BTLA、CTLA-4基因多态性与肝癌TACE联合靶向治疗疗效及预后相关性.pdf

昆明医科大学学报2 0 2 3，44(9)：12 6 135Journal of Kunming Medical UniversityBTLA、C T LA-4基因多态性与肝癌TACE联合靶向治疗疗效及预后相关性D0I:10.12259/j.issn.2095-610X.S20230927CN 53-1221/R陆小华，袁洪新（南通大学附属医院介入放射科，江苏摘要】目的探讨B及T淋巴细胞弱化因子(BTLA)、细胞毒性T淋巴细胞抗原-4(CTLA-4)基因多态性与肝癌经动脉插管化疗栓塞(TACE)联合靶向治疗疗效及预后相关性。方法选取2 0 2 1年1月至2 0 2 1年12 月期间南通大学附属医院介入放射科收治的新发8 5例肝癌患者作为研究对象，均行TACE联合靶向治疗，根据RECIST实体瘤疗效评价标准将患者分为治疗有效组（n=58)、疾病进展组（n=27)，比较2 组临床资料、BTLA、CTLA-4基因多态性，分析BTLA、C T LA-4基因多态性与疗效的相关性，并于治疗后18 个月电话随访生存状况，对比不同基因型患者生存状况，分析其与生存预后的相关性。结果CTLA-4rs231775、r s 7 336 18、rs3087243、r s 45538 0 8 位点、BTLArs2171513、r s 3112 2 7 0、r s 198 2 8 0 9、r s 16 8 596 2 9位点基因型均符合Hardy-Weinberg遗传平衡定律；治疗有效组BTLArs3112270位点AG、G G 基因型比例、rs1982809位点GA、A A 比例、rs16859629位点CC基因型、CTLA-4rs231775位点AA基因型比例、rs3087243位点GA、A A 基因型低于疾病进展组，BTLArs3112270位点AA基因型比例、rs1982809位点GG比例、rs16859629位点TT基因型、CTLA-4rs231775位点GG基因型比例、rs3087243位点GG基因型高于疾病进展组（PG、r s 198 2 8 0 9G A、r s 16 8 596 2 9 T C、C T LA-4 r s 2 317 7 5 A G、r s 30 8 7 2 43 G A与疗效相关（P0.05)；BT LA r s 3112 2 7 0 位点AA、A G 基因型患者18 个月生存率高于GG基因型患者，rs1982809位点GG、G A 基因型患者18 个月生存率高于AA基因型患者，rs16859629位点TT基因型患者18 个月生存率高于TC、CC基因型患者(P0.05)；CT LA-4r s 2 317 7 5位点 GG、G A 基因型患者18 个月生存率高于AA基因型患者、rs3087243位点GG基因型18 个月生存率高于GA、A A 基因型患者(PG、r s 198 2 8 0 9 G A、r s 16 8 596 2 9 T C、CT LA-4 r s 2 317 7 5 A G、r s 30 8 7 2 43GA与生存预后相关（P0.05）。结论BTLA基因rs3112270、r s 198 2 8 0 9、r s 16 8 596 2 9、CT LA-4基因rs231775、r s 30 8 7 2 43位点多态性与肝癌TACE联合靶向治疗疗效、预后密切相关，以期为预估TACE治疗提供参考。关键词BTLA；CT LA-4；基因多态性；肝癌；TACE；靶向治疗；疗效；预后；相关性中图分类号】R735.7文献标志码 A文章编号2 0 95-6 10 X(2023)09-0126-10The Analysis of the Correlation between BTLA,CTLA-4 GenePolymorphisms and the Efficacy and Prognosis of TACECombined Targeted Therapy for Liver Cancer东南通，2 2 6 0 0 1)LU Xiaohua,YUAN Hongxin(Dept.of Interventional Radiology,Affiliated Hospital of Nantong University,Nantong Jiangsu 226001,China)Abstract Objective To investigate the correlation between gene polymorphisms of B and T lymphocyteattenuator(BTLA)and cytotoxic T lymphocyte antigen-4(CTLA-4)and the efficacy and prognosis of targeted收稿日期 2 0 2 3-0 4-13基金项目南通市科技局基础科学研究公众健康基金资助项目（JC2021196）作者简介 陆小华（198 2），男，江苏南通人，医学硕士，副主任医师，主要从事肿瘤及血管相关疾病的介人治疗及机制研究工作。