修回日期: 2005-08-19
接受日期: 2005-08-26
在线出版日期: 2005-09-28
原发性肝癌(primary hepatic cancer, PHC)是世界范围内第8位最常见的恶性肿瘤, 在我国肝细胞癌(hepatocellular carcinoma, HCC)占其91.5%. HCC是肿瘤病因学中的重要类型, 肝硬化、病毒性肝炎、化学致癌物及环境因素等所造成的慢性肝脏损害都可诱发HCC.HCC恶性度高, 容易复发及转移, 预后差, 而且早期诊断较困难, 延误了最佳治疗时期. HCC的生物标志物对于HCC的早期诊断、监测肿瘤进展、疗效判定、复发和存活率的判定十分重要. 因此, 寻找有效的HCC生物标志物是医学工作者多年以来的努力方向, 并取得了很大的进展. 甲胎蛋白(alpha-fetoprotein, AFP)是临床上诊断HCC最常用的指标, 其敏感性和特异性分别为60%和90%, AFP-L3、AFU、DCP及anti-p53等也都有各自的优缺点, 新近发现SCCA-IgM IC在HCC患者有表达, 其敏感性及特异性均较高, 可能不久以后会成为HCC早期诊断的重要依据.
引文著录: 王嘉倍, 刘连新. 肝细胞癌生物标志物的研究进展. 世界华人消化杂志 2005; 13(18): 2251-2256
Revised: August 19, 2005
Accepted: August 26, 2005
Published online: September 28, 2005
N/A
- Citation: N/A. N/A. Shijie Huaren Xiaohua Zazhi 2005; 13(18): 2251-2256
- URL: https://www.wjgnet.com/1009-3079/full/v13/i18/2251.htm
- DOI: https://dx.doi.org/10.11569/wcjd.v13.i18.2251
肝细胞癌(hepatocellular carcinoma, HCC)是世界范围内第四位常见的恶性肿瘤[1]. 每年大约有250 000名患者死于肝细胞癌[2]. 肝细胞癌的发病机制至今仍未清楚. 其在临床上恶性度较高, 死亡率在消化道恶性肿瘤中排第三位. 所以早期发现、早期治疗是提高患者生存率的关键, 而大多数患者就诊时已届中晚期, 失去了最佳治疗时机. HCC患者死亡的原因多为肿瘤破裂所致的大出血、食道胃底静脉曲张破裂出血、肝性脑病、多脏器功能衰竭及肝肾综合征等. 因此寻找有助于HCC早期诊断的生物标志物具有很高的临床价值. 本文就一些HCC生物标志物加以阐述.
SCCA是一种丝氨酸蛋白酶抑制剂, 可存在于正常鳞状上皮的棘状层和颗粒层, 但其在上皮起源的肿瘤细胞中典型表达[3-4]. 其同源体SCCA-1和SCCA-2可保护肿瘤细胞免于凋亡, SCCA-1还可促进肿瘤生长[5]. 最近有报道[6]在所有手术切除的HCC标本中经免疫组化均发现了过度表达的鳞状细胞癌抗原(SCCA)异质体(SCCA-1, SCCA-2和SCCA-PD), 而在正常肝脏组织中未发现此类物质.
Beneduce et al[7]对50例HCC患者, 50例肝硬化患者, 50例慢性肝炎患者及73例正常对照组的健康人的血清SCCA-IgM IC进行了检测, 研究发现: 由SCCA及IgM组成的循环免疫复合物在73名正常对照组中没有被检测到, 而在50例HCC患者中有35例(70%)检测到了SCCA-IgM IC; 在50例肝硬化患者中有13例(26%)检测到了SCCA-IgM IC; 在50例慢性肝炎患者中有9例(18%)检测到了SCCA-IgM IC. 研究还发现, SCCA-IgM IC水平与AFP水平无关联性, 因为在相同的50例HCC患者中有21例(42%)检测到AFP显著升高(>200 μg/L). Giannelli et al[8]的研究还发现, SCCA-IgM IC在HCC肿瘤组织中的表达要比癌周组织中强得多, 而且在转移的肿瘤结节中也可以发现明显的SCCA-IgM IC的表达. 但并没有发现SCCA-IgM IC的表达与HCC的组织学分期及肿瘤大小之间的关联性. 因此, SCCA-IgM IC作为一种新的血清生物标志物, 其敏感性及特异性均较高, 对微小的转移灶也有较高的敏感性, 其单独或与AFP联合应用, 可极大的提高对HCC诊断的敏感性及特异性.
甲胎蛋白是在胎儿发育过程中由胎肝合成的, AFP在胚胎的12-14 wk合成达高峰, 血清浓度可达1-3 g/L, 胎儿出生后, 基因表达即关闭, 出生1年后, 血清浓度降至正常水平, 约为5.8 μg/L. 但当肝细胞发生病变时, AFP基因表达开放, 于血清中可检测到AFP浓度显著升高, 因此AFP成为诊断肝癌的生物标志物.
1.2.1 AFP: Abelev在1968年首次将AFP作为HCC的生物标志物加以研究[9]. 在当前, 临床上诊断HCC最常用的仍然是AFP, 最初其阳性值设定为>400 μg/L, AFP检测具有较高的敏感性, 30-60%的HCC患者表现为AFP的显著升高, 但AFP诊断HCC仍有一定的局限性, 因为有相当多的肝硬化患者的血清AFP值在200 μg/L以上. Gupta et al[10]发现AFP对丙型肝炎患者的敏感性和特异性分别是41%-65%和80%-94%. 此外, 部分良性肝病, 生殖系统和胃肠道的一些恶性肿瘤中AFP值也升高. 而且在乙肝表面抗原阴性的HCC患者中AFP水平明显低于乙肝表面抗原阳性或者丙肝表面抗原阳性的患者[11].
尽管AFP作为HCC生物标志物有一定的缺陷, 但其通常与超声检查联合应用来筛查HCC[12-13]. 尽管有些临床医生认为AFP对HCC的筛查没有作用[14], 但He et al[15]研究证明AFP与超声联合应用筛查HCC的敏感性几乎可达到100%.
另外, 有研究[16-17]证明AFP水平与肿瘤大小、组织学分级、肝内转移和门静脉癌栓形成等肿瘤各项指标无关.
1.2.2 AFP异质体(AFP variants): 随着生物化学及其相关分析技术的进展与应用, 发现AFP分子与外源性凝集素的亲和力不同, 即存在不均一性的糖链异质性. 应用不同的凝集素亲和电泳可以把它们分成若干个组分. 也可以用等电聚焦技术来分离AFP组分[18-19]. 研究发现总AFP中包含三种组分: AFP-L1, 来自良性肝病, 是AFP的主要组分; AFP-L2来自孕妇; AFP-L3为HCC细胞所特有.
Yoshida et al[20]以AFP-L3百分比大于15%作为正常与否的临界值, 并发现AFP-L3所占百分比与HCC的分期及肿瘤的大小有着很高的关联性, 而这些是AFP所不具备的. Khien et al[21]测定了65例HCC患者、25例慢性肝病患者的AFP-L3, HCC患者中敏感性为96.9%, 特异性为92.0%, 准确性为95.5%. AFP水平与AFP-L3水平无明显相关性. 在非恶性肝病中, 肝细胞不表达AFP-L3. 约35%小肝癌(<2cm)患者血清中可检测出AFP-L3, 与影像学比较可提前9-12 mo发现HCC[22]. Tada et al[23]研究还发现, 病理检查发现HCC患者门静脉及肝静脉受浸润者血清的AFP-L3的表达要比单纯HCC患者的AFP-L3表达强得多, 而门静脉及肝静脉受浸润往往是预后不良的提示. AFP-L3降低与否还可以作为HCC患者治疗的评估指标[24]. 所以, AFP-L3不仅可以早期诊断HCC, 而且还可以评估HCC患者的治疗及预后. 因此, AFP-L3作为AFP异质体是新一代的HCC生物标志物. 有着很高的临床使用价值.
1.2.3 AFP mRNA: HCC可以通过检测血清AFP mRNA进行早期诊断及判断预后, 因为AFP mRNA是肝癌细胞表达的第一步, 是从病变处脱离入血的微量肝癌细胞的标志, 它反映了循环血中HCC肿瘤细胞的存在及肝外转移的发生[25]. 应用逆转录聚合酶链反应(RT-PCR)可以检测到外周血中微量的HCC肿瘤细胞的存在[26]. 但AFP mRNA的临床价值仍然存在争议.
