐珠单抗治疗14例EGFR突变型肺腺癌脑转移的疗效观察

  目的  观察和评估奥希替尼联合贝伐珠单抗治疗14例EGFR突变型肺腺癌脑转移患者的疗效及安全性。  方法  回顾性分析2017年1月至2019年12月广州市胸科医院收治的伴有EGFR突变的肺腺癌脑转移患者共14例,所有患者均给予奥希替尼及贝伐珠单抗联合治疗,每3周为1个周期,观察其脑转移病灶疗效并评估不良反应。  结果  14例患者中11例（78.6%）脑转移病灶为部分缓解,2例疾病稳定（14.3%）,1例疾病进展（7.1%）,客观缓解率为78.6%（11/14）,疾病控制率为92.9%（13/14）。颅内中位无进展生存期（median progression-free survival,mPFS）为12.5个月（95%CI:8.53~15.87）。最常出现的不良反应为贫血（57.1%）、高血压（50.5%）以及蛋白尿（28.6%）,未出现因不良反应停药事件发生。  结论  奥希替尼联合贝伐珠单抗治疗EGFR突变型肺腺癌脑转移疗效较好,不良反应可控,但仍需大样本临床试验进一步验证。      

低剂量阿帕替尼治疗卡瑞利珠单抗致反应性毛细血管增生症的疗效分析

  目的  评价低剂量阿帕替尼治疗卡瑞利珠单抗致反应性毛细血管增生症（reactive cutaneous capillary endothelial proliferation,RCCEP）的疗效和安全性。   方法  回顾性分析不同抗血管生成药物联合卡瑞利珠单抗治疗晚期实体肿瘤140例的RCCEP发生率。观察250 mg和125 mg阿帕替尼两个剂量组治疗50例单药卡瑞利珠单抗诱发RCCEP的疗效和安全性。  结果  140例晚期实体瘤使用卡瑞利珠单抗联合不同抗血管生成药物的RCCEP发生率:贝伐珠单抗组为93.3%,安罗替尼组为72.7%,呋喹替尼组为47.6%,瑞戈非尼组为70.6%,阿帕替尼组为14.0%。阿帕替尼较其它抗血管生成药物显著降低RCCEP发生率:贝伐珠单抗组 vs. 阿帕替尼组（P<0.000 1）;安罗替尼组 vs. 阿帕替尼组（P<0.000 1）;呋喹替尼组 vs. 阿帕替尼组（P=0.005）;瑞戈非尼组 vs. 阿帕替尼组（P<0.000 1）。观察50例晚期实体瘤不同剂量阿帕替尼治疗卡瑞利珠单抗诱发RCCEP的疗效和安全性,结果提示42例250 mg阿帕替尼治疗组有效率为97.6%,8例125 mg阿帕替尼治疗组有效率为87.5%;两个剂量组均未出现≥G3的治疗相关不良事件。   结论  阿帕替尼较其它抗血管生成药物更显著降低卡瑞利珠单抗的RCCEP发生率,低剂量阿帕替尼可有效安全地缓解RCCEP。      

贝伐珠单抗联合化疗治疗胃肠肿瘤的不良事件观察

  目的  观察贝伐珠单抗（Bevacizumab,AVASTIN,Bev）与化疗药物联合治疗胃肠道肿瘤患者的不良事件,以便合理、安全的使用贝伐珠单抗,避免严重不良事件的发生。  方法  回顾性分析贝伐珠单抗（Bev）联合常规化疗治疗胃肠肿瘤77例,收集整理患者既往史,治疗前及治疗开始直至停止治疗后8周或死亡期间症状、体征和实验室检查,分析其在胃肠肿瘤患者治疗中的安全性。  结果  患者均为不能手术的局部进展期或转移性胃癌和结直肠癌,其中结直肠癌65例,胃癌12例,均采用贝伐珠单抗联合化疗。男性36例,女性41例,中位年龄49岁。77例患者中不良事件发生率为89.6%（69/77）,3/4级不良事件（adverse even,AE）及严重不良事件（serious adverse even,SAE）发生率为26.0%（20/77）。分层分析不同情况下3/4级AE和SAE发生率,高龄组（≥65岁）为44.4%（4/9）,低龄组（< 65岁）为23.5%（16/68）;男性为25%（9/36）,女性为26.8%（11/41）;一线使用化疗联合贝伐珠单抗患者组为30.6%（11/36）,二线及以上使用化疗联合贝伐珠单抗为22.0%（9/41）,均无统计学差异。贝伐珠单抗联合两药化疗方案,其AE均以以血液学毒性和消化道反应（恶性、呕吐）为主,发生率50%~60%。贝伐珠单抗联合单药化疗AE发生率5%~8%。高血压、蛋白尿、出血及伤口愈合不良均为偶发、轻度。SAE 1例。  结论  贝伐珠单抗联合化疗治疗胃肠恶性肿瘤AE发生率无明显增加,性别、年龄以及使用贝伐珠单抗的时机（一线或二线及以上使用）等,其不良事件的发生率均无明显差异,贝伐珠单抗联合化疗治疗胃肠肿瘤患者耐受性良好。      

