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IL17在肝癌患者血清中表达增高对肝癌诊断的研究

2014-10-24范莎莎等

关键词:甲胎蛋白回归肝癌

范莎莎等

摘 要 为了早期发现并监测肝癌,通过ELISA法检测肝癌组与正常组血清IL17细胞因子水平,运用ROC统计学分析比较其与AFP诊断肝癌的诊断效能,logistic回归获得IL17和AFP两者联合诊断的模型预测肝癌.结果发现肝癌组的IL17质量浓度(5.76±2.35 ng/L)较对照组(2.98±2.53 ng/L)明显增高(P*<0.001),ROC分析IL17和AFP预测肝癌的曲线下面积(AUC)分别为0.820和0.884,两者对诊断均有统计学意义.通过Logistic 逐步回归得出的AFP和IL17回归模型PRE,预测肝癌的ROC曲线下面积为0.963,诊断的敏感度(87.5%)、特异度(82.1%)较两指标单独诊断时明显提高.因此本研究获得的诊断模型可以用于临床对肝癌的联合诊断.

关键词 肝癌;IL17;logistic 回归;受试者诊断特征曲线;甲胎蛋白

中图分类号 R7357文献标识码 A文章编号 10002537(2014)03001905

肝细胞癌(Hepatocellular Carcinoma, HCC)是最常见的肝原发恶性肿瘤, 因为发病率较高、发现困难、治疗方法少等原因,在导致人类死亡的各种疾病中位居前列[1].目前,原发性肝细胞癌在全球范围内致死率位列第五位,而在一些非洲和亚洲国家,原发性肝细胞癌在肿瘤性死因中已经位居首位[23].肝癌早期转移和术后复发是肝癌患者生存率低的主要原因.目前有效的肝癌诊断标志物寥寥无几,当前的检测技术仍然不能及时发现和有效检测早期阶段的肝癌[45].

超声和AFP检测是最常用的筛查肝癌方法,然而超声检测依赖于医师的专业知识和设备实际灵敏度,难以对肿块的良恶性予以评估和诊断.AFP被认为是用于筛查和早期诊断肝癌最实用且可行的方法,40%~60%HCC患者的AFP值增高,然而还有很大一部分肝癌患者其AFP值没有增高[67],因此AFP用作唯一的工具来诊断肝癌仍然存在较大的漏诊率[89].

IL17具有促肿瘤生长、转移的作用.动物实验研究表明IL17具有促进多种肿瘤生长的作用,这些肿瘤包括纤维肉瘤、结肠癌、皮肤癌、非小细胞肺癌等[1012].研究认为IL17能通过聚集血管内皮细胞而促进肿瘤血管的形成,也能促进成纤维细胞产生血管内皮生长因子、前列腺素、趋化因子、巨噬细胞炎性蛋白2,转化生长因子的产生促进肿瘤细胞的粘附和运动,为肿瘤血管的形成和癌细胞播散提供条件[1213].

基于以上IL17与肿瘤和肝癌的各种相关报道,结合已有的AFP的临床价值,检测IL17在肝癌血清中的表达情况及评估与AFP的相关性显得非常有意义,这样更有利于系统地分析和检验二者的检验效能,以便更好地对其进行利用.

1 方法

1.1 实验分组

筛选2013年9月和2013年12月在湖南省人民医院就诊的肝癌病人和健康体检者,其中肝癌患者入组条件为:未经过任何治疗,经病理或者临床诊断为肝癌的患者.共有84名受试者参与,并分为以下两组:健康对照组,包括32名,年龄范围为21~72岁(平均年龄43±15岁);肝癌组共52名,男45例,女7例,年龄范围为27~80岁(平均年龄54±12岁),各组人员在入院后进行相应治疗之前,完成CT、超声、血生化常规、血清AFP检测,各组实验对象资料见表1.

1.2 标本收集

用真空采血针取患者晨起空腹3 mL静脉全血,4 ℃ 4 000 r/min离心10 min,提取离心后的血清.血清保存在-80 ℃,保存至同批测定.

1.3 酶联免疫吸附实验(ELISA)

酶联免疫吸附实验(Enzymelinked Immunosorbent Assay)检测人血清IL17质量浓度.使用人IL17酶联免疫吸附试剂盒( CSBE14917H, Cusabio Biotech),检测浓度下限(LLD)为7.8 ng/L.批内精密度(化验内精度)CV%<8%,批间精密度(检测精度之间)CV%<10%.Biotech 酶标仪检测各样本孔吸光值.Curve Expert 1.4软件计算和绘制ELISIA标准品曲线,并计算各孔的质量浓度.

1.4 应用Roche电化学发光法测定血清AFP质量浓度

按试剂盒说明书处理样本后,吸取10 μL标本与生物素化抗AFP单克隆抗体和钉标记的抗AFP抗体混匀,加入链霉亲合素包被的微粒,反应混合液吸到测量池中,微粒通过磁铁吸附到电极上,电极加电压后产生化学发光,通过光电倍增管进行测定.检测结果由Elecsys软件自动测出.

1.5 软件分析

应用SPSS17.0统计软件进行统计分析.应用受试者诊断特征曲线(receiver operator characteristic curve ROC)评估指标的预测效能,得到曲线下面积(Area Under the Curve, AUC).最大约登指数计算临界值(Cutoff points),约登指数(Youden index)=敏感度+特异度-1.各组均值比较,两独立样本t检验.logistic逐步回归分析.所有结果以双尾检验P<0.05认为有统计学差异.Medcalc软件绘制ROC曲线.

2 结果

2.1 正常组与肝癌组血清IL17的质量浓度

图1 正常组与肝癌组血清IL17质量浓度

Fig.1 Serum levels of IL17 in HCC group and control group

结果显示IL17在肝癌组中质量浓度明显高于正常组,分别为5.76±2.35 ng/L和2.98±2.53 ng/L(P*<0.001),差异有统计学意义.两独立样本t检验.见图1.

2.2 血清IL17诊断肝癌组与正常组的效能分析

以IL17的血清质量浓度为变量做ROC曲线,检测其作为肝癌血清诊断标志物的可能性,结果显示IL17和AFP分别单独作为标志物对肝癌与正常组进行区分时,其曲线下面积AUC分别为0820 (P<0.001)和0.884 (P<0.001),见图2和表2,二者作为血清标志物均具有统计学意义.IL17诊断的特异度较AFP低,但敏感性较AFP增加.选择截断点IL17为2.455 ng/L时,敏感度为67.3%,特异度为87.5%.当AFP为32.4 μg/L截断点时约登指数最大,其敏感度为61.5%,特异度为100%,如表3.若选择以20 μg/L的诊断指标为截断点,其敏感度为61.5%,特异度为90.6%.

3 讨论

原发性肝癌的早期诊断对提高患者的5年生存率以及生存质量十分重要.在中国,AFP目前仍然被作为筛查和早期诊断肝癌的主要指标,主要由于其应用的方便和指标具备一定的特异性,肝癌患者约60%血清中的AFP较正常人增高[14],但是AFP的敏感度和特异度对于临床诊断来说还远远不够,即使联合已有的指标DCP或AFPL3时,诊断肝癌患者的敏感度也仅为78.3%[1415].因此,寻找更加敏感和特异的肝癌诊断指标有着非常重要的临床意义,也是目前肝癌研究的重点方向.

本研究应用了受试者诊断特征曲线分析,判断指标的潜在诊断价值.在AUC>0.5的情况下,AUC越接近于1,说明诊断效果越好.通过ROC分析,结果显示肝癌患者IL17血清质量浓度为2.455 ng/L临界点时,其曲线下面积(AUC=0.820)有统计学意义.目前关于IL17与肝癌转移的报道在国内外还非常少,仅有IL17增高与肝癌复发相关的类似研究,当IL17大于0.9 ng/L时,肝癌患者术后的复发概率增高,远期生存质量较差,预测肝癌术后复发的敏感度为63.3%,特异度为91.1%[16].另外也有报道178例肝癌患者,肿瘤内IL17质量浓度与肝癌术后复发及生存相关,是预后的不良因素,认为IL17质量浓度与肿瘤的微血管密度正相关,可能的机制为IL17能够促进血管生成,导致肿瘤进展[17].

Logistic回归分析是一类数据挖掘技术,可以从大量的医学信息和临床数据中提取有用的信息和知识,建立某种疾病的诊断或治疗模型.临床医生进行诊断时如果利用计算机辅助诊断的logistic回归模型,其准确率可得到提高.本研究构建的IL17和AFP的logistic回归模型其诊断肝癌的效能较IL17和AFP均增高,该模型在国内外的文献中还未见报道.

本研究发现在肝癌血清中IL17质量浓度较正常组增高,因此IL17可作为诊断肝癌的标志物,其敏感度较AFP高.同时获得了IL17和AFP两指标联合诊断的logistic回归模型,其ROC的曲线下面积AUC(0964)较两指标单独诊断的曲线下面积均高,且敏感度和特异度都提高.本研究还有待于进一步扩大样本,验证其logistic回归诊断模型的诊断效能.

参考文献:

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[7] CHEAH Y L, CHOW P K H. Liver transplantation for hepatocellular carcinoma: an appraisal of current controversies[J]. Liver Cancer, 2012,1(34):183189.

[8] BRAILLON A. Is the American association for the study of liver diseases recommendation for hepatocellular carcinoma screening a culdesac?[J]. World J Gastroenterol, 2013,19(21):33693370.

[9] DANIELE B, BENCIVENGA A, MEGNA A S, et al. Alphafetoprotein and ultrasonography screening for hepatocellular carcinoma[J]. Gastroenterology, 2004,127(5 Suppl 1):S108S112.

[10] ZIPPRICH A, GARCIATSAO G, ROGOWSKI S, et al. Prognostic indicators of survival in patients with compensated and decompensated cirrhosis[J]. Liver Int, 2012,32(9):14071414.

[11] ACCOLLA R S, LOMBARDO L, ABDALLAH R, et al. Boosting the MHC Class IIrestricted tumor antigen presentation to CD4+ T helper cells: a critical issue for triggering protective immunity and reorienting the tumor microenvironment toward an antitumor state[J]. Front Oncol, 2014,4:32.

[12] RAN Q, HAO P, XIAO Y, et al. CRIF1 interacting with CDK2 regulates bone marrow microenvironmentinduced G0/G1 arrest of leukemia cells[J]. PLoS One, 2014,9(2):e85328.

[13] MIYAHARA Y, ODUNSI K, CHEN W, et al. Generation and regulation of human CD4+ IL17producing T cells in ovarian cancer[J]. Proc Natl Acad Sci U S A, 2008,105(40):1550515510.

[14] BERTINO G, ARDIRI A, MALAGUARNERA M, et al. Hepatocellualar carcinoma serum markers[J]. Semin Oncol, 2012,39(4):410433.

[15] YAMAMOTO K, IMAMURA H, MATSUYAMA Y, et al. AFP, AFPL3, DCP, and GP73 as markers for monitoring treatment response and recurrence and as surrogate markers of clinicopathological variables of HCC[J]. J Gastroenterol, 2010,45(12):12721282.

[16] 刘立国.术前血清中细胞因子含量与肝癌切除术后预后相关性探讨[D].北京:北京协和医院, 2012.

[17] 李宝华,何蓓晖,潘高辉. 白细胞介素17在原发性肝癌中的表达及意义[J]. 中国卫生检验杂志, 2012(10):24052406,2409.

(编辑 王 健)

[7] CHEAH Y L, CHOW P K H. Liver transplantation for hepatocellular carcinoma: an appraisal of current controversies[J]. Liver Cancer, 2012,1(34):183189.

[8] BRAILLON A. Is the American association for the study of liver diseases recommendation for hepatocellular carcinoma screening a culdesac?[J]. World J Gastroenterol, 2013,19(21):33693370.

[9] DANIELE B, BENCIVENGA A, MEGNA A S, et al. Alphafetoprotein and ultrasonography screening for hepatocellular carcinoma[J]. Gastroenterology, 2004,127(5 Suppl 1):S108S112.

[10] ZIPPRICH A, GARCIATSAO G, ROGOWSKI S, et al. Prognostic indicators of survival in patients with compensated and decompensated cirrhosis[J]. Liver Int, 2012,32(9):14071414.

[11] ACCOLLA R S, LOMBARDO L, ABDALLAH R, et al. Boosting the MHC Class IIrestricted tumor antigen presentation to CD4+ T helper cells: a critical issue for triggering protective immunity and reorienting the tumor microenvironment toward an antitumor state[J]. Front Oncol, 2014,4:32.

[12] RAN Q, HAO P, XIAO Y, et al. CRIF1 interacting with CDK2 regulates bone marrow microenvironmentinduced G0/G1 arrest of leukemia cells[J]. PLoS One, 2014,9(2):e85328.

[13] MIYAHARA Y, ODUNSI K, CHEN W, et al. Generation and regulation of human CD4+ IL17producing T cells in ovarian cancer[J]. Proc Natl Acad Sci U S A, 2008,105(40):1550515510.

[14] BERTINO G, ARDIRI A, MALAGUARNERA M, et al. Hepatocellualar carcinoma serum markers[J]. Semin Oncol, 2012,39(4):410433.

[15] YAMAMOTO K, IMAMURA H, MATSUYAMA Y, et al. AFP, AFPL3, DCP, and GP73 as markers for monitoring treatment response and recurrence and as surrogate markers of clinicopathological variables of HCC[J]. J Gastroenterol, 2010,45(12):12721282.

[16] 刘立国.术前血清中细胞因子含量与肝癌切除术后预后相关性探讨[D].北京:北京协和医院, 2012.

[17] 李宝华,何蓓晖,潘高辉. 白细胞介素17在原发性肝癌中的表达及意义[J]. 中国卫生检验杂志, 2012(10):24052406,2409.

(编辑 王 健)

[7] CHEAH Y L, CHOW P K H. Liver transplantation for hepatocellular carcinoma: an appraisal of current controversies[J]. Liver Cancer, 2012,1(34):183189.

[8] BRAILLON A. Is the American association for the study of liver diseases recommendation for hepatocellular carcinoma screening a culdesac?[J]. World J Gastroenterol, 2013,19(21):33693370.

[9] DANIELE B, BENCIVENGA A, MEGNA A S, et al. Alphafetoprotein and ultrasonography screening for hepatocellular carcinoma[J]. Gastroenterology, 2004,127(5 Suppl 1):S108S112.

[10] ZIPPRICH A, GARCIATSAO G, ROGOWSKI S, et al. Prognostic indicators of survival in patients with compensated and decompensated cirrhosis[J]. Liver Int, 2012,32(9):14071414.

[11] ACCOLLA R S, LOMBARDO L, ABDALLAH R, et al. Boosting the MHC Class IIrestricted tumor antigen presentation to CD4+ T helper cells: a critical issue for triggering protective immunity and reorienting the tumor microenvironment toward an antitumor state[J]. Front Oncol, 2014,4:32.

[12] RAN Q, HAO P, XIAO Y, et al. CRIF1 interacting with CDK2 regulates bone marrow microenvironmentinduced G0/G1 arrest of leukemia cells[J]. PLoS One, 2014,9(2):e85328.

[13] MIYAHARA Y, ODUNSI K, CHEN W, et al. Generation and regulation of human CD4+ IL17producing T cells in ovarian cancer[J]. Proc Natl Acad Sci U S A, 2008,105(40):1550515510.

[14] BERTINO G, ARDIRI A, MALAGUARNERA M, et al. Hepatocellualar carcinoma serum markers[J]. Semin Oncol, 2012,39(4):410433.

[15] YAMAMOTO K, IMAMURA H, MATSUYAMA Y, et al. AFP, AFPL3, DCP, and GP73 as markers for monitoring treatment response and recurrence and as surrogate markers of clinicopathological variables of HCC[J]. J Gastroenterol, 2010,45(12):12721282.

[16] 刘立国.术前血清中细胞因子含量与肝癌切除术后预后相关性探讨[D].北京:北京协和医院, 2012.

[17] 李宝华,何蓓晖,潘高辉. 白细胞介素17在原发性肝癌中的表达及意义[J]. 中国卫生检验杂志, 2012(10):24052406,2409.

(编辑 王 健)

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