Glypican-3蛋白在肝癌患者血清中的表达及意义

目的检测肝细胞癌（HCC）患者血清Glypican-3（GPC3）和AFP蛋白的表达水平,比较其诊断价值,分析血清GPC3与临床因素的关系。方法采用ELISA法分别检查HCC30例,其他肝脏肿瘤8例,其他肝脏疾病13例,正常人25例血清标本中GPC3及AFP的表达。结果血清GPC3蛋白诊断HCC的敏感性和特异性分别为50．0％和92．O％,联合AFP蛋白检测使诊断敏感性由50．0％提高至73．3％。GPC3蛋白水平升高提示HCC患者临床分期晚、肿瘤分化程度低、肝硬化明显。结论GPC3蛋白是具有前景的特异性HCC血清标志物,可用于HCC的诊断,并可能与肿瘤侵袭相关。     

肝细胞癌患者血清DcR3水平与临床意义

目的:探讨血清DcR3水平对HCC的诊断价值及临床意义.方法:采用ELISA检测67例HCC、8例肝硬化、17例胆囊炎患者和28例正常人血清DcR3水平; 化学发光法测定血清AFP水平; 免疫组织化学二步法检测HCC癌组织中DcR3蛋白的表达.结果:HCC组和肝硬化组血清DcR3水平均明显高于正常对照组( P<0.01), HCC血清DcR3水平与伴有肝硬化、包膜浸润和复发转移有关( P<0.05). DcR3蛋白在HCC癌组织中的表达与血清水平呈正相关( r = 0.395, P<0.01), 但DcR3血清阳性率明显高于癌组织IHC( P<0.05). AFP与DcR3联合检测对HCC的诊断灵敏度可由单项检测的82%和76%提高到93%.结论:血清DcR3升高在HCC的发生发展及浸润转移中起重要作用, 通过监测高危人群以及肝癌患者血清中的DcR3水平, 同时联合检测AFP, 可能对HCC的筛查、诊断和判断预后有一定意义.     

肝细胞癌癌组织和癌旁肝组织Ep-CAM和C-Kit表达及其意义探讨

目的探讨肝细胞癌(HCC)患者癌组织和癌旁肝组织C-Kit蛋白和肿瘤干细胞标志上皮细胞黏附分子(EpCAM)蛋白表达的变化。方法 2014年3月～2017年1月我院经手术切除治疗的HCC患者癌组织和癌旁肝组织标本90份,采用免疫组化染色法检测癌组织和癌旁肝组织Ep-CAM蛋白和C-Kit蛋白表达情况,并比较不同分化、有无包膜、不同病灶大小、术前不同血清甲胎蛋白(AFP)水平癌组织Ep-CAM蛋白和C-Kit蛋白表达阳性率的差异。结果在本组90例HCC患者肝组织中,癌组织Ep-CAM蛋白和C-Kit蛋白表达阳性率分别为65.6%和74.4%,均显著高于癌旁组织的11.1%和4.4%,差异具有统计学意义(P0.05);50例Ⅲ级和Ⅳ级组织学分化、49例术前血清AFP400 ng/ml、28例发生血管浸润的癌组织Ep-CAM蛋白表达阳性率分别为76.0%、79.6%和82.1%,显著高于40例Ⅰ级和Ⅱ级组织学分化、41例血清AFP≤400 ng/ml和62例未发生肿瘤血管浸润的癌组织(分别为52.5%、48.8%和58.1%);Ⅲ级和Ⅳ级组织学分化、术前血清AFP400 ng/ml、发生血管浸润的癌组织C-Kit蛋白表达阳性率分别为86.0%、87.8%和89.3%,也显著高于Ⅰ级和Ⅱ级组织学分化、血清AFP≤400 ng/ml和未发生肿瘤血管浸润的癌组织(分别为60.0%、58.5%和67.7%),差异均具有统计学意义(P0.05)。结论 HCC患者癌组织Ep-CAM蛋白和C-Kit蛋白表达阳性率显著增高,并与肿瘤组织学分级、术前血清AFP水平和是否发生肿瘤血管浸润有关,其临床意义还有待进一步探讨。     

磷脂酰肌醇蛋白聚糖3在肝穿刺活检标本中鉴别诊断的意义

目的:探讨磷脂酰肌醇蛋白聚糖3(GPC3)在肝细胞癌(HCC)等不同肝病组织中的表达情况及其对肝癌早期诊断的意义.方法:采用免疫组织化学方法检测126例肝穿刺活检标本(包括13例极早期HCC、44例早期HCC、16例不典型增生、29例肝硬化和24例肝炎)及57例中晚期HCC手术标本中GPC3和甲胎蛋白(AFP)的表达,...     

