by Xijun Min, Dr Yan Li, Liuyan Chen, Chengwei Yao, Dr Xi Qin

As a traditional tumour marker for the diagnosis of early primary hepatic carcinoma (PHC), alpha fetoprotein (AFP) has a low positive rate. Serum glypican 3 (GPC3) is a novel biomarker, and the combined examination of GPC3 and AFP will provide a new strategy for the diagnosis of PHC. We aimed to compare and evaluate the clinical value of GPC3 and AFP alone or in combination for the diagnosis of PHC. The concentrations of AFP and GPC3 in serum were measured in 181 healthy people, 121 patients with chronic hepatitis B and 143 patients with confirmed PHC by chemiluminescence and non-competitive ELISA solid-phase immunoassay. The Spearman rank correlation test was utilized to analyse the correlation of AFP and GPC3. The ROC curve was drawn and the diagnostic value of GPC3 and AFP in PHC were analysed and compared. There was a significant correlation between GPC3 and AFP (Rs=0.757, P<0.05). The positive rate of GPC3 was 59.38% in patients with confirmed PHC whose AFP was negative. The sensitivity and accuracy of combination detection (88.73%, 90.56%) were significantly higher than those of GPC3 alone (74.83%, 88.54%) and AFP (85.24%, 81.80%) (P<0.05). The Youden index (0.7841) of the combined detection was significantly higher than that of the individual application of GPC3 (0.699) and AFP (0.496) (P<0.05). Our results suggest that the single detection of GPC3 has higher clinical value than that of AFP. Combined GPC3 and AFP detection has higher sensitivity and accuracy than single detection, and has higher application value in clinical diagnosis of PHC.

Introduction
Primary hepatic carcinoma (PHC) is among the most common malignant tumours. In recent years, its incidence ranks fifth among all malignant tumour types, with about 900 000 new cases occurring annually in the world [1,2]. In China, PHC ranks fourth in the incidence and second in the mortality of common malignant tumours, respectively [3,4]. The early symptoms of PHC are not obvious, and about 60% of cases are found at an advanced stage, often when tumour metastasis has occurred [5]. Therefore, early detection, early diagnosis and early treatment are vital for the improvement of the survival rate of PHC patients. Currently, the most widely applied means of clinical diagnosis of early PHC is the detection of alpha fetoprotein (AFP) in serum combined with imaging examination. However, liver inflammation and cirrhosis sometimes interfere with the early diagnosis of PHC, and therefore the optimum time for treatment can be missed [6]. AFP has been used in the clinical diagnosis and prognosis evaluation of PHC: research demonstrated that 400μg/L is considered to be the best clinical cut-off value for PHC in patients with suspected chronic liver disease, the sensitivity and specificity of AFP are 49–71% and 49–86% respectively, and the AFP level of 32–59% patients with hepatic carcinoma is within the standard scope [7]. Therefore, the early diagnosis of hepatic carcinoma is prone to be either missed or misdiagnosed.

At present, AFP, AFP L3 (Lens culinaris-agglutinin-reactive fraction of AFP) and DCP [des-gamma carboxyprothrombin, also known as protein induced by vitamin K absence/antagonist-II (PIVKA-II)] have been identified as serum molecular markers for the diagnosis of hepatic carcinoma. The “Guidelines for the diagnosis and treatment of hepatocellular carcinoma (2019 edition)” propose that in addition to AFP, AFP L3, DCP and cell-free plasma microRNA can also be identified as molecular markers for early diagnosis of hepatic carcinoma, especially for people with negative serum AFP [8]. The American Association for the Study of Liver Diseases (AASLD), the European Association for the Study of the Liver (EASL) and the Japanese Society of Hepatology (JGH) suggest that although AFP, AFP L3 and DCP tests cannot serve as indispensable conditions for the confirmation of the diagnosis of hepatic carcinoma, they can play a major role in diagnosis and recurrence monitoring [9–11]. Nevertheless, these tumour markers are not sensitive to small hepatic carcinoma with lumps ≤2cm in diameter, and patients with chronic liver disease are also likely to have positive test results. A study by Edoo and colleagues shows that the joint detection of AFP, carcinoembryonic antigen (CEA) and cancer antigen (CA)19-9 for the screening and diagnosis of hepatic carcinoma is not better than AFP alone [12].

Therefore, there is an urgent need to find a novel diagnostic indicator of significant clinical value that is more sensitive and specific than AFP for the diagnosis of early hepatic carcinoma, especially for hepatic carcinoma with negative AFP. Serum glypican 3 (GPC3) is a novel biomarker. Studies have detected high expression of GPC3 in more than 70% of hepatic carcinomas, even in the early stage of hepatic carcinoma progression, but not in normal adult tissues. GPC3 also has a bearing on the metastasis and prognosis of hepatic carcinoma, and is expected to be applied as a new target for early diagnosis, prognosis evaluation and immunotherapy of hepatic carcinoma [13,14]. El-Saadany et al. conducted a study on 80 patients with primary hepatic carcinoma in Egypt, by adopting flow cytometry (FCM) and ELISA for the detection of GPC3 and AFP, respectively; they concluded that GPC3 was more sensitive for the diagnosis of hepatic carcinoma than AFP and the combined detection of GPC3 and AFP showed high sensitivity (98.5%) and specificity (97.8%) [15]. In recent years, it has been found that a single test analyte cannot meet the diagnostic needs of early hepatic carcinoma, which implies that the combination of multiple test indicators is of great utility for improving the positive detection rate. Our study took hepatic carcinoma patients, chronic hepatitis B patients and healthy people as the research subjects. The purpose of this study was to explore the clinical diagnostic value of GPC3 and AFP and their combination in primary hepatic carcinoma, as well as the clinical diagnostic significance of GPC3 in AFP-negative hepatic carcinoma.

Patients and methods

Clinical data
This study included 143 patients with hepatic carcinoma (who had not been treated surgically), who were hospitalized at Hainan Cancer Hospital (Haikou, China) from July to November 2018. Without tumour, node, metastasis (TNM) staging, the included patients received B-ultrasound and CT examination. The diagnosis of hepatic carcinoma was in accordance with the “Guidelines for diagnosis and treatment of primary liver cancer in China (2017 edition)” [16]. The control group was made up of 121 patients with chronic hepatitis B at the same period, all cases were confirmed by Hepatitis B serologic test and clinical examination. Among them, 25 cases were among those with positive HBsAg, HBeAg, HBcAb markers, 96 cases were among those with positive HBsAg, HBeAb, HBcAb markers. The control group was composed of 181 healthy participants. The group of confirmed hepatic carcinoma patients included 108 males and 35 females, average age 56.4±12.7 years. The group of chronic hepatitis B included 106 males and 15 females, average age 51.85±14.1 years. The diagnosis of viral hepatitis B reached the diagnostic specification of the “Guideline of prevention and treatment for chronic hepatitis B (2015 update)” [17]. The healthy population group included 147 males and 34 females, average age 54.11±13.4 years. There was no statistically significant difference between the groups in terms of age, sex and other general characteristics (P>0.05).

This study was approved by the Ethics Committee of Hainan Cancer Hospital, and informed consent forms were signed by all subjects enrolled.

Specimen collection and pretreatment
Fasting venous blood was taken from all subjects in the morning. After natural agglutination, all samples were centrifuged at 3700rpm for 10min, and the serum was separated and stored at −20°C for further experiments.

Determination of serum AFP and GPC3 concentration
AFP detection kit (cat. no. 37104503, Roche Applied Science) was used to detect the concentration of AFP by chemiluminescence assay (COBAS-e-602, Roche Applied Science), AFP ≥7ng/ml was positive. GPC3 detection kit (cat. no. 41651U, CanAg Diagnostics) was used to determine the content of GPC3 in serum by non-competitive ELISA solid-phase method, GPC3 ≥0.095ng/ml was positive. Absorbance was measured at 450nm using a microplate reader (MULTISKAN-FC, Thermo Fisher Scientific). The test operation steps and quality controls had been operated in strict accordance with the instructions of the kits.

