蛋白质飞行质谱技术在原发性肝癌诊断中的应用

目的研究原发性肝癌患者与健康人血清蛋白质表达的差异性,寻找对原发性肝癌更有效的肿瘤诊断标志物。方法应用表面增强激光解析电离飞行时间质谱（SELDI—TOF—MS）技术检测50例原发性肝癌患者、50名健康对照者血清中的蛋白质谱,寻找有意义的蛋白质谱。同时采用双抗体夹心法检测血清甲胎蛋白（AFP）。结果原发性肝癌患者血清4个蛋白质峰与健康对照组比较差异有统计学意义。分别为：3354．71,8825．80（高表达）,4345．08,13715．01（低表达）。从中筛选出质荷比（M／z）分别为3354．71、8825．80差别最显著的蛋白质峰,使用这两个蛋白质峰作为原发性肝癌的诊断模式,其灵敏度、特异度分别为90％（45／50）,94％（47／50）。100例患者中,AFP阳性27例,灵敏度为54％（27／50）,特异度为100％（30／30）。结论SELDI—TOF—MS蛋白质芯片技术在原发性肝癌的诊断及肿瘤特异性蛋白质生物标志的筛选中具有一定的价值,灵敏度和特异度较高,操作简单,正确诊断指数（r=83）优于AFP（r=53）。     

蛋白质芯片技术应用于肝癌诊断的初步研究

 【摘要】　目的　应用表面增强激光解析电离飞行时间质谱（SELDI-TOF-MS）技术和蛋白质芯片从肝癌患者血清中筛选可用于肝癌诊断的标志蛋白质。方法　运用SELDI-TOF-MS技术及CM10蛋白质芯片检测46例原发性肝癌患者和64名健康人血清,获得蛋白质指纹图谱,采用Biomarker Wizard软件选出肝癌患者与健康人血清中的表达差异蛋白质,评价其灵敏度、特异度和诊断效能,确定最佳标志蛋白质。结果　肝癌组与健康对照组血清中有16个蛋白质的表达差异在2倍以上,并且差异有统计学意义（P＜0.05）。其中质荷比为6845.70的蛋白诊断效能最高,其灵敏度为89.1 %（41／46）,特异度87.5 %（56／64）,且该蛋白质与肿瘤大小有相关性（r＝-0.363,P＜0.05）。经数据库搜索该蛋白质很可能是免疫球蛋白重链可变区片段。结论　应用SELDI-TOF-MS技术诊断肝癌,具有灵敏度高,特异性好,快速简便的优点,质荷比为6845.70的蛋白质可能是肝癌患者血清中的特异性标志物。     

应用SELDI-TOF-MS技术建立卵巢浆液性乳头状囊腺癌诊断的血浆蛋白质组学模型

目的：通过蛋白质芯片-质谱技术结合生物信息学的支持向量机方法，筛选卵巢浆液性乳头状囊腺癌患者差异表达的蛋白质峰，探讨建立数学逻辑的卵巢浆液性乳头状囊腺癌诊断模型及其意义。方法：采用表面增强激光解离飞行时间质谱（surface-enhanced laser desorption/ionization time of flight mass spectrometry,SELDI-TOF-MS）技术，运用CM10蛋白质芯片（一种弱阳离子芯片）分别检测26例卵巢浆液性乳头状囊腺癌和51例对照组（其中卵巢囊肿12例、子宫肌瘤31例、卵巢良性囊腺瘤8例）的血浆标本，对所得到的质谱数据采用Biomarker Wizard软件分析，初步筛选蛋白质峰，结合生物信息学的支持向量机（support vector machines，SVM）方法建立并测试卵巢浆液性乳头状囊腺癌患者以及对照组的血浆蛋白质谱诊断模型。结果：应用SELDI-TOF-MS在CM10芯片上捕获到的蛋白质，经过Biomarker Wizard软件分析筛选出了卵巢浆液性乳头状囊腺癌患者与对照组的71个差异蛋白质峰（P〈0．01），利用SVM方法再次筛选获得7个蛋白质峰组成卵巢浆液性乳头状囊腺癌的蛋白质谱最优化模型，这7个蛋白质中质荷比（m／z）为4099、4477、4123、4081和3938的表达上调，质荷比（m／z）为8785和13783的表达下降。经三倍交叉验证后用盲法测定，所建模型用于卵巢浆液性乳头状囊腺癌患者与对照组鉴别的敏感度和特异度分别为84．62％和96．08％，阳性预测值为92．21％。结论：蛋白质芯片-质谱技术可以快速、有效地筛选出卵巢浆液性乳头状囊腺癌患者血浆差异蛋白质，结合SVM可建立有效的蛋白质质谱诊断模型，对卵巢癌诊断方法的建立具有潜在意义。     