Ijichi et al[27]对87例HCC患者进行研究, 检测术前及术后的血清AFP mRNA水平, 并跟踪调查这些患者术后28 mo的复发情况. 在术前有31例患者(36%)血清AFP mRNA阳性, 术后有30例患者(34%)血清AFP mRNA阳性, 术后28 mo有46例患者(53%)复发. HCC患者术前及术后AFP mRNA均为阳性的患者有极高的复发率(85%), 并有远处转移及多部位复发的趋势. 术后AFP mRNA阳性患者的复发间期要明显短于阴性患者, 所以术后AFP mRNA水平与肿瘤术后转移间隔时间呈正相关. Cillo et al[25]还发现循环血中AFP mRNA的水平与胆汁郁积指数、结节大小及动脉侵入有显著关联性. 因此AFP mRNA不仅可以早期预测HCC, 而且还可以评估HCC患者的预后及复发.
AFU是广泛分布于人体组织细胞、血液、体液中的溶酶性酸性水解酶, 系统命名为α-L-岩藻糖苷岩藻糖水解酶(EC3, 2, 1, 51). 法国学者Deugnier于1984年首先提出AFU可能成为诊断原发性肝癌的一项有用指标.
Giardina et al[28]对132例肝硬化患者进行了长达8年的跟踪调查发现: 在超声诊断HCC前6-9 mo, 有43.7%的HCC患者血清AFU值显著升高, 而且对于AFP阴性的HCC患者血清AFU也有较高的敏感性(57.1%). 但是, 研究还发现AFU与肿瘤大小无关联性, 而AFP则与肿瘤大小有显著关联性. 因此, 血清AFU对早期的HCC诊断有很大的帮助, 临床上应对血清AFU升高的肝硬化患者提高警惕. 而且, 鉴于AFU检测方法简单、快速、方便, 适合大规模人群普查早期HCC, 临床上应该大力推广[29].
TPS是从组织多肽抗原(TPA)中纯化而来, 其实质是细胞角蛋白18片段上的M3决定簇[30], 在细胞周期的S晚期和G2期, 伴随着DNA、蛋白质的合成, TPS被合成并于细胞分裂后不久释放入血或体液中. 所以, 血清TPS水平常反映肿瘤细胞的分裂增殖活性.
Yao et al[31]的研究发现: TPS仅与肿瘤大小有关. 他们分别对69例HCC患者, 57例健康人, 56例慢性肝炎患者和49例肝硬化患者应用单克隆TPS IRMA测定其血清TPS水平, 同时还测定了AFP值. 结果显示: TPS血清水平仅高于正常对照组(P = 0.001), 与肝硬化及肝炎组无显著差异. TPS与AFP无显著相关性. 血清TPS仅与肿瘤大小之间存在显著相关性(P = 0.005); AFP与肿瘤大小, 门脉癌栓及肿瘤分化程度之间存在显著相关性(P<0.05). 血清TPS与HCC肿瘤侵袭性之间无显著相关性. 但与肝功能受损程度相关性显著, 导致在部分肝硬化患者或肝炎患者中出现了高水平表达. 因此, TPS虽然尚不能够独立诊断HCC, 但是对肝病患者血清TPS的升高应注意结合其它相关的临床指标加以具体分析, 以便能够达到早期诊断HCC的目的.
p53基因定位于17P13.1, 长约20 kb, 含11个外显子, 转录mRNA长约2.8 kb, 产物p53蛋白由393个氨基酸组成. 野生型p53基因克隆或其cDNA的表达, 可抑制其它癌基因对细胞的转化, 一旦其发生了突变或缺失, 即可诱发多种肿瘤. 长时间接触真菌毒素及黄曲霉毒素B1均可诱发p53基因突变[32]. 可通过检测anti-p53水平来加以判断.
Alves et al[33]应用免疫组化方法对54例HCC患者, 30例肝硬化患者的anti-p53进行检测. 在Ⅲ-Ⅳ级的HCC患者中有58.3%检测到了anti-p53的免疫表达, 在Ⅰ-Ⅱ级的HCC患者中有22.2%的患者显示阳性结果, 平均阳性率为35.2%. 其数据介于美国与非洲报道的数据之间(6%-18%, 40-50%). 而在癌旁组织及30例肝硬化患者中没有检测到anti-p53的免疫表达. 因此, anti-p53的检测对HCC的诊断具有一定的价值, 可作为一种新的HCC生物标志物, 但由于其与美国和非洲所报道的数据有所差别, 而且其对于其它的胃肠道恶性肿瘤也有较高的阳性率: 胆管癌(100%), 胰腺癌(75%), 结肠癌(70%), 食管癌(60%)和胃癌(35%). 所以其仍需进一步的研究以供临床应用.