Statistical analysis
SPSS 17.0 statistical software was used for the statistical analysis of data. The measurement data were processed by analysis of variance or t-test, and the counting data were analysed by nonparametric test. P<0.05 was used to express that the difference is significant. Spearman correlation analysis was used, P<0.05 was used to express that the difference is statistically significant. Receiver operating characteristic (ROC) curve was used to determine the best critical value of GPC3 in the diagnosis of hepatic carcinoma, and AUC was used to evaluate the diagnostic value of GPC3 and AFP in primary hepatic carcinoma.

Results

Comparison of GPC3 and AFP levels among groups
The levels of GPC3 and AFP were 0.21±0.19ng/ml and 19.50±11.45ng/ml respectively in the group with confirmed hepatic carcinoma. The levels of GPC3 and AFP were 0.07±0.03ng/ml and 3.25±1.16ng/ml of chronic hepatitis B patients with positive HBsAg, HBeAg, HBcAb markers. The levels of GPC3 and AFP of chronic hepatitis B patients with positive HBsAg, HBeAb, HBcAb markers were 0.07±0.05ng/ml and 2.69±0.96ng/ml respectively. The levels of GPC3 and AFP of healthy subjects were 0.04±0.01 ng/ml and 1.96±1.52ng/ml respectively. The levels of serum GPC3 and AFP of patients with confirmed hepatic carcinoma were significantly higher than those in chronic hepatitis B patients with positive HBsAg, HBeAg, HBcAb markers, chronic hepatitis B patients with positive HBsAg, HBeAb, HBcAb markers and the healthy subjects (P<0.05). In the control group, among patients with chronic hepatitis B, the serum GPC3 level of those with positive HBsAg, HBeAb, HBcAb markers was not statistically significant compared with those with positive HBsAg, HBeAg, HBcAb markers (P>0.05), but showed an upward trend. Compared with healthy subjects, the GPC3 and AFP levels of chronic hepatitis B patients with positive HBsAg, HBeAb, and HBcAb markers and those with positive HBsAg, HBeAg, HBcAb markers showed no significant difference. The results are shown in Table 1.

Table 1. Comparison of GPC3 and AFP levels among groups (mean±SD)

Comparison of positive rates of GPC3 and AFP in the confirmed hepatic carcinoma group
Among 143 patients with confirmed hepatic carcinoma, 107 cases were GPC3 positive (≥0.095ng/ml), the positive rate was 74.83%, and 79 cases were AFP positive (≥7ng/ml), the positive rate was 55.24%. The positive rate of GPC3 was significantly higher than that of AFP, the difference was statistically significant (χ2 = 27.618, P=0.002, P<0.05). 69 cases were positive for both GPC3 and AFP, and the positive rate was 48.25%; Among the 64 patients with confirmed hepatic carcinoma with negative AFP, 38 were positive for GPC3, with a positive rate of 59.38%. The results are shown in Table 2

Table 2. Comparison of GPC3 and AFP positive rates in the confirmed hepatic carcinoma group

Correlation analysis between GPC3 and AFP
Results of Spearman correlation matrix analysis showed that the correlation between GPC3 and AFP was statistically significant (Rs =0.757, P<0.05). ROC curve analysis of single and combined detection and diagnosis of hepatic carcinoma by GPC3 and AFP
Based on the serum GPC3 and AFP indicators of the confirmed hepatic carcinoma group and the control group, the receiver operating characteristic curve was formulated and analysed. The area under the ROC curve (AUC) of GPC3, AFP and combined detection for the diagnosis of hepatic carcinoma were 0.937, 0.847 and 0.859 respectively. The findings suggested that the high value of single and combined detection for the diagnosis of hepatic carcinoma. The AUC of the combined detection was higher than that of single AFP and lower than that of single GPC3. The 95% confidence intervals of GPC3, AFP and combined detection were 0.914–0.961, 0.808–0.887 and 0.822–0.896 respectively. The results are shown in Figure 1.