HS-AFP在肝癌早期诊断及鉴别诊断中的价值

目的：探讨肝癌特异性甲胎蛋白（HS—AFP）对肝细胞癌的诊断及鉴别诊断价值。方法：对105例肝癌患者及151例良性肝病血清HS—AFP及AFP进行检测，并对10例HS—AFP阳性的良性肝病患者进行15个月随访观察。HS—AFP采用不连续缓冲系统PAGE结合Westernblot分离检测，AFP采用化学发光法测定。结果：105例肝癌患者HS—AFP和AFP〉200μg／L阳性率分别为60．0％和50．5％（P〉0．05），良性肝病患者HS—AFP阳性率显著低于AFP〉200μg／L的阳性率（P〈0．05）。AFP处于50～400μg／L范围的肝癌组HS—AFP阳性率为77．1％，良性肝病组阳性率为13．6％（P〈0．01），151例良性肝病中有10例HS-AFP出现阳性，其中3例于随访期间检出肝癌。11例小体积肝癌HS—AFP阳性率（45．5％）高于AFP〉200μg／L的阳性率（18．2％）。结论：HS—AFP对肝癌的诊断价值优于AFP浓度。对肝癌高危人群监测HS—AFP有助于肝癌的早期诊断。对血清AFP中低浓度升高的良恶性肝病有较高的鉴别诊断价值、     

血清蛋白质质谱模型在大肠癌诊断中的应用

目的 建立蛋白质芯片技术检测血清蛋白质质谱的方法,探讨基于人工神经网络的血清蛋白质质谱模型在大肠癌诊断中的应用价值。方法 应用表面增强激光解吸电离飞行时间质谱仪(SELDI-TOF-MS),测定了147例血清标本(其中大肠癌55例,健康人92例)的蛋白质质谱,用随机抽取的87例标本(大肠癌32例,健康人55例)作为训练组,进行训练与交叉验证,将筛选出来的5910,8930,4476和8817的4个质荷比峰作为输入,建立人工神经网络预测模型。并用另外测试组(大肠癌23例,健康人37例)的血清标本盲法验证该模型。结果 利用从训练组得出的基于人工神经网络的血清蛋白质质谱模型,对测试组的60例(包括Dukes A)未知血清进行预测,得到该方法对大肠癌的检出率为82．6％(19／23),排除率为91．9％(34／37)。结论 蛋白质芯片技术检测血清蛋白质质谱法在大肠癌的诊断中较以往的传统方法具有更高的检出率和排除率,值得进一步研究与应用。     

荧光检测外周血AFP mRNA的表达与肝癌的相关性研究

目的：研究定量检测原发性肝癌患者外周血单个核细胞（PBMC）甲胎蛋白信使核糖核酸（AFP mRNA)表达的意义。方法：应用荧光定量聚合酶链反应（FQ－PCR）定量检测51例原发性肝癌，10例转移性肝癌，18例非肝癌的恶性肿瘤，16例慢性肝病患者的外周血单个核细胞（PBMC）甲胎蛋白mRNA。结果：AFP mRNA在非肝癌的恶性肿瘤患者无异常表达，在转移性肝癌（20％，2／10），慢性肝病（6．3％，1／16），原发性肝癌（37．3％，19／5）、AFP mRNA的基因拷贝数和肝癌TNM分期[1]，门静脉癌栓，肝外转移显著相关，而与肿瘤大小及血清AFP值无相关性。结论：巢式FQ－PCR检测肝癌患者外周血AFP mRNA异常预示有血源性转移的可能，可作为肝癌的诊断，肿瘤分期和有无肝外转移及愈合的指标。     

就医指南

原发性肝癌的标志——甲胎蛋白(AFP) AFP正常值低于7ng／mL。肝细胞性肝癌发生时明显升高，因此常常以AFP作为检测原发性肝癌的诊断指标，标准为高于500ng／mL，阳性率可达70％-90％。卵巢癌等患者血清中水平也升高，而在消化道癌及肺癌中少见。怀孕时可一时性升高，慢性肝炎及肝硬化患者血清中经常在中等水平。     

