Values of circulating GPC-3 mRNA and alpha-fetoprotein in detecting patients with hepatocellular carcinoma Min Yao, Deng-Fu Yao, Yin-Zhu Bian, Wei Wu, Xiao-Di Yan, Dan-Dan Yu, Li-Wei Qiu, Jun-Ling Yang, Hai-Jian Zhang, Wen-Li Sai and Jie Chen
Nantong, China
Author Affiliations: Department of Immunology, Medical School of Nantong University, Nantong 226001, China (Yao M); Research Center of Clinical Medicine, Affiliated Hospital of Nantong University, Nantong 226001, China (Yao DF, Wu W, Yan XD, Yu DD, Qiu LW, Yang JL, Zhang HJ, Sai WL and Chen J); Department of Oncology, Affiliated Yancheng Hospital of Nantong University, Yancheng 224001, China (Bian YZ)
Corresponding Author: Deng-Fu Yao, MD, PhD, Research Center of Clinical Medicine, Affiliated Hospital of Nantong University, 20 West Temple Road, Nantong 226001, China (Tel: 86-513-85052297; Fax: 86-513-85052254; Email: yaodf@ahnmc.com)
© 2013, Hepatobiliary Pancreat Dis Int. All rights reserved.
doi: 10.1016/S1499-3872(13)60028-4
Acknowledgements: We are grateful to Dr. Hua Huang (Department of Pathology, Affiliated Hospital of Nantong University) for his excellent technical assistance in the immunohistochemical studies, and thank T. FitzGibbon, MD, for comments on earlier drafts of the manuscript.
Contributors: YM, YDF and BYZ proposed and wrote the first draft. All authors contributed to the design and interpretation of the study and further drafts. YDF is the guarantor.
Funding: The study was supported in part by grants-in-Aid from the Projects of Jiangsu Medical Science (HK201102, H200925), the Priority Academic Program Development of Jiangsu Higher Education Institution (PAPD), and the Program of Nantong Society Undertaking and Technological Innovation (HS2011012), China.
Ethical approval: The study was approved by the Institutional Ethics Committee of both institutions adhered to the ethical guidelines of the 1975 Helsinki Declaration.
Competing interest: No benefits in any from have been received or will be received from a commercial party related directly or indirectly to the subject of this article.
BACKGROUND: The prognosis of hepatocellular carcinoma (HCC) is poor and its early diagnosis is of the utmost importance. This study aimed to investigate the values of glypican-3 (GPC-3) expression in the liver and sera and its gene transcription for diagnosis and monitoring of metastasis of HCC.
METHODS: Liver GPC-3 was analyzed in HCC tissues from 36 patients by immunohistochemistry and Western blotting. GPC-3 mRNA from circulating peripheral blood mononuclear cells from 123 HCC patients or 246 patients with other diseases or 36 HCC tissues was amplified by RT-PCR, quantitative real-time PCR, and confirmed by DNA sequencing. Circulating GPC-3 level was detected by ELISA.
RESULTS: The increasing expression of GPC-3 was observed from non-cancerous to cancerous tissues, with brown granule-like staining localized in tumor parts of atypical hyperplasia and HCC formation. The positive rate of GPC-3 was 80.6% in HCC, 41.7% in their paracancerous tissues, and none in distal cancerous tissues (P<0.001), with no significant difference in differentiation grade and tumor number except for size (Z=2.941, P=0.003). Serum GPC-3 was detected only in HCC (52.8%) and significant difference was found between GPC-3 and tumor size (χ2=6.318, P=0.012) or HBV infection (χ2=23.362, P<0.001). Circulating GPC-3 mRNA was detected in 70.7% of HCC tissues, with relation to TNM stage, periportal cancerous embolus, and extra-hepatic metastasis (P<0.001). The combination of circulating GPC-3, GPC-3 mRNA and alpha-fetoprotein is of complementary value for HCC diagnosis (94.3%).
