الفهرس 
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Abstract
Hepatocellular carcinoma (HCC, also called malignant hepatoma) is one of the most common malignant tumors and carries a poor survival rate. Hepatocellular carcinoma is a disease prevalent in many populations worldwide. It initiates many economic and health problems in management modalities and leads to increasing mortality rates. Worldwide, trials have attempted to discover specific early markers for detection and prediction of the disease, hoping to set more precise strategy for liver cancer prevention.
In Egypt, the incidence of Hepatocellular carcinoma is predicated to increase significantly over the next decades due to the high prevalence of hepatitis C virus (HCV).
This study was designed to evaluate MAGE- transcript, SNP of COX-2 gene, serum GPC-3, and AFP and AFU level in blood as tumor- specific biomarkers for diagnosis of HCC patients.
This study was carried out on 200 individuals (85 females and 115 males) with age ranged from (19 to 69 years). They were selected from Clinical Oncology and Internal Medicine Outpatient clinics, Faculty of Medicine, Zagazig University Hospital. They were divided into the following groups: Group I: consists of 25 healthy individuals (13 males and 12 females) aged from 19-49 years served as control. Group II: consists of 50 HCV infected patients without complications (33 males and 17 females) aged from 22 to 61 years. Group III: consists of 50 HCV infected patients with Cirrhosis (32 males and 18 females) aged from 42-68 years. Group IV: consists of 75 HCV infected Patients complicated
with cirrhosis and HCC (45 localized and 30 metastatic) (37 males and 38 females) aged from 43-69 years.
The following measures were carried out:
Serum AFP and Glypican-3 by ELISA technique, serum AFU by colorimetric method and MAGE-1 m-RNA determination in blood and identification of SNP in COX-2 gene promoter by RT-PCR.
Six ml venous blood was withdrawn from each individual; three ml of blood was collected in heparinized tube for RNA extraction for determination of MAGE-1 m-RNA and for DNA extraction for identification of SNP in COX-2 gene promoter. The other three ml of blood was collected in anticoagulant –free tube, then left for 10 minutes in water bath at 37 ºC until clot, then centrifuged at 2000 rpm for 10 minutes for separation of serum which was transferred into another tube and kept frozen at -20 ºC to detect AFP, AFU and Glypican-3.
The RT-PCR for determination of MAGE-1 m-RNA is based on: RNA extraction, reverse transcription of the target RNA to generate c-DNA, PCR amplification of the target cDNA using MAGE-1 specific primers and detection of the amplified product by running in 2.3 % agarose gel.
The RT-PCR for determination of SNP of Cyclooxygenase-2 gene is based on: DNA extraction, PCR amplification of the target cDNA using Cox-2 specific primers and detection of the amplified product by running in 2.0 % agarose gel.
The quantitative determination of the cancer antigen AFP concentration in the human serum was determined using AFP immunoassay test. The AFP ELISA test is based on the principle of a solid phase enzyme-linked immunoassay.
The quantitative determination of the cancer antigen GPC-3 concentration in the human serum was determined according to KAMIYA BIOMEDICAL COMPANY - U.S.A. using GPC-3 immunoassay test Cat. No. KT-50507, Human Glypican 3 (GPC-3) ELISA.
The quantitative determination of the tumor marker AFU concentration in the human serum is based on the enzymatic cleavage of the synthetic substrate P-nitro phenyl α- L- Fucopyranoside to P-nitro phenol and L-fucose. The yellow color of P-nitro phenol in alkaline medium can be measured quantitatively at 405 nm.
Determination of MAGE-1 m-RNA by RT-PCR assay in our study revealed that: In group I (control group), MAGE-1 m-RNA could not be detected in PBMCs from the 25 healthy donors. Also, similar results were obtained in group II (HCV without cirrhosis) and group III (HCV complicated with cirrhosis). In group IV (a) (localized HCC), the positive rate of MAGE-1 transcript in PBMCs was 22.2% (10 out of 45), whereas in group IV (b) (metastatic HCC), MAGE-1 was detected in 70% (21 out of 30). The positive results of MAGE-1 in all HCC patients were 46.1%.
By using Fisher’s exact test, the frequency of the MAGE-1 transcript detected in PBMCs was significantly higher in metastatic HCC group than localized HCC one (P=0.021). Also, high significant frequency of MAGE-1 m-RNA was detected in group IV (b) than group I, II, III (P<0.05). No significant difference of MAGE-1 expression was detected between group IV(a) and group I,II,III (P>0.05).The distribution of MAGE-1 m-RNA expression among healthy, hepatic, cirrhotic and HCC patients indicates that MAGE-1 m-RNA is tumor-specific marker, and could be detected only in samples of HCC patients.
The results of SNP of Cox-2 showed no significant differences in the characteristics of subjects among the three groups tested for the -1195 G/A polymorphism, the GG, GA, AA genotype frequencies were 10 (13%), 49 (66%), 16 (21%) in HCC patients (group IV), respectively 23 ( 46 %), 17 (34%), 10(20%) in group III, respectively 25 (50%), 12 (24%), 13 (26%) in group II, respectively; and 13 (52%), 5 (20%), 7 (28%) in healthy control group I, respectively (p < 0.000). The -1195G allelic frequencies in four groups were 69 (46%), 63(63%), 62(62%) and 31(62%), respectively; and -1195A allelic frequencies were 81(54%), 37(37%), 38(38%) and 19(38%), respectively (Table 3). -1195A alleles carriers had a higher risk for HCC group compared with cirrhosis and hepatitis C groups.
