血清异常凝血酶原复合物、甲胎蛋白、铁蛋白检测对HBV相关肝细胞癌的辅助诊断意义

文 君， 李 嘉， 王春妍， 马 莹

(天津市第二人民医院 肝病Ⅱ科， 天津 300193)

血清异常凝血酶原复合物、甲胎蛋白、铁蛋白检测对HBV相关肝细胞癌的辅助诊断意义

文 君， 李 嘉， 王春妍， 马 莹

(天津市第二人民医院 肝病Ⅱ科， 天津 300193)

目的探讨血清异常凝血酶原复合物(PIVKA-Ⅱ)、甲胎蛋白(AFP)、铁蛋白(FER)单项及联合检测在HBV相关肝细胞癌(HCC)辅助诊断中的意义。方法收集2016年6月-2017年2月于天津市第二人民医院住院的40例HBV相关HCC、41例乙型肝炎肝硬化、44例慢性乙型肝炎患者及36例健康体检者的血清，并检测PIVKA-Ⅱ、AFP、FER水平，分别分析三者单独及联合检测诊断HBV相关HCC的受试者工作曲线下面积(AUC)、敏感度和特异度。非正态分布的计量资料组间比较采用非参数Kruskal-WallisH检验， 进一步两两比较采用Mann-WhitneyU检验。采用二元logistic 进行逐步回归分析产生三项指标联合预测概率的新变量pre。结果血清AFP及PIVKA-Ⅱ水平在乙型肝炎肝硬化、慢性乙型肝炎、HBV相关HCC及健康对照组间的差异均具有统计学意义(χ2值分别为51.446、59.613，P值均<0.001)。AFP水平在HBV相关HCC组显著高于乙型肝炎肝硬化组、慢性乙型肝炎组及健康对照组(Z值分别为 -4.609、-6.026、-6.031，P值均<0.001)，乙型肝炎肝硬化AFP水平也明显高于对照组(Z=-2.30，P=0.021)；PIVKA-Ⅱ水平在HBV相关HCC组显著高于乙型肝炎肝硬化组、慢性乙型肝炎组及健康对照组(Z值分别为 -6.080、-6.595、-5.608，P值均<0.001)，慢性乙型肝炎组PIVKA-Ⅱ水平也明显高于对照组(Z=-2.153，P=0.031)；FER水平在HBV相关HCC患者组显著高于慢性乙型肝炎患者(Z=-2.177，P=0.029)。单项指标检测诊断HBV相关HCC时，AFP的敏感度最高(79.49%)，但FER的特异度最高(94.28%)；双项目检测方案中，AFP/PIVKA-Ⅱ方案的敏感度最高(89.74%)，FER+AFP和FER+PIVKA-Ⅱ方案特异度较高(97.14%)；三项目检测时，FER/AFP/PIVKA-Ⅱ方案的敏感度较高(92.31%)，FER+AFP+PIVKA-Ⅱ特异度较高(97.14%)。结论PIVKA-Ⅱ、AFP、FER三项联合检测可提高单独检测的敏感度及特异度。血清PIVKA-Ⅱ和AFP对HCC诊断具有较高的临床应用价值，单项即可很好辅助诊断，二者联合未提高诊断率。

癌， 肝细胞； 铁蛋白质类； 甲胎蛋白类； 异常凝血酶原； 诊断

原发性肝癌是我国最常见的恶性肿瘤之一，在我国该病发生率和病死率均占全球的50%以上，由于起病隐匿，进展较迅速，确诊时很多患者已经达到局部晚期或发生远处转移，因此早期发现及早期治疗是提高疗效的关键。其中肝细胞癌(HCC)占原发性肝癌的90%以上，是最常见的一种病理类型。HBV感染在HCC的发生、发展中起重要作用。目前我国HCC主要依靠影像学改变、肿瘤标志物的检测以及相应临床表现来诊断。血清异常凝血酶原(protein induced by vitamin K absence or antagonist-Ⅱ, PIVKA-Ⅱ)即γ-羧基凝血酶原，也被称为维生素K缺乏或拮抗剂诱导的蛋白质，为肝脏合成凝血酶原过程中凝血酶原前体未完全羧化形成的异常产物[1]。研究[2-3]显示PIVKA-Ⅱ已逐渐成为临床上诊断HCC的特异性肿瘤标志物，具有较高的诊断特异度，能较好地反映HCC的病理特征及预后。AFP也是诊断HCC的重要指标和特异度强的肿瘤标志物，但根据对国内外多篇文献的 Meta分析发现其对HCC诊断敏感度仅为54%～63%，特异度为82%～89%[4]。铁蛋白(ferritin, FER)是主要的贮铁蛋白，对调节铁的储存及代谢和体内平衡至关重要，且可保护细胞免受氧化损伤，其中血清FER水平可作为体内储铁情况的指标。近年来，研究[5]表明HCC的发生与FER水平异常紧密相关。本研究旨在通过对HBV相关HCC患者进行PIVKA-Ⅱ、AFP、FER检测，了解其对HCC的诊断效果及价值，探讨3 种指标联合检测对HCC辅助诊断的意义。