CONCLUSION: Both GPC-3 overexpression and GPC-3 mRNA abnormality could be used as markers for the diagnosis of HCC and monitoring its metastasis.
(Hepatobiliary Pancreat Dis Int 2013;12:171-179)
KEY WORDS: hepatocellular carcinoma; glypican-3; immunohistochemistry; quantitative real-time PCR; gene expression; sequencing
Introduction
Hepatocellular carcinoma (HCC) is a common malignancy, but the outcomes of its treatment are poor.[1, 2] The incidence of HCC is closely related to the population infected with HBV in China.[3, 4] The reported data have shown the geographic distribution of HCC patients, about 80% of HCC patients with HBsAg-positive, in the inshore area of the Yangtze River.[5, 6] Thus HCC prognosis is poor and early diagnosis is of utmost importance. Although circulating alpha-fetoprotein (AFP) level is a useful diagnostic marker for HCC, the false-negative rate with AFP level alone may be as high as 40% for patients with early stage HCC, and the AFP level may be lower in 15%-30% of patients with advanced HCC.[7, 8] New tumor specific markers,[9, 10] such as circulating hepatoma-specific gamma-glutamyl transferase (HS-GGT),[5] hepatoma-specific AFP (HS-AFP or AFP-L3),[11, 12] miRNA,[13] and Golgi glycoprotein 73,[14, 15] have been used to improve the sensitivity and specificity of early detection and prognosis of the tumor. However, the overall results are not satisfactory.[16, 17]
Glypican-3 (GPC-3) is a membrane anchored heparin sulfate proteoglycan normally expressed in fetal liver and placenta, but not in normal adult liver.[18] It is an oncofetal antigen as a reliable biomarker for HCC and hasn't been observed in benign liver lesions by in situ hybridization or immunohistochemistry.[19] High grade dysplastic nodules typically express GPC-3 in a weak and focal fashion, although the results have not been consistent in different series.[20, 21] GPC-3 expression in hepatocarcinogenesis was previously investigated by rat hepatoma models, with the brown GPC-3 positive expression mainly distributed in cytosol and membrane. The incidence of GPC-3 expression was 100% in precancerous and cancerous tissues, and its abnormality was associated with hepatocyte malignant transformation, indicating that it is an early biomarker for HCC.[22, 23] However, little is known about the GPC-3 gene transcription from liver tissues and circulating peripheral blood mononuclear cells (PBMCs) and extra-hepatic metastatic cells of HCC. In the present study, the level of GPC-3 and its RNA expression in the liver and circulating PBMCs from HCC patients were analyzed by RT-PCR, quantitative real-time PCR, immunohistochemistry or ELISA to estimate the clinical value of GPC-3 as a tumor marker in diagnosis of HCC and its metastasis.
Methods
Patients
Patients enrolled in this study were divided into six groups: HCC (123 patients), liver cirrhosis (70), chronic hepatitis (70), acute hepatitis (56), non-liver tumor (50), and healthy individuals as controls (30). Profiles of the groups and their clinical data are shown in Table 1. The median age of the HCC patients was 44 years (36-81). Of the HCC patients, 89 (72.4%) had a history of HBV infection, and 5 had HCV infection. Child-Pugh classification showed 60 patients in Child-Pugh A, 45 in Child-Pugh B, and 18 in Child-Pugh C, respectively. Serum AFP concentration higher than 20 ng/mL was taken as a positive result. Patients also included 196 patients with benign liver diseases (including liver cirrhosis, chronic and acute hepatitis) and 50 patients with non-liver tumors (20 patients with bile duct carcinoma, 22 with gastric cancer, and 8 with pancreatic carcinoma). Among the 70 patients with liver cirrhosis, 38 were classified in Child-Pugh A, 18 in Child-Pugh B, and 14 in Child-Pugh C, respectively. The 30 healthy controls with negative HBV markers (HBsAg, HBcAb, and HBV DNA) and normal alanine aminotransferase levels were from the Nantong Central Blood Bank, China.