Figure 1. ROC curve of GPC3 and AFP
AFP, alpha-fetoprotein; GPC3, glypican-3; GPC3 and AFP, combined detection of GPC3 and AFP; ROC, receiver operating characteristic.

Comparison of sensitivity, specificity, accuracy and Youden index of GPC3 and AFP alone and in combination
We then further compared the sensitivity, specificity, accuracy and Youden index of GPC3, AFP and combined detection, so as to decide its diagnostic value. Regard the clinical diagnosis results as the gold standard and list the quadruple table of each index for calculation. The results demonstrated that the sensitivity and accuracy of combined detection of GPC3 and AFP were significantly higher than those of GPC3 and AFP alone (P<0.05); The Youden index of combined detection was significantly higher than that of GPC3 and AFP alone (P<0.05); The specificity of GPC3 alone (95.03%) was significantly higher than that of AFP alone and combined detection (P<0.05). The results are shown in Tables 3–6.

Table 3. Quadruple table of the value of GPC3 in diagnosis of hepatic carcinoma

Table 4. Quadruple table of the value of AFP in the diagnosis of hepatic carcinoma

Table 5. Quadruple table of the value of GPC3 combined with AFP in the diagnosis of hepatic carcinoma

Table 6. Comparison of diagnostic value between GPC3 and AFP and combined detection

Discussion
In the current diagnostic approaches of hepatic carcinoma, compared with assessment by imaging, serum tumour markers can be expressed earlier with high specificity, which plays a role in early monitoring and early warning of the occurrence of hepatic carcinoma. AFP is still recognized as the major serological marker for the clinical diagnosis of hepatic carcinoma in the world so far. However, AFP comes with some limitations as a clinical diagnostic indicator of hepatic carcinoma, such as low sensitivity, which makes it easy to miss a diagnosis of early hepatic carcinoma [18]. Nevertheless, other indicators currently applied cannot completely replace AFP as a serological marker for the diagnosis of hepatic carcinoma. In this regard, efforts are needed to find diagnostic indicators more appropriate than AFP and to establish a proper reference interval proven to be of high value for the diagnosis of hepatic carcinoma, especially for the early diagnosis of AFP-negative hepatic carcinoma. GPC3, a novel immune marker with high specific expression in hepatic carcinoma cells, has emerged as a research focus in this field.

GPC3 is a type of glycan protein distributed on the surface of the cell membrane, which is anchored on the cell surface by glycosylphos-phatidylinositol. It is a member of the heparan phosphate proteoglycan family. As a membranous interstitial protein, the expression of GPC3 is consistent with that of some growth factors and their receptors, and is able to regulate cell morphology and various cell behaviours through a series of signal transduction cascades, such as proliferation, migration, adhesion, differentiation and survival [19]. In the development of hepatic carcinoma, GPC3 can stimulate the growth of cancer cells by activating the classic Wnt/β catenin signalling pathway, and activate the Wnt/β catenin signalling pathway by mediating the binding of fibroblast growth factor (FGF) to cells, as well as participating in the regulation of a variety of tumour-related signaling pathways by preventing the degradation of insulin like growth factor (IGF) and inhibiting the hedgehog (Hh) signalling pathway [20,21]. Current studies suggest that GPC3 plays an important role in the occurrence and development of hepatic carcinoma. In addition to hepatic carcinoma, recent studies have indicated that GPC3 is also highly correlated to the occurrence and development of colorectal cancer, ovarian cancer, melanoma, breast cancer and yolk sac tumours [22–26].