应用SELDI-TOF-MS技术筛选肺腺癌患者血清诊断标志物

目的 探讨肺腺癌患者及正常人血清中蛋白质质谱的不同,筛选出肺腺癌血清诊断标志物.方法 用WCX2蛋白芯片结合表面增强激光解吸电离飞行时间质谱(SELDI-TOF-MS)技术,检测24例肺腺癌和10例正常人血清蛋白质谱,筛选出差异表达蛋白质.结果 本实验共检测到86个有效的蛋白质波峰,其中m/z位于分子质量2000～10 000的波峰有78个.筛选出m/z分子质量为8129.55,2022.18,3271.91,3933.44,3504.49,3811.71的6个血清肿瘤标志物.结论 SELDI-TOF-MS技术是寻找肺腺癌血清诊断标志物的有效工具.利用蛋白组学和生物信息学及相关技术,将有利于建立新的疾病诊断模式--蛋白质指纹图谱.     

应用SELDI蛋白质芯片技术筛选肺腺癌血清标志物的研究

目的 通过SELDI蛋白质芯片技术筛选肺腺癌特异血清标志物。方法 采用表面增强激光解析离子化一飞行时间质谱技术（SELDI-TOF-MS），选用弱阳离子加疏水膜芯片对15例肺腺癌患者，30例健康人血清进行检测，筛选在肺腺癌患者血清中差异表达的蛋白质。结果 在质荷比0—20000范围内，共检测到180个蛋白峰，建立了由8个差异表达蛋白质组成的肺腺癌诊断模型。这8个蛋白质中有6个在肺腺癌中表达上调，2个表达下调。软件分析结果显示在预测组中诊断肺腺癌的敏感性为93．33％（14／15）、特异性为100．00％（30／30）；对检测组进行双盲检测，敏感性为73．33％（11／15）、特异性为86．67％（26／30）。结论 由8个差异表达蛋白及其特定组合构成的诊断模型可以区分肺腺癌和健康人。SELDI蛋白质芯片技术能直接筛选出肺腺癌患者血清中相对特异的潜在标志物，具有较好的临床应用价值。     

表面增强激光解吸技术预测胃癌术后转移复发的临床研究

 目的　应用表面增强激光解吸（SELDI）技术筛选胃癌术后转移相关的血清蛋白质组指纹并建立预测模型。方法　应用CM10弱阳离子芯片结合表面增强飞行时间质谱（SELDI-TOF-MS）技术检测60例胃癌根治术后患者和26名健康对照血清样本的蛋白质谱，经过2年随访分为转移组（22例）和无转移组（38例），利用Biomarker Wizard软件比较各组间的血清蛋白质指纹图谱，Biomarker Pattern软件建立预测模型。结果　术后转移患者和健康对照相比有 74个蛋白质峰差异有统计学意义，术后无转移患者和健康对照相比有69个蛋白质峰差异有统计学意义，术后转移患者和无转移患者相比有14个蛋白质峰有显著性差异，质荷比（m／z）为4768和8841的两个蛋白质组成的诊断模型可将无转移胃癌患者与转移胃癌患者准确的分组，在学习模式下，灵敏度和特异度分别为95.46 %（21／22），86.84 %（33／38），准确度为90 %（54／60）。在测试模式下，灵敏度和特异度分别为81.82 %（18／22）和84.21 %（32／38），准确度为83.33 %（50／60）。结论　SELDI-TOF-MS技术可筛选出胃癌转移复发相关的蛋白质组指纹，在m／z为4768和8841的两个蛋白质峰建立的决策树模型可用于对术后胃癌患者转移、复发的早期预测。     

SELDI-TOF MS proteinchip technology for screening of serum markers of HBV-induced hepatocellular carcinoma