GPC3、GP73在肝细胞癌临床诊断中的价值

目的评价磷酯酰肌醇蛋白聚糖3(GPC3)、高尔基体蛋白73(GP73)的高表达对肝细胞癌(HCC)诊断价值。方法对39例HCC、31例肝硬化患者,基线时收集外周血及肝组织,采用ELISA法检测血清GPC3、GP73的表达量,实时定量逆转录PCR法(qRT-PCR)检测外周血单个核细胞(PBMC)及肝组织中GPC3 mRNA和GP73 mRNA的相对表达量;ROC曲线分析诊断的灵敏度、特异度等指标。结果 HCC组GPC3、GP73测量值显著高于肝硬化组(P0.001);GPC3诊断HCC的截断值9.3μg/L,ROC曲线下面积(AUC)=0.956,灵敏度为89.74%,特异度为96.77%,阳性预测值为97.2%,阴性预测值为88.2%,阳性似然比(+LR)27.82,阴性似然比(-LR)0.11;GP73诊断HCC的截断值77.68 ng/mL,AUC=0.937,灵敏度为92.31%,特异度为83.87%,阳性预测值为87.8%,阴性预测值为89.7%,+LR:5.72,-LR:0.092。结论 GPC3、GP73在HCC患者中表达明显增高,且灵敏度显著优于常规AFP,GPC3、GP73的特异度与常规AFP相近,因此GPC3、GP73有望成为一种新的较好、可靠的HCC无创诊断指标。     

肝细胞癌组织Glypican3和Sonic Hedgehog蛋白表达及其功能分析*

目的 探讨肝细胞癌(HCC)组织Glypican3(GPC3)和Sonic Hedgehog(Shh)蛋白表达及其功能分析。方法 取96例HCC患者癌组织和癌旁肝组织,体外培养HepG2、MHCC97H、Huh7和SMMC7721细胞,采用免疫组织化学染色法检测组织GPC3和Shh蛋白表达水平,采用免疫荧光染色和蛋白印迹法检测体外培养的肝癌细胞GPC3和Shh蛋白表达,应用String数据库预测GPC3和Shh蛋白之间相互作用。结果 在96例HCC癌组织,GPC3和Shh阳性率分别为77.1%和68.8%,而两种蛋白高表达率分别为60.4%和53.1%;高分化HCC癌组织GPC3蛋白阳性率为85.7%,显著高于低分化组的50.0%(P<0.05), Shh蛋白阳性率为81.0%,显著高于低分化组的40.0%(P<0.05);存在微血管侵犯(MVI)的HCC癌组织GPC3蛋白阳性率为72.7%,显著高于无MVI组的50.0%(P<0.05),Shh蛋白阳性率为65.9%,显著高于无MVI组的42.3%(P<0.05);体外培养的HepG2、MHCC97H、Huh7和SMMC7721四种肝癌细胞胞浆GPC3和Shh蛋白共表达,两种蛋白表达趋势一致。结论 GPC3和Shh蛋白可能影响HCC肿瘤细胞分化和MVI形成,并且两种蛋白可能存在共表达及相互作用。     

应用实时荧光定量RT-PCR法建立肝癌分子诊断指数

目的: 筛选肝细胞癌(hepatocellular carcinoma,HCC)中表达水平较正常肝细胞差异大、特异性好的基因, 建立肝癌分子诊断指数, 以期从分子病因学角度实现对肝癌的早期诊断.方法: 随机选择38例手术切除组织标本, 其中正常肝5例、肝炎4例、肝硬化(LC)12例、HCC和癌旁组织17例, 实时荧光定量RT-PCR检测9个基因的表达, 以管家基因G3PDH为对照, 2-△△CT法计算目的基因相对表达量, 依据表达异常的基因个数建立分子诊断指数.结果: GPC3等6个基因在正常肝、肝炎、LC与HCC组的两组或多组间存在差异( P<0.05);GPC3、E2F1从肝炎组到LC、HCC组呈递增趋势, HGF、CLDN10在LC组表达量升高, 而在HCC组明显下降, PTEN、PRDM2、MGMT从肝炎组到LC、HCC组呈递减趋势; 9个基因在LC组分子诊断指数平均值为1.58, 在HCC组平均值为5.24, 二者差异显著; GPC3、E 2 F 1 、M M P 2 从癌旁到癌呈递增趋势,CLDN10、HGF、PTEN、DLC1、PRDM2、MGMT从癌旁到癌呈递减趋势.结论: 通过筛选更多基因的组合分析, 分子诊断指数有望成为鉴别诊断早期肝癌的一种有效方法.     

AU结合因子1对肝细胞癌患者磷脂酰肌醇蛋白聚糖3水平的影响

目的探讨肝细胞癌(HCC)患者中AU结合因子1(AUF1)对磷脂酰肌醇蛋白聚糖3(GPC3)表达的影响及其机制。方法LCI-HCC基因表达数据下载自GSE14520,最终纳入214例有随访信息的乙型肝炎相关HCC患者。35例HCC及配对癌旁样本选取自2009年—2013年在河南省肿瘤医院接受常规根治性手术的HCC患者。采用免疫组化检测GPC3和AUF1蛋白在HCC中的表达及相关性;在HCC细胞系中敲减或者过表达AUF1后,采用Western Blot和实时荧光定量PCR检测GPC3表达;利用RNA结合蛋白免疫沉淀实验和RNA稳定性检测研究AUF1调控GPC3表达的机制。计量资料两组间比较采用t检验,计数资料两组间比较采库中,肝癌组织GPC3的表达均显著高于癌旁组织(P值均0.05);TCGA数据库中,GPC3高表达与肝癌患者的不良预后有关(P0.05)。免疫组化结果显示,GPC3和AUF1蛋白均在肝癌组织中呈高表达,阳性表达率分别为77.1%(27/35)和74.3%(26/35)。体外实验结果表明,在肝癌细胞系HepG2和Huh-7中敲减AUF1明显降低了GPC3表达(P值均0.05),而过表达AUF1则上调GPC3表达(P0.05)。AUF1蛋白可与GPC3 mRNA结合,敲减AUF1导致GPC3 mRNA稳定性降低。结论 AUF1是调控GPC3基因转录后修饰的重要调节因子,AUF1和GPC3蛋白的异常高表达可能参与肝癌的发生和发展。     

肝细胞癌组织SOCS1和STAT3蛋白的表达意义

目的:探讨SOCS1和STAT3蛋白在HCC组织中的表达、相互关系及在HCC发生发展中的意义.方法:应用免疫组化方法检测48例HCC组织和癌周肝组织、肝硬化(liver cirrhosis,LC)组织(n=11)及正常肝组织中(n=11)SOCS1和STAT3的表达水平.结果:癌周肝组织中SOCS1蛋白表达强度显著高于HCC组织,SOCS1蛋白在LC组织及正常肝组织中全部呈阴性表达.HCC组织和癌周肝组织STAT3蛋白阳性表达率显著高于LC组织和正常肝组织;SOCS1在瘤体大小间的表达有显著性差异(P<0.01).STAT3在表达AFP阴性和阳性癌组织组间有显著性差异(P<0.05).HCC组织中SOCS1和STAT3表达具有显著等级正相关(rs=0.431,P<0.01).结论:SOCS1和STAT3表达与HCC的发生密切相关,且两者之间的表达强度具有显著等级正相关.     

