Immunohistochemical study on p53, H-rasp21, c-erbB-2 protein and PCNA expression in HCC tissues of Han and minority ethnic patients

Abstract

AIM: To find out the difference of human primary liver carcinogenesis between Han and minority ethnic patients in Xinjiang.

METHODS: Expression of p53, c-erbB-2, H-rasp21 protein and proliferating cell nuclear antigen (PCNA) in tumor tissues of 50 patients (Han 38, minority 12) with primary hepatic carcinoma was detected by immunohistochemistry (LSAB).

RESULTS: The positive frequency of p53, c-erbB-2, H-rasp21 and PCNA expression was 46.0% (23/50), 70.0% (35/50), 68.0% (34/50) and 8 2.0% (41/50) in tumor tissues; 4.0% (2/50), 22.0% (11/50), 64.0% (32/50) and 52.0% (26/50) in peritumors respectively and a significant difference, except for H-rasp21, of oncogene alteration was found (P ＜ 0.05) between tumor and non-tumorous tissues. Combined the three oncogenes alteration, 26% (13/50) tumor tissues had positive immunoreactivity, but in peritumor and normal livers it was negative. The positive rate of p53, c-erbB-2 and H-rasp21 protein expression was 39.5% (15/38), 60.5% (23/38) and 39.5% (15/38) in tumors of Han patients; 66.7% (8/12), 100% (12/12) and 75.0% (9/12) in minorities respectively, with statistical difference (P ＜ 0.05).

CONCLUSION: Overexpression of p53, c-erbB-2 and H-rasp21 in human primary liver carcinoma is an important biomarker of genetic alteration. The different frequency of these oncogenetic changes may reflect some environmental or/and ethnic hereditary factors affecting the liver carcinogenesis. The special life style of Han, Uygur, Kazak and Mongolia nationalities in Xinjiang may also be related to the etiopathogenesis of this disease.

Key Words: liver neoplasms; p53; H-rasp21; c-erbB-2; proliferating cell nuclear antigen; immunohistochemistry

INTRODUCTION

Hepatocellular carcinoma (HCC) is one of the major cancers with highest mortality in the world. There is a striking variety of HCC incidence rates between countries, with a highest to lowest of 112.5 for males and 54.7 for females. The high-risk populations are clustered in Sub-Sahara Africa and eastern Asia[1]. Although many etiological factors, e.g., HBV, aflatoxin B1, heavy alcohol drinking, etc. have been established[2-4], the exact mechanisms of HCC carcinogenesis is still poorly understood. The natural environment, multiple ethnic groups with different hereditary background, and special life style in Xinjiang are quite different from other areas in China, which may implicate the etiopathogenesis of HCC.

A great number of studies have demonstrated that human HCC hepatocarcinogenesis is a multistepwise and multigenetic alteration induced by various pathogens with the involvement of multifactorial etiology. Alterations of cytogenes, including mutation, amplification, allelic loss of oncogenes and tumor suppressor genes, are most common in many forms of human cancers, including HCC. Overexpression of p53 protein, c-erbB-2, c-myc and H-ras oncoprotein and proliferating cell nuclear antigen (PCNA) were observed in the tumor tissues of patients or experimental abnormals with HCC[1-10]. In order to understand the possible mechanism of hepatocarcinogenesis and if there is any difference among various ethnic patients with HCC, we detected the expressions of p53 protein, c-erbB-2 and H-rasp21 oncoprotein, and PCNA in tumor, peritumor and normal liver tissues by immunohistochemistry.

MATERIALS AND METHODS

Patients

A series of 50 patients with HCC, including 38 Han, 12 minorities (Uygur 8, Kazak 2 and Mongolia 2); males 34, femals 16; aged from 18 to 72 (average 50.1) years, was collected from the 1st Teaching Hospital of Xinjiang Medical University from 1991 to 1995.

Specimens

All the tumor and peritumor tissues of 50 cases and 10 specimens of normal liver tissue as a control were fixed in 100 mL/L formalin and embedded in paraffin for routine histopathologic examination, and these paraffin blocks were obtained from the pathologic record. The blocks including 50 tumors, 50 peritumors and 10 non-tumors were cut into five sections of 4 μm in thickness and underwent H.E. and immunohistostaining, respectively.

Immunohistochemical staining

Four-μm thick sections were dewaxed, and rehydrated according to routine procedures. Nonspecific binding was blocked by incubation with bovine albumin for 10 min and endogeneous peroxidase activity was blocked with 30 mL/L hydrogen peroxide for 5 min. The sections were pretreated in a microwave oven at 750 W for 10 min in citrate buffer (pH6.0), and then incubated for 5 min in microwave oven with primary antibodies, including monoclonal anti-p53 protein (Do-7), anti-H-rasp21 oncoprotein, anti-PCNA (P-c 10) (1∶40) and polyclonal anti-c-erbB-2 oncoprotein (1∶200) (Dako), respectively. We used the LSAB kits (Dako) for immunostaining, the secondary antibodies and reagents were treated by the routine procedures in microwave oven. The colour was developed using 3-amino-9-ethyicarbazol (AEC). The sections were counterstained with Meyer′s hematoxylin.