The incidence of hepatocellular carcinoma (HCC) is highest among primary liver cancer. HBV and HBV-induced liver cirrhosis may lead to HCC (1). At present, it is difficult to diagnose HCC at early stage or to differentiate HCC. Therefore, it is much needed to explore and develop a simple, rapid diagnostic method, which has higher sensitivity and specificity for HCC at early stage (2, 3). Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) is a novel non-electrophoresis-based proteomic technology. SELDI offers the advantages of rapid and simple examination as well as high specificity and sensitivity (4, 5). To our knowledge,there has been little study reported using SELDI-TOF-MS technology to investigate HCC. In this study, 25 cases of HCC patients not receiving any therapy, 25 cases receiving the interposition chemotherapy and 50 cases of the healthy people were tested by Weak cationic exchange (WCX2) protein chip and SELDI-TOF-MS. The differentially expressed peptides or proteins were analyzed by BioMarker Wizard software. At the different M/Z value range, seven peptides/ proteins were obviously different among these three groups. Four peptides including 6489.48Da, 6662.34Da, 8593.75Da and 8720.23Da were up-regulated in healthy controls, two including 7777.27Da and 9250.00Da were up-regulated in the patients with HCC without receiving any therapy and one protein of 16200.17Da was up-regulated in the patients with HCC receiving the interposition chemotherapy. Using Biomarker Pattern software, one pattern including two peptides (7777.27Da, 9250.00Da) can separate HCC without receiving any therapy from normal controls. This diagnosis pattern gave the much-improved sensitivity of 92% and the specificity of 100%. Through searching protein database, these seven peptides or proteins were identified as possible Galanin related peptide, Pro-neuregulin-4 protein, small inducible cytokine A15 precursor, 9 kDa protein, CSL-zincfinger protein 1, mitochondrial hinge protein, actin related protein, respectively. Using SELDI-TOF MS, the method of sieving the tumor markers from HCC becomes quick and valid. These differentially expressed peptides or proteins could be biomarkers of HCC in the serum and drug targets for treating HCC.     

Serum N-glycan profiling as a diagnostic biomarker for the identification of hepatitis B virus-associated hepatocellular carcinoma

BackgroundChanges in N-glycosylation of proteins are thought to play a key role in cancer. This study aims to investigate the changes in the serum N-glycan profiles of patients with hepatitis B virus (HBV)-related liver disease, and to evaluate the role of N-glycan markers in the noninvasive diagnosis of hepatocellular carcinoma (HCC).MethodsSerum samples were available for 21 patients with HCC, 20 patients with liver cirrhosis (LC), 20 patients with chronic hepatitis B (CHB), and 20 healthy subjects. Serum N-glycans were released and analyzed using DNA sequencer-assisted fluorophore-assisted carbohydrate electrophoresis (DSA-FACE). Serum AFP was determined by electrochemiluminescence (ECL) (AFP reference value range: <10 ng/mL).ResultsThere were characteristic changes in the serum N-glycan profiles of patients with HBV-related liver disease, including NA2FB, NA3, and NA3Fb. NA2FB was the most abundant in LC patients, while NA3Fb abundance was the highest in HCC patients. For HCC screening in patients, especially in patients with LC, the sensitive of Log peak 9 (94.4%) and Log (peak 9/peak 7) (88.9%) were better than alpha-fetoprotein (AFP) (33.3–61.1%), and their specificity was similar to that of AFP. The receiver operating characteristic (ROC) curve showed that the accuracy of Log peak 9 (AUC: 0.81±0.07) and Log (peak 9/peak 7) (AUC: 0.87±0.06) was better than that of AFP (AUC: 0.72±0.09), while the accuracy of AFP combined with the above 2 indexes was better than that of a single index. Moreover, Log (peak 9/peak 7) combined with AFP (AUC: 0.89±0.06) had the best accuracy in the diagnosis of HCC.ConclusionsOur research indicates that N-glycan may serve a new, valuable, and noninvasive alternative method for diagnosing HCC, and it may be a supplement to AFP in the diagnosis of HCC in patients with HBV-related liver disease.     

GP73在肝细胞癌诊断中的价值

目的:探讨GP73(Golgi protein-73)在肝细胞癌诊断中的价值.方法:收集外周血血清504例,其中肝细胞癌144例、肝硬化50例、乙型病毒性肝炎100例、乙型肝炎病毒携带者84例、其他恶性肿瘤50例、肝良性肿瘤26例和健康志愿者50例;应用双抗体夹心酶联免疫定量测定方法和电化学发光法检测血清中GP73和甲胎蛋白(α-fetoprotein,AFP)的表达水平.结果:肝细胞癌组血清GP73的表达水平显著高于其他各组(P＜0.05);受试者工作特征(receiver operating characteristic,ROC)曲线设定GP73临界值为64 ng/mL时,GP73诊断肝细胞癌的灵敏度和特异度分别为83.3％和88.3％,显著高于AFP(72.2％和76.7％),差异有统计学意义(P＜0.05);血清GP73联合AFP检测诊断肝细胞癌的灵敏度可达94.4％.结论:血清GP73的表达水平用于诊断肝细胞癌的灵敏度和特异度优于AFP,血清GP73联合AFP检测可提高肝细胞癌的诊断率.     