HBV相关肝细胞癌患者血清N-糖组图谱改变与肝组织糖基转移酶表达变化的关系

目的本研究通过对HBV相关肝细胞癌(HCC)患者血清N-聚糖检测及肝癌组织与癌旁组织中糖基转移酶基因表达水平比较分析,探索HCC患者血清N-聚糖变化的可能机制。方法收集解放军总医院2018年—2019年HBV相关HCC手术患者(34例)的肝癌和癌旁组织及正常肝组织标本,同时采集血清标本。从34例HCC患者中随机选择8例HCC患者血清标本作为HCC试验组,20例健康成年人血清标本作为对照组。应用DSA-FACE法分析HCC试验组与对照组血清N-聚糖图谱。采用荧光定量PCR法检测34例HBV相关HCC患者癌组织和癌旁组织中8种糖基转移酶基因(FUT3、FUT4、FUT6、FUT7、FUT8、Gn-TⅢ、Gn-TⅣa和Gn-TⅤ) mRNA表达水平,并应用蛋白印迹法检测相应蛋白表达水平。计量资料两组间比较采用独立样本t检验。结果与对照相比,8例HCC患者血清中N-聚糖峰9 (peak9,NA3Fb)的丰度明显升高(t=-2.514,P 0.05)。糖基转移酶FUT8、Gn-TⅣa和Gn-TⅤ基因mRNA表达水平在癌组织和癌旁组织间有差异,其中癌组织中FUT8和Gn-TⅤ基因的mRNA与蛋白表达水平显著高于癌旁组织(mRNA:1.50±0.34 vs 0.65±0.11,t=-2.354,P=0.022; 3.57±0.64 vs 1.33±0.16,t=-3.384,P=0.001)(蛋白:0.70±0.11 vs 0.083±0.017,t=9.555,P=0.001; 1.33±0.19 vs 0.60±0.15,t=5.097,P=0.007);癌组织中GnTⅣa基因的mRNA表达水平显著高于癌旁组织(mRNA:2.90±0.47 vs 1.68±0.19,t=-2.403,P=0.019),蛋白表达水平与癌旁组织无显著差异(蛋白:0.52±0.24 vs 0.24±0.11,t=1.833,P=0.141)。癌组织中这些糖基转移酶表达改变与血清中N-聚糖丰度变化一致。结论 HBV相关HCC患者血清中一些N-聚糖水平变化可能与肝癌组织中糖基转移酶基因GnT-V、GnT-IVa和FUT8表达上调密切相关。     

Glypican-3: a novel serum and histochemical marker for hepatocellular carcinoma

BACKGROUND & AIMS: Early detection of hepatocellular carcinoma (HCC) is critical for successful treatment. However, the differential diagnosis between HCC and benign hepatic lesions is sometimes difficult and new biochemical markers for HCC are required. It has been reported that glypican-3 (GPC3) messenger RNA (mRNA) is significantly increased in most HCCs compared with benign liver lesions or normal liver. The goal of this study is to determine whether GPC3 is also overexpressed at the protein level and whether GPC3 is detectable in the serum of patients with HCC. METHODS: GPC3 was assessed in liver tissue sections by immunohistochemistry and in serum by enzyme-linked immunosorbent assay. Serum alpha-fetoprotein (AFP) level was also measured in the same patients. RESULTS: Immunohistochemical studies showed that GPC3 is expressed in 72% of HCCs (21 of 29), whereas it is not detectable in hepatocytes from normal liver and benign liver diseases. Consistent with this, GPC3 was undetectable in the serum of healthy donors and patients with hepatitis, but its levels were significantly increased in 18 of 34 patients (53%) with HCC. In addition, only 1 of 20 patients with hepatitis plus liver cirrhosis displayed elevated levels of serum GPC3. Interestingly, in most cases, there was no correlation between GPC3 and AFP values. Thus, at least 1 of the 2 markers was elevated in 82% of the patients with HCC. CONCLUSIONS: GPC3 is specifically overexpressed in most HCCs and is elevated in the serum of a large proportion of patients with HCC. The simultaneous determination of GPC3 and AFP may significantly increase the sensitivity for diagnosis of HCC.     

Evaluation for clinical utility of GPC3, measured by a commercially available ELISA kit with Glypican‐3 (GPC3) antibody,as a serological and histological marker for hepatocellular carcinoma

Aims: We evaluated the clinical utility of glypican‐3 (GPC3), which has been proposed as a potential novel tumor marker for hepatocellular carcinoma (HCC), as a serological and histological marker for HCC. Methods: The serum GPC3 level was compared between 200 patients with HCC and 200 patients with chronic liver disease (CLD). In addition, the expression of GPC3 was examined with immunohistochemistry on 38 resected specimens from patients with HCC. A commercially available GPC3 antibody was used for these analyses. Results: The median values of serum GPC3 in patients with HCC and with CLD were 924.8 pg/mL and 1161.6 pg/mL, respectively. We found no elevation of serum GPC3 level in patients with HCC in comparison with those with CLD; rather the level was higher in patients with CLD (P < 0.0001). In immunohistochemical analysis, 14 of 38 (36.9%) HCC tissues were positive for GPC3, whereas no corresponding non‐cancerous tissue was positive. The positivity for GPC3 tended to increase with pathologic decreased differentiation of HCC. Conclusions: We did not find serum GPC3 level, measured by a commercially available ELISA kit with GPC3 antibody, to be useful in the diagnosis of HCC. However, we did observe increased GPC3 staining in HCC tissue with moderate or poor differentiation, suggesting that GPC3 is produced by HCC tumors. This lack of utility could have been due to the measuring procedure used in the present study. Further evaluation of GPC3 in HCC with other measuring procedures is needed.     