Assessment of staining reaction

Only tumor cells with distinct nuclear immunostaining in both p53 protein and PCNA were considered positive. Tumors were recorded as positive for c-erbB-2, H-rasp21 oncoprotein if more than 5% of tumors showed distinct membrance and cytoplasm staining. The immunoreactivity was registered semiquantitatively. The positive cells were scored as (-) negative; (+) 1-5 positive cells per high magnification (400 fold); (++) 6.20 positive cells; (+++) more than 21 positive cells found in p53 protein and PCNA immunostaining respectively.

Statistics

The immunoreactivity for p53, c-erbB-2, H-rasp21 and PCNA parameters were considered to represent discrete values in various lesions. The Chi square test (χ²) was used and P ＜ 0.05 was accepted as a statistically significant difference.

RESULTS

The immunostaining p53 protein and PCNA appeared red-brown in nucleus and tumor or peritumor cells with various densities were distribuled unevenly (Figure 1, Figure 2). The immunoreactivity of c-erbB-2 and H-rasp21 oncoprotein showed distinct membrance and cytoplasma staining (Figure 3, Figure 4). Table 1 summarizes the different frequencies of immunostaining positive cells for p53 protein, H-rasp21, c-erbB-2 and PCNA in tumor and non-tumor tissues.

Table 1 Expression of p53 protein, c-erbB-2, H-rasp21 and PCNA in various liver lesions.

Histology	n	p53		c-erbB-2		H-ras		PCNA
		n	%	n	%	n	%	n	%
Tumor (A)	50	23	46.0	35	70.0	34	68.0	41	82.0
Peritumor (B)	50	2	4.0a	11	22.0a	32	64.0c	26	52.0a
Cirrhosis	33	2	6.0	9	27.3	23	70.0	18	54.5
Non-cirrhosis	17	0	0	2	11.8	9	53.0	8	47.0
Normal liver (C)	10	0	0	0	0	3	30.0	0	0

^aP ＜ 0.05, vs A, C;

^cP ＜ 0.05, vs C; χ² test.

Figure 1 Strongly positive immunoperoxidase staining of the p53 protein with the anti-p53 mAb Do-7 in HCC, predominantly in nuclei. Absence of staining in bnontumorous liver. LSAB method, × 100

Figure 2 HCC with diffuse PCNA staining using mAb PC10. LSAB method, × 200

Figure 3 Cholangiocellular carcinoma showing extensive strongly membrance and plasma positivity for c-erbB-2 using poly-Ab code N. A485. LSAB method, × 400

Figure 4 Diffuse H-rasp21 using mAb Ncc-Ras-001 staining in HCC and nontumorious tissues. LSAB method, × 400

There was a significant difference among three oncogenes expression respectively except PCNA between Han and minorities (P ＜ 0.05). The oncoprotein expression was lower in Han than in the three minorities. Table 2 summarizes the three oncogenes expression in tumor and non-tumor tissues and distribution of different nationalities.

Table 2 Expression of p53, c-erbB-2, H-rasp21 and PCNA in HCC of different nationalities.

Nationalities	n	p53		c-erbB-2		H-ras		PCNA
		n	%	n	%	n	%	n	%
Han	36	15	39.5	23	60.0	15	39.5	32	84.2
Minority	12	8	66.7a	12	100.0a	9	75.0a	9	75.0
Uygur	8	5	62.5	8	100.0	6	75.0	5	62.5
Kazak	2	1	50.0	2	100.0	2	100.0	2	100.0
Mongolia	2	2	100.0	2	100.0	1	50.0	2	100.0

^aP ＜ 0.05, vs Han, χ² test.

Table 3 shows the combined expression of three oncogenes in HCC of different nationalities. The frequency was higher in minorities (41.7%) than in the Hans (21.1%), without statistical difference (P ＞ 0.05).

Table 3 The nationality distribution of the combined expression of three oncogenes in HCC.

Nationalities	n	p53+c-erbB-2+H-rasp21
		n	%
Han	38	8	21.1
Minority	12	5	41.7

DISCUSSION

Alterations of oncogenes and tumor suppressor genes were critical events in hepatocarcinogenesis induced by HBV/HCV, chemical carcinogens, etc. have been demonstrated by biopathological and experimental researches[7-10]. Cerutti[11] and Tong[12] reported that the mutagenesis of H-ras proto-oncogene and p53 tumor suppressor gene may be the most important events change in hepatocellular carcinomas. Fujimoto et al[13] observed some allelic loss of tumor suppressor genes in Chinese patients with hepatocarcinomas. Scorsone et al[14] demonstrated that p53 gene mutations clustered at codon 249 in HBV positive hepatocarcinoma patients in China. Recently, Ming et al[15] also found that frequency of 249 codon mutation of p53 gene was much higher in high prevalent area of hepatocellular carcinomas than that in the low-risk area in China. More than 95% (20/21) cancer specimens exhibited strong intranuclear accumulation of p53 protein, which can be detected by immunohistology. However, Biersing et al[16] and Vesey et al[17] found little or no point mutations of p53 gene in human hepatocarcinoma in Swedish and Australian patients. Therefore, the overexpression of p53 protein in hepatocarcinoma specimens can be used as the mutant p53 biopathological mark in tumor tissues. Liu et al[18] and Zu et al[19] found that the frequency of intranuclear positive immunoreactivity of p53 protein was significantly different between high-risk and low-risk HCC areas, and HBxAg can enhance p53 protein accumulation, suggesting that HBxAg was capable of binding p53 and formed a protein-protein complex which might reduce or inactivate the antiproliferative activity of p53 and played an important role in the pathogenesis of HBV-associated hepatocarcinomas. In this study, the overexpression of p53 protein revealed very significant difference between tumor (46%) and peritumor (4%) tissues, and negative in norm al liver samples (P ＜ 0.05), indicating that p53 gene mutations played similar roles in hepatocarcinogenesis in Xin Jiang regardless of the etiological factors. Su et al[20], Nakapoulou-et al[21] and Yu et al[22] have reported that c-erbB-2 oncogene was related to the dysregulation of proliferation and differentiation of the hepatocytes in hepatotumorigenesis, and over expression in various pathological lesions had different frequencies. We observed that overexpression of c-erbB-2 oncoprotein in tumor (70%) and in peritumor (22%) and in normal liver tissues also showed very distinct difference (P ＜ 0.05), suggesting that c-erbB-2 oncogene and tumor suppressor genes jointly participate in the occurrence and development of HCC, and related to the abnormal regulation even malignant transformation of hepatocytes induced by various pathogenes. Ushijima et al[23], Imai et al[24], Tamano et al[25] found that mutations of ras oncogene may be the early events, and the expression in tumor or non-tumor tissues can be detected with different rates. Lin et al[7] found that the expression of N-ras in liver tumor was correlated to the differentiated status of altered liver cells. In this study, there was no statistical significance between tumor (68%) and peritumor (64%) tissues in the expression of H-ras oncoprotein. Positive cells were also found in 30% of normal liver specimens, reasonably which can not be explained. Proliferating cell nuclear antigen (PCNA), as an index of cellular proliferative status was determined in various lesions. Waga et al[26], Maeda et al[27] found that p53 tumor suppressor gene can control the cyclin-dependent kinases to regulate DNA replication involving PCNA interaction by p21 protein pathway. The overexpression of PCNA with high frequency was usually used as a reliable marker for assessment of tumor progress, premalignant evolution and clinical prognosis of patients with various malignancies. We found that the positive frequency of PCNA in tumor (82%) was significantly higher than in peritumor (52%) tissues (P ＜ 0.05), indicating that the non-neoplastic tissues surrounding the hepatocarcinomas may have premalignant lesions including cellular hyperplasia, dysplasia, even in situ carcinoma. Therefore, these multiple molecular indexes in combination could be used as the biomolecular standard for diagnosis of malignant and benign lesions. The inactivation of tumor suppressor gene p53, Rb, p16, etc. was demonstrated in different forms, and implied the pathogenesis of human malignant diseases. Slagle et al[28] and Shao et al[29] found the loss of heterozygosity on chromosome 17p near the p53 gene or 1p in HBV-positive HCC in China.

It is noteworthy that the alteration of p53 tumor suppressor gene and c-erbB-2 and H-ras oncogenes in different ethnic patients with HCC revealed significant difference. The expression of p53 protein and c-erbB-2 and H-ras oncoprotein in carcinoma specimens was higher in the minority than in Han patients by immunohistochemical detection (P ＜ 0.05). Moreover, the three combined abnormal expression of p53 protein and c-erbB-2 and H-ras oncoprotein intumor tissues was higher in minority (41%) than in Han (21%) patients. This may reflect, to a certain extent, the differences of ethnic gene-susceptibility to various carcinogenes and effect on hepatocarcinogenesis. The pathogenesis of primary hepatic carcinoma is a multistage process with the involvement of multifactorial etiology, and the gene-environment interactions are involved in the development of HCC in humans. In order to understand the exact difference concerning the etiopathogenesis of HCC among different ethnic groups, further studies with more samples are needed, especially in minority ethnic patients.