贝伐珠单抗诱导肿瘤血管正常化的时间窗及与紫杉醇联合治疗肺癌荷瘤鼠的实验研究

目的 探讨贝伐珠单抗诱导肿瘤血管正常化的时间窗及贝伐珠单抗联合紫杉醇对小鼠肺腺癌移植瘤的抑瘤效果。方法 选取成功构建的人肺腺癌A549裸鼠皮下移植瘤模型54只,实验分为两部分：第一部分荷瘤小鼠24只随机分为两组：对照组和贝伐珠单抗组各12只,分别腹腔注射生理盐水和贝伐珠单抗5 mg／kg,于给药后选取第1、3、5、8天共4个时间点,每个时间点各3只,测量瘤体体积及裸鼠体质量,采用Western blotting和免疫荧光法分别检测瘤体内血管内皮生长因子（VEGF）水平和微血管密度（MVD）。第二部分小鼠30只随机分为四组：对照组、紫杉醇单药组和贝伐珠单抗单药组各5只及联合组15只。联合组于贝伐珠单抗给药当天及给药后第3、5天各选取5只腹腔注射紫杉醇,紫杉醇和贝伐珠单抗的剂量分别为3 mg／kg和5 mg／kg,于给药后选取第3、7、10、14、17、20天共6个时间点测量瘤体体积,21天后处死裸鼠称取瘤体质量,采用Western blotting和免疫组化法分别检测瘤体VEGF水平和MVD。结果 在第一部分实验中,与对照组相比,贝伐珠单抗组给药后肿瘤的生长得到抑制,以第三天抑制效应最显著,此时瘤体的体积最小,瘤体内VEGF含量表达减少,瘤体MVD也相应减少。在第二部分实验中,与对照组相比,贝伐珠单抗不同时间点联合紫杉醇给药均可显著抑制肿瘤生长,以贝伐珠单抗联合紫杉醇第三天给药组抑制效应更为显著,且瘤体的体积、质量、VEGF含量及MVD均较其他联合给药组少。结论 贝伐珠单抗诱导的血管正常化时间窗可能在给药后第1～3天,在该时间窗内联合紫杉醇可达到最大的抗肿瘤效应。     

含贝伐珠单抗维持治疗晚期结直肠癌患者疗效与安全性的Meta分析

目的 系统评价含贝伐珠单抗不同联合方案治疗晚期结直肠癌的疗效与安全性。方法 计算机检索PubMed、EMbase、Web of Science、Cochrane Library 数据库截至2017年7月31日有关贝伐珠单抗维持治疗晚期结直肠癌的随机对照试验。按纳入排除标准筛选文献、资料提取和评价质量,采用RevMan 5.3 软件进行Meta 分析。 结果 共纳入8项研究,2 644例晚期结直肠癌患者。Meta分析显示,贝伐珠单抗维持治疗组（包括单药和联合化疗）前者无疾病进展生存期（PFS）和总生存期（OS）均优于无治疗组（HR=0.65,95% CI：0.53~0.78,P<0.001;HR=0.83,95% CI：0.71~0.98,P=0.020）,但前者3~4级感觉神经障碍及高血压发生率高于后者（P=0.001,0.008）。贝伐珠单抗维持治疗组与持续治疗组对比,两者的PFS和OS相当（HR=1.05,95% CI：0.56~1.71,P=0.830;HR=1.11,95% CI：0.92~1.35,P=0.270）,而后者3~4级感觉神经障碍、疲乏和手足综合征明显高于前者（P＜0.001,0.020,0.040）。与单药贝伐珠单抗组比较,贝伐珠单抗抗抗联合厄洛替尼组能改善PFS和OS（HR=0.81,95% CI：0.67~0.96,P=0.020;HR=0.81,95% CI：0.67~0.99,P=0.040）,但贝伐珠单联合厄洛替尼组3~4级皮疹发生率明显高于单药贝伐珠单抗组（P<0.001）。结论 贝伐珠单抗单药、联合低毒化疗药物或厄洛替尼,均可改善晚期结直肠癌患者PFS和OS,患者可耐受,贝伐珠单抗维持治疗可作为晚期结直肠癌维持阶段的优选治疗方案。     