Diagnostic value of glypican-3 in serum and liver for primary hepatocellular carcinoma

AIM:To evaluate the diagnostic value of glypican-3(GPC3) in serum and liver for primary hepatocellular carcinoma(HCC).METHODS:Serum levels of GPC3 and α-fetoprotein(AFP) were measured in 75 patients with primary HCC and 32 patients with liver cirrhosis.Expression of GPC3 and AFP in 58 HCC and 12 cirrhotic specimens was detected with immunohistochemical staining.RESULTS:When the cut-off value of serum GPC3 was set at 300 ng/L,its sensitivity and specificity for HCC were 47.0% and 93.5%,respectively.Among the...     

肝癌组织和血清中钙囊素的表达及其意义

目的 研究人肝癌组织及血清中钙囊素(S100A11)的表达及其临床意义.方法 免疫组织化学法检测46例肝癌及其癌旁组织中S100A11表达,比较肝癌与癌旁组织中S100A11的阳性率,分析肝癌组织中S100A11表达水平与各临床参数之间的关系;同时检测肝癌(62例)、肝硬化(32例)、慢性肝炎(30例)患者及健康体检者(28名)血清中S100A11浓度,比较S100A11浓度与甲胎蛋白(AFP)、γ-谷氨酰转肽酶同工酶Ⅱ(GGT-Ⅱ)诊断肝癌的灵敏性及特异性.结果 S100A11在肝癌组织中的表达阳性率(78.3%)显著高于癌旁组织(19.6%,P<0.01),其表达水平与分化程度相关,分化程度越低表达水平越高.根据ROC曲线,当确定诊断界值为7.3μg/L时,S100A11在肝癌患者血清中的阳性率为30.6%,明显高于肝硬化、慢性肝炎及健康人(P值均<0.05).肝癌患者血清中S100A11与AFP、GGT-Ⅱ均无相关性,联合检测三项指标对肝癌诊断有互补性,可使诊断敏感性提高至84.5%.结论 S100A11可能与肝癌的发生发展有关.联合检测S100A11与AFP和GGT-Ⅱ可提高对肝癌的诊断敏感度.

Abstract：

Objective To explore the expression of calcium binding protein (S100A11) and its clinical significances in human hepatocellular carcinoma (HCC) tissue and blood plasma. Methods The expressions of S100A11 in 46 cases of HCC tissues and their paracancerous tissues were detected by immunohistochemistry. The relationship between S100A11 expression level in HCC tissues and clinical parameters was analyzed. The S100A11 expression levels in blood plasma of HCC patients (62 cases), liver cirrhosis patients (32 cases), chronic hepatitis patients (30 cases) and healthy subjects (30 cases) were detected. The sensitivity and specificity of S100A11, alpha fetoprotein (AFP) and γ-glutamyl transpeptidase Ⅱ (GGT-Ⅱ ) in HCC diagnosis were compared. Results The positive rate of S100A11 in HCC tissue (78. 3%) was significantly higher than that in paracancerous tissues (19. 6%) (P<0. 05). The expression level was correlated with the degree of differentiation, the lower differentiation degree with the higher expression level. According to ROC curve, if the cutoff points for diagnosis was set at 7. 3 μg/L, the positive rate of S100A11 in HCC patients blood plasma was 30. 6% , which was significantly higher than that in the blood plasma of patients with liver cirrhosis, patients with chronic hepatitis and healthy persons (P<0. 05). There was no correlation between S100A11 and AFP or GGT-Ⅱ in the blood plasma of HCC patients. These three indicators were complementary in HCC diagnosis, and the diagnostic sensitivity increased to 84.5% with combined detection. Conclusions S100All may be related to HCC genesis and development. The HCC diagnostic sensitivity may be increased with combined detection of S100All ,AFP and GOT- Ⅱ.     