Liver specimens
Fresh surgical specimens including paracancerous (2 cm to cancer) and distal cancerous (5 cm to cancer) tissues were obtained from 36 patients (31 men and 5 women; aged 35-69 years, mean 50.9±9.8) at our hospital from March 2010 to January 2011. The specimens were snap frozen in liquid nitrogen and stored at -80 �� until use. HCC diagnosis was based on the criteria proposed by the National Collaborative Cancer Research of China[24] in addition to Edmondson grading, and TNM staging, and histological examination. Each specimen was divided into three parts for immunohistochemical analysis, GPC-3 mRNA, and pathological examination (HE staining). The diagnosis of HCC was confirmed histologically in all patients. Written informed consent was obtained from all the patients, and the study was approved by the Ethics Committee of both institutions.
Immunohistochemical analysis
UltraSensitive™ S-P (the streptavidin-peroxidase method) kit and monoclonal antibody of anti-GPC-3 (BioMosaics, USA) were purchased from Fuzhou Maixin Biotechnology Development Company, China. Liver specimens were fixed in 10% neutral formalin and embedded in paraffin. Four-micron thick sections from the tissues were cut, deparaffinized in xylene, and dehydrated in a gradient of ethanol solution. At last, they were restained with hematoxylin and examined. The negative control included empty control with normal rabbit IgG instead of primary antibodies or with second antibodies only. The negative control included 0.01 mol/L PBS instead of primary, second antibodies, and S-P reagent. The expression of liver GPC-3 was semi-quantitatively evaluated on the basis of the percentage of positive cells, and classified as follows: diffusely positive staining (+++) when positive cells were more than 50% of the total cells; moderately positive staining (++), 16%-50%; weakly positive staining (+), 5%-15%; and negative staining (-) less than 5%. The results of staining were evaluated by two independent pathologists without knowledge of the clinicopathologic features, and any difference in interpretation was resolved by consensus. Duplicate tissue cores for each tumor showed high levels of homogeneity for staining intensity and percentage of positive cells. The higher score was taken as the final score in case of a difference between duplicate tissue cores.
Preparation of PBMCs
Ficoll (Cedarlane, Canada) 2.5 mL was added to each sample, and centrifuged at 2000 rpm for 20 minutes. PBMCs were collected from the Ficoll/plasma interface, washed in normal saline for 3 times, and pelleted using low speed centrifugation. The cells (2×105/tube) were stored at -85 �� for total RNA isolation.
RNA isolation and cDNA synthesis
Liver tissues (50 mg) were washed with 1.0 mL of TRIzol reagent (Promega, USA). Total RNA was isolated according to the protocol of the manufacturer. RNA purity was estimated from the ratio of absorbance (A) readings at 260 and 280 nm, with an A260/280 ratio between 1.8 and 2.0. For synthesis of cDNA, 1 µg of total RNA was denatured in the presence of random hexamers (100 pmol/L, Promega, USA) and reverse-transcriptase (GIBCO, BRL, USA) at 23 �� for 10 minutes, at 42 �� for 60 minutes, at 95 �� for 10 minutes, on ice for 5 minutes, and then stored at -20 �� for PCR amplification.
Primer design and nested-PCR amplification
GPC-3 primers were designed according to the GenBank sequence (NM_001164617) fragment (nt757-878) and synthesized by Shanghai Sangon Bioscience and Technology Co., Ltd., China. The primers were GPC-3-1: 5'-TGC CTG ATT CAG CCT TGG AC-3' (nt757-776) and GPC-3-2: 5'-CCT AGT GAC TTG CAG TGA CTT G-3' (nt857-878), and the size of amplified product was 122bp. And the internal control primers were 5'-CAC TGG CGT CTT CAC CAC CAT-3' (nt396-416) and 5'-GTG CAG GAG GCA TTG CTG AT-3' (nt541-560), and the product size was 165bp as a control. The PCR was done using cDNA as a template with primers. The cycles were as follows: hot start at 94 �� for 5 minutes and then at 94 �� for 10 seconds, at 50 �� for 30 seconds, at 72 �� for 1 minute for 38 cycles, extended at 70 �� for 10 minutes, and stored at 4 ��. The amplified products were separated by electrophoresis on 2% agarose gel and stained with ethidium bromide, and detected with the ChemiDoc XRS+ system (BIO-RAD).
