الفهرس 
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Abstract
Hepatocellular carcinoma is a common malignancy worldwide and causing one million deaths per year. The incidence of hepatocellular carcinoma is increasing in many countries. The rate of hepatocellular carcinoma is increasing in Egypt where the major risk factors are chronic infections with hepatitis B and C viruses. Hepatitis C virus seems to play a predominant role compared with hepatitis B virus. The overall survival of patients with hepatocellular carcinoma is grim because most patients are diagnosed late, when curative treatment is not possible. Liver biopsy is important in diagnosis of hepatocellular carcinoma but complications of liver biopsy are reported in 0.06% to 0.32% of patients, and typically occur within the first few hours after the biopsy. Complications include hemorrhage, bile peritonitis, penetration of viscera, and pneumothorax. Rarely, death occurs as a direct result of liver biopsy. About one third of patients experience pain at the site of entry, in the right upper quadrant, or in the right shoulder. Tumor markers in the early detection of tumors are promising tools that could improve the control and treatment of tumors. While alpha-fetoprotein is a commonly used tumor marker in the detection of hepatocellular carcinoma, its sensitivity and specificity are insufficient to detect hepatocellular carcinoma in all patient samples. Many research groups are evaluating the sensitivity of available tumor markers and also are investigating the development of novel markers. The interest in p53 arises from the realization that the mutations of this gene are the most frequent and consistent genetic alterations in human cancers. Roughly, about 50% of all cancers have a defective p53 gene. Immunohistochemical demonstration of the p53 tumor protein may be useful in predicting prognosis of several types of tumors. However, the subjective characteristics of the technique are the limiting factor in the interpretation of the results. Because the accumulation of mutant p53 protein can be released into the extra cellular environment, such as serum. The ELISA is a convenient non-invasive technique that can be widely used in different laboratories. In fact, c-Myc is the most commonly overexpressed gene in human cancers. The association of c-Myc with liver carcinogenesis was first identified by the high expression of c-Myc in chronic liver disease and HCC and the frequent c-Myc amplification in liver cancer tissue, which is commonly seen in patients at younger age and with poor prognosis. The aim of the present study was the detection of some tumor markers for hepatocellular carcinoma associated with HCV infection. The material of this study consists of liver tissues and serum samples of 80 HCV infected patients with different pathological disorders. Also, serum samples were collected from twenty (20) healthy volunteers as a negative control group. In the present study, tissues from those HCV infected patients were stained with H&E and classified pathologically into 2 groups: - Chronic hepatitis C patients with HCC (40 patients). - Chronic hepatitis C patients without HCC (40 patients). Firstly we studied the clinical characters of these patients, their age were ranged from 20 to 65 years with mean age of 40.39 ± 8.579. They were 45 males and 35 females. Some of liver function tests including aspartate aminotransferase and alanine aminotransferase, albumin and bilirubin were measured for all serum samples included in this study using standard methodologies. Liver biopsies were processed for diagnostic purposes to detect p53 and c- Myc antigens by using indirect immunoperoxidase technique. The AFP, p53 and c- Myc antigens were detected in all serum samples using (ELISA). The following results were obtained:- -positive cytoplasmic immunostaining for both p53 and c. Myc was detected in HCC specimens. There was no immunohistochemical evidence in normal liver specimen and negative controls. -By using SDS-PAGE serum samples were electrophoresied and stained by the coomassie Brilliant blue R-250 for the identification of targeted antigen. - The immunoblotting (Western blot) analysis for p53, showed a molecular weight of 53 kDa for the single immunoreactive band in the sera of the hepatic patients only. And showed a molecular weight of 62 kDa for the single immunoreactive band in the sera of the hepatic patients for c. Myc antigen. - In this study, α-fetoprotein detected by ELISA in serum samples of 80 hepatic patients. The AFP was detected in HCC patients with detection rate 43%, 57% of HCC patients were negative AFP at cutoff 200 ng/ml. In chronic hepatitis C patients and healthy individuals the value of AFP was below 200 ng/ml. - Detection of p53 antigen by ELISA revealed that the cut-off was found to be (0.4 O.D.). -ELISA was detected p53 antigen in 26 out of 40 HCC patients with detection rate 65%. And detected in 12 Out of 40 CHC patients with detection rate 30 %. There was no samples from healthy individuals positive for p53 antigen. -The levels of p53 antigen (O. D) in patients with HCC (0.447±0.09) was higher than levels of p53 in chronic hepatitis (0.331± 0.10) and healthy individuals (0.181±0.11). An extremely significant difference (P=0.0001) were shown in levels of P53 antigen (O.D) between healthy individuals (0.181±0.11) vs chronic group (0.331± 0.10) and HCC group (0.447±0.09). Interestingly patients with HCC had extremely higher (p=0.0001) levels of P53 (O.D) compared with those with chronic patients. - Over expression of c- Myc oncoprotein was found and detected using ELISA in 29 out of 40 HCC patients, with a detection rate of 72.5%, 15 out of 40 CHC patients with detection rate 37.5%. - The levels of c- Myc antigen (O.D) in patients with HCC (0.384±0.11), was higher than levels of c- Myc in chronic hepatitis (0.290± 0.04) and healthy individuals (0.150±0.03). Overall significance of differences among the HCC patients and both the chronic hepatitis C group and healthy individuals was determined by ANOVA test for c. Myc (O.D) and P value was 0.0001. - Using ROC curve, we assessed and compared the diagnostic accuracy of AFP, c- Myc and p53 as markers for liver cancer in chronic HCV. The area under ROC curve of AFP, c- Myc and p53 for discriminating patients with liver cancer from those chronic hepatitis C patients and (p value) were 0.690 (<.0001); 0.740;(<.0001) and 0.750 ; (<.0001) respectively. - The best linear combinations of blood markers was selected by MDA using the minimum Wilks Lambda test for construction of score equations based on three markers (p53 , AFP and c- Myc). However, discriminant Score = [- 0.455 (numeric constant) + 0.001 × AFP + 1.390 × p53 (O.D) + 0.921 × c- Myc (O.D)] were selected. The areas under the ROC curve for discriminant score was 0.928 (P < 0.0001; extremely significant). Sensitivity, specificity, efficiency, positive predictive and negative predictive values were 94 %, 73 %, 81 %, 67 % and 96 %; respectively; at a discriminant cut-off score = 0.4 (i.e. less than 0.4 indicated patients without HCC and greater than 0.4 indicated liver HCC). From the above results obtained by the current study it could be concluded that:- In conclusion, detection of p53 & c- Myc antigens using ELISA may act as a good diagnostics markers for HCC and might be helpful in differential diagnosis between HCC patients and chronic hepatitis C patients.