Diagnostic role of serum glypican-3 in differentiating hepatocellular carcinoma from non-malignant chronic liver disease and other liver cancers

Background and Aims:  The role of glypican-3 (GPC3), a novel serum marker, in differentiating hepatocellular carcinoma (HCC) from non-malignant chronic liver disease and other malignant space-occupying lesions in the liver is largely unknown. The aims of this study were to evaluate its diagnostic role and clinical correlations in patients with HCC.
Methods:  Six groups were studied which included 40 healthy subjects, 50 patients with chronic hepatitis (CH), 50 patients with liver cirrhosis (LC), 100 patients with HCC, 50 patients with intrahepatic cholangiocarcinoma (ICC) and 50 patients with metastatic carcinoma (MCA). Serum GPC3 levels were measured by using a sandwich enzyme-linked immunosorbent assay method.
Results:  Fifty-three percent of HCC patients had elevated serum GPC3 levels with values ranging 35.5–7826.6 ng/mL. The serum marker was undetectable in other groups except one patient (2%) with LC and another patient (2%) with MCA. In most cases of HCC, elevated GPC3 values did not correlate with α-fetoprotein (AFP) levels. Detectable GPC3 was significantly correlated with the presence of viral hepatitis markers but was not correlated with tumor size and stage of HCC. Serum GPC3 was superior to AFP in detecting small HCC (56.3% and 31.3%, respectively). A combination of serum GPC3 and AFP yielded an improved sensitivity for detecting small HCC to 75%.
Conclusion:  Serum GPC3 is highly specific for detecting HCC. The combined use of serum GPC3 and AFP provides a potentially promising tool to better differentiate HCC from benign liver disorders, as well as from other liver cancers.     

Glypican-3与原发性肝癌的关系研究进展

近年来有关Glypican-3（GPC3）与原发性肝癌（HCC）关系的研究逐年增多,发现GPC3无论在原发性肝癌病人的血清中还是肿瘤组织中均有较高的表达,对原发性肝癌的诊断具有较高的灵敏性和特异性。其表达水平与原发性肝癌的预后也有一定的关系。同时,GPC3也为原发性肝癌的治疗带来了新的思路。     

Expression and significance of tumor-related genes in HCC

AIM: To investigate the expression and clinical significance of DEK, cyclin D1, insulin-like growth factor Ⅱ (IGF-Ⅱ), glypican 3 (GPC3), ribosomal phosphoprotein 0 (rpPO) mRNA in hepatocellular carcinoma (HCC) and its paraneoplastic tissues. METHODS: The expression of mRNAs of DEK, cyclin D1, IGF-Ⅱ, GPC3 and rpP0 mRNA was detected in HCC and its paraneoplastic tissues by multiplex RT-PCR. RESULTS: By the simplex RT-PCR, the overexpression of mRNAs of DEK, cyclin D1, IGF-Ⅱ, GPC3, rpP0 mRNA in HCC and its paraneoplastic tissues was 78.1%, 87.5%, 87.5%, 75.0%, 81.3% and 15.6%, 40.6%, 37.5%, 21.9%, 31.3% respectively (P<0.05). By the multiplex RT-PCR, at least one of the mRNAs was detected in all HCC samples and in 75.0% of paraneoplastic samples (P>0.05). However, all these five mRNAs were found in 68.8% of HCC samples, but only in 9.4% of paraneoplastic tissues (P<0.05). The positive expression of mRNAs of DEK, cyclin D1, IGF-Ⅱ, GPC3, rpP0 in well- and poorly-differentiated HCC was 89.0%, 66.7%, 66.7%, 66.7%, 77.8% and 73.9%, 95.7%, 95.7%, 95.7%, 82.6%, respectively (P>0.05). The expression of these genes in HCCs with α-feto protein (AFP) negative and positive was 90.0%, 80.0%, 90.0%, 90.0%, 90.0% and 72.7%, 86.3%, 77.3%, 90.9%, 68.2% respectively (P>0.05). CONCLUSION: The expression of DEK, cyclin D1, IGF-Ⅱ, GPC3, rpP0 mRNA in HCC is much higher in HCC than in its paraneoplastic tissues. Multiplex RT-PCR assay is an effective, sensitive, accurate, and cost-effective diagnostic method of HCC.