DNA sequencing
The 122bp amplified product of the GPC-3 gene was purified with Montage PCR centrifugal filter devices (Millipore, USA) according to the protocol instructions. DNA (1 µg) was used for sequencing reaction preparation and directly sequenced in a MegaBACE DNA sequencer with the DYEnamic ET Dye Terminator Cycle Sequencing Kit (Amersham Biosciences, USA). The sequences were edited using the Version 3.0 program and aligned with the original GPC-3 sequences.
Western blotting
Liver tissues (100 µg) were lysed in the sodium dodecyl sulfonate (SDS) sample buffer solution, separated with 10% of SDS polyacrylamide gel, and electro-transferred to nitrocellulose membranes. After blocking with 5% nonfat dry milk in the buffer [10 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 0.05% Tween 20], the membranes were probed with anti-GPC-3 (1:500, Maixin Biotechnology Development Company, Chain), followed by incubation with horse radish peroxidase-conjugated anti-rabbit immunoglobulin G secondary antibodies (1:2000, Maixin, Chain). The antibody binding was then visualized with enhanced chemiluminescence reagent (Beyotime Institute of Biotechnology, China), and the images detected with the ChemiDoc XRS+system using the Quantity One (v4.62, BIO-RAD, USA) software.
Quantitative fluorescent PCR
GPC-3 cDNA templates were diluted 10-fold for use. For quantitative fluorescent PCR with primers (GPC-3-1 and GPC-3-2) and SYBR Premix Ex TaqTM kit (TAKARA, Japan), the reaction system contained the following components: GPC-3 cDNA templates 2 µL, SYBR Premix Ex TaqTM 12.5 µL, each 0.5 µL of forward (10 µm) and reverse (10 µm) primer, and sterile water (9.5 µL). The PCR mixture of GPC-3 was amplified with an ABI PRISM7900H sequence detection system using the GAPDH gene as a control, with reaction condition at 95 �� for 2 minutes, and then at 95 �� for 12 seconds, at 60 �� for 40 seconds for a total of 40 cycles. The standard amplification curves of GPC-3 and GAPDH primers were generated according to the cycles of threshold (CT) from cDNA samples. Finally, the CT values were determined, and ΔCT=the CT value of GPC-3 minus the CT value of GAPDH. The circulating GPC-3 mRNA from PBMC in healthy subjects was not amplified and the CT values were under zone.
Detection of serum GPC-3 by ELISA
Serum GPC-3 was detected using an ELISA kit (Cusabio Biotech, USA) according to the manufacturer's instructions. In brief, 50 µL of serum or standard was separately put into each well, added 50 µL of HRP-conjugate, and incubated for 2 hours at 37 ��. Fifty µL of substrate A and B was added to each well for 15 minutes at 37 ��, and the plate was washed according to the routine method. Finally, 50 µL of stop solution was added and absorbance was read at 450 nm. The reference value of circulating GPC-3 in healthy subjects was under zone ng/mL.
Statistical analysis
The sensitivity and specificity of the test were calculated according to the following formulas: sensitivity= a/(a+c); and specificity=d/(b+d), where a denotes true-positive cases, b, false-positive cases, c, false-negative cases, and d true-negative cases.[25] Data were expressed as mean±SD or median (range). Differences between groups were assessed by Student's t test or a Fisher's exact test. A P≤0.05 was considered to be statistically significant.