The results of GPC-3 revealed that the mean level of GPC-3 in patients with HCC (1529.5 ± 1695.6 ng/ml) was significantly higher than that in Cirrhotic (15.5 ± 7.8 ng/ml), hepatic patients without complications (1.5 ± 0.43 ng/ml) and control group (1.0 ± 0.37 ng/ml) ( P< 0.001). A significant positive increase of serum GPC-3 was detected in cirrhotic group compared with control and hepatic patients without complications group (P< 0.05), while there is no significant difference between control and hepatic patients without complications group (P< 0.05).
The results of AFP showed highly significant difference of the mean values of serum AFP among all studied groups (P< 0.001). The mean level of AFP in patients with HCC (511.7 ± 1195.1 ng/ml) was significantly higher than that in Cirrhotic (22.4 ± 24.3 ng/ml), hepatic patients without complications (14.2 ± 8.6 ng/ml) and control group (3.9 ± 2.0 ng/ml) ( P< 0.001). A significant positive increase of serum AFP was detected in cirrhotic group compared with control and hepatic patients without complications group (P< 0.05), while there is no significant difference
between control and hepatic patients without complications group (P< 0.05).
Results of AFU showed a highly significant difference of the mean values of serum AFU among all studied groups (P< 0.001). The mean level of AFU enzyme in patients with HCC (35.099 ± 19.07 U/L; ranged from 8.11 to 71.34 U/L) was significantly higher than that in Cirrhotic (16.54 ± 7.83 U/L; ranged from ranged from 5.32 to 32.54 U/L), hepatic patients without complications (4.04 ± 0.978 U/L; ranged from 2.57 to 6.48 U/L) and control group (4.91 ± 1.41 U/L; ranged from 2.54 to 7.21 U/L) ( P< 0.001).
Results of GPC-3, AFP and AFU showed that GPC-3, AFP and AFU were higher than normal value in (75 out of 75), (65 out of 75) and (68 out of 75) serum samples of HCC patients with sensitivity 100%, 86.7 % and 90.7 % respectively. (0 out of 75), (10 out of 75) and (7 out of 75) samples of HCC patients showed GPC-3, AFP and AFU within normal range. (99 out of 125), (115 out of 125) and (107 out of 125) samples of non malignant individuals (control, hepatic without complications and cirrhotic groups) were within normal range of GPC-3, AFP and AFU with specificity 79.2 %, 92 % and 85.6 % respectively, while were higher than normal value in (26 out of 125), (10 out of 125) and (18 out of 125) respectively.
Results of MGE-3 transcript showed that MAGE-1 m-RNA could not be detected in 25 healthy control group, also, similar results were obtained in hepatic patients without complications and cirrhotic group with specificity 100 %, while in HCC group the positive rate of MAGE-1 transcript was 41.3 % (31 out of 75) and negative rate was 58.7 % (44 out of 75) with sensitivity 41.3 % and specificity 100 %.
Also, our results revealed that combined measurements of serum GPC-3, AFP and AFU in Patients with HCC showed positive GPC-3, AFU and AFP in 70/75 patients and negative results in 5/ 75 patients.
Our results showed that MAGE-1 transcript was negatively correlated to age, GPC-3, AFP and AFU (r=- 328, P<0.000), (r=- 0.580, P< 0.000), (r=- 0.372, P< 0.000) and (r=- 0.316, P< 0.000).
Also, AFP was positively correlated to age, AFU and GPC-3 (r= 0.141, P< 0.046), (r= 0.186, P< 0.008) and (r= 0.509, P< 0.000), negatively correlated toMAGE-1 (r=- 0.372, P< 0.000).
Our results showed that there was a positive correlation between AFP and AFU level (r= 0.186, P< 0.008) this will increase the diagnostic positive rate of the patients with HCC.
Also, AFU was positively correlated to age, AFP and GPC-3 (r= 0.538, P< 0.000), (r= 0.186, P< 0.008) and (r= 0.383, P< 0.000), negatively correlated to MAGE-1 (r=- 0.316, P< 0.000).
Also, GPC-3 was positively correlated to age, AFP and AFU (r= 305, P<0.000), (r= 0.509, P< 0.000) and (r= 0.383, P< 0.000), negatively correlated to MAGE-1 (r=- 0.580, P< 0.000).
Conclusion:
MAGE-1 is appropriate tumor-specific marker; there was no expression of MAGE transcripts detected in the surrounding non-cancerous tissues, nor in the livers of cirrhosis with sensitivity 41.3 % and specificity 100 %. Because it showed low sensitivity so that combination with other markers can be help in early diagnosis of HCC.
Expression of Cox-2 in well-differentiated HCC is generally higher than less-differentiated HCC or histologically normal liver, suggesting that
Cox-2 may be involved in hepatocarcinogenesis. Expression of Cox-2 has been demonstrated in liver generation after partial hepatoectomy. SNPs are one of the most common forms of human genetic variation. SNPs in the promoter region of genes may affect either the expression or the activity of enzymes and therefore may be mechanistically associated with cancer risk.
GPC-3 can be used as a potential biomarker for the diagnosis of early HCC and can be used in screening of HCC as they found that the serum GPC-3 level was higher than 300 ng/ml in 50 % of early HCC.
Moreover serum AFP is a widely used marker for HCC. However, serum AFP levels are increased in patients with liver diseases other than HCC; it represents a liver cell-specific, not a tumor-specific marker.
Also, AFU was found to be increased in HCC than other groups and can be used as tumor marker in the diagnosis of HCC.
Addition of GPC-3and AFU to AFP gives a significant improvement in detection of HCC in patients with cirrhosis. Therefore, combination of multiple markers may be more valuable in the diagnosis of HCC.