1 对象与方法

1.1 研究对象 选取本院2016年6月-2017年2月住院患者与健康体检者共161例，包括男97例，女64例，年龄18～74岁，平均(44.8±14.2)岁。其中HBV相关HCC患者40例，男23例，女17例，平均(58.5±10.1)岁；乙型肝炎肝硬化患者41例，男21例，女20例，平均(48.7±12.1)岁；慢性乙型肝炎患者44例，男32例，女12例，平均(33.0±10.2)岁。HBV相关HCC、乙型肝炎肝硬化、慢性乙型肝炎诊断标准符合《原发性肝癌诊疗规范》[6]和《慢性乙型肝炎防治指南》[7]中的诊断标准。健康体检者36例，男21例，女15例，平均(39.6±8.9)岁。

1.2 标本要求及检测方法 采集所有受试者空腹静脉血4 ml，室温放置1 h，2000 r/min离心8 min，分离后的血清于-80 ℃冰箱保存待检。PIVKA-Ⅱ 采用化学发光酶免疫检测法，试剂盒购自日本富士瑞必欧公司；AFP采用化学发光免疫法检测，AXSYM免疫化学发光仪及配套试剂购自美国雅培公司；血清FER采用免疫比浊法检测，试剂盒购自上海景源生物公司。

2 结果

2.1 不同组间血清FER、AFP及PIVKA-Ⅱ水平 血清AFP及PIVKA-Ⅱ水平在乙型肝炎肝硬化组、慢性乙型肝炎组、HBV相关HCC组及健康对照组间的差异均具有统计学意义(P值均<0.001)。AFP水平在HBV相关HCC组显著高于乙型肝炎肝硬化组、慢性乙型肝炎组及健康对照组(Z值分别为 -4.609、-6.026、-6.031，P值均<0.001)，乙型肝炎后肝硬化AFP水平也明显高于对照组(Z=-2.30，P=0.021)；PIVKA-Ⅱ水平在HBV相关HCC组显著高于乙型肝炎肝硬化组、慢性乙型肝炎组及健康对照组(Z值分别为 -6.080、-6.595、-5.608，P值均<0.001)，慢性乙型肝炎组PIVKA-Ⅱ水平也明显高于对照组(Z=-2.153，P=0.031)。FER水平在各组间差异无统计学意义(P>0.05)(表1)。

2.2 FER、AFP及PIVKA-Ⅱ单项及联合检测对HBV相关HCC的辅助诊断价值 单项指标检测诊断HBV相关性HCC时，AFP的敏感度最高(79.49%)，但FER的特异度最高(94.28%)；双项目检测方案中，AFP/PIVKA-Ⅱ方案的敏感度最高(89.74%)，FER+AFP和FER+PIVKA-Ⅱ方案特异度较高(97.14%)；三项目检测时， FER/AFP/PIVKA-Ⅱ方案的敏感度较高(92.31%)，FER+AFP+PIVKA-Ⅱ特异度较高(97.14%)(表2)。