Results
Hepatic GPC-3 expression and clinical impact
The expression and hepatocyte distribution of GPC-3 in different parts of HCC tissues were analyzed by immunohistochemistry with human anti-GPC-3 antibody (Fig. 1). The positive GPC-3 expression showed brown particles in cytosol and cell membrane with only a few cellular nuclei. The GPC-3 staining of HCC tissues was significantly higher than that of their paracancerous or distal cancerous tissues (Fig. 1). When more positive cells were stained, they were often diffusely localized in HCC tissues. Moreover, positive GPC-3 was markedly increased and expressed in HCC tissues (Fig. 1D).
The positive rate of GPC-3 expression and its intensity in HCC, paracancerous and distal cancerous tissues are shown in Table 2. The incidence of GPC-3 expression in the HCC group (80.6%, 29/36) was significantly higher than that in the paracancerous (41.7%, 15/36; χ2=1.455, P<0.001) or distal cancerous groups (0%, 0/36; χ2=48.558, P<0.001). It was also significantly higher in the paracancerous group than in the distal cancerous group (χ2=18.947, P<0.001). The intensity of GPC-3 expression in the HCC group was strongly positive (44.4%), moderately positive (19.4%), and weakly positive (16.7%), which were significantly higher than those in the paracancerous (Z=4.405, P<0.001) and distal cancerous groups (Z=6.679, P<0.001), respectively. In the paracancerous group the values were significantly higher than those in the distal cancerous group (Z=4.293, P<0.001). The clinicopathological characteristics were not significantly different among GPC-3 intensity, differentiation degree, age, gender, tumor number, and AFP level except for tumor size (≥3 cm group vs <3 cm group, P=0.008) and HBV infection (data not shown).
GPC-3 gene transcription in HCC
GPC-3 mRNA from the liver and circulating PBMCs in patients with liver diseases was amplified by RT-PCR (Fig. 2). The fragment of GPC-3 mRNA in PBMCs, cancerous tissues, and paracancerous tissues from HCC patients can be amplified and confirmed by sequencing, but not in distal cancerous tissues and PBMCs from patients with benign liver diseases.
The relative quantities of GPC-3 mRNA expression in PBMCs from patients with benign liver diseases by quantitative real-time PCR are shown in Fig. 3. The dissociation curves of both GPC-3 and GAPDH had a single peak, indicating that the two pairs of primers were specifically amplified. The relative quantity results of GPC-3 gene transcription from circulating PBMCs in patients with liver diseases are summarized in Table 3. The incidence of circulating GPC-3 mRNA was 70.7% in HCC, 0.0%-1.4% in benign liver diseases, and 2.0% in non-liver tumors. Significant differences (P<0.001) were observed between HCC and each of the study groups in circulating GPC-3 gene transcription. The non-GPC-3 expression in benign liver diseases was detected except of two patients with cirrhosis and one with non-liver tumor. There was no false-positive in patients with acute hepatitis or chronic hepatitis or in healthy subjects.
The pathological characteristics of circulating GPC-3 mRNA in PBMCs from HCC patients are shown in Table 4. The incidence of GPC-3 mRNA was significantly higher in HCC patients with I-II vs III-IV staging (χ2=5.511, P=0.019), HBV infection (χ2=50.571, P<0.001), and small tumor (χ2=4.520, P=0.034). It was especially true in patients with periportal cancer embolus (100% vs 54.4%, χ2=28.347, P<0.001) or extra-hepatic metastasis (100% vs 37.9%, χ2=57.019, P<0.001), but higher than that in patients without periportal cancer embolus or extra-hepatic metastasis. No significant difference was found in AFP level (χ2=0.098, P=0.754), tumor number (χ2=0.165, P=0.685) and Child-Pugh classification (χ2=1.005, P=0.605).
