Influence of Glypican-3 as Anewly Diagnostic Biomarker in Earlydetection of Hepatocellular Carcinoma among Saudi Patients

1Assistant Prof of Virology and Molecular biology, Health Sciences Research Center (HSCR), Princess Nourah bint Abdulrahman University (PNU), Kingdom Saudi Arabia (KSA). 2Prof of Genetic, Biology department, Princess Nourah bint Abdulrahman University (PNU), Kingdom Saudi Arabia (KSA). 3Prof of Oncology and Hematology, college of Medicine, King Saud University (KSU), Kingdom Saudi Arabia (KSA). 4Assistant Prof of molecular biology, Princess Nourah bint Abdulrahman University (PNU), Kingdom Saudi Arabia (KSA). 5Prof Biochemistry, Central Labe, King Saud University (KSU). 6Assistant Prof of Hepatopathology, Faculty of Medicine, EL-Azher University and Specialist Physician, Oncology Clinic-Medical Affaies, Tawam Hospital, AL Ain, UAE.

According to Saudi Arabia, cancer registry, hepatic malignancieswas 4.8% of all newly detected cases of all other types of malignance.HCC ratio 2.1: 1a male to female and it accounts for 83% and 80% of all Hepatocellular carcinoma cases in males and females, respectively 1 The chronic liver diseases are play main role in development of liver cancer.So, many genetic changes is hypothesized in HCC patients during long time.Many studies demonstrate that the infection of HBV and HCV is the main etiology agent of Liver cancer 2,3 .Butin Saudi Arabia, Hepatitis C is also common with a prevalence rate of 1% to 3% of the population which further increases the risk of HCC 3, 4. The main problems in most surveillance programs of hepatocellular carcinoma (HCC) that is highly cost.In fact low percent of patients with HCC are able to treatments and transplantation of liver, but most of them are poor and can't follow-up the treatment.And many clinical diagnosis depend on serum alpha fetoprotein (s AFP) that is normal levels in about 30% of liver cancer.On other hand AFP is significant in casestumor size is >3 to 5, But it was in significant in HCC with small size < 2 5,6 .Therefore, depend on AFP as the famous biomarker for detection and follow-up of HCC isn't accurate and for early surveillance of HCCneeded to a new diagnostic markers 7 .
Glypican-3 (GPC-3) is type of heparin sulfate proteoglycans cell that bind to the cell membrane and interacts with many growth factors in the migration, proliferation, and modulation of cell survival in different tissues which contributing to development of Liver cancer 7,8 .Recently viewed some studies reported that GPC-3 levels are elevated inin serum or liver tissues of Hepatocellular carcinoma patients but is negative in primary liver lesion, hepatic cirrhosis and hepatitis 9 .In addition, GPC-3 detectedinHCC cases AFP-negative 10 .GPC-3 could use as new biomarker for early detection of Hepatocellular carcinoma 8,10 .
The present study advocateto compare between the sensitivity and specificity of sGPC-3 andGPC-3 mRNA by using ELISA and RT-PCR in diagnostic Hepatocellular carcinoma patients from cirrhotic patients and normal controls and compared them with AFP levels in the same study groups.