通信作者袁洪新，E-mail：j o p x 6 0 8 7 2 1c n.c o m第9期therapy combined with transcatheter arterial chemoembolization(TACE)for liver cancer.Methods Eighty-fivenew hepatocellular carcinoma patients admitted to the Department of Interventional Radiology of Nantong UniversityHospital between 2021-01-01 and 2021-12-31 were selected for the study,and all of them were treated withTACE combined with targeted therapy.According to the RECIST solid tumor efficacy evaluation criteria,thepatients were divided into the effective treatment group(n=58)and the disease progression group(n=27).Theclinical data,BTLA,CTLA-4 gene polymorphisms between the 2 groups were compared and the correlationbetween BTLA,CTLA-4 gene polymorphisms and efficacy were analyzed,And follow-up survival status by phoneat 18 months after the treatment was conducted to compare the survival status of patients with different genotypes,and analyze their correlation with survival prognosis.Results CTLA-4 rs231775,rs733618,rs3087243,rs4553808,BTLA and the genotypes of rs2171513,rs3112270,rs1982809 and rs16859629 all conformed toHardy-Weinberg law of genetic balance.The BTLA rs3112270 AG,GG genotype ratio,rs1982809 GA,AA ratio,rs16859629 CC genotype,CTLA-4 rs231775 AA genotype ratio,rs3087243 GA,AA genotype ratio in thetreatment effective group were all lower than those in the disease progression group,while the proportion of AAgenotype at BTLA rs3112270,GG genotype at rs1982809,TT genotype at rs16859629,GG genotype at CTLA-4rs231775,and GG genotype at rs3087243 were all higher than those in the disease progression group(P G,rs1982809 G A,rs16859629 T C,CTLA-4 rs231775 A G,rs3087243 G A were all associated with the efficacy(P 0.05);The 18 monthsurvival rate of BTLA rs3112270 AA and AG genotype patients was higher than that of GG genotype patients,rs1982809 GG and GA genotype patients were higher than that of AA genotype patients,and rs16859629 TTgenotype patients are higher than those of TC and CC genotype patients(P 0.05);The 18 month survival rate ofpatients with CTLA-4 rs231775 GG and GA genotypes was higher than that of patients with AA genotypes,and the18 month survival rate of GG genotype at rs3087243 was higher than that of patients with GA and AA genotypes(P G,rs1982809 G A,rs16859629T C,CTLA-4 rs231775 A G,rs3087243 