贝伐珠单抗联合化疗对复治晚期非鳞非小细胞肺癌患者的疗效及预后分析

  目的  探讨贝伐珠单抗联合化疗对复治晚期非鳞非小细胞肺癌（non-squamous non-small cell lung cancer,NSNSCLC）患者的疗效和安全性,分析影响预后的因素。  方法  回顾性分析2013年2月至2017年6月北京胸科医院收治的41例复治晚期NSN? SCLC患者的病例资料。其中腺癌38例,其他病理类型3例。19例患者为二线治疗,22例患者为二线以上治疗。表皮生长因子受体（epidermal growth factor receptor,EGFR）突变阳性18例,突变阴性23例。评价贝伐珠单抗联合化疗的疗效和安全性,对可能影响预后的因素进行单因素和多因素分析。  结果  所有患者均接受化疗联合贝伐珠单抗的治疗,化疗的平均周期数为3.1个,贝伐珠单抗治疗的平均周期数为5.0个。41例患者均可评价疗效。全组患者客观缓解率（objective response rate,ORR）为12.2%,疾病控制率（disease control rate,DCR）为82.9%。二线治疗与二线以上治疗的患者疗效接近,ORR分别为10.5%、13.6%（P=0.572）,DCR分别为89.5%和77.3%（P=0.271）,差异无统计学意义。中位无进展生存期（progression-free survival,PFS）和中位总生存期（overall survival,OS）分别为4.6个月（95%CI:3.619~5.581）、11.9个月（95%CI:9.797~14.003）。单因素分析提示EGFR突变、贝伐珠单抗治疗周期数 > 4个及女性患者获得更长的生存（χ2=19.673,P < 0.001;χ2=6.820,P=0.009;χ2=6.374,P=0.012）。多因素分析显示,EGFR突变状态、贝伐珠单抗治疗周期数为影响患者预后的独立危险因素（HR=0.129,P=0.001;HR=0.336,P=0.012）。常见的不良反应有骨髓抑制、出血、高血压、蛋白尿等,多数为1~2级。  结论  贝伐珠单抗联合化疗对复治晚期NSNSCLC患者疗效确切,不良反应可耐受,EGFR突变阳性、贝伐珠单抗使用4个周期以上的患者预后较好。      

阿美替尼联合贝伐珠单抗治疗晚期NSCLC伴原发EGFR T790M突变：3例病案报道及文献复习

随着检测技术的发展，非小细胞肺癌患者（non-small cell lung cancer, NSCLC）伴原发表皮生长因子受体（epidermal growth factor receptor, EGFR）T790M突变的检出率不断增加，而针对原发EGFR T790M突变NSCLC的一线治疗尚无标准。本文中我们报道了3例晚期NSCLC伴EGFR敏感突变及原发T790M突变的治疗经验，3例患者一线初治均为阿美替尼联合贝伐珠单抗。其中，1例在治疗3个月后因出血风险停用贝伐珠单抗并于10个月后更换为奥希替尼；1例患者在治疗13个月后更换为奥希替尼并停用贝伐珠单抗。3例患者最佳疗效均达部分缓解（partial response,PR）。随访至2022年10月，2例患者一线治疗后进展，无进展生存期（progression-free survival, PFS）分别为11个月和7个月；1例患者治疗后持续反应，治疗时间已达19个月。2例患者基线伴有多发脑转移，一线治疗后颅内病灶最佳疗效均为PR，颅内PFS分别为14个月和未达到（16+个月）。3例患者在治疗期间未见新的不良反应，未发生3级以上不良反...     