Material and Methods
In the current study conduct on 300 blood samples, 145 collected from HCC and 105 samples collected from chronic liver cirrhosis (CLC)  11 .Theconcentration ofGlypican-3 was evaluated from the curve.The normal range of serum Glypican-3 was 0-55 ng/mL and the abnormal malignant rang was detected in the case of Glypican-3 >100 ng/ mL 11 .
AFP was detected by usingthe Cobas601 electrochemiluminescence immunoassay analyzer.It indicated positive in the case of serum AFP > 7 µg/L.detection of Glypican-3 mrna by rt-PCr PCR was performed using Dream Taq Green PCR Master Mix (ThermoScientific, Fermentas).The temperatures protocol of reaction was performed at 25ÚC for 10 min then 120 min respectively, and 85ÚC for 5 min and kept at 4ÚC.The reaction for â actin was performed in 25 µl reaction containing 1 µl cDNA, 1x master mix, 25 pmole of each primer.
For amplified Glypican-3, the cyclic condition consisted of initial denaturation at 94ÚC for 5 min, followed by 35 cycles of denaturation at 94°C for 30 sec, annealing at 58°C for 45 sec, elongation at 72°C for1 min and final elongation at 72°C for 10 min and the primer used was: forward, 5'-TTCTCAACAACGCCAATA-3', and reverse, 5'-GATGTAGCCAGGCAAAGC-3'.The PCR products were visualized on 2% agarose and were 452 bp for b-actin and 256 bp for target gene of Glypican-3 11 .statistical analysis T h e s t a t i s t i c a l a n a l y s i s i n t h i s studywasdone byusing IBMSPS Sversion20 (SPSSInc.,Chicago,IL).Where qualitative data were expressedasfrequenciesandpercentages.Mann-Whitney test (non-parametric t-test) usingin comparison between two groups for qualitative data.Kruskal-Wallis test (non-parametric ANOVA) thenpost-Hoc"Schefetest" on rank of variables was used for pair-wise comparison between three groups.Medians and ranges were using for Numerical data.Spearman-rho method was used to test correlation betweennumericalvariables. For prediction of cutoffvalueswas used Receiver Operating Characteristic (ROC) curve.Sensitivity,specificity,positivepredictive value(PPV)andnegative predictive values(NPV) were calculatedforallmarkersused.Thep-value<0.05 was considered significant.
The highest results of sGPC-3 were detected in HCC with HBV and HCV positive compared to HBV and HCV negative patients.It was 96% (120/125), 90% (18/20) in positive and negative hepatitis virus HCC patients respectively.While for AFP, the highest results were measured in HBV and HCV positive patients.
In contrast, sGPC-3 was 10.5 % positivity in cirrhosis group (11/105); where 12 % (9/75) in positive hepatitis B and C patientsand it was 6.7  This results showed that level of sGPC-3 was higher and significant in HCC in positive hepatitis B and C patientsthan negative hepatitis viruses cases (p<0.001).While in CLC there is no significant difference between positive and negative HBV, HCV patients (p = 0. 612).
The detection value of GPC-3 was higher than that of AFP was 0.457 to 0.496, and could use these marker in early diagnosis of HCC.Moreover, GPC-3 level was elevated in the serum of individuals with HCC who had serum AFP level less than 400 ng/ml.So could use these markers in early diagnosis of early HCC in Saudi patients.
This result demonstrated that sensitivity and specificity of AFP is not accuracy in primary HCC compared to accuracy in new tumor marker (GPC-3), which not detected in healthy group and it can distinguish between primary HCC and CLC patients.Further we need more investigated the combination of the two markers (GPC-3, AFP) in continuous follow-up of primary HCC and CLC.