According to this study, after comparing the positive rate of GPC3 and AFP in the confirmed hepatic carcinoma group, the positive rate of GPC3 (74.83%) was significantly higher than that of AFP (55.24%). The simultaneously positive rate of GPC3 and AFP was only 48.25%, which was lower than that of GPC3 and AFP alone. Among 143 patients diagnosed with hepatic carcinoma, 64 were AFP negative, among which the positive rate of GPC3 was 59.38%. These results showed that GPC3 has clinical diagnostic significance for AFP negative hepatic carcinoma patients, which is consistent with the results of Liu [27]. The correlation between GPC3 and AFP had been verified by correlation analysis in this study. AFP is considered to have some limitations as a traditional diagnostic indicator of hepatic carcinoma, whereas the detection rate of AFP-negative liver cancer patients can be increased by more than a half by applying the GPC3 approach. Therefore, GPC3 has its superiority as a novel biomarker for the diagnosis of hepatic carcinoma in comparison with AFP.

Another result on the receiver operating characteristic curve (ROC) showed that the AUC of GPC3 alone (0.937) was significantly higher than that of AFP alone (0.847) and combined detection (0.859), and the AUC of combined detection was slightly higher than that of AFP. The 95% confidence intervals of GPC3, AFP and combined detection were 0.914–0.961, 0.808–0.887 and 0.822–0.896, respectively. All these suggested that single and combined detection have certain accuracy and high diagnostic value in the diagnosis of primary hepatic carcinoma. As a novel diagnostic indicator, GPC3 provides better diagnostic effect and higher reliability than the traditional detection indicator AFP.

This study also compared the sensitivity, specificity, accuracy and Youden index of the novel diagnostic indicator GPC3 with the traditional diagnostic indicator AFP and combined detection. The results showed that taking the clinical diagnosis results as the gold standard, the sensitivity and accuracy of combined detection (88.73%, 90.56%) were significantly higher than that of GPC3 (74.83%, 88.54%) and AFP (55.24%, 81.80%) alone, and the specificity of GPC3 alone (95.03%) was significantly higher than that of AFP alone (94.37%) and combined detection (89.68%) (P<0.05). As a comprehensive reflection of the diagnostic value of each index in the diagnostic experiment, the Youden index of joint detection (0.7841) was significantly higher than that of GPC3 (0.699) and AFP (0.496) alone. All these results show that in the clinical diagnosis of primary hepatic carcinoma, the combined detection of GPC3 and AFP has higher sensitivity and accuracy and higher clinical application value than detection of a single marker. The sensitivity, accuracy, specificity and Youden index of GPC3 alone are higher than those of AFP alone, indicating that GPC3, as a novel diagnostic indicator of hepatic carcinoma, is more applicable than AFP, and is more sensitive and accurate in the diagnosis of hepatic carcinoma with reduced missed diagnosis rate and misdiagnosis rate. It has strong clinical application value and is expected to be used as a novel tumour marker for the diagnosis of PHC. This study confirmed the role of GPC3 as a novel immune marker for the diagnosis of primary hepatic carcinoma, its clinical application value is better than the traditional diagnostic indicator AFP. The combined detection of GPC3 and AFP can improve the diagnostic rate of primary hepatic carcinoma. Given that the sample volume is of restrained size and the tumour staging research hasn’t been carried out, this study has its own limitations. Our team will focus on the application research of GPC3 in the early diagnosis of primary hepatic carcinoma in the following studies, so as to provide a theoretical and practical basis for screening novel immune markers applicable for clinical application and promotion. [/av_textblock] [av_textblock size='' av-medium-font-size='' av-small-font-size='' av-mini-font-size='' font_color='' color='' id='' custom_class='' av_uid='av-23o2c4' admin_preview_bg=''] References

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The authors
Xijun Min†1, Yan Li†1, Liuyan Chen2, Chengwei Yao2, Xi Qin*2
1 College of Pharmacy, Hainan Medical University, Haikou, 571199, China
2 Department of Laboratory Medicine, Hainan Cancer Hospital, Haikou, 570310, China

† Authors contributed equally to this work.

* Corresponding author
E-mail: 1291602544@qq.com

Editor’s comment
This article presenting original research data has not been peer-reviewed.
Please address any questions to the corresponding author.