贝伐珠单抗治疗复发胶质母细胞瘤疗效和预后分析

  目的   探讨贝伐珠单抗治疗复发胶质母细胞瘤的疗效及预后因素。  方法  回顾性分析2011年12月至2020年7月中山大学肿瘤防治中心接受贝伐珠单抗治疗的81例成人复发胶质母细胞瘤患者的临床资料,评价其疗效和不良反应,以总生存期为预后指标进行单因素和Cox比例风险模型多因素分析。  结果  81例患者的客观有效率为62.9%,疾病控制率为80.2%,90.1%的患者生存质量状况（KPS）评分和神经症状有改善。中位无进展生存期（median progression-free survival,mPFS）和中位总生存期（median overall survival,mOS）分别为4.4个月（95%CI:4.0～4.8）和7.8个月（95%CI:6.8～8.8）。单因素分析显示用药时机（首次或≥2次复发时用药）、剂量（5 mg/kg,6～9 mg/kg,10 mg/kg）和是否联合化疗对mOS无影响,多因素分析显示KPS评分和MGMT启动子甲基化状态是影响mOS的独立预后因素。不良事件主要为1～2级。  结论  贝伐珠单抗可以改善成人复发胶质母细胞瘤患者的生存质量,治疗耐受性好。KPS评分和MGMT启动子甲基化状态是影响mOS的独立预后因素,贝伐珠单抗在复发后的用药时机、剂量、是否联合化疗对mOS无影响。      

消岩汤联合卡培他滨和贝伐珠单抗在结直肠癌维持治疗中的临床研究

  目的  探讨消岩汤联合卡培他滨和贝伐珠单抗在结直肠癌（colorectal cancer,CRC）维持治疗中的疗效和安全性。  方法  回顾性选取2016年1月至2021年12月于天津中医药大学第一附属医院接受一线标准化疗后进入维持治疗阶段的晚期CRC患者120例,分为治疗组（消岩汤联合卡培他滨和贝伐珠单抗维持治疗）60例和对照组（卡培他滨和贝伐珠单抗维持治疗）60例。比较两组间的无进展生存期（progression-free survival,PFS）,总生存期（overall survival,OS）和安全性。  结果  两组患者在基线特征方面差异无统计学意义,治疗组显著延长了CRC患者的PFS（10.9个月 vs. 9.2个月,P=0.03）,但OS未见显著获益（21.2个月 vs. 19.4个月,P=0.87）。消岩汤可改善患者神疲乏力（P=0.01）、恶心呕吐（P=0.01）、腹胀（P=0.02）的症状,且能减少恶心呕吐（P=0.04）及腹泻（P=0.02）的不良反应。  结论  消岩汤联合卡培他滨和贝伐珠单抗维持治疗可显著提高晚期CRC患者的PFS,减轻胃肠道系统不良反应且安全性良好。      

基于网络药理学和动物实验探讨青蒿鳖甲汤抗血管新生的作用机制

目的:基于网络药理学预测青蒿鳖甲汤抗血管新生的潜在分子机制,并用Lewis肺癌小鼠移植瘤模型对STAT3/VEGF信号通路进行实验验证。方法:使用TCMSP、BATMAN-TCM数据库及文献筛选青蒿鳖甲汤的有效成分,利用Swiss Target Prediction平台预测成分靶点基因。GeneCards、OMIM、TTD等3个数据库检索血管新生靶点基因,与中药成分靶点基因匹配后,使用STRING数据库和Cytoscape 3.7.1软件进行蛋白质间相互作用（protein-protein interaction,PPI）分析,筛选关键靶点基因;Metascape平台对关键靶点基因进行基因本体（gene ontology,GO）功能富集分析及京都基因与基因组百科全书（Kyoto encyclopedia of genes and genomes,KEGG）通路富集分析。在动物实验中,C57BL/6J雄性小鼠接种Lewis肺癌细胞,建立皮下移植瘤模型,分为模型组,青蒿鳖甲汤高（16.0 g/kg）、中（8.0 g/kg）、低（4.0 g/kg）剂量组,贝伐株单抗组（15 mg/kg）,分别给予相应剂量青蒿鳖甲汤灌胃或贝伐株单抗腹腔注射。14天后处死,取皮下瘤组织,称量瘤重,计算瘤体积和抑瘤率。免疫组化法检测血管标志物CD31表达情况,计算微血管密度（microvascular density,MVD）;HE染色法观察瘤组织病理形态;Western blot法检测pSTAT3、STAT3及VEGF蛋白表达水平。结果:获取青蒿鳖甲汤有效成分61个及靶点基因523个,血管新生相关靶点基因1 229个,交集靶点基因176个,关键靶点基因22个,其中VEGF是青蒿鳖甲汤发挥抗血管新生作用的首位关键靶点基因,信号通路以癌症相关信号通路为主,如HIF-1、VEGF、JAK-STAT等。动物实验结果表明,与模型组相比,青蒿鳖甲汤中、高剂量组瘤重、瘤体积均显著降低（P＜0.05或P＜0.01）,高剂量组MVD降低（P＜0.01）;各给药组肿瘤细胞体积缩小,核固缩、碎裂现象多见,可见不同程度的坏死区及出血;高剂量组VEGF、pSTAT3蛋白表达水平及pSTAT3/STAT3水平均显著降低（P＜0.05）,中、低剂量组VEGF、pSTAT3蛋白水平虽有降低趋势,但差异无统计学意义。结论:青蒿鳖甲汤可以通过多成分-靶点-通路抑制血管新生,其作用机制可能与抑制STAT3/VEGF信号通路有关。     

坎地沙坦对小鼠肝癌移植瘤及新生血管生成的抑制作用

目的 探讨血管紧张素Ⅱ 1型受体拮抗剂坎地沙坦对小鼠肝癌移植瘤生长及血管生成的影响。方法 建立肝癌H22细胞小鼠移植瘤模型。将48只小鼠随机分为生理盐水组（0.2ml d1～d8）、低剂量坎地沙坦组（2mg／kg,0.2ml d1～d8）、高剂量坎地沙坦组（20mg／kg,0.2ml d1～d8）和氟尿嘧啶组（25mg／kg,0.2ml d1～d6）,每组12只。第9天处死小鼠取瘤组织,测瘤重,计算抑瘤率;免疫组化法检测移植瘤组织内血管内皮生长因子（VEGF）和CD34表达,测定微血管密度（MVD）。另取40只小鼠以相同条件进行平行实验,每组10只,观察小鼠的生存时间。结果 高剂量坎地沙坦组及氟尿嘧啶组的抑瘤率分别为35.3％和50.6％,均高于低剂量坎地沙坦组的20.7％,差异有统计学意义（P＜0.05）;与生理盐水组相比,低剂量（5.083±1.240）与高剂量坎地沙坦组（4.083±1.165）VEGF分值均显著降低（P＜0.05）,且高剂量组降低更明显（P＜0.05）;与生理盐水组相比,低剂量（20.633±2.171）与高剂量（17.150±2.713）坎地沙坦组MVD均显著降低（P＜0.01）,且高剂量组降低更明显（P＜0.05）;高剂量坎地沙坦组及氟尿嘧啶组小鼠的生命延长率分别为39.5％和30.6％,均高于低剂量坎地沙坦组的20.4％（P＜0.05）。结论 坎地沙坦可抑制小鼠肝癌移植瘤的生长,其机制可能与抑制肿瘤新生血管生成有关。     

三氧化二砷联合TNP-470对裸鼠人肝癌移植瘤的影响

目的:探讨三氧化二砷(As2O3)联合烟曲霉的提取物烟曲霉醇(TNP-470)对裸鼠人肝癌移植瘤生长的影响及其作用机制。方法:人肝癌转移模型(HCC)-LCI-D20原位移植瘤裸鼠32只,随机分4组(每组8只)给药:As2O3联合TNP-470组、As2O3组、TNP-470组、生理盐水(NS)对照组,给药21天后,对裸鼠移植瘤大小、肿瘤微血管密度(MVD)、免疫组化、透射电镜结果进行观察。结果:As2O3组、TNP-470组及联合组的平均瘤重及平均瘤体积均低于对照组(P<0.05);计算两药相互作用系数(CDI)值:瘤重CDI(0.95)<1,体积CDI(0.89)<1,两药有协同抑制移植瘤生长的作用。As2O3组抑制移植瘤VEGF、EGFR的表达;TNP-470组抑制移植瘤EGFR的表达,对移植瘤VEGF的表达无影响;联合组明显抑制移植瘤EGFR的表达,抑制移植瘤VEGF的表达作用等同As2O3组。裸鼠原位移植瘤的光镜、电镜下观察结果有意义;各用药组裸鼠原位移植瘤的MVD值均低于对照组(P<0.05)。结论:As2O3联合TNP-470协同抑制裸鼠人肝癌移植瘤的血管新生,能有效抑制移植瘤生长。     

大肠腺瘤和腺癌组织中缺氧诱导因子-1α的表达及其与VEGF、微血管密度的关系

背景与目的:缺氧诱导因子-1α(hypoxia-inducible factor 1 alpha,HIF-1α)是肿瘤细胞适应缺氧而产生的一种核转录因子,在促进肿瘤新生血管生成中起重要作用。本研究旨在探讨大肠腺瘤和腺癌组织中HIF-1α的表达及其与血管内皮生长因子(vascular endothelial growth factor,VEGF)、微血管密度(microvessel density,MVD)的关系。方法:采用原位杂交技术检测HIF-1α mRNA,应用免疫组织化学方法检测VEGF蛋白的表达,用CD34单克隆抗体标记血管内皮细胞并计数MVD。结果:大肠腺癌组织HIF-1α mRNA阳性表达率为67.8％(42/62),腺瘤组织为44.4％(8/18)。腺癌组织从Dukes’A期到Dukes’C+D期HIF-lα mRNA表达阳性率不断增加(P<0.05)。HIF-1α mRNA表达平均阳性率为:腺瘤44.4％;腺癌Dukes’A期41.2％,Dukes’B期72.2％,Dukes’C+D期81.5％。腺癌组VEGF阳性表达率高于腺瘤组(59.7％ vs 33.3％,P<0.005).HIF-1α表达与VEGF呈正相关(r_s=0.768,P<0.01),与MVD呈正相关(r_s=0.683,P<0.05)。结论:HIF-1α及其靶基因 VEGF的过度表达与肿瘤新生血管形成呈正相关,这在大肠腺瘤癌变及大肠腺癌发展过程中可能起重要作用。     

Triple therapy with osimertinib,bevacizumab and cetuximab in EGFR-mutant lung cancer with HIF-1α/TGF-α expression

Osimertinib, a third generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, is the standard treatment for patients with lung cancer harboring EGFR T790M; however, acquired resistance is inevitable due to genetic and epigenetic changes in cancer cells. In addition, a recent randomized clinical trial revealed that the combination of osimertinib and bevacizumab failed to exhibit superior progression-free survival compared with osimertinib alone. The present study aimed to investigate the effect of triple therapy with osimertinib, bevacizumab and cetuximab in xenograft tumors with different initial tumor volumes (conventional model, 200 mm³ and large model, 500 mm³). The results demonstrated that osimertinib significantly inhibited tumor growth in both the conventional and large models; however, maximum tumor regression was attenuated in the large model in which hypoxia-inducible factor-1α (HIF-1α) and transforming growth factor-α (TGF-α) expression levels increased. Although the combination of osimertinib and bevacizumab exerted a greater inhibitory effect on tumor growth compared with osimertinib in the conventional model, the effect of this combination therapy was attenuated in the large model. TGF-α attenuated sensitivity to osimertinib in vitro; however, this negative effect was counteracted by the combination of osimertinib and cetuximab, but not osimertinib and bevacizumab. In the large xenograft tumor model, the triple therapy induced the greatest inhibitory effect on tumor growth compared with osimertinib alone and its combination with bevacizumab. Clinical trials of the triple therapy are required for patients with lung cancer with EGFR mutations and HIF-1α/TGF-α.     

Audit of Molecular Mechanisms of Primary and Secondary Resistance to Various Generations of Tyrosine Kinase Inhibitors in Known Epidermal Growth Factor Receptor-Mutant Non-small Cell Lung Cancer Patients in a Tertiary Centre

AimsPresently, three generations of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are approved against oncogene addicted EGFR-mutant non-small cell lung cancer (NSCLC). Patients with actionable EGFR mutations invariably develop resistance. This resistance can be intrinsic (primary) or acquired (secondary).Materials and methodsThis was a retrospective study carried out between January 2016 and April 2021 analysing 486 samples of NSCLC for primary and secondary resistance to first- (erlotinib, gefitinb), second- (afatinib) and/or third-generation (osimertinib) TKIs in EGFR-mutant NSCLCs by next generation sequencing (NGS). Tissue NGS was carried out using the Thermofischer Ion Torrent? Oncomine? Focus 52 gene assay; liquid biopsy NGS was carried out using the Oncomine Lung Cell-Free Total Nucleic Acid assay. All cases were previously tested for a single EGFR gene with the Therascreen® EGFR RGQ PCR kit.ResultsThe results were divided into four groups: (i) group 1: primary resistance to first- and/or second-generation TKIs. This group, with 21 cases, showed EGFR exon 20 insertions, dual, complex mutations and variant of unknown significance, de novo MET gene amplification besides other mutations. (ii) Group 2: primary resistance to third-generation TKIs. This group showed two cases, with one showing dual EGFR mutation (L858R and E709A) and EGFR gene amplification. (iii) Group 3: secondary resistance to first- and second-generation TKIs. This group had 27 cases, which were previously reported negative for EGFR T790M by single gene testing. Significant findings were MET gene amplification in four cases, with one also showing MET exon 14 skipping mutation. Three cases showed small cell change and one showed loss of primary mutation. (iv) Group 4: secondary resistance to third-generation TKIs. The latter group was further subgrouped into group 4A: secondary resistance to osimertinib (third-generation TKI) when offered as second-line therapy after first- and second-generation TKIs on detection of T790M mutation. This group had 15 cases. EGFR T790M mutation was lost in 10 (10/15; 67%) cases and was retained in five cases. Patients with T790M loss experienced early resistance (6.9 months versus 12.6 months mean, P = 0.0024) compared with cases that retained T790M. Two cases gained MET amplification as the resistance mechanisms. Other mutations that were found when EGFR T790M was lost were in FGFR3, KRAS, PIK3CA, CTNNB1, BRAF genes. One case had EML4-ALK translocation. Two cases showed driver EGFR deletion 19, retained T790M and C797S mutation in Cis form. Group 4B: secondary resistance to osimertinib (when given as first-line therapy) in EGFR-mutant NSCLC. This group had three cases. The duration of osimertinib treatment ranged from 11 to 17 months. Two patients showed additional C797S mutation along with primary EGFR mutation.ConclusionThis study shows the wide spectrum of primary and secondary EGFR resistance mechanisms to first, second and third generation of TKIs and helps us to identify newer therapeutic targets that could carry forward the initial advantage offered by EGFR TKIs.     

HIF-1α在乳腺癌合并2型糖尿病患者中的表达

目的:探讨乳腺癌合并2型糖尿病中缺氧诱导因子-α(HIF-1α)的表达及其对血管内皮生长因子(VEGF)、微血管密度(MVD)的影响.方法:回顾性分析我院2011年1月-2014年12月收治的118例乳腺癌合并2型糖尿病患者(实验组)和非糖尿病的乳腺癌患者的临床资料(对照组),比较两组基本临床病理特征差异,采用免疫组化SP法检测两组患者HIF-1α及VEGF的表达,采用CD34对血管内皮细胞进行标记并计算MVD.结果:实验组中肿瘤直径大于2cm的人数远多于对照组,差异有统计学意义(χ2=4.434,P=0.035);对照组和实验组淋巴结转移率分别为43.75%(56/128)、59.32%(70/118),实验组的淋巴结转移率远高于对照组(χ2=5.959,P=0.015);实验组的HIF-1α阳性表达率为80.51%,显著高于对照组的HIF-1α阳性表达率(64.84%),差异存高度统计学意义(χ2=7.533,P=0.006),实验组的VEGF阳性表达率为86.44%,高于对照组的VEGF阳性表达率(68.75%),差异存高度统计学意义(χ2=10.929,P=0.001);实验组的MVD计数高于对照组,差异存高度统计学意义(t=2.883,P=0.004).实验组中HIF-1α阳性表达组VEGF阳性表达率为90.53%(86/95),HIF-1α阴性表达组的VEGF阳性表达率为69.57%(16/23),HIF-1α阳性表达组明显高于HIF-1α阴性表达组,两组比较差异有统计学意义(χ2=6.941,P=0.008),两组的MVD计数分别为121.35±32.46,102.74±34.86,差异存统计学意义(t=2.432,P=0.017).结论:2型糖尿病能促进乳腺癌组织微血管的增生,加速肿瘤组织的转移与增长,是乳腺癌患者预后差及临床分期晚的危险因素.     

重组人血管内皮抑制素对小鼠肿瘤和心肌中血管结构及血管生成相关因子表达的影响

目的 观察重组人血管内皮抑制素对小鼠肿瘤及心肌中微血管影响的差异.方法 40只小鼠随机分为空白对照组(未荷瘤,生理盐水100 μl/d)、药物对照组(未荷瘤,重组人血管内皮抑制素400 μg/d)、模型组(荷瘤,生理盐水100 μl/d)和实验组(荷瘤,重组人血管内皮抑制素 400 μg/d),分别处理28 d.实验前后称量小鼠体重,测量移植瘤体积.采用免疫组化法检测小鼠心脏和移植瘤组织中基质金属蛋白酶(MMP)-2、MMP-9、缺氧诱导因子1α(HIF-1α)以及血管内皮生长因子(VEGF)蛋白的表达情况,计数微血管密度(MVD).以CD34与Masson双染法观察微血管结构的变化.结果 实验组小鼠肿瘤体积的增加值为(48.18±37.31)mm3,低于模型组[(113.80±73.27)mm3,P＜0.05];各组小鼠体重变化的差异无统计学意义(P＞0.05).应用重组人血管内皮抑制素后,移植瘤组织中MMP-9和VEGF蛋白的表达显著降低,移植瘤组织中MMP-2、HIF-1α以及心肌组织中MMP-2、MMP-9、HIF-1α和VEGF蛋白的表达均无显著变化;移植瘤组织的MVD显著降低,胶原覆盖血管的比例升高,而心肌组织的MVD和结构几乎无变化.结论 重组人血管内皮抑制素可通过下调移植瘤组织中MMPs和VEGF蛋白的表达,降低MVD,抑制移植瘤的生长,并可使移植瘤血管趋向成熟,但不能降低心肌组织中MMPs的表达和成熟血管的MVD.

Abstract：

Objective To compare the effect of rh-endostatin on micrangium in tumor and myocardial tissue in nude mice. Methods Nude mice were randomized into 4 groups(10 mice in each group), blank control group (without tumor burden, received NS 100 μl·d-1 injection), drug control group (without tumor burden, received rh-endostatin 400 μg·d-1 injection), model group (with tumor burden, received NS 100 μl·d-1 injection) and treatment group (with tumor burden, received rh-endostatin 400 μg·d-1 injection) for 28 days. The tumor volume and body weight of the mice were measured before and after administration. The expression of CD34, MMP-2, MMP-9, HIF-1α and VEGF in the myocardium and tumor were detected by immunohistochemistry. The vascular structure was observed by immunoenzymatic CD34 and Masson double staining. Results The increase of tumor volume of the treatment group[(48.18±37.31) mm3]was significantly lower than that in the model group [(113.80±73.27) mm3). The changes of body weight was not significant different among the four groups. After treated with rh-endostatin, the expressions of MMP-9 and VEGF in tumors were significantly down-regulated, but the expressions of MMP-2 and HIF-1α in the tumor were not. The microvessel density (MVD) in the tumors of treatment group was significantly decreased compared with that of model group. The proportion of tumor vessels covered by collagen in the treatment group was increased compared with that of the model group. However, MVD and micrangium in myocardium were not changed significantly. Conclusion Rh-endostatin can decrease the expression of MMP-9, VEGF and MVD, inhibit the tumor growth and normalize tumor micrvangium in tumor but not weaken the MMPs and MVD of mature micragium in myocadium.     

Antitumor and vascular effects of apatinib combined with chemotherapy in mice with non-small-cell lung cancer

Objective The aim of this study was to investigate the antitumor and vascular effects of apatinib use combined with chemotherapy on mice with non-small-cell lung cancer (NSCLC). Methods First, 60 tumor-bearing nude mice were randomly divided into control, low-dose, and high-dose groups. Four nude mice per group were sacrificed before administration and on days 1, 3, 7, and 10 after administration. HIF-1α expression in tumor tissues was detected. Second, 32 nude mice were randomly divided into control, premetrexed, synchronous, and sequential groups. The weights and tumor volumes of mice were recorded. Results (1) HIF-1α expression decreased significantly on days 3 and 7 after low-dose apatinib treatment. There was no significant difference in HIF-1α expression in the high-dose apatinib group (P > 0.05). MMP-2 and MMP-9 expression levels in the low-dose apatinib group were significantly lower than those in the control group (P < 0.05). (2) In the low-dose apatinib group, the microvessel density increased gradually from days 3 to 7 post-treatment, while that in the high-dose apatinib group decreased significantly. (3) The inhibitory effect of sequential therapy using low-dose apatinib and pemetrexed was optimal, while that of synchronous treatment was not better than that of pemetrexed usage alone. Sequential treatment using low-dose apatinib and pemetrexed exerted the best antitumor effect. (4) The expression levels of p-AKT, p-mTOR, p-MEK, and p-ERK in the sequential group were significantly lower than those in the other three groups (P < 0.05). Conclusion Apatinib usage involves certain considerations, such as dose requirements and time window for vascular normalization during lung cancer treatment in nude mice, suggesting that dynamic contrast-enhanced magnetic resonance imaging and other tests can be conducted to determine the vascular normalization window in patients with lung cancer and to achieve the optimal anti-vascular effect.