G A were all associated with the survival prognosis(P 0.05).Conclusion The polymorphisms of BTLA gene rs3112270,rs1982809,rs16859629,CTLA-4 gene rs231775,rs3087243 are closely related to the efficacy and prognosis of TACE combined with the targeted therapy for livercancer and these will provide the reference for predicting TACE treatment.Key words BTLA;CTLA-4;Gene polymorphism;Liver cancer;TACE;Targeted therapy;Curative陆小华，等BTLA、C T LA-4基因多态性与肝癌TACE联合靶向治疗疗效及预后相关性effect;Prognosis;Correlation127肝癌目前在我国是第四大恶性肿瘤，其致死率位居第二，仅次于肺癌，肝癌早期隐匿，疾病进展较快，绝大多数患者确诊时已经处于中晚期或伴有严重肝硬化，此时已经失去最佳手术治疗时机，而介人治疗是其有效替代最佳治疗方案 1-2 。经动脉插管化疗栓塞(transcatheter arterialchemoembolization，T A CE)是目前治疗中晚期肝癌患者常用手段，可有效杀伤肿瘤病灶，改善机体免疫功能、生活质量 3。临床评价TACE疗效和预后主要依靠影像学数段，从肿瘤数目、大小等表观学方面进行评价 4，但从微观基因方面评价疗效和预后的研究少见。人类基因组计划研究显示，不同个体间相同基因组序列同源度达99%以上，其余约1%为遗传变异，多见于单碱基改变，即单核苷酸多态性（single nucleotide polymorphism,SNP)5。最近研究发现，B及T淋巴细胞弱化因子(Band T lymphocyte attenuator，BT LA)、细胞毒性T淋巴细胞抗原-4(cytotoxicT-lymphocyte antigen-4，CT LA-4)等基因多态性与多种肿瘤易感性密切相关，其基因位点突变可增加肿瘤发生 6-7 。基于以上背景，本研究创新性观察BTLA、C T LA-4基因多态性与肝癌TACE联合靶向治疗疗效及预后相关性。1资料与方法1.1病例选择选取2 0 2 1年1月至2 0 2 1年12 月南通大学附属医院介入放射科收治的新发8 5例肝癌患者作为研究对象，其中男6 5例，女2 0 例；年龄1283665岁，平均（52.355.34)岁；Child-Pugh分级：7 5例A级，10 例B级；BCLC分期：2 0 例B期，6 5例C期。纳人标准：符合原发性肝癌诊疗规范（2 0 19年版）8 诊断标准，经病理学检查确诊；均行TACE联合靶向治疗；美国东部肿瘤协作组体能状态评分均为0 1分；Child-Pugh分级为A级、B级；BCLC分期为B期、C期；TACE前白细胞4.0 10%L、血红蛋白8.0g/L；无全身性感染疾病；近3个月无免疫抑制、激素药物治疗史。排除标准：大量腹水经治疗后不能消除；近3个月出现消化道出血；既往存在肝移植、放化疗或系统性等治疗；严重脾功能亢进；其他恶性肿瘤；预计生存期 6 个月；免疫系统疾病；妊娠期或哺乳期女性。本研究获得南通大学附属医院医学伦理委员会批准（批号：2020-K024-01)。1.2治疗方法所有受试者均在 Innova IGS540 DSA血管造影X射线机（美国GE公司）行TACE治疗。操作步骤：常规碘伏消毒、1%利多卡因局部麻醉，采用Seldinger改良法经右侧股动脉穿刺置鞘，置人5FRH导管至腹腔干、肠系膜上动脉，行碘克沙醇造影，确定肿瘤供血动脉。用Progreat微导管或Merit微导管超选进人肝脏肿瘤动脉供血分支中，灌注表柔比星0 30 mg、洛铂2 0 30 mg化疗药物，予表柔比星10 mg与碘化油混制乳剂1025mL栓塞，至肿瘤内碘油沉积致密后终止栓塞。最后再次造影，显示肿瘤供血动脉分支栓塞满意后结束手术。所有人组患者均在TACE治疗后3 7 d开始口服索拉非尼（40 0 mgbid)或仑伐替尼（6 0 kg患者，8 mg，q d；6 0 k g 患者，12mg，q d)，如患者出现药物相关严重不良事件则适当减少剂量或停药观察。疗效评估：治疗1个月后，根据RECIST实体瘤疗效评价标准进行疗效评估 9，完全缓解：肿瘤病灶完全消失，部分缓解：病灶直径减小50%，稳定：病灶直径减小 2 5%或出现新病灶。有效组：完全缓解、部分缓解、稳定；疾病进展组：进展。1.3BTLA、C T LA-4基因多态性检测采集2 组患者静脉血3mL，经抗凝处理后，按照DNA抽提试剂盒（上海生工公司）提取患者DNA，并对其进行检测定量。