J Cancer Res Clin Oncol (1996) 122:763-766 �9 Springer-Verlag 1996

Bin H e �9 Z h a o - Y o u T a n g �9 K a n g - D a Liu �9 G e Z h o u

Analysis of the cellular origin of hepatocellular carcinoma by p53 genotype

Received: 11 April 1996/Accepted: 5 June 1996

Abstract The polymerase chain reaction and HaeIII enzymatic digestion were used to study the seventh exon of the p53 gene in 29 primary and recurrent hepatomas in paraffin-embedded samples from 11 pa- tients. The mutation rate of the p53 gene and its geno- types in samples of primary and recurrent tumours and multiple nodules were investigated. The cellular origins of hepatocellular carcinoma were analysed by p53 genotype, p53 mutation rates were found to be 69.0% (20/29) in the primary and recurrent tumours, 58.8% (10/17) in tumours with a single nodule and 83.3% (10/12) in tumours with multiple nodules. The p53 genotypes were found to be different in 6 pairs of primary and recurrent tumours, and another 5 pairs had the same p53 genotypes. The samples with multiple nodules in the same patients had the same p53 geno- types. Seven recurrences were of multicentric origin and four were of unicentric origin. It is suggested that the recurrent lesions developed from both unicentric and multicentric origins.

Key words HCC - p53 - Genotype �9 Mutation

Abbreviat ