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Value of serum anti-p53 antibodies as a prognostic factor in Egyptian patients with hepatocellular carcinoma
Available online at www.sciencedirect.com
Clinical Biochemistry 41 (2008) 1131 – 1139
Value of serum anti-p53 antibodies as a prognostic factor in Egyptian patients with hepatocellular carcinoma Mohamed M. Atta a , Samir A. El-Masry b,⁎, Mohamed Abdel-Hameed a , Hosam A. Baiomy a , Naglaa E. Ramadan a b
a Department of Hepatology, Gastroenterology and Infectious Diseases, Faculty of Medicine, Benha University, Egypt Department of Molecular Biology, Genetic Engineering and Biotechnology Research Institute, Minufiya University, Sadat City, P.O 79, Egypt
Received 30 December 2007; received in revised form 15 May 2008; accepted 4 June 2008 Available online 20 June 2008
Abstract Objective: p53 antigen is an oncoprotective antigen and when damaged, leads to production of anti-p53 and also predisposes to various cancers, including hepatocellular carcinoma (HCC). Serum anti-p53 has been proven to have a prognostic value in patients with HCC. The objective of this study was to determine the prevalence and prognostic utility of serum anti-p53 in Egyptian patients with HCC. Methods: Forty one patients with HCC, 26 patients with liver cirrhosis and 29 healthy controls were included in this study. For all the studied groups, we studied the clinical data, abdominal ultrasound (US) findings, biochemical liver function tests, serum alpha-fetoprotein (AFP) levels detected by enzyme immunoassay (EIA) kit and anti-p53 antibody levels by a modified enzyme-linked immunosorbent assay (ELISA). The severity of liver disease was assessed by Child–Pugh and MELD scores. Tumor characteristics were detected by (US) with or without computed tomography (CT) scan. These characteristics included tumor size, number and site. Tumor staging was done using Okuda, Cancer Liver Italian Program (CLIP) and Tokyo staging systems. Also, the overall survival of patients with HCC with reference to p53 antibody level was studied. Results: The mean age of HCC patients was 57.95 ± 8.41. There was a male predominance among HCC patients with male-to-female ratio of 3.6:1. Anti-p53 antibodies were detected in the sera of 68.3% of HCC patients, 50% of liver cirrhosis patients and 17.2% of healthy control subjects. The data showed that HCC patients had a significantly higher mean anti-p53 antibody values (p = 0.0001), than both liver cirrhosis patients and healthy control groups. Our results revealed that anti-p53 has a positive significant correlation with AFP (p = 0.002), severity of liver disease [Child Pugh score (p = 0.02) and MELD score (p = 0.0003)], tumor size (p b 0.0001), tumor number (p = 0.003) and tumor staging systems [Okuda (p = 0.04), CLIP (p = 0.006) and Tokyo (p b 0.0001)]. Also, our results revealed that serum anti-p53 antibodies had a significant association with overall survival of patients with HCC (p = 0.019) with a shorter survival time in anti-p53 positive status patients and with higher anti-p53 antibody levels within 19 months follow up. Conclusion: The detection of anti-p53 antibodies may be suitable for assessing the prognosis of HCC patients. The higher percentage of positivity of anti-p53 antibodies in Egyptian control subjects than reported elsewhere needs further thorough investigation. © 2008 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. Keywords: ELISA; Anti-p53 antibodies; Hepatocellular carcinoma
Introduction Hepatocellular carcinoma (HCC) is the fifth commonest malignant tumor all over the world [1–4] representing more than 5% of all cancers [5], and the third most common cause of
⁎ Corresponding author. Molecular Biology Department, Genetic Engineering and Biotechnology Research Institute, Minufiya University, Sadat City, Egypt, P.O. 79. Tel.: +20 50 6392880. E-mail address: [email protected] (S.A. El-Masry).
cancer-related deaths [1,6]. In Egypt, HCC was considered as a common malignant tumor [7], accounting for about 4.7% of chronic liver disease patients [4]. The prognosis of HCC is generally grave [3], approximately 75% of patients with HCC present with advanced, unresectable disease and some element of hepatic dysfunction [8] and in Egypt most patients presented at a late stage in approximately 85% of cases [7]. HCC can only be cured if diagnosed at an early stage [9], so there is a pressing need either to prevent the tumor or to diagnose it at a pre-symptomatic stage, when surgical interventions are still possible [10].
0009-9120/$ - see front matter © 2008 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2008.06.006
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The p53 gene is a tumor suppressor gene that is located on the short arm of chromosome 17. It encodes a 53-kiloDalton nuclear phosphoprotein (p53 protein) that functions as an inhibitor of cellular proliferation and transformation [11]. Mutations in the p53 gene are the most common genetic changes in human cancers and there is evidence that supports a high level of p53 alteration in HCC [12]. Abnormalities of the p53 gene can lead to the production of anti-p53 antibody in serum of cancer patients, serum anti-p53 antibody could be a useful prognostic factor in patients with HCC [13]. The analysis of anti-p53 antibodies by ELISA may be a useful non-invasive laboratory procedure for detection of humoral response to the p53 alterations [14]. In this study, we aimed to evaluate the prognostic value of serum anti-p53 antibodies in patients with HCC. Patients and methods Patients This study was approved by the Ethics and Research Committee of the Benha faculty of Medicine, Benha University, Egypt. Serum samples were obtained from sixty seven patients with chronic liver disease, divided into two groups: Group (I) included forty one patients with HCC, admitted to Benha University Hospital between April 2005 to December 2006. Patients with cancers other than HCC or metastatic liver cancer were excluded. Group (II) included twenty six patients with liver cirrhosis and without any evidence of HCC. Twenty nine healthy adults were recruited as controls (Group III). All patients included in this study had the procedure thoroughly explained to them. HCC was diagnosed by abdominal US and serum AFP, with or without triphasic CT scan and/or liver histopathology. AFP was assayed by an enzyme immunoassay (EIA) Kit (Roche Mannheim, Germany). The clinical/pathological data of the patients were recorded, including age, sex, viral infections {Hepatitis C Virus (HCV) and Hepatitis B Virus (HBV)}, alcohol intakes, biochemical liver function test results, and AFP levels. Severity of liver disease was assessed by Child– Pugh and MELD scores. Tumor characteristics were detected by Abdominal US with or without CT scan (including tumor size, number, site, halo sign and neovascularization). Tumor staging was done using {Okuda [15], CLIP [16] and Tokyo [17] staging systems}. Type of therapy, and patient survival time (defined as the period from initial presentation until death) were also recorded. Ten milliliters of venous blood were collected from all subjects and serum was separated and stored at −70 °C, until use. Detection of anti-p53 antibodies using Enzyme-Linked Immunosorbent Assay (ELISA) ELISA procedure was established in our laboratories, to estimate anti-p53 antibodies levels in the sera of HCC patients and control groups, as the method described previously by Engvall and Perlmann [18] with some modifications. The procedure in brief is as follows: polystyrene microtiter plates (Nunc Maxisorp, flat bottom) were coated with 100 μL of recombinant wild-type human p53 protein (Sigma Chemical
Company, USA) in the concentration of 5 μg/mL in Carbonate buffer, 0.06 M, pH 9.6. The coated plate was incubated overnight at room temperature under humidified atmosphere. The plates were washed four time with phosphate buffered saline (PBS) containing 0.1% Tween 20 (PBS-T). The nonspecific sites in the wells were blocked with 0.2% non-fat milk for 2 h at 37 °C. After 4 washes with PBS-T, the plates were incubated with 1:1000 dilution of the sera from patients and control groups. After 2 h incubation at 37 °C and washing, antihuman IgG whole molecule alkaline phosphatase conjugate (Sigma Chemical company, USA), at dilution 1:500 in PBS-T containing 0.2% non-fat milk, was added as the secondary antibody. At the end of 2 h incubation at 37 °C and washing, the color was developed by the addition of 100 μL of the substrate, para-nitrophenyl phosphate (Sigma Chemical Company, USA) to each well. After arresting the reaction with 50 μL of 3 N HCl, the optical density reading was taken in the microplate spectrophotometer (EL311 microplate autoreader, Bio-Tek instruments, USA) at 405 nm wave length. Cutoff level of ELISA above or below which the tested samples were considered positive or negative was calculated as the mean concentrations of 29 serum samples from healthy individuals + 3 standard deviations (SD). Statistical analysis Statistical package (SPSS, version 10.0) was used for data management. Descriptive statistics was presented as mean ± standard deviations for continuous variables, number and percentage for categorical variables (frequency distribution). Unpaired Student t-test (two sided) was used to test the significance of difference between the mean value of studied groups and chi-square test was used for comparison of categorical variables. Pearson correlation test was used to identify the correlation between anti-p53 and different clinicopathological variables. Univariate analysis was used to identify the defendant variables for anti-p53. CoX regression analysis was performed to identify the influence of anti-p53 on overall survival. The significance level was set at p b 0.05. Results The demographic features and characteristics of the two patients' groups were summarized in Table 1. A total of 96 adults, which comprised 41 patients with HCC, 26 patients with liver cirrhosis and 29 apparently-normal control subjects were studied. The mean age of HCC patients was 57.9 ± 8.4 years with a range between 42 and 74 years. In liver cirrhosis patients the mean age was 50.7 ± 8.9 years with a range between 35 and 65 years, while that of the healthy controls the mean age was 38.5 ± 12.8 years with a range between 21 and 52 years. There was a significant difference in the mean ages of HCC patient group, liver cirrhosis (p = 0.003) and healthy controls (p = 0.001). There was male predominance among the patients with HCC, 32 men (78.0%) versus 9 women (22.0%), with a male-to-female ratio of 3.6:1. But, in the liver cirrhosis patients there was 14 (53.8%) males versus 12 (46.2) females, with a
M.M. Atta et al. / Clinical Biochemistry 41 (2008) 1131–1139 Table 1 Demographic features and characteristics of the studied patient groups Characteristics
HCC patients (n = 41)
Liver cirrhosis (n = 26)
p value
Age (years) Range Mean ± SD
42–74 y 57.9 ± 8.4
35–65 y 50.7 ± 8.9
0.003
Sex Male Female
32 (78.0%) 9 (22.0%)
14 (53.8%) 12 (46.2%)
0.036
Residence Urban Rural
14 (34.1%) 27 (65.9%)
1 (3.8%) 25 (96.2%)
0.003
Occupation Farmer Non-farmer
20 (48.8%) 21 (51.2%)
9.0 (34.6%) 17.0 (65.4%)
0.188
Etiology Smoking Alcohol intake HBV HCV
19 (46.3%) 2 (4.9%) 1 (2.4%) 39 (95.1%)
9 (34.6%) 1 (3.8%) 3 (11.5%) 22 (84.6%)
0.245 0.668 0.159 0.152
Severity of liver cirrhosis Child–Pugh score Class A Class B Class C
15 (36.6%) 8 (19.5%) 18 (43.9%)
2 (7.6%) 12 (46.2%) 12 (46.2%)
0.008 0.02 0.856
MELD score Early (6–11) Intermediate (12–18) Advanced (19–40)
8 (19.5%) 15 (36.5% 18 (43.9%)
4 (15.4) 12 (46.1%) 10 (38.5%)
0.66 0.43 0.65
SD: Standard Deviation, HBV: hepatitis B Virus, HCV: hepatitis C Virus, MELD: Model of End stage Liver Diseases.
male-to-female ratio of 1.2:1. Similar values were seen among the healthy controls, with 16 men (55.2%) versus 13 women (44.8%), with a male-to-female ratio of 1.2:1. There was a significant difference in the sex ratios of HCC patients, liver cirrhosis patients (p = 0.036) and healthy controls (p = 0.034). Concerning the residence, either rural or urban, 65.9% of HCC cases had a rural residence in comparison with cirrhotic cases (96.2%) which was statistically significant (p = 0.003). In addition, a positive history for farming was found in 48.8% of HCC cases, 34.6% of cirrhotic cases with no statistical significance (p = 0.188). Regarding HCC etiology, the results showed that the frequency of HBV positivity had no statistically significant difference between the studied patients. It was detected only in 2.4% in HCC cases and in 11.5% in cirrhotic cases. HCV was present in 95.1% of HCC cases and 84.6% of cirrhotic cases with no statistically significant difference between the two groups. The severity of liver cirrhosis assessed by Child–Pugh classification among the studied patients showed that 36.6% of HCC cases were Child A which was significantly higher (p = 0.008) than cirrhotic cases (7.6%), while 19.5% of HCC cases were Child B compared to 46.2% in cirrhotic cases with statistically significant difference (p = 0.02). As regards Child C,
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no statistical significant difference between the two groups. There was no statistically significant difference among the two groups as regards the severity of liver disease assessed by MELD score (Table 1). Tumor imaging characteristics of HCC patients were illustrated in Table 2. Abdominal US showed the dominant occurrence of HCC on top of cirrhosis (100%) and a higher percentage of portal vein thrombosis (PVT) (24.4%) and the higher incidence of the focal lesion to be single (63.4%), large (83%) and affecting the right lobe (60.1%). Neovascularization was present in 95.1% of HCC cases and halo sign in 65.9% of the patients. There was no statistically significant difference between abdominal US and CT scan features of the studied HCC patient group. Regarding Okuda, CLIP, and Tokyo staging systems, most of HCC patients were relatively at advanced stage of the disease. As regards AFP levels, the data revealed that HCC patients had the highest mean value (188 ± 264 ng/mL), with a significantly higher correlation (p = 0.0001), than the mean of AFP values in both liver cirrhosis patients (7.8 ± 7.5 ng/mL) and healthy control (2.6 ± 1.6 ng/mL) groups. In the receiver operating curve (ROC), the area under curve (AUC) for AFP was 92% when we use 24.3 ng/mL as a cutoff point, with a sensitivity of 63% and a specificity of 90%. If the AFP cutoff is raised to 200 ng/mL the sensitivity drops to 34% and the specificity is 100% and if raised to 400 ng/mL the sensitivity Table 2 Tumor-related findings of the HCC patient group Characteristics
HCC patients (n: 41)
Percentage (%)
7/15/19
17.0/36.6/46.3
26/15
63.4/36.6
25/7/9
60.1/17/21.9
25/2/14
60/4.9/34.1
12/13/4/12
29.3/31.7/9.8/29.3
39 27
95.1 65.9
13/18/10
31.7/43.9/24.4
11/23/7
26.8/56.1/17.1
14/27
34.1/65.9
AFP(ng/mL) levels Negative b200 ng/mL 200-400 ng/mL/> 400 ng/mL
27 7/7
65.8 17.1/17.1
Anti-p53 antibodies Positive/Negative
28/13
68.3/31.7
Ultrasonographic features Tumor size (cm) b3 cm/3–5 cm/N 5 cm No. of nodules Single/2 or more Site of tumor Right lobe/Left lobe/Both Shape Rounded /oval /Ill-defined Echogenicity Hypoechoic/Hyperechoic/ Isoechoic/Mixed Neovascularization Halo sign Tumor staging systems Okuda stage I/II/III CLIP stage Early/ Intermediate / Advanced Tokyo stage Early (0 – 3)/Advanced (4 or more)
CLIP: Cancer of the Liver Italian Program, AFP: Alpha-fetoprotein.
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Fig. 3. Anti-p53 antibody positivity in HCC patient group (68.3%), liver cirrhosis patient group (50%) and healthy control group (17.2%) using indirect ELISA and 0.04 OD cutoff point. Fig. 1. Receiver operating curve (ROC) for AFP is represented by grey line with an area under curve (AUC) = 92%, and the ROC for anti-p53 antibodies is represented by the black line with an AUC = 72% among HCC patient group.
drops to 17% and the specificity is 100%. For p53 the cutoff point that gives an AUC of 72% was 0.63 optical densities (OD) with a sensitivity of 63% and a specificity of 91%, at a cutoff level of 0.4 OD, the sensitivity was 68.3% while the specificity is 32% (Fig. 1). There was a significant positive correlation (p = 0.002) between AFP and anti-p53 antibodies among patients with HCC as shown in Fig. 2. The frequency of anti-p53 antibodies, with a cutoff point of 0.4 OD, was higher than that reported worldwide. For patients with HCC, the results showed that anti-p53 antibodies were positive in 28 of 41 HCC patients (68.3%) and in 13 of 26 liver cirrhosis patients (50%) and in 5 of 29 healthy controls subjects (17.2%) as shown in Fig. 3. The data showed that HCC patients had the highest mean anti-p53 antibody values (0.73 ± 0.32), with a significantly higher correlation (p = 0.0001), than the mean of anti-p53 antibody values in both liver cirrhosis patients (0.5 ± 0.2) and healthy control (0.39 ± 0.05) groups.
Regarding the severity of liver disease among HCC patients as assessed by Child–Pugh and MELD scores. The data showed that there was a positive significant correlation between antip53 antibodies levels and Child–Pugh score (P = 0.002) as well as MELD score (p = 0.003) (Fig. 4A, B). But with AFP levels, There was no statistically significant correlation between AFP values and Child score (p = 0.8), MELD (p = 0.1) score among HCC cases. The correlation between anti-p53 and tumor size and number, among HCC patient group, revealed that there was a positive significant correlation with the tumor numbers (p = 0.003) and tumor size (P b 0.0001) as shown in Figs. 5A, B. While there was no correlation with AFP levels in both tumor number (p = 0.21) and size (p = 0.1). Fig. 6 showed the correlation between anti-p53 antibodies levels and different staging systems among HCC patients. The results illustrated that there was a significant correlation with Okuda (p = 0.04) (Fig. 6A), CLIP (p = 0.006) (Fig. 6B) and Tokyo (p bP b 0.0001) (Fig. 6C) staging systems. In regard to the liver functions tests for HCC patient group, we found that there is a
Fig. 2. Correlation between AFP and anti-p53 antibodies showed a highly significant positive correlation (p = 0.0001) among HCC patient group.
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Fig. 4. Correlation between anti-p53 antibody values and Child–Pugh score (A) (p = 0.002) and MELD score (B) (p = 0.003) among HCC group.
positive significant correlation between anti-p53 antibodies and asparatate aminotransferase (AST) (p = 0.04), alkaline phophatase (ALP) (p = 0.04) and total serum bilirubin (p = 0.04). But there is a negative correlation with serum albumin (p = 0.03). In relation to prognosis, we studied the overall survival of patients with HCC with reference to p53 antibodies levels. The results revealed that serum anti-p53 antibodies had a significant association with overall survival of patients with HCC (p = 0.019). There was a shorter survival time (mean, 8.7 months) in the p53 positive status patients and a longer survival time (mean, 12.15 months) in the p53 negative status patients (Fig. 7). In detail, the data showed that, there was a shorter survival time (mean, 7.8 months) in the p53 category (0.93–1.37 ng/mL) group and longer survival time (mean, 9.76 months) in the p53 category (0.48–0.88 ng/mL) group and the longest survival time (mean, 11.78 months) in the p53 category (0.23–0.46 ng/mL) group (Fig. 8).
Discussion Mutations in the p53 gene are the most common genetic changes in human cancers and there is evidence that supports a high level of p53 alteration in HCC [12]. Abnormalities of the p53 gene can lead to the production of anti-p53 antibody in serum of cancer patients. The prevalence of anti-p53 in our study was high in HCC patients (68.3%) and it is in contrast with the results obtained by the other studies. A study of 130 European patients with HCC showed a prevalence of 7% [19]. In Africa, Akere and Otegbayo [20] found a prevalence 12.2% in Nigerians patients with HCC. Similarly, a low prevalence was reported by Soini et al. [21], Sitruk et al. [22], Tangkijvanich et al. [23] and De Benedetti et al. [24] among patients with HCC. Even though the sample sizes varied in these studies, the same ELISA method was used for qualitative analysis of serum anti-p53.
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Fig. 5. Correlations of p53 antibody values and tumor number (p = 0.003) and size (p b 0.0001) among HCC group.
On the other hand, there are some reports that showed a high prevalence of anti-p53 antibodies and they are in agreement with our findings. Ryder et al. [25] at the Institute of Liver Studies, Kings College Hospital, London, found a prevalence of 50% of serum anti-p53 antibodies among HCC patients. Their study concluded that serum anti-p53 could be of value in the diagnosis and characterization of patients with HCC. A high prevalence of serum anti-p53 antibodies were also observed by Parasole et al. [26] 57.8%. The reasons for the differences though not yet fully understood, may be related to some yet unidentified biogeographical differences in the study populations. The findings of anti-p53 antibodies in sera of five healthy control individuals (17.2%) and in sera of 13 patients with liver cirrhosis (50%) were higher than that observed by Dong-Dong and Xi-Ran [27] in their report which showed that p53 was detected in 20% in liver cirrhosis patients and only 7% healthy control individuals. In Africa, Akere and Otegbayo [20] reported that anti-p53 was detected in 8.9% Nigerian healthy controls. Statistically, our study showed a significant correlation between AFP levels and anti-p53 antibodies levels among patients with HCC, liver cirrhosis and control subjects. This may be due to the fact that AFP repression was mediated by wild-type p53 through a binding site during hepatic differentia-
tion and mutant p53 protein was unable to repress AFP-driven gene expression in the process of tumorigenesis [28]. Also, there was a significant positive correlation between p53 and AST, ALP and total bilirubin, while Shiota et al. [13] reported just association but not correlation between anti-p53 antibodies and serum bilirubin only and such correlations were not mentioned by other investigators yet. Concerning the severity of liver disease assessed by Child Pugh and MELD scores, our study revealed that there was a significant correlation between anti-p53 antibodies and both scores. These results disagree with the findings of both Parasole et al. [26] and Shiota et al. [13] who reported that there was no significant correlation between p53 and Child Pugh score. The correlation between anti-p53 antibodies and MELD score was not studied before by any investigator. Also, our results revealed that there was a significant correlation between anti-p53 antibodies and tumor number and size among HCC patients and also, these results disagree with the results of Parasole et al. [26] who reported that there was no correlation between anti-p53 and tumor size and number while Shiota et al. stated that anti-p53 antibodies correlated with the number of the tumor but not with the size. Ryder et al. [25] reported that anti-p53 was detectable in 50% of small tumors. The extent of correlation between anti-p53
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Fig. 6. Correlations of anti-p53 antibodies with the three staging systems. Okuda (A), CLIP(B) and Tokyo (C) staging among HCC group.
antibodies and tumor number and size and its clinical value is still debated. Regarding the staging systems, we found that there was a significant correlation between anti-p53 levels and different staging systems (Okuda, CLIP and Tokyo), but this finding
disagrees with Charuruks et al. [14] who found no correlation between anti-p53 antibodies and Okuda staging, while Parasole et al. [26] reported a significant correlation between anti-p53 antibodies and CLIP score. As for Tokyo staging system, which is a new staging system for HCC proposed by Tatishi et al. [17]
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and its correlation with anti-p53 antibodies was not studied before by any investigator. Our results are supported by Guan et al. [11] who reported that the prevalence of anti-p53 was correlated with the degree of malignancies, suggesting the possibility of its clinical application, especially for detection of high grade or high stage tumors. In the present study, anti-p53 antibodies had a significant association with overall survival of patients with HCC where overall survival was shorter in patients with higher anti-p53 antibodies levels and with anti-p53 positive status respectively. This finding was in agreement with Shiota et al. [13], Charuruks et al. [14] and Parasole et al. [26]. Statistically, the univariate analysis for our data revealed that anti-p53 was significantly a determining factor for HCC positivity, mortality, severity of liver disease (MELD, Child), tumor number, tumor size, tumor morphology, also, for AFP, different staging systems (Okuda, CLIP, Tokyo), but not for age, sex and PVT. These results enhanced the use of anti-p53 as a marker for HCC and this observation is documented by Guan et al. [11] who stated that, for predictive evaluation of HCC, p53 as a factor should be considered together with other significant factor, particularly tumor size, Child–Pugh score and vascular invasion and are consistent with the results of Ryder et al., [25] who concluded that anti-p53 is a convenient and specific test for detection of humoral response to alterations in p53 gene expression and could be of value in the diagnosis and characterization of patients with HCC. Also, Charuruks et al. [14] and Guan et al. [11] documented that detection of serum p53 my represent a suitable non-invasive investigation in assessing the clinical implications and prognosis of patients with HCC. Similarly, Shiota et al. [13] concluded that serum anti-p53 antibodies could be a useful prognostic factor in patients with HCC. We conclude that there was a high prevalence of anti-p53 antibodies among Egyptian patients with HCC which correlated well with both severity and mortality, and these results may enhance the use of anti-p53 as a prognostic tool for patients with
Fig. 7. Overall survival by anti-p53 status among HCC patient group. There was a shorter survival time (mean, 8.7 months) in the anti-p53 positive status group (grey line) and a longer survival time (mean, 12.15 months) in the anti-p53 negative status group (black line).
Fig. 8. Overall survival comparing with anti-p53 value among HCC patient group. There was a shorter survival time (mean, 7.8 months) in the p53 category (0.93–1.37 ng/mL) group (dashed line) and longer survival time (mean, 9.76 months) in the p53 category (0.48–0.88 ng/mL) group (grey line) and the longest survival time (mean, 11.78 months) in the p53 category (0.23–0.46 ng/ mL) group (black line).
HCC. The higher percentage of anti-p53 in Egyptian patient groups as well as in Egyptian control subjects raises the question of the role of environmental carcinogens other than viral hepatitis such as chemicals, aflatoxins or other pollutants which needs further large scale investigation. References [1] Liovet JM, Fuster J, Bruix J. The Barcelona approach: diagnosis, staging and treatment of hepatocellular carcinoma. Liv Transplant 2004;10:115–20. [2] Marrero JA, Fontana RJ, Barrat A, et al. Prognosis of hepatocellular carcinoma of 7 staging system in an American cohort. Hepatology 2005;41:707–16. [3] Lopez JB. Recent development in the first detection of hepatocellular carcinoma. Clin Biochem 2005;26:65–79. [4] El-Zayadi AR, Badran HM, Barakat EM, et al. Hepatocellular carcinoma in Egypt: a single center study over a decade. World J Gastroenterol 2005;11:5193–8. [5] Michielsen PP, Francque SM, Dongan JL. Viral hepatitis and hepatocellular carcinoma. World J Sur Oncol 2005;3:27. [6] Grieco A, Pompili M, Caminiti G, et al. Prognostic factors for survival in patients with early intermediate hepatocellular carcinoma undergoing nonsurgical therapy: comparison of Okuda, CLIP and BCLC staging systems in a single Italian centre. Gut 2005;54:411–8. [7] Abdel-Gafar Y, Sleem H, Tawfik M, et al. Percutaneous ethanol injection in large size and multiple HCC: two years follow-up in 165 patients. Med J Cairo Univ 2002;70 suppl II December. [8] Vauthy BJ, Lauwers GY, Esnaola NF, et al. Simplified staging for hepatocellular carcinoma. J Clin Oncol 2002;20:1527–36. [9] Okochi O, Hibi K, Uemura T, et al. Detection of mitochondrial DNA alterations in the serum of heptocellular carcinoma patients. Clin Can Res 2002;8:2875–8. [10] Kew MC. Hepatic tumors and cysts. In: Feldman M, Friedman LS, Sleisenger MH, editors. Gastrointestinal and liver disease: pathophysiology, diagnosis, management. 7th ed. Philadelphia, London, New York, St. Louis, Sydney: Saunders; 2002. p. 1577–2802. [11] Guan YS, He Q, La Z. Roles of p53 in carcinogenesis, diagnosis and treatment of hepatocellular carcinoma. J Can Molec 2006;2:191–7. [12] Harris CC, Hollstein M. Clinical implications of the p53 tumor suppressor gene. N Engl J Med 1993;329:1318–27 Comment in: N Engl J Med 1994; 330: 864–5.
M.M. Atta et al. / Clinical Biochemistry 41 (2008) 1131–1139 [13] Shiota G, Kishimoto Y, Suyama A. Prognostic significance of serum antip53 antibody in patients with hepatocellular carcinoma. J Hepatol 1997;27: 661–8. [14] Charuruks N, Tangkuvanich P, Voravud V. Clinical significance of p53 antigen and anti-p53 antibodies in the sera of hepatocellular carcinoma patients. J Gastroenterol 2001;36:830–6. [15] Okuda K, Ohtsuki T, Obta H, et al. Natural history of hepatocellular carcinoma and prognosis in relation to treatment study of 850 patients. Cancer 1985;56:918–28. [16] The Cancer of the Liver Italian Program (CLIP) investigators. A new prognostic system for hepatocellular carcinoma: a retrospective study of 435 patients: the Cancer of the Liver Italian Program (CLIP) investigators. Hepatology 1998;28:751–5. [17] Tateishi R, Yoshida H, Shiina S, et al. Proposal of a new prognostic model for hepatocellular carcinoma: an analysis of 403 patients. Gut 2005;54: 419–25. [18] Engvall E, Perlmann P. Enzyme Linked Immunosorbent Assay (ELISA): quantitative assay of IgG. Immunochemistry 1971;8:871–8. [19] Saffroy R, Lelong JC, Azoulay D, et al. Clinical significance of circulating anti-p53 antibodies in European patients with hepatocellular carcinoma. Br J Cancer 1999;79:604–10. [20] Akere A, Otegbayo JA. Evaluation of the pattern and prognostic implications of anti-p53 in hepatocellular carcinoma. Singapore Med J 2007;48:41–4.
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[21] Soini Y, Chia SC, Bennett WP, et al. An aflatoxin-associated mutational hotspot at codon 249 in the p53 tumor suppressor gene occurs in hepatocellular carcinoma from Mexico. Carcinogenesis 1996;17:1007–12. [22] Sitruk V, Vaysse J, Chevret S, et al. Prevalence and prognostic value of serum anti-p53 in hepatocellular carcinoma. A study of 159 patients. Gastroenterol Clin Biol 2000;24:1159–63. [23] Tangkijvanich P, Janchai A, Charuruks N, et al. Clinical associations and prognostic significance of serum anti-p53 antibodies in Thai patients with hepatocellular carcinoma. Asian Pac J Allergy Immunol 2000;18:237–43. [24] De Benedetti VM, Welsh JA, Trivers GE, et al. p53 is not mutated in hepatocellular carcinomas from Alaska natives. Can Epidem Biomarkers Prev 1995;4:79–82. [25] Ryder SD, Rizzi PM, Volkmann M, et al. Use of specific ELISA for the detection of antibodies directed against p53 protein in patients with hepatocellular carcinoma. J Clin Pathol 1996;49:295–9. [26] Parasole R, Izzo F Perrone F, et al. Prognostic value of serum biological markers in patients with hepatocellular carcinoma. Clin Can Res 2001;7:3504–9. [27] Dong-Dong LD, Xi-Ran ZX. Plasma 249 ser p53 mutation in patients with hepatocellular carcinoma residing in a high risk area. J Cell Mol Med 2003; 7:89–92. [28] Lee KC, Crowe AJ, Barton MC. P53-mediated repression of alphafetoprotein gene expression by specific DNA binding. Mol and Cell Biol 1999;19:1279–88.
Clinical Biochemistry 41 (2008) 1131 – 1139
Value of serum anti-p53 antibodies as a prognostic factor in Egyptian patients with hepatocellular carcinoma Mohamed M. Atta a , Samir A. El-Masry b,⁎, Mohamed Abdel-Hameed a , Hosam A. Baiomy a , Naglaa E. Ramadan a b
a Department of Hepatology, Gastroenterology and Infectious Diseases, Faculty of Medicine, Benha University, Egypt Department of Molecular Biology, Genetic Engineering and Biotechnology Research Institute, Minufiya University, Sadat City, P.O 79, Egypt
Received 30 December 2007; received in revised form 15 May 2008; accepted 4 June 2008 Available online 20 June 2008
Abstract Objective: p53 antigen is an oncoprotective antigen and when damaged, leads to production of anti-p53 and also predisposes to various cancers, including hepatocellular carcinoma (HCC). Serum anti-p53 has been proven to have a prognostic value in patients with HCC. The objective of this study was to determine the prevalence and prognostic utility of serum anti-p53 in Egyptian patients with HCC. Methods: Forty one patients with HCC, 26 patients with liver cirrhosis and 29 healthy controls were included in this study. For all the studied groups, we studied the clinical data, abdominal ultrasound (US) findings, biochemical liver function tests, serum alpha-fetoprotein (AFP) levels detected by enzyme immunoassay (EIA) kit and anti-p53 antibody levels by a modified enzyme-linked immunosorbent assay (ELISA). The severity of liver disease was assessed by Child–Pugh and MELD scores. Tumor characteristics were detected by (US) with or without computed tomography (CT) scan. These characteristics included tumor size, number and site. Tumor staging was done using Okuda, Cancer Liver Italian Program (CLIP) and Tokyo staging systems. Also, the overall survival of patients with HCC with reference to p53 antibody level was studied. Results: The mean age of HCC patients was 57.95 ± 8.41. There was a male predominance among HCC patients with male-to-female ratio of 3.6:1. Anti-p53 antibodies were detected in the sera of 68.3% of HCC patients, 50% of liver cirrhosis patients and 17.2% of healthy control subjects. The data showed that HCC patients had a significantly higher mean anti-p53 antibody values (p = 0.0001), than both liver cirrhosis patients and healthy control groups. Our results revealed that anti-p53 has a positive significant correlation with AFP (p = 0.002), severity of liver disease [Child Pugh score (p = 0.02) and MELD score (p = 0.0003)], tumor size (p b 0.0001), tumor number (p = 0.003) and tumor staging systems [Okuda (p = 0.04), CLIP (p = 0.006) and Tokyo (p b 0.0001)]. Also, our results revealed that serum anti-p53 antibodies had a significant association with overall survival of patients with HCC (p = 0.019) with a shorter survival time in anti-p53 positive status patients and with higher anti-p53 antibody levels within 19 months follow up. Conclusion: The detection of anti-p53 antibodies may be suitable for assessing the prognosis of HCC patients. The higher percentage of positivity of anti-p53 antibodies in Egyptian control subjects than reported elsewhere needs further thorough investigation. © 2008 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. Keywords: ELISA; Anti-p53 antibodies; Hepatocellular carcinoma
Introduction Hepatocellular carcinoma (HCC) is the fifth commonest malignant tumor all over the world [1–4] representing more than 5% of all cancers [5], and the third most common cause of
⁎ Corresponding author. Molecular Biology Department, Genetic Engineering and Biotechnology Research Institute, Minufiya University, Sadat City, Egypt, P.O. 79. Tel.: +20 50 6392880. E-mail address: [email protected] (S.A. El-Masry).
cancer-related deaths [1,6]. In Egypt, HCC was considered as a common malignant tumor [7], accounting for about 4.7% of chronic liver disease patients [4]. The prognosis of HCC is generally grave [3], approximately 75% of patients with HCC present with advanced, unresectable disease and some element of hepatic dysfunction [8] and in Egypt most patients presented at a late stage in approximately 85% of cases [7]. HCC can only be cured if diagnosed at an early stage [9], so there is a pressing need either to prevent the tumor or to diagnose it at a pre-symptomatic stage, when surgical interventions are still possible [10].
0009-9120/$ - see front matter © 2008 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2008.06.006
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The p53 gene is a tumor suppressor gene that is located on the short arm of chromosome 17. It encodes a 53-kiloDalton nuclear phosphoprotein (p53 protein) that functions as an inhibitor of cellular proliferation and transformation [11]. Mutations in the p53 gene are the most common genetic changes in human cancers and there is evidence that supports a high level of p53 alteration in HCC [12]. Abnormalities of the p53 gene can lead to the production of anti-p53 antibody in serum of cancer patients, serum anti-p53 antibody could be a useful prognostic factor in patients with HCC [13]. The analysis of anti-p53 antibodies by ELISA may be a useful non-invasive laboratory procedure for detection of humoral response to the p53 alterations [14]. In this study, we aimed to evaluate the prognostic value of serum anti-p53 antibodies in patients with HCC. Patients and methods Patients This study was approved by the Ethics and Research Committee of the Benha faculty of Medicine, Benha University, Egypt. Serum samples were obtained from sixty seven patients with chronic liver disease, divided into two groups: Group (I) included forty one patients with HCC, admitted to Benha University Hospital between April 2005 to December 2006. Patients with cancers other than HCC or metastatic liver cancer were excluded. Group (II) included twenty six patients with liver cirrhosis and without any evidence of HCC. Twenty nine healthy adults were recruited as controls (Group III). All patients included in this study had the procedure thoroughly explained to them. HCC was diagnosed by abdominal US and serum AFP, with or without triphasic CT scan and/or liver histopathology. AFP was assayed by an enzyme immunoassay (EIA) Kit (Roche Mannheim, Germany). The clinical/pathological data of the patients were recorded, including age, sex, viral infections {Hepatitis C Virus (HCV) and Hepatitis B Virus (HBV)}, alcohol intakes, biochemical liver function test results, and AFP levels. Severity of liver disease was assessed by Child– Pugh and MELD scores. Tumor characteristics were detected by Abdominal US with or without CT scan (including tumor size, number, site, halo sign and neovascularization). Tumor staging was done using {Okuda [15], CLIP [16] and Tokyo [17] staging systems}. Type of therapy, and patient survival time (defined as the period from initial presentation until death) were also recorded. Ten milliliters of venous blood were collected from all subjects and serum was separated and stored at −70 °C, until use. Detection of anti-p53 antibodies using Enzyme-Linked Immunosorbent Assay (ELISA) ELISA procedure was established in our laboratories, to estimate anti-p53 antibodies levels in the sera of HCC patients and control groups, as the method described previously by Engvall and Perlmann [18] with some modifications. The procedure in brief is as follows: polystyrene microtiter plates (Nunc Maxisorp, flat bottom) were coated with 100 μL of recombinant wild-type human p53 protein (Sigma Chemical
Company, USA) in the concentration of 5 μg/mL in Carbonate buffer, 0.06 M, pH 9.6. The coated plate was incubated overnight at room temperature under humidified atmosphere. The plates were washed four time with phosphate buffered saline (PBS) containing 0.1% Tween 20 (PBS-T). The nonspecific sites in the wells were blocked with 0.2% non-fat milk for 2 h at 37 °C. After 4 washes with PBS-T, the plates were incubated with 1:1000 dilution of the sera from patients and control groups. After 2 h incubation at 37 °C and washing, antihuman IgG whole molecule alkaline phosphatase conjugate (Sigma Chemical company, USA), at dilution 1:500 in PBS-T containing 0.2% non-fat milk, was added as the secondary antibody. At the end of 2 h incubation at 37 °C and washing, the color was developed by the addition of 100 μL of the substrate, para-nitrophenyl phosphate (Sigma Chemical Company, USA) to each well. After arresting the reaction with 50 μL of 3 N HCl, the optical density reading was taken in the microplate spectrophotometer (EL311 microplate autoreader, Bio-Tek instruments, USA) at 405 nm wave length. Cutoff level of ELISA above or below which the tested samples were considered positive or negative was calculated as the mean concentrations of 29 serum samples from healthy individuals + 3 standard deviations (SD). Statistical analysis Statistical package (SPSS, version 10.0) was used for data management. Descriptive statistics was presented as mean ± standard deviations for continuous variables, number and percentage for categorical variables (frequency distribution). Unpaired Student t-test (two sided) was used to test the significance of difference between the mean value of studied groups and chi-square test was used for comparison of categorical variables. Pearson correlation test was used to identify the correlation between anti-p53 and different clinicopathological variables. Univariate analysis was used to identify the defendant variables for anti-p53. CoX regression analysis was performed to identify the influence of anti-p53 on overall survival. The significance level was set at p b 0.05. Results The demographic features and characteristics of the two patients' groups were summarized in Table 1. A total of 96 adults, which comprised 41 patients with HCC, 26 patients with liver cirrhosis and 29 apparently-normal control subjects were studied. The mean age of HCC patients was 57.9 ± 8.4 years with a range between 42 and 74 years. In liver cirrhosis patients the mean age was 50.7 ± 8.9 years with a range between 35 and 65 years, while that of the healthy controls the mean age was 38.5 ± 12.8 years with a range between 21 and 52 years. There was a significant difference in the mean ages of HCC patient group, liver cirrhosis (p = 0.003) and healthy controls (p = 0.001). There was male predominance among the patients with HCC, 32 men (78.0%) versus 9 women (22.0%), with a male-to-female ratio of 3.6:1. But, in the liver cirrhosis patients there was 14 (53.8%) males versus 12 (46.2) females, with a
M.M. Atta et al. / Clinical Biochemistry 41 (2008) 1131–1139 Table 1 Demographic features and characteristics of the studied patient groups Characteristics
HCC patients (n = 41)
Liver cirrhosis (n = 26)
p value
Age (years) Range Mean ± SD
42–74 y 57.9 ± 8.4
35–65 y 50.7 ± 8.9
0.003
Sex Male Female
32 (78.0%) 9 (22.0%)
14 (53.8%) 12 (46.2%)
0.036
Residence Urban Rural
14 (34.1%) 27 (65.9%)
1 (3.8%) 25 (96.2%)
0.003
Occupation Farmer Non-farmer
20 (48.8%) 21 (51.2%)
9.0 (34.6%) 17.0 (65.4%)
0.188
Etiology Smoking Alcohol intake HBV HCV
19 (46.3%) 2 (4.9%) 1 (2.4%) 39 (95.1%)
9 (34.6%) 1 (3.8%) 3 (11.5%) 22 (84.6%)
0.245 0.668 0.159 0.152
Severity of liver cirrhosis Child–Pugh score Class A Class B Class C
15 (36.6%) 8 (19.5%) 18 (43.9%)
2 (7.6%) 12 (46.2%) 12 (46.2%)
0.008 0.02 0.856
MELD score Early (6–11) Intermediate (12–18) Advanced (19–40)
8 (19.5%) 15 (36.5% 18 (43.9%)
4 (15.4) 12 (46.1%) 10 (38.5%)
0.66 0.43 0.65
SD: Standard Deviation, HBV: hepatitis B Virus, HCV: hepatitis C Virus, MELD: Model of End stage Liver Diseases.