正常肝脏组织可以在肝细胞内质网内将DCP羧化使之成为有活性的凝血酶原, 一旦机体需要, 凝血酶原即可转变成凝血酶参与机体的各项生理功能. 但HCC患者肝细胞发生癌变, 内质网失去其正常功能, 不能将DCP羧化成有活性的凝血酶原, 从而血清中DCP含量增高.
1984年Liebman首次报道91%的肝癌患者血清中DCP的水平升高. 血清DCP水平与HCC患者是否有动脉侵入有很大关联性, 血清DCP水平>300 AU/L的HCC患者绝大多数伴有动脉侵入, 而DCP水平与肿瘤结节数量之间没有发现关联性[34]. 有研究表明, DCP作为HCC的生物标志物具有48-62%的敏感性和81-98%的特异性[35]. 袁联文 et al[36]测定了东京大学医学部附属病院肝胆胰外科1995至1997年间收治的92例HCC患者血清DCP和AFP水平, 发现DCP诊断HCC的准确率, 敏感性和特异性分别为56%, 46%和92%, AFP阳性率为41%, 且两者在诊断上无明显相关性, 即血清DCP在诊断HCC不仅与AFP同样有效, 而且具有较高的特异性. HCC患者血清DCP水平与癌灶大小呈正相关, 且术前血清DCP高的患者年生存率明显低于血清DCP低的患者[37].
由于DCP和AFP之间在诊断HCC上没有关联性, 而且单独的DCP或AFP都不是诊断HCC的完美指标, 所以联合这两种生物标志物来提高对HCC的诊断率是一种合乎逻辑的方法. Marrero et al[38]研究发现联合应用DCP和AFP在鉴别HCC和其他肝脏疾病方面并不优于单独应用DCP. 可是联合应用DCP和AFP-L3对于早期诊断HCC有较大的帮助[39].
GPC3是一种硫酸类肝素蛋白多糖[40], 它可以借助磷脂酰基醇糖基锚定在细胞膜上. GPC3在绝大多数HCC患者有表达, 而在正常肝组织及良性肝脏病变中不表达, GPC3通过增强自分泌或旁分泌Wnt基因表达信号来刺激体内和体外HCC肿瘤细胞生长[41]. GPC3能够黏附Wnt基因, 促进Wnt基因高表达, Wnt基因的表达产物能够促进多种肿瘤组织生长. 有研究证明, HCC患者的GPC3 mRNA表达增强[42,43]. 并且由Sung et al[44]证明培养基上的HCC细胞可以分泌GPC3. 根据Capurro et al[45]的研究结果, 只有1/20的肝硬化患者在血清中检测到GPC3. 所以检测血清GPC蛋白含量或GPC mRNA含量对于HCC的诊断具有较大帮助.
在75%的HCC患者中可以检测到GPC3 mRNA的过量表达, 仅在3.2%的正常人血清中检测到GPC3 mRNA的高表达[46]. 免疫组化实验证实在72%的HCC患者肝脏组织中可以检测到GPC3蛋白[47]; 而且, 应用酶联免疫标记试验(ELISA)在40-53%HCC患者的循环血中可以检测到GPC3蛋白[47,48]. 此外, GPC3的表达与肿瘤组织的分级及大小无关, 与AFP无关联性[49]. 因此, 在临床上检测GPC3可以对HCC患者进行早期诊断, 鉴于其对AFP阴性的HCC患者也有较高的敏感性, 可将其与AFP联合应用.
GGT及GGT-mRNA GGT可以催化谷胱甘肽中的γ-谷氨酸残基向氨基酸肽链或水转移. GGT在肝脏主要定位于Kupffer细胞、门脉周围血管和胆管内皮细胞, 在肝细胞为阴性. 可是发生癌变的肝细胞GGT均为阳性. 应用聚丙烯酰胺凝胶电泳可将GGT分离出9-11条区带, 其中Ⅰ'、Ⅱ和Ⅱ'为肝癌特异性GGT[50]. 其检测HCC的阳性率可达75%, 假阳性<5%, 血清GGT浓度与AFP和肿瘤大小无关联性, 其对AFP阴性的HCC患者的阳性率可达75%, 从而可以弥补AFP的不足. 所以, GGT可以作为早期诊断HCC的一个极其敏感的酶学指标[51].
GGT-mRNA分三种亚型(A、B、C亚型), 应用RT-PCR技术可检测患者外周血中的GGT-mRNA, A亚型主要存在于正常肝脏组织中, HCC组织中主要含有B亚型. GGT-mRNA亚型转化与GGT基因异常表达、甲基化状态改变和肝癌的发生关系密切.