disCussion
In fact, the surveillance programs of HCC has evolved significantly over the past few decades 14.Unfortunately, the most of itsdepend on ultrasoundand alpha fetoprotein (AFP) 15.However, in somereports recorded that the sensitivity for diagnosis of Hepatocellular carcinoma approximately 25%-60%, and its specificity is low and AFP was estimated in patients with CLC range between 11%-47% and 15%-58% in non -cirrhosischronic hepatitis patients 2 .In addition, 30% HCC patients present with normal AFP.Thus, the needed for new tumor markers that can differentiated among Hepatocellular carcinoma and primary hepatic necrosis it is much important for specific diagnosisand early surveillance of HCC 16, 17.The data in this study demonstrated that, Glypican-3 mRNA in HCC washigher sensitive between the studied markers for detection liver cancer in Saudi patients (100% positivity rate), followed by Serum Glypican-3 (95%), then AFP (80%).And also in cirrhosis group, GPC-3 mRNA showed the highest positivity (30%), then sGPC-3 (10.5%).
In contrast, by comparison of sGlypican-3 and AFP between the all studied groups showed significant difference between HCC, CLC and healthy control groups.This demonstrated that the continuous measurement of GPC-3 could provide a significant improved test for detection of HCC in both cases with/nothepatitis viruses,this is in accordance with other researchers 10,15,18,19,20,21 .And the influence of Glypican-3 as a newly tumor biomarker for liver cancer and early detection of primary HCC has been recently confirmed by El-Shenawy et al. 22 and Iman Attia et al. 23 .
The present study documented the clinical utility of Glypican-3 mRNA as sensitive quantitative assay for screening HCC.Our results in agreements with youssef et al. 24 , Yan et al., 25 , Gomaa et al, 26 .Some studies reported that, in HCC patients, s GPC-3 and AFP levels increased and negative in other benign liver cases 10,18,19 .The absence of serum GPC-3 in healthy individuals was reported by some other studies 15,20, 21, 22.The results in this study, demonstrated that sGlypican-3 in Hepatocellular carcinoma group was significantly differed from CLC, health control subject, that pointed to in Saudi patients, serum Glypican-3 was high significantly in HCC as is can considered as newly tumor biomarker for early diagnosis and surveillance of HCC.And that because release of GPC-3 protein by hepatoma cells in first stage HCC cases,sGPC-3 was significantly elevated in HCC.These results indicated that the hepatitis cirrhotic cases also had Glypican-3 elevated, that agreement with the study's results of youssef et al. 24 , Yan et al., 25 , Gomaa et al, 26 .So, Glypican-3 have specific sensitive inSaudi patients withhepatic disorders.
Recently, several studies demonstrated that Glypican-3 ishighly sensitive and specificand be favorite than AFP in diagnosis and early surveillance of HCC, 21 .The comparison of ROC curves for GPC-3 displayed that GPC-3 was high accurate than AFP in the diagnosis of HCC.The sensitivity of sGPC-3 was 95% and AFP was 80% in HCC consistent with the findings of by El-Shenawy et al. 22 , Iman Attia et al. 23 and youssef et al. 24 .
As regards Glypican-3mRNA level by RT-PCR, our results showed that GPC-3mRNA was estimated in all Hepatocellular carcinoma group (100%), in cirrhosis patients was (30%) and was absent completely in healthy controls subject.Our results were in constant with youssef et al. 24 , Yan et al., 25 , Gomaa et al, 26 Yongle et al 27 and Sung et al 28 , these studies reported similar result reported that Glypican-3mRNA is significantly up regulated in Hepatocellular carcinoma compared to normal and benign liver samples, and hence Glypican-3 mRNA could be useful as diagnostic molecular tumor marker for early diagnosis of Hepatocellular carcinoma.
Regarding the one hundred five CLC cases, included in the present study, eighteen of them were positive for Glypican-3mRNA.These patients were surveillance for 12 months.Thefifteen who were positive hepatitis viruses also having moderate elevation in serum Glypican-3 and AFP levels has been detected as HCC after 6 months of follow up.These results could support that Glypican-3 could be used for screening and early diagnosis of HCC among CLC patients.This suggestion was agreement with El-Saadany et al 15 and Iman Attia et al. 23 , where reported that sGPC-3 levels was estimated during the follow up of their patients with liver cirrhosis and Hepatocellular carcinoma developed during follow-upselected cases with detect a significant change of serum AFP levels.
The low positivity of Glypican-3 (10.5 % ) in cases of HBV and HCVwithout hepatic malignant (cirrhosis group (11/105)in this study was 12 % (9/75) in positive hepatitis B and C patients and it was 6.7 % (2/30) in non-hepatitis viruses cases (negative HBV and HCV)give evidence that the high specificity of Glypican-3 in HCC versus non HCC hepatitis cases, as positivity of HBV or HCV infection will not give false positive result especially in Saudi patients, where there is continuous prevalence of hepatitis viral infection.Liu et al., (2010) reported that GPC-3 was present in the serum of HCC patients, but was undetectable in all patients with hepatitis as well as healthy individuals 9.And Liet al., (2013) reported that Glypican-3 was highly expressed in acute and chronic hepatitis patients, indicating that such protein was less sensitive in detection of hepatic diseases but could be helpful as their serum biomarker 11 .While Gaoet al., (2015) demonstrated that serum Glypican-3 was evaluated in thechronic hepatitis patients, cirrhosis, and hepatocellular carcinoma 20 .
By comparing between two techniques used in this study,the sensitivity of GPC-3by RT-

table 1 .
The demographic feature of HCC and CLC patients *LC (Liver cirrhosis), N/A (not detected).

table 2 .
The positivity rate and median levels of serum GPC-3, GPC-3mRNA and compare with AFP.View it in a separate window