male-to-female ratio of 1.2:1. Similar values were seen among the healthy controls, with 16 men (55.2%) versus 13 women (44.8%), with a male-to-female ratio of 1.2:1. There was a significant difference in the sex ratios of HCC patients, liver cirrhosis patients (p = 0.036) and healthy controls (p = 0.034). Concerning the residence, either rural or urban, 65.9% of HCC cases had a rural residence in comparison with cirrhotic cases (96.2%) which was statistically significant (p = 0.003). In addition, a positive history for farming was found in 48.8% of HCC cases, 34.6% of cirrhotic cases with no statistical significance (p = 0.188). Regarding HCC etiology, the results showed that the frequency of HBV positivity had no statistically significant difference between the studied patients. It was detected only in 2.4% in HCC cases and in 11.5% in cirrhotic cases. HCV was present in 95.1% of HCC cases and 84.6% of cirrhotic cases with no statistically significant difference between the two groups. The severity of liver cirrhosis assessed by Child–Pugh classification among the studied patients showed that 36.6% of HCC cases were Child A which was significantly higher (p = 0.008) than cirrhotic cases (7.6%), while 19.5% of HCC cases were Child B compared to 46.2% in cirrhotic cases with statistically significant difference (p = 0.02). As regards Child C,
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no statistical significant difference between the two groups. There was no statistically significant difference among the two groups as regards the severity of liver disease assessed by MELD score (Table 1). Tumor imaging characteristics of HCC patients were illustrated in Table 2. Abdominal US showed the dominant occurrence of HCC on top of cirrhosis (100%) and a higher percentage of portal vein thrombosis (PVT) (24.4%) and the higher incidence of the focal lesion to be single (63.4%), large (83%) and affecting the right lobe (60.1%). Neovascularization was present in 95.1% of HCC cases and halo sign in 65.9% of the patients. There was no statistically significant difference between abdominal US and CT scan features of the studied HCC patient group. Regarding Okuda, CLIP, and Tokyo staging systems, most of HCC patients were relatively at advanced stage of the disease. As regards AFP levels, the data revealed that HCC patients had the highest mean value (188 ± 264 ng/mL), with a significantly higher correlation (p = 0.0001), than the mean of AFP values in both liver cirrhosis patients (7.8 ± 7.5 ng/mL) and healthy control (2.6 ± 1.6 ng/mL) groups. In the receiver operating curve (ROC), the area under curve (AUC) for AFP was 92% when we use 24.3 ng/mL as a cutoff point, with a sensitivity of 63% and a specificity of 90%. If the AFP cutoff is raised to 200 ng/mL the sensitivity drops to 34% and the specificity is 100% and if raised to 400 ng/mL the sensitivity Table 2 Tumor-related findings of the HCC patient group Characteristics
HCC patients (n: 41)
Percentage (%)
7/15/19
17.0/36.6/46.3
26/15
63.4/36.6
25/7/9
60.1/17/21.9
25/2/14
60/4.9/34.1
12/13/4/12
29.3/31.7/9.8/29.3
39 27
95.1 65.9
13/18/10
31.7/43.9/24.4
11/23/7
26.8/56.1/17.1
14/27
34.1/65.9
AFP(ng/mL) levels Negative b200 ng/mL 200-400 ng/mL/> 400 ng/mL
27 7/7
65.8 17.1/17.1
Anti-p53 antibodies Positive/Negative
28/13
68.3/31.7
Ultrasonographic features Tumor size (cm) b3 cm/3–5 cm/N 5 cm No. of nodules Single/2 or more Site of tumor Right lobe/Left lobe/Both Shape Rounded /oval /Ill-defined Echogenicity Hypoechoic/Hyperechoic/ Isoechoic/Mixed Neovascularization Halo sign Tumor staging systems Okuda stage I/II/III CLIP stage Early/ Intermediate / Advanced Tokyo stage Early (0 – 3)/Advanced (4 or more)
CLIP: Cancer of the Liver Italian Program, AFP: Alpha-fetoprotein.
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Fig. 3. Anti-p53 antibody positivity in HCC patient group (68.3%), liver cirrhosis patient group (50%) and healthy control group (17.2%) using indirect ELISA and 0.04 OD cutoff point. Fig. 1. Receiver operating curve (ROC) for AFP is represented by grey line with an area under curve (AUC) = 92%, and the ROC for anti-p53 antibodies is represented by the black line with an AUC = 72% among HCC patient group.
drops to 17% and the specificity is 100%. For p53 the cutoff point that gives an AUC of 72% was 0.63 optical densities (OD) with a sensitivity of 63% and a specificity of 91%, at a cutoff level of 0.4 OD, the sensitivity was 68.3% while the specificity is 32% (Fig. 1). There was a significant positive correlation (p = 0.002) between AFP and anti-p53 antibodies among patients with HCC as shown in Fig. 2. The frequency of anti-p53 antibodies, with a cutoff point of 0.4 OD, was higher than that reported worldwide. For patients with HCC, the results showed that anti-p53 antibodies were positive in 28 of 41 HCC patients (68.3%) and in 13 of 26 liver cirrhosis patients (50%) and in 5 of 29 healthy controls subjects (17.2%) as shown in Fig. 3. The data showed that HCC patients had the highest mean anti-p53 antibody values (0.73 ± 0.32), with a significantly higher correlation (p = 0.0001), than the mean of anti-p53 antibody values in both liver cirrhosis patients (0.5 ± 0.2) and healthy control (0.39 ± 0.05) groups.
Regarding the severity of liver disease among HCC patients as assessed by Child–Pugh and MELD scores. The data showed that there was a positive significant correlation between antip53 antibodies levels and Child–Pugh score (P = 0.002) as well as MELD score (p = 0.003) (Fig. 4A, B). But with AFP levels, There was no statistically significant correlation between AFP values and Child score (p = 0.8), MELD (p = 0.1) score among HCC cases. The correlation between anti-p53 and tumor size and number, among HCC patient group, revealed that there was a positive significant correlation with the tumor numbers (p = 0.003) and tumor size (P b 0.0001) as shown in Figs. 5A, B. While there was no correlation with AFP levels in both tumor number (p = 0.21) and size (p = 0.1). Fig. 6 showed the correlation between anti-p53 antibodies levels and different staging systems among HCC patients. The results illustrated that there was a significant correlation with Okuda (p = 0.04) (Fig. 6A), CLIP (p = 0.006) (Fig. 6B) and Tokyo (p bP b 0.0001) (Fig. 6C) staging systems. In regard to the liver functions tests for HCC patient group, we found that there is a
Fig. 2. Correlation between AFP and anti-p53 antibodies showed a highly significant positive correlation (p = 0.0001) among HCC patient group.