VEGF由血管内皮细胞分泌, 在正常血管的生长发育中起着重要作用. 但VEGF同时也能促进肿瘤组织内血管的生长. 由VEGF调节的肿瘤相关的血管生长对于HCC的生长及转移非常重要[52]. 晚期HCC患者肿瘤组织多表现出血管增多的特性. VEGF和它的受体(VEGFR-1和VEGFR-2)交互作用, 在HCC的发展过程中起着关键性作用[53]. HCC患者血清VEGF的浓度要比健康者显著升高; 但在HCC患者和非HCC肝病患者之间却未发现显著区别. 免疫组化分析显示VEGF蛋白在高分化的HCC细胞中和HCC患者正常肝组织中均显著表达; 而在低分化的HCC中, VEGF在瘤内血管的内皮细胞中的表达要明显强于在肿瘤细胞中的表达[54]. 此外, 血清VEGF的浓度与CT和MR成像信号的强度和性质之间有关联性[55-56].
鉴于VEGF的血清浓度会在HCC出现前显著升高, 并且VEGF的分布状态会随着HCC的发展而显著改变, 因此, 检测患者血清VEGF浓度也是早期预测HCC的一项重要手段, 但由于其特异性较低, 所以应结合其它标志物共同使用.
TGF-β1是一大类多功能的生长因子, 哺乳动物中只表达TGF-β1, 2, 3, 其中TGF-β1含量最多. TGF-β1参与正常和转化细胞的生长和分化. TGF-β1可以抑制免疫系统, 从而促进癌细胞生长、浸润及远处转移. TGF-β1 mRNA在HCC患者有高表达, 尤其在小HCC与高分化的HCC的肿瘤组织中. 对38例小HCC(<3 cm)患者血清TGF-β1检测时发现其值显著升高. 将血清TGF-β1阳性标准定为800 ng/L时, 其诊断小HCC的特异性超过95%, 敏感性为68%[57].
TGF-β1是HCC肿瘤发生的原始标志物[58]. 由于AFP对小HCC的敏感性较低(24%), TGF-β1在诊断小HCC比AFP具有更高的敏感性及特异性, 所以TGF-β1可以作为一种生物标志物对可疑HCC患者进行早期检测.
丛生蛋白也叫载脂蛋白J, 是一种糖蛋白异二聚体, 存在于大多数动物的组织和体液中[59]. 有报道丛生蛋白在应激反应[60]、细胞凋亡和肿瘤发生中起到了重要作用. 丛生蛋白在人体多种恶性肿瘤中过度表达, 包括乳腺[61]、肾脏[62]、膀胱[63]、胰腺[64]、结肠[65]和淋巴[66]的恶性肿瘤. 丛生蛋白在肿瘤转化及恶化过程中所起的作用并没有得到明确阐述; 由于丛生蛋白经过翻译后修饰可以得到不同的亚型, 所以产生了很多有争议的结果[67]. 但是, 丛生蛋白在人类的几种恶性肿瘤中起到重要作用这一点是明确的. 经过免疫组化染色分析丛生蛋白在HCC患者中有89%的阳性率[68], 经过化学治疗的HCC患者丛生蛋白有上调趋势[69]. 其对HCC诊断的临床使用价值有待于进一步研究.
甘氨酰脯氨酸二肽氨基肽酶(GPDA)在1987年第一次被用作HCC的生物标志物来研究, 最近Ni et al[70]证明GPDA的同工酶GPDA-F在HCC患者血清呈持续阳性, 他们认为此生物标志物对于诊断无AFP分泌的HCC有较高的敏感性.
我国是HCC的高发区, 这与我国肝炎病毒感染较重, 传播广泛有关, 尤其是HBV、HCV的感染, 病毒性肝炎与HCC的发生密切相关. 所以, 防治HCC是医学工作者们面临的一项重要任务. 如果能够较早的发现并确定诊断HCC, 及早进行治疗, 那样将会极大地提高生存率. 可是目前所发现的与HCC有关的生物标志物虽然很多, 但尚无一种生物标志物能够独立确诊所有的HCC, 而且这些生物标志物之间不能起到关联互补的作用, 这就给防治HCC的工作带来了极大的困难. 因此应该有效地利用已知的HCC生物标志物, 并不断地深入研究探索新的HCC生物标志物, 从而有效地对HCC做到早期发现, 早期诊断, 早期治疗, 并做好预后评估, 防治复发.
电编:李琪 编辑:张海宁
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