2.3 FER、AFP及PIVKA-Ⅱ 单项及联合检测诊断HBV相关HCC的ROC曲线分析 单项检测时，FER的AUC为0.630(0.499～0.762)，AFP的AUC为0.908(0.836～0.980)，PIVKA-Ⅱ的AUC为0.879(0.799～0.959)(图1)。以FER、AFP、PIVKA-Ⅱ检测结果为检验变量，以诊断结果为状态变量，用向后条件方法做二元logistic回归分析，得出HBV相关HCC概率预测模型：P=1/[1+e-(-5.562+0.383AFP-0.068PIVKA-Ⅱ)]，算出各个体预测值的预测变量pre，其中FER没有纳入模型中。对上述logistic回归模型进行似然比检验，结果显示差异有统计学意义(χ2=67.425，P<0.001)。以预测变量pre为检验变量，诊断结果为状态变量，做AFP及PIVKA-Ⅱ二者联合检测的ROC曲线分析，其AUC为0.941(0.879～1.000)，高于各指标单项检测，但与AFP及PIVKA-Ⅱ相比，AUC差异没有统计学意义(Z值分别为0.351、0.618，P值均>0.05)。

表1 各组血清AFP、FER、PIVKA-Ⅱ水平比较[M(P25～P75)]

注：与HBV相关HCC组比较，1)P<0.001；与健康对照组比较，2)P<0.05

表2 血清FER、AFP及PIVKA-Ⅱ单项及联合检测对HBV相关性HCC的辅助诊断价值比较

注：“+”表示联合检测，“/”表示其中任一项

图1 FER、AFP及PIVKA-Ⅱ单项及AFP联合PIVKA-Ⅱ检测诊断HBV相关HCC的ROC曲线

3 讨论

PIVKA-Ⅱ是HCC在缺乏或对维生素K摄取及利用障碍时，由依赖维生素K的γ-谷氨酰羧化酶及辅酶活性降低、肝癌细胞内质网产生凝血酶原前体蛋白N端谷氨酸羧化不全所致，这些不成熟的凝血酶原释放入血，具有刺激癌细胞生长作用。PIVKA-Ⅱ具有敏感度、特异度高的优点，且其浓度与HCC的组织分化、病灶大小、门静脉浸润及临床分期等有关。大量临床资料显示，PIVKA-Ⅱ水平与平均肝癌体积、瘤灶数量、肿瘤大小均呈正相关，即随肿瘤的增大而升高，也随肿瘤TNM分期增高而升高；与平均生存时间呈负相关，与HCC的恶性程度呈正相关性。手术切除瘤灶后，PIVKA-Ⅱ水平下降低于临界值可能说明手术效果良好，但如果血清 PIVKA-Ⅱ水平再次升高，可能预示HCC复发或远处转移。动态监测 PIVKA-Ⅱ变化，能较好地协助临床正确评价HCC的发生、发展、浸润转移或复发等变化。PIVKA-Ⅱ已成为临床用于评价患者预后的一个重要指标[8-15]。AFP作为HCC的经典肿瘤标志物，在半个多世纪的临床应用中发现其敏感度和特异度均受到临床需求的挑战，如AFP阴性易导致HCC的漏诊和误诊，又因AFP水平在约30%的肝硬化、慢性肝病等非HCC患者中也有不同程度的升高[16]，致使伴有肝硬化的部分HCC患者经有效治疗后血清AFP仍处于较高水平[17]，从而影响HCC的疗效监测等。FER在肝脏合成，在肝内的含量最多，肝细胞损伤或被破坏时就会释放大量的FER。 有研究[18]表明，肝细胞、巨噬细胞、癌细胞可以分泌合成FER，且体内血清FER主要来源于巨噬细胞，因此肝癌细胞增殖可使FER分泌增加。

本研究表明血清AFP及PIVKA-Ⅱ水平在乙型肝炎肝硬化、慢性乙型肝炎、HCC及健康对照组间的差异均具有统计学意义。与其他研究不同，本研究中与另外几组比较HCC组FER水平较高，但差异无统计学意义，可能与机体感染肝炎病毒时，肝细胞发生炎症反应，血清合成FER增多，同时肝细胞炎症可引起肝细胞实质性损害，从而对血清FER清除能力下降，导致FER水平明显升高[19]，故各组间无统计学差异。此外，亦有可能与选取病例数不足，样本量不足，或未进行HCC的更为细致的临床分期相关，需进一步的临床研究来证实。本研究表明，AFP、PIVKA-Ⅱ在HBV相关HCC辅助诊断中具有重要价值，两者单项检测即可很好的预测诊断HCC，联合检测不会提高HBV相关HCC的诊断率。单项指标检测诊断HBV相关HCC时，敏感度最高者为AFP，特异度最高者为FER，这与大量临床报道相符合[20-21]。双项目检测方案中，敏感度最高方案为AFP/PIVKA-Ⅱ；三项目检测时，敏感度较高方案为FER/AFP/PIVKA-Ⅱ，特异度较高方案为FER+AFP+PIVKA-Ⅱ。总之，三项联合检测可以起到互补作用，可显著提高HBV相关HCC的检出率，与单项检测相比诊断效能大大提高。