Detection of serum GPC-3 levels
Serum GPC-3 with AFP level in HCC patients was analyzed (Table 5). Only GPC-3 was overexpressed in HCC patients (52.8%) and was 0.0%-1.4% in patients with benign liver diseases or 2.0% in patients with non-liver tumors. There was significant difference between the HCC group and each of the study groups (P<0.001). The non-GPC-3 expression was detected in sera of patients with benign liver diseases but one patient with cirrhosis. There were no false-positive results in patients with acute hepatitis or chronic hepatitis or in healthy subjects. However, in comparison with AFP for HCC diagnosis, the higher false-positive results (14.3%-20.0%) of AFP could be found in patients with benign liver diseases, although there was a higher positive rate for serum AFP (70.73%, 87/123). According to tumor size, the frequency of serum GPC-3 abnormality was significantly higher (χ2=7.78, P<0.01) in the <3 cm (12/15, 80.0%) than in the ≥3 cm (45/108, 41.7%) (data not shown).
Diagnostic value of combined GPC-3 and AFP
The values of combined circulating GPC-3, GPC-3 mRNA and AFP for the diagnosis of HCC in 369 patients (liver diseases, non-liver tumors) are shown in Table 6. The positive rate in HCC patients was 52.8% in GPC-3, 70.7% in GPC-3 mRNA, and 70.7% in AFP, respectively. The percentage of abnormalities after analysis of the combined three markers was 94.3% (116/123) in HCC, 21.4% (15/70) in liver cirrhosis, 17.1% (12/70) in chronic hepatitis, 14.3% (8/56) in acute hepatitis, and 2.0% (1/50) in non-liver tumors, respectively. Total sensitivity, specificity, diagnostic accuracy, positive predictive value, and negative predictive value were 94.3%, 85.4%, 88.3%, 76.3%, and 96.8%, respectively. GPC-3 and GPC-3 mRNA in the diagnosis of HCC were superior to AFP level in specificity, positive predictive value, and diagnostic accuracy.
Discussion
HCC is one of the most common malignancies worldwide and early detection of HCC remains a key goal for the improvement of its poor prognosis. Screening programs of patients at risk, such as chronic carriers of HBV and individuals with cirrhotic HCV are justified.[26, 27] Early diagnosis and treatment (operation) of small-size HCC are of the utmost importance for prolonging the survival of the patients.[28, 29] HCC could be detected by biological markers and imaging technologies. AFP is a useful tumor marker for HCC, but it has a higher false-negative or false-positive rate.[12] Hepatoma-specific or complementary values are proposed for HCC diagnosis as predictive markers of HCC, especially for the diagnosis of small-size HCC with false-negative AFP. GPC-3 can be up-regulated in HCC, modulate cell-cycle progression, and promote cellular migration and invasiveness of HCC cells.[30] In this study, the expressions of the liver and circulating GPC-3 at protein or gene transcription level were investigated to explore their clinical values as tumor markers in diagnosis and hematogenous metastasis of HCC.
GPC-3 links to the cellular membrane through a glycosylphosphatidylinositol anchor and plays an important role in regulating cell growth and is closely related to HCC.[31] The two subunits are produced by cleavage between Arg358 and Ser359, which generates a N-terminal fragment of 40 kD (soluble) and a C-terminal fragment of 30 kD (combined with membrane).[32, 33] A recent study has shown that GPC-3 can induce oncogenesis through activation of the insulin-like growth factor-II signaling pathway.[34] In this study, its expression with carcinoembryonic nature was brown, nested distribution and deep stained in HCC tissues, and it was present in the cytoplasm and cell membrane. The positive rate of hepatic GPC-3 was 80.6% in HCC, 41.7% in its surrounding tissues, and no expression in distal cancerous tissues. The intensity of GPC-3 expression in HCC tissues was significantly higher than that in their surrounding or distal cancerous tissues. A significant difference of GPC-3 expression was observed in the different parts of HCC tissues, indicating that GPC-3 derived from HCC tissues is a specific tumor biomarker.