获取CTLA-4基因rs231775、r s 7 336 18、r s 30 8 7 2 43、r s 45538 0 8 位点、BTLA 基 因 rs2171513、r s 3112 2 7 0、r s 19 8 2 8 0 9、昆明医科大学学报rs16859629T位点，设计并合成SNP位点扩增引物，PCR体系如下：2 LDNA模板（10 ng/L)，上下游引物各1L，15L2PCRReadyMix，在PCR仪进行扩增，程序为：94预变性4min、94变性30 s、6 0 退火30 s、7 2 延伸30 s，共35次循环，7 2 终延伸10 min，4保存。反应结束后，采用1%琼脂糖胶电泳检测PCR产物，确定产物大小，用AMPureXP磁珠纯化、回收。最后使用HiSeq XTen测序仪进行测序。采用panel高通量数据分析基因分型。1.4观察指标（1)比较2 组临床资料，包括性别、肿瘤最大径、年龄、肿瘤数目、BMI、Ch i ld-Pu g h 分级、BCLC分期、肝外转移、血管侵犯；（2)分析BTLA、C T LA-4基因多态性检测结果，CTLA-4rs231775、r s 7 336 18、r s 30 8 7 2 43、r s 45538 0 8 位点、BTLA rs2171513、r s 3112 2 7 0、r s 19 8 2 8 0 9、r s 16 8 59629T位点均符合Hardy-Weinberg遗传平衡定律；（3)比较2 组BTLA、C T LA-4基因位点基因型；(4)单因素、多因素分析BTLA、C T LA-4基因多态性与疗效的相关性；（5)比较不同BTLA、CTLA-4基因位点基因型患者生存预后状况；（6)单因素、多因素分析BTLA、CT LA-4基因多态性与生存预后的相关性。1.5统计学处理临床病历资料收集使用EXCEL2019，数据处理使用软件SPSS25.0，计数资料用n(%)表示，采用检验，等级资料用Ridit检验；计量资料呈方差齐性、近似服从呈正态分布以（x土s）表示，采用t检验；采用K-M法绘制生存曲线，单因素、Logistic多因素回归分析BTLA、CT LA-4基因多态性与疗效、预后相关性。P0.05)；肿瘤最大径、肿瘤数目、Child-Pugh分级、BCLC分期、肝外转移、血管侵犯差异有统计学意义(P0.05)，见表1。2.2BTLA、CT LA-4基因多态性检测结果经TaqManqRT-PCR检测显示，BTLArs2171513位点基因型为GG、G A、A A；r s 3112 2 7 0位点基因型为AA、A G、G G；r s 19 8 2 8 0 9 位点基第44卷第9期资料性别男女年龄(岁)BMI(kg/m)肿瘤最大径(cm)肿瘤数目(个)Child-Pugh分级A级B级BCLC分期B期C期肝外转移有无血管侵犯有无*P0.05)；治疗有效组BTLArs3112270位点AG、G G 基因型比例、rs1982809位点GA、A A比例、rs16859629位点CC基因型、CTLA-4rs231775位点AA基因型比例、rs3087243位点GA、A A 基因型低于疾病进展组，BTLArs3112270位点AA基因型比例、rs1982809位点GG比例、陆小华，等BTLA、C T LA-4基因多态性与肝癌TACE联合靶向治疗疗效及预后相关性Tab.1 Comparison of clinical data between two groups n(%)/(X s)治疗有效组(n=58)43(74.14)15(25.86)52.13 4.7621.39 1.253.82 0.512.75 0.4455(94.83)3(5.17)18(31.03)40(68.97)8(13.79)50(86.21)24(41.38)34(58.62)129表12 组临床资料比较n（%)/（x s)】疾病进展组(n=27)22(81.48)5(18.52)52.82 5.1121.18 1.175.03 0.743.38 0.5320(74.07)7(25.93)2(7.41)25(92.59)10(37.04)17(62.96)18(66.67)9(33.33)rs16859629位点TT基因型、CTLA-4rs231775位点GG基因型比例、rs3087243位点GG基因型高于疾病进展组，差异有统计学意义（PA、CT LA-4 r s 7 336 18 C T、rs4553808AG与疗效无关（P0.05)；BT LArs3112270A G、r s 19 8 2 8 0 9 G A、r s 16 8 59 6 2 9 T C、C T LA-4 r s 2 317 7 5A G、r s 30 8 7 2 43G A 与疗效相关(P0.05)；BT LA r s 3112 2 7 0 位点AA、A G 基因型患者18 个月生存率高于GG基因型患者，P0.5520.4570.6080.5450.7360.4648.7770.001*5.7530.001*5.7760.001*5.7160.017*5.9630.015*4.7130.030*130位点rs2171513GGGAAArs3112270AAAGGGrs1982809GGGAAArs16859629TTTCCC项目rs2171513GGGAAArs3112270AAAGGGrs1982809GGGAAArs16859629TTTCCC*P0.05。