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Fig. 4. Correlation between anti-p53 antibody values and Child–Pugh score (A) (p = 0.002) and MELD score (B) (p = 0.003) among HCC group.
positive significant correlation between anti-p53 antibodies and asparatate aminotransferase (AST) (p = 0.04), alkaline phophatase (ALP) (p = 0.04) and total serum bilirubin (p = 0.04). But there is a negative correlation with serum albumin (p = 0.03). In relation to prognosis, we studied the overall survival of patients with HCC with reference to p53 antibodies levels. The results revealed that serum anti-p53 antibodies had a significant association with overall survival of patients with HCC (p = 0.019). There was a shorter survival time (mean, 8.7 months) in the p53 positive status patients and a longer survival time (mean, 12.15 months) in the p53 negative status patients (Fig. 7). In detail, the data showed that, there was a shorter survival time (mean, 7.8 months) in the p53 category (0.93–1.37 ng/mL) group and longer survival time (mean, 9.76 months) in the p53 category (0.48–0.88 ng/mL) group and the longest survival time (mean, 11.78 months) in the p53 category (0.23–0.46 ng/mL) group (Fig. 8).
Discussion Mutations in the p53 gene are the most common genetic changes in human cancers and there is evidence that supports a high level of p53 alteration in HCC [12]. Abnormalities of the p53 gene can lead to the production of anti-p53 antibody in serum of cancer patients. The prevalence of anti-p53 in our study was high in HCC patients (68.3%) and it is in contrast with the results obtained by the other studies. A study of 130 European patients with HCC showed a prevalence of 7% [19]. In Africa, Akere and Otegbayo [20] found a prevalence 12.2% in Nigerians patients with HCC. Similarly, a low prevalence was reported by Soini et al. [21], Sitruk et al. [22], Tangkijvanich et al. [23] and De Benedetti et al. [24] among patients with HCC. Even though the sample sizes varied in these studies, the same ELISA method was used for qualitative analysis of serum anti-p53.
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Fig. 5. Correlations of p53 antibody values and tumor number (p = 0.003) and size (p b 0.0001) among HCC group.
On the other hand, there are some reports that showed a high prevalence of anti-p53 antibodies and they are in agreement with our findings. Ryder et al. [25] at the Institute of Liver Studies, Kings College Hospital, London, found a prevalence of 50% of serum anti-p53 antibodies among HCC patients. Their study concluded that serum anti-p53 could be of value in the diagnosis and characterization of patients with HCC. A high prevalence of serum anti-p53 antibodies were also observed by Parasole et al. [26] 57.8%. The reasons for the differences though not yet fully understood, may be related to some yet unidentified biogeographical differences in the study populations. The findings of anti-p53 antibodies in sera of five healthy control individuals (17.2%) and in sera of 13 patients with liver cirrhosis (50%) were higher than that observed by Dong-Dong and Xi-Ran [27] in their report which showed that p53 was detected in 20% in liver cirrhosis patients and only 7% healthy control individuals. In Africa, Akere and Otegbayo [20] reported that anti-p53 was detected in 8.9% Nigerian healthy controls. Statistically, our study showed a significant correlation between AFP levels and anti-p53 antibodies levels among patients with HCC, liver cirrhosis and control subjects. This may be due to the fact that AFP repression was mediated by wild-type p53 through a binding site during hepatic differentia-
tion and mutant p53 protein was unable to repress AFP-driven gene expression in the process of tumorigenesis [28]. Also, there was a significant positive correlation between p53 and AST, ALP and total bilirubin, while Shiota et al. [13] reported just association but not correlation between anti-p53 antibodies and serum bilirubin only and such correlations were not mentioned by other investigators yet. Concerning the severity of liver disease assessed by Child Pugh and MELD scores, our study revealed that there was a significant correlation between anti-p53 antibodies and both scores. These results disagree with the findings of both Parasole et al. [26] and Shiota et al. [13] who reported that there was no significant correlation between p53 and Child Pugh score. The correlation between anti-p53 antibodies and MELD score was not studied before by any investigator. Also, our results revealed that there was a significant correlation between anti-p53 antibodies and tumor number and size among HCC patients and also, these results disagree with the results of Parasole et al. [26] who reported that there was no correlation between anti-p53 and tumor size and number while Shiota et al. stated that anti-p53 antibodies correlated with the number of the tumor but not with the size. Ryder et al. [25] reported that anti-p53 was detectable in 50% of small tumors. The extent of correlation between anti-p53
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Fig. 6. Correlations of anti-p53 antibodies with the three staging systems. Okuda (A), CLIP(B) and Tokyo (C) staging among HCC group.
antibodies and tumor number and size and its clinical value is still debated. Regarding the staging systems, we found that there was a significant correlation between anti-p53 levels and different staging systems (Okuda, CLIP and Tokyo), but this finding
disagrees with Charuruks et al. [14] who found no correlation between anti-p53 antibodies and Okuda staging, while Parasole et al. [26] reported a significant correlation between anti-p53 antibodies and CLIP score. As for Tokyo staging system, which is a new staging system for HCC proposed by Tatishi et al. [17]
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and its correlation with anti-p53 antibodies was not studied before by any investigator. Our results are supported by Guan et al. [11] who reported that the prevalence of anti-p53 was correlated with the degree of malignancies, suggesting the possibility of its clinical application, especially for detection of high grade or high stage tumors. In the present study, anti-p53 antibodies had a significant association with overall survival of patients with HCC where overall survival was shorter in patients with higher anti-p53 antibodies levels and with anti-p53 positive status respectively. This finding was in agreement with Shiota et al. [13], Charuruks et al. [14] and Parasole et al. [26]. Statistically, the univariate analysis for our data revealed that anti-p53 was significantly a determining factor for HCC positivity, mortality, severity of liver disease (MELD, Child), tumor number, tumor size, tumor morphology, also, for AFP, different staging systems (Okuda, CLIP, Tokyo), but not for age, sex and PVT. These results enhanced the use of anti-p53 as a marker for HCC and this observation is documented by Guan et al. [11] who stated that, for predictive evaluation of HCC, p53 as a factor should be considered together with other significant factor, particularly tumor size, Child–Pugh score and vascular invasion and are consistent with the results of Ryder et al., [25] who concluded that anti-p53 is a convenient and specific test for detection of humoral response to alterations in p53 gene expression and could be of value in the diagnosis and characterization of patients with HCC. Also, Charuruks et al. [14] and Guan et al. [11] documented that detection of serum p53 my represent a suitable non-invasive investigation in assessing the clinical implications and prognosis of patients with HCC. Similarly, Shiota et al. [13] concluded that serum anti-p53 antibodies could be a useful prognostic factor in patients with HCC. We conclude that there was a high prevalence of anti-p53 antibodies among Egyptian patients with HCC which correlated well with both severity and mortality, and these results may enhance the use of anti-p53 as a prognostic tool for patients with
Fig. 7. Overall survival by anti-p53 status among HCC patient group. There was a shorter survival time (mean, 8.7 months) in the anti-p53 positive status group (grey line) and a longer survival time (mean, 12.15 months) in the anti-p53 negative status group (black line).
Fig. 8. Overall survival comparing with anti-p53 value among HCC patient group. There was a shorter survival time (mean, 7.8 months) in the p53 category (0.93–1.37 ng/mL) group (dashed line) and longer survival time (mean, 9.76 months) in the p53 category (0.48–0.88 ng/mL) group (grey line) and the longest survival time (mean, 11.78 months) in the p53 category (0.23–0.46 ng/ mL) group (black line).
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