Metastasis is the final stage in tumor progression and is thought to be responsible for up to 90% of HCC deaths associated with cancerous cells which enter the circulation,[8, 11] eventually grow into lethal tumor in distal organs. Metastasis reflects inherent differences within the disseminating cells of distinct tumors. GPC-3 gene transcription is closely related to hepatocyte malignant transformation, with an increasing tendency from normal to precancerous to cancerous development, and the cell differentiation is in parallel with total RNA expressions in hepatocarcinogenesis. It could be examined in cancerous tissues or circulating PBMCs from HCC patients, and not in distal cancerous liver tissues or the cells from benign liver diseases. The incidence of hepatic GPC-3 mRNA is increased dynamically during HCC formation.[22] The higher positive rate is found in HCC patients with I-II stage, HBV infection, and small tumor, especially in HCC patients with periportal cancer embolus or extra-hepatic metastasis, suggesting that the up-regulation of circulating GPC-3 mRNA is a more sensitive and specific biomarker for monitoring the metastasis of HCC.
Studies[35, 36] found the efficacy of GPC-3 as a diagnostic tool in HCC. The reported sensitivity ranges from 75% to 85% in larger series. Higher specificity and diffuse staining indicate that GPC-3 is of great utility in HCC diagnosis by liver biopsies.[21] Although AFP is an accepted tumor marker to diagnose HCC now, the positive rate is only about 70% and the false-negative rate is 30%.[5, 8] In the early diagnosis of HCC, a slight increase of AFP level is difficult to differentiate benign liver diseases or non-liver tumors. In this study, the incidence of GPC-3 mRNA in HCC patients with I-II vs III-IV stage, HBV infection, and small tumor, especially in those with periportal cancer embolus or extra-hepatic metastasis was higher than in those without periportal cancer embolus or without extra-hepatic metastasis. No significant difference was found in AFP level, tumor number and Child-Pugh classification. The level of circulating GPC-3 was significantly higher in HCC patients than in those with benign liver diseases or non-liver tumors. The findings indicate that the abnormality of GPC-3 level could be a useful molecular marker for HCC diagnosis.
The overexpression of the liver or circulating GPC-3 at protein or gene transcription level could be examined only in HCC patients except for few cases of liver cirrhosis or non-liver tumors. It is not significantly related to AFP level. The detection of GPC-3 and AFP markers is of complementary value in enhancing the sensitivity and specificity of HCC diagnosis. In this study, the sensitivity of three markers in 123 HCC patients was 52.8%, 70.7%, and 70.7% respectively, but the total positive rates of these markers could rise up to 94.3%. In HCC and LC patients, the sensitivity, specificity, diagnostic accuracy, positive predictive value, and negative predictive value (%) (n=193) were 52.8%, 97.1%, 68.9%, 97.0%, and 53.9% for GPC-3; 70.7%, 97.1%, 80.3%, 97.7%, and 65.3% for GPC-3 mRNA; and 70.7%, 80.0%, 74.0%, 86.1%, and 60.8% for AFP, suggesting that GPC-3 and GPC-3 mRNA are superior to AFP in the diagnosis of HCC except for serum GPC-3 sensitivity.
In this study, there was no significant relationship between GPC-3 expression intensity and histological differentiation grade or the number of tumors and AFP level. GPC-3 gene transcription level is significantly related to tumor size, the high expression rate in small HCC or differentiation of benign from malignant liver diseases. Although GPC-3 is promising, its limitations should be considered in some HCC patients without GPC-3 expression. Scattered and weak GPC-3 expression could be examined in paracancerous tissues (cirrhotic nodules or inflammatory tissues).
In conclusion, the detection of GPC-3 and its gene transcription in the hepatoma-specificity is superior to serologic AFP alone. It is efficacious in differential diagnosis and monitoring of hematogenous metastasis of HCC.[37, 38] The combined circulating GPC-3 and AFP markers increase the sensitivity of HCC diagnosis. Further studies help to identify GPC-3 specifically, develop new methods, explore the mechanism in hepatocarcinogenesis,[4, 6] and know how to target GPC-3 sites or RNA interference-mediated suppression of GPC-3 expression or its antibody for HCC therapy.[39] However, the specific biomarkers with a high sensitivity for early CC seem to be more practical so far.[40]
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Received February 8, 2012
Accepted after revision August 1, 2012 |