亲和层析微柱法测定肝癌特异性AFP及其在肝癌诊断中的临床价值

Objective: To establish a convenient and economic method to determine hepatoma-specific α-fetoprotein (HS-AFP) for diagnosis of hepatocellular carcinoma (HCC). Methods: HS-AFP from serum of HCC patients was separated by a mini-column Lens culinaris agglutinin (LCA)-affinity chromatography. The levels of serum total AFP and separated HS-AFP were detected by radioimmunoassay (RIA). Results: Circulating AFP was separated into three peaks (AFP-1, AFP-2, and AFP-3) by LCA-affinity chromatography. During the elution course, the AFP-1 and AFP-2 could be eluted with TE buffer. HS-AFP (AFP-3) from sera of HCC patients was eluted dearly on the LCA-sepharose gel mini-column with a solution containing a-methyl-D-mannoside. It was a part of total AFP and only found in sera of HCC patients. A ratio of more than 15% for HS-AFP to total AFP in serum was considered as a specific marker for HCC diagnosis with higher sensitivity (92.7%) and specificity(88.2%). Conclusion: The new assay for circulating HS-AFP analysis is more sensitive, repeatable, and convenient. Its clinical application would be useful to early diagnosis of HCC.     

组织hTERT基因及GGTmRNA-H亚型与血清AFP对小肝癌早期诊断意义的比较

目的 :研究小肝癌组织中端粒酶逆转录酶hTERT基因及谷氨酰转移酶GGTmRNA H亚型表达 ,通过与小肝癌血清AFP的比较 ,探讨其在小肝癌早期诊断中的意义。方法 :应用RT PCR法检测 65例原发性肝癌患者癌组织hTERT与GGTmRNA H亚型的表达 ,同时通过放免法检测血清AFP阳性表达情况。结果 :在 65例原发性肝癌中 ,癌组织hTERT基因的阳性例数 5 6例 ( 86 2 % ) ,GGTmRNA H亚型的阳性例数为 61例 ( 93 8% ) ,肝组织血清AFP阳性 40例 ( 61 5 % ) ,P <0 0 5 ;hTERT基因在2 0例小肝癌癌组织阳性表达例数为 16例 ,阳性率为 80 0 % ;GGTmRNA H亚型阳性表达例数为 18例 ,阳性率为 90 0 % ;与血清AFP组比较 ,差异有统计学意义 ,P <0 0 5。结论 :肝癌组织中hTERT基因、GGTmRNA H亚型联合检测可弥补AFP测定的不足 ,对提高肝癌早期诊断具有重要意义。hTERT基因及GGTmRNA H亚型的检测比AFP更敏感、特异性更高 ,有望成为小肝癌早期诊断的理想指标     

Quantitative analysis of hepatoma-specific α-fetoprotein （HS-AFP） by a new mini-column affinity chromatography and its clinical value in diagnosis of hepatocellular carcinoma

Objective： To establish a convenient and economic method to determine hepatoma-specific α-fetoprotein （HS- AFP） for diagnosis of hepatocellular carcinoma （HCC）. Methods： HS-AFP from serum of HCC patients was separated by a mini-column Lens culinaris agglutinin （LCA）-affinity chromatography. The levels of serum total AFP and separated HS-AFP were detected by radioimmunoassay （RIA）. Results： Circulating AFP was separated into three peaks （AFP-1, AFP-2, and AFP-3） by LCA-affinity chromatography. Dunng the elution course, the AFP-1 and AFP-2 could be eluted with TE buffer. HSAFP （AFP-3） from sera of HCC patients was eluted clearly on the LCA-sepharose gel mini-column with a solution containing α-methyI-D-mannoside. It was a part of total AFP and only found in sera of HCC patients. A ratio of more than 15% for HS-AFP to total AFP in serum was considered as a specific marker for HCC diagnosis with higher sensitivity （92.7%） and specificity （88.2%）. Conclusion： The new assay for circulating HS-AFP analysis is more sensitive, repeatable, and convenient. Its clinical application would be useful to early diagnosis of HCC.     

Diagnostic value of protein induced by vitamin K absence (PIVKAII) and hepatoma-specific band of serum gamma-glutamyl transferase (GGTII) as hepatocellular carcinoma markers complementary to alpha-fetoprotein

Serum protein induced by vitamin K absence or antagonist II (PIVKAII), hepatoma-specific band of serum gamma-glutamyl transferase (GGTII), and alpha-fetoprotein (AFP) levels were determined in 120 patients with hepatocellular carcinoma (HCC) and 90 patients with cirrhosis. The mean serum concentration of PIVKAII in HCC patients was higher than that in cirrhotic patients. A total of 53.3% of patients (64 out of 120) with HCC had PIVKAII levels above 40 mAU ml(-1). However, only 13 patients with cirrhosis had higher PIVKA II levels. Of 32 small HCC patients, 13 (40.6%) had PIVKAII values above 40 mAU ml(-1). An increased concentration of AFP (i.e. 20 ng ml(-1)) was observed in 70 out of 120 (58.3%) patients with HCC and in 33 out of 90 (36.7%) patients with cirrhosis. Positive GGTII was found in 74.0% (89 out of 120) cases of HCC (sensitivity), in 16 of 90 cases of cirrhosis, and 14 of 32 (43.8%) small HCC patients had GGTII positive. No significant correlation was found between serum levels of AFP and PIVKAII. Combining the information from PIVKAII, AFP, and GGTII significantly increases the sensitivity over AFP alone. PIVKAII and GGTII are useful tumour markers complementary to AFP for diagnosis of HCC.     

Diagnostic Value of Glypican-3 for Hepatocellular Carcinomas

Background: Hepatocellular carcinoma (HCC) is a common and dangerous malignancy in many parts of the world,and especially in Egypt. Early diagnosis is the most important step in successful HCC management. However mostcases are detected at late stage making effective intervention impossible. Aim: The aim of this study was to evaluatethe potential of Glypican-3 (GPC-3) to aid in diagnosis of HCC, especially in patients with low serum alpha-fetoprotein(AFP). Subjects and methods: Serum GPC-3 was assessed by flow-cytometry and serum AFP by enzyme-linkedimmunosorbent assay (ELISA) in 40 HCC patients with AFP< 400ug\l. (GI), 40 HCC patients with AFP> 400ug\l.(GII) and 20 healthy controls (GIII). Results: GPC-3 was found to be significantly elevated in HCC as compared tohealthy subjects (GI 38.2±22. 5, GII 50.2±22.6, and GIII 2.24±1.19), with sensitivities of 85% for GI and 84% for GIIand specificities of 95% for GI and 92% for GII. AFP showed respective sensitivities of 50% and 79%, and specificitiesof 80% and 90%, for HCC diagnosis. The combination of GPC-3 with AFP achieved the highest sensitivity (98.5%) andspecificity (97.8%). Conclusion: Serum GPC-3 has a better sensitivity than AFP for the diagnosis of HCC. Combinationof two markers appears warranted for greatest accuracy     

Elevation of Serum MAGE-4 Protein Levels and Prediction of Hepatocellular Carcinogenesis in Patients with Liver Cirrhosis

Early detection of hepatocellular carcinoma (HCC) is clinically important because advanced HCC limits treatment modalities for the cancer. We have previously reported that serum levels of MAGE-4 protein are strongly associated with the development of HCC. The present study was designed to determine whether elevated serum MAGE-4 protein levels can predict hepatocellular carcinogenesis in patients with liver cirrhosis before clinical diagnosis. Among 62 cirrhotic patients, 28 patients were diagnosed with HCC during the follow-up period. The levels of MAGE-4 protein and a-fetoprotein (AFP) were significantly elevated in cirrhotic patients with HCC. Univariate and multivariate analyses suggest that elevated serum MAGE-4 protein is more significant than AFP. Importantly, retrospective analysis of prefrozen sera of cirrhotic patients revealed a transient or continuous elevation of serum MAGE-4 protein levels in 14 of 28 cirrhotic patients with HCC (50%) before clinical diagnosis. In contrast, elevated serum MAGE-4 protein levels were observed in 3 of 33 cirrhotic patients without HCC (9%), and in 2 of 34 hepatitic patients (6%). These results indicate that elevated serum MAGE-4 protein levels can be a predictive marker of hepatocellular carcinogenesis in cirrhotic patients, thereby enabling us to treat patients at an earlier stage.