rs1982809位点GG、G A 基因型患者18 个月生存率高于AA基因型患者，rs16859629位点TT基因型患者18 个月生存率高于TC、CC基因型患者昆明医科大学学报表2 8 5例患者BTLA、C T LA-4基因型分布情况Tab.2Distribution of BTLA and CTLA-4 genotypes in 85 patientsBTLA基因型n5625445346483077285Tab.3 Comparison of BTLA and CTLA-4 genotypes between the two groups n(%)BTLA基因型治疗有效组疾病进展组(n=27)u(n=58)0.78840(68.97)16(59.26)16(27.59)9(33.33)2(3.45)2(7.41)41(70.69)4(14.81)17(29.31)17(62.96)0(0.00)6(22.22)40(68.97)8(29.63)17(29.31)13(48.15)1(1.72)6(22.22)51(87.93)16(59.26)7(12.07)1(3.70)0(0.00)10(37.04)第44卷CTLA-4基因型频率位点rs23177565.88GG29.41GA4.71AArs73361852.94CC40.00CT7.06TTrs308724356.47GG35.29GA8.24AArs455380884.71AA9.41AG5.88GG表32 组BTLA、C T LA-4基因型比较n(%）P0.4314.6380.001*3.3470.001*2.4640.014*n314212945314832546336CTLA-4基因型治疗有效组 疾病进展组项目(n=58)rs231775GGGAAArs733618CCCTTTrs3087243GGGAAArs4553808AAAGGG(P0.05)；CT LA-4 r s 2 317 7 5位点 GG、G A 基因型患者18 个月生存率高于AA基因型患者、rs3087243位点GG基因型18 个月生存率高于GA、频率36.4749.4114.1210.5952.9436.4756.4737.655.8854.1238.827.06(n=27)2.9200.004*25(43.10)6(22.22)31(53.45)11(40.74)2(3.45)10(37.04)6(10.34)3(11.11)31(53.45)14(51.85)21(36.21)10(37.04)43(74.14)5(18.52)15(25.86)17(62.96)0(0.00)5(18.52)33(56.90)13(48.15)21(36.21)12(44.44)4(6.90)2(7.41)P0.0190.9454.491Ars3112270A Grs1982809G Ars16859629T CCTLA-4基因rs231775A Grs733618C Trs3087243GArs4553808AG*P0.05。AA基因型患者，差异有统计学意义（P0.05),见表5、图1 2。2.6BTLA、CT LA-4基因多态性与生存预后的相关性在进行Logistic多因素回归分析时，将肿瘤最大径、肿瘤数目、Child-Pugh分级、BCLC分Tab.5Comparison of survival and prognosis of patients with different BTLA and CTLA-4 genotypesBTLA基因型项目nrs2171513GGGAAArs3112270AAAGGGrs1982809GGGAAArs16859629TTTCCCP0.05。陆小华，等BTLA、CT LA-4基因多态性与肝癌TACE联合靶向治疗疗效及预后相关性表4 BTLA、C T LA-4基因多态性与疗效的相关性Tab.4Correlation between BTLA and CTLA-4 gene polymorphisms and therapeutic effectLogistic多因素回归分析OR95%CI1.2110.417 3.5195.3961.794 16.2286.6022.061 21.1474.6751.53714.2205.8281.80918.7730.9780.325 2.9416.0372.260 16.1281.2400.249 6.177表5不同BTLA、CT LA-4基因型患者生存预后状况比较生存率P1.1150.5435371.70(38/53)2564.00(16/25)450.00(2/4)4280.95(34/42)3461.76(21/34)616.67(1/6)4776.60(36/47)2867.86(19/28)714.29(1/7)7077.14(54/70)728.57(2/7)50.00(0/5)131单因素分析P0.4170.001*0.001*0.001*0.001*0.623 A、CT LA-4 r s 7 336 18 C T、rs4553808AG与生存预后无关(P0.05)；BT LArs3112270AG、r s 198 2 8 0 9G A、r s 16 8 596 2 9T C、C T LA-4 r s 2 317 7 5A G、r s 30 8 7 2 43G A 与生项目nrs231775GGGAAA11.1630.004*10.9280.004*18.4020.001*OR1.7406.2527.6995.4415.6501.1226.4861.065304012rs733618CCCTTTrs3087243GGGAAArs4553808AAAGGG95%CI0.523 5.7912.041 19.1532.274 26.0651.88215.7302.005 15.9240.246 5.1132.175 19.3400.196 5.782CTLA-4基因型生存率86.67(26/30)70.00(28/40)16.67(2/12)966.67(6/9)4365.12(28/43)3073.33(22/30)4686.96(40/46)3151.61(16/31)50.00(0/5)4369.77(30/43)3366.67(22/33)666.67(4/6)P0.5180.001*0.001*0.001*0.001*0.2640.001*0.187P19.5020.001*0.5630.75522.1520.001*0.0910.956132存预后相关(PArs3112270A Grs1982809GArs16859629TCCTLA-4基因rs231775AGrs733618C Trs3087243GArs4553808AG*PG、r s 2 7 0 5535A G 位点可增加乳腺癌发生风险。王居已 2 0 研究显示，BTLArs1982809GA、r s 16 8 596 2 9T C 基因多态性可增加非小细胞肺癌细胞易感性，但rs2171513GA、r s 3112 2 7 0 A G 可降低非小细胞肺癌易感性。本研究通过分析BTLA_rs2171513、r s 3112 2 7 0、rs1982809、r s 16 8 596 2 9T 位点，发现2 组BTLArs3112270、r s 198 2 8 0 9、r s 16 8 596 2 9位点基因型陆小华，等BTLA、C T LA-4基因多态性与肝癌TACE联合靶向治疗疗效及预后相关性表6 BTLA、C T LA-4基因多态性与生存预后的相关性Tab.6(Correlation between BTLA and CTLA-4 gene polymorphism and survival prognosisHR1.1564.3365.5876.6206.5881.1577.9060.907133单因素分析Logistic多因素回归分析95%CIP0.187 7.1440.0951.509 12.4570.001*1.91416.3080.001*2.069 21.1820.001*2.397 18.1050.001*0.201 6.6650.1022.522 24.781G、r s 19 8 2 8 0 9 G A、r s 16 8 59 6 2 9TC与肝癌疗效、预后密切相关。先前有报道显示，接受铂类药物化疗可损伤患者DNA，引起碱基序列改变，导致基因对DNA损伤修复不及时，不能及时修复化疗药物引起的NA损伤，增加化疗药物耐药性，影响治疗效果 2 1。本研究中TACE联合靶向治疗，化疗药物采用表柔比星、洛铂，前者为细胞周期非特异性药物，可迅速进人细胞核与DNA结合，有效抑制核酸合成和有丝分裂进程，阻止RNA合成；而后者是第3代铂类化疗药物，不仅影响DNA合成、复制，还可影响原癌基因c-mye表达。CTLA-4是T淋巴细胞表面较为重要共刺激基因，与CD28属同一家族，主要位于染色体2q33区域，可与CD28竞争结合B7-1/B7-2分子，从而发挥负向调节T淋巴细胞增殖分化作用，还可抑制细胞因子分泌、阻止细胞周期进展 2 2 。CTLA-4也是肿瘤免疫检查点基因，在肿瘤免疫过程中发挥重要作用，目前已有研究显示，CTLA-4基因多态性与肿瘤、自身免疫疾病遗传和环境易感性中存在关联性，可增加肿瘤、疾病患病风险 2 3-2 4。如 Liu 等 2 5 研究显示，CTLA-4 rs231775与转移性肾细胞癌疗效、预后密切相关。Qin等 2 6 通过分析CTLA-4 rs231775、r s 30 8 7 2 43、rs4553808、r s 57 42 90 9、r s 7 336 18 位点，发现HR0.9024.4395.1237.2626.0580.9426.6420.83495%CI0.165 4.9361.441 13.6721.65915.8202.183 24.1592.116 17.3420.188 4.7242.183 20.2100.176 3.952P0.2360.001*0.001*0.001*0.001*0.221G、rs3087243GA与疗效、预后相关，这与Qin等 2 6 研究结果不一致。分析原因：可能是因为2 种化疗方式、化疗药物、CTLA-4基因位点较多等有关，机体内基因调控机制较为复杂，相同基因位点在不同肿瘤中表现不一。综上所述，本研究证实BTLA基因rs3112270、rs1982809、r s 16 8 596 2 9位点、CTLA-4基因rs231775、r s 30 8 7 2 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