Proliferating cell nuclear antigen and P53 protein expression and prognosis in primary liver cancer

Abstract

AIM: To study the individual difference and prognosis of primary liver cancer (PLC) after hepatectomy.

METHODS: Two hundred specimens obtained from PLC patients were examined immunohistochemically using anti-P53 and anti-PCNA monoclonal antibodies. The mutations of p53 in exons 5-8 of the gene were examined using the polymerase chain reaction/single-stranded conformational polymorphism (PCR/SSCP) method in 60 of the 200 specimens.

RESULTS: The results showed that the labeling index of proliferating cell nuclear antigen (PCNA) and expression of p53 had a parallel correlation (P < 0.001). The high labeling index of PCNA or overexpression of p53 was associated with a high risk of tumor recurrence, more aggressive growth and poor survival.

CONCLUSION: Immunohistochemical assessment of PCNA and P53 can provide very useful information for the prognosis of PLC.

Key Words: Prognosis; P53 protein; Liver neoplasms; Monoclonal antibodies; Immunohistochemstry; Polymerase chain reaction

INTRODUCTION

With the development of diagnostic techniques, many cases of primary liver cancer (PLC) can be resected at an early stage[1,2]. Because of the biologic behavior of PLC, the recurrence rate in the patients who underwent hepatectomy is very high[3-5]. Some authors have described the factors influencing HCC recurrence, such as tumor size, capsule formation, differentiation of tumor cells, clinicopathologic stage, proliferating cell nuclear antigen (PCNA), and mutation of p53[5-9]. To study the individual difference and prognosis of PLC after hepatectomy, 200 specimens obtained from PLC patients were examined immunohistochemically using anti-P53 and anti-PCNA monoclonal antibodies. The mutations of p53 in exons 5-8 of the gene were examined using the polymerase chain reaction/single-stranded conformational polymorphism (PCR/SSCP) method in some of the 200 specimens.

MATERIALS AND METHODS

Patients

From August 1992 to December 1995, 200 PLC patients who underwent hepatectomy were recruited to this study. One hundred and eighty-eight cases were male and 12 cases were female with ages ranging from 20 to 72 years. One hundred and thirty-four cases underwent radical hepatectomy and 66 cases underwent palliative resection. Forty-seven cases had small tumor(s) (diameter < 5 cm), and hepatocellular carcinoma was confirmed in all of the patients.

Immunohistochemical staining

Formalin-fixed and paraffin-embedded specimens were examined immunohistochemically using anti-PCNA monoclonal antibody Pc-10 and anti-P53 monoclonal antibody DO-7 (LSAB kit, Dako). The deparaffinized tissue sections had to be treated in a microwave oven before the immunohistochemical staining procedure for the P53[9,10].

The criteria of nuclear labeling index for PCNA and expression of p53 in this study were as follows: the positive nuclei number was semiquantitatively evaluated by counting the number of positive nucleus in 8-10 randomly-chosen medium power (× 160 magnification) fields; four degrees of positive nuclei for PCNA were identified:“+” ＜ 24%, “++” = 25%-50%, “+++” = 51%-74%, “++++” ＞ 75%; four degrees for P53 were identified: “-” no positive cell, “+” ＜ 30%,“++” = 31%-70%, “+++” ＞ 71%.

PCR/SSCP

PCR/SSCP method[11,12] was used to examine the mutations of p53 in exons 5-8 of the gene in 60 of the 200 specimens.

Statistical analysis

The data were analyzed statistically by the chi-square test and the spearman rank correlation coefficient test. The Bio Medical Data Processing (BMDP) Statistical Software was used to compare the survival rates of the patients in different groups (Breslow and Mantel-Cox). P-values < 0.05 were considered statistically significant.

RESULTS

Labeling index of PCNA and mutations of p53 gene in PLC

Using the PCR/SSCP method, we found mutations in 20 (33.3%) of the 60 cases. Eighteen mutations were found in exon 7 and two in exon 8. No mutations were found in exon 5 or 6. The P53 protein expression was also positive in the 20 cases. There were no significant differences in labeling index of PCNA or expression of P53 between the small and large tumors (P > 0.05) (Tables 1 and 2).

Table 1 Proliferating cell nuclear antigen labeling index and tumor size.

	Cases	PCNA labeling index
		Low index (+, ++)	High index (+++, ++++)
Small tumor	47	32	15a
Large tumor	152	88	65a

^aP > 0.05, small tumor vs large tumor. PCNA: Proliferating cell nuclear antigen.

Table 2 P53 expression and tumor size.

	Cases	P53 expression
		Low expression (-, ++)	Overexpression (++,+++)
Small tumor	47	31	16a
Large tumor	153	90	63a

^aP > 0.05, small tumor vs large tumor.

The labeling index of PCNA correlated with expression of P53 (r = 0.3814, t = 3.9004, P = 0.0003).

Recurrence and labeling index of PCNA and expression of P53

By June 1996, tumor recurrence occurred in 38 of the 134 patients who underwent radical resection. PCNA high labeling index (+++, ++++) accounted for 71.1% (27/38). Overexpression of P53 accounted for 55.3% (21/38). In 96 cases who had not tumor recurrence, PCNA high labeling index accounted for 27.1% (26/96). Overexpression of P53 accounted for 33.3% (32/96). There were significant differences in high PCNA labeling index and P53 overexpression between the recurrence and non-recurrence groups (P < 0.01) (Tables 3 and 4).

Table 3 Proliferating cell nuclear antigen labeling index and tumor recurrence.

	Cases	PCNA labeling index
		Low index (+, ++)	High index (+++, ++++)
Recurrence	38	11	27e
No recurrence	96	42	26e

^eP > 0.001, recurrence vs no recurrence. PCNA: Proliferating cell nuclear antigen.

Table 4 p53 expression and tumor recurrence.

	Cases	p53 expression
		Low expression (-, ++)	Overexpression (++, +++)
Recurrence	38	17	21e
No recurrence	96	64	32e

^eP > 0.001, recurrence vs no recurrence.

Survival rates and labeling index of PCNA and expression of p53

The different survival rates of the patients with low or high labeling index of PCNA, low expression or overexpression p53 who underwent palliative hepatectomy are listed in Tables 5 and 6.

Table 5 Comparison of the survival rates of patients with low or high labeling index of proliferating cell nuclear antigen after palliative hepatectomy.

PCNA index	Cases	Survival rate (%)
		6 mo	12 mo	18 mo	Median time
Low index (+, ++)	39	89.7	76	69	14
High index (+++, ++++)	27	50.9	11.8	3.9	5

PCNA: Proliferating cell nuclear antigen.

Table 6 Comparison of the survival rates of patients with weak expression or overexpression P53 after palliative hepatectomy.

PCNA index	Cases	Survival rate (%)
		6 mo	12 mo	18 mo	Median time
Low index (-, ++)	39	81.4	63.5	55.3	11
High index (++, +++)	27	52.9	11.8	5.9	6

P = 0.0079, Mantel-cox; P = 0.0051 Breslow. PCNA: Proliferating cell nuclear antigen

DISCUSSION

Some authors reported that the labeling index of PCNA and overexpression of P53 might provide information about clinical outcomes. PCNA labeling index correlated with the degree of histologic differentiation. Labeling index of PCNA was significantly low in small tumors and encapsulated tumors and high in large tumors and tumors without capsule[7-9]. But Ng et al[10] reported that the presence of p53 mutations did not show a significant association with tumor size, sex, age, tumor invasiveness, microsatellite lesion, venous permeation, cirrhosis or encapsulation[7,8]. This study showed that the high labeling index of PCNA and overexpression of p53 were frequent in large tumors, but there was no statistical difference (P > 0.05).

Recurrence was closely related to the biologic behavior. Labeling index of PCNA was correlated with the recurrence. The tumor with a high index of PCNA had more aggressive growth and recurrence, resulting in low survival rates[9-12]. This study indicated that tumors with a high labeling index of PCNA and overexpression of p53 tended to have a high risk of recurrence.

Even some small tumors may show rapid recurrence and poor survival rate. In this study, 3 cases of small tumor and 11 cases of large tumor had both a high labeling index of PCNA and overexpression of p53. They underwent radical hepatectomy, but all of them experienced tumor recurrence 3-5 mo later after the operation. A low recurrence rate was found in the tumor with low labeling index, and even after recurrence, the tumor grew slowly and had a good survival. For the patients with a high risk of recurrence, hepatic arterial chemoembolization 3-4 wk after hepatectomy could decrease the recurrence rates and improve the survival[6]. This study also showed that the immunohistochemical assessment of PCNA and P53 could provide very useful information for the prognosis of PLC.