综上，在条件允许的情况下，上述3项指标的联合检测对HBV相关HCC的确定诊断、减少漏诊具有较高的临床应用价值。 当然，以上各项检测手段仅能作为辅助方式，HBV相关HCC的诊断尚需病原学、影像学、病理学的综合诊断。如条件允许，可进一步研究上述3项指标在治疗前后的对比，为HBV相关HCC治疗疗效评价提供依据。

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(本文编辑：王 莹)

ValueofserumPIVKA-Ⅱ,alpha-fetoprotein,andferritininassistingthediagnosisofHBV-relatedhepatocellularcarcinoma

WENJun,LIJia,WANGChunyan,etal.

(SecondDepartmentofHepatology,TianjinSecondPeople′sHospital,Tianjin300193,China)

ObjectiveTo investigate the value of serum PIVKA-Ⅱ, alpha-fetoprotein (AFP), and ferritin (FER) measured alone or in combination in assisting the diagnosis of HBV-related hepatocellular carcinoma (HCC).MethodsA total of 40 patients with hepatitis B virus (HBV)-related HCC, 41 patients with liver cirrhosis after hepatitis B, 44 patients with chronic hepatitis B (CHB), and 36 controls who underwent physical examination were enrolled. Their serum samples were collected and the serum levels of PIVKA-Ⅱ, AFP, and FER were measured. The area under the ROC curve (AUC), sensitivity, and specificity of PIVKA-Ⅱ, AFP, and FER measured alone or in combination in the diagnosis of HBV-related HCC were analyzed. The non-parametric Kruskal-WallisHtest was used for comparison of non-normally distributed continuous data between groups, and the Mann-WhitneyUtest was used for further comparison between two groups. A binary logistic stepwise regression analysis was used to determine the new variable pre of predicted probability of combined measurement of these three indices.ResultsThere were significant differences in the serum levels of AFP and PIVKA-Ⅱ between the liver cirrhosis group, CHB group, HBV-related HCC group, and healthy control group (χ2=51.446 and 59.613, bothP<0.001). The HBV-related HCC group had a significantly higher serum level of AFP than the liver cirrhosis group, CHB group, and healthy control group (Z=-4.609, -6.026, and -6.031, allP<0.001), and the liver cirrhosis group also had a significantly higher serum level of AFP than the healthy control group (Z=-2.30,P=0.021). The HBV-related HCC group had a significantly higher serum level of PIVKA-Ⅱ than the liver cirrhosis group, CHB group, and healthy control group (Z=-6.080, -6.595, and -5.608, allP<0.001), and the CHB group had a significantly higher serum level of PIVKA-Ⅱ than the healthy control group (Z=-2.153,P=0.031). The HBV-related HCC group had a significantly higher serum level of FER than the CHB group (Z=-2.177,P=0.029). When measured alone, AFP had the highest sensitivity in the diagnosis of HBV-related HCC (79.49%), and FER had the highest specificity (94.28%). When any two of these indices were measured, PIVKA-Ⅱ/AFP had the highest sensitivity (89.74%), and FER+AFP and FER+PIVKA-Ⅱ had a high specificity (97.14%). FER/AFP/PIVKA had a sensitivity of 92.31% and the combined measurement of FER, AFP, and PIVKA-Ⅱ had a specificity of 97.14%.ConclusionCombined measurement of PIVKA-Ⅱ, AFP, and FER can improve the sensitivity and specificity of single measurement. Serum PIVKA-Ⅱ and AFP have a high clinical value in the diagnosis of HCC; single measurement can well assist the diagnosis, and combined measurement does not increase the diagnostic rate.

carcinoma, hepatocellular; ferritins; alpha-fetoproteins; protein induced by vitamin K absence or antagonist-Ⅱ; diagnosis

10.3969/j.issn.1001-5256.2017.09.020

2017-03-20；

2017-05-22。

文君(1985-)，女，主治医师，主要从事肝病学及内镜学研究。

李嘉,电子信箱: 18622663700@163.com。

R735.7

：A

：1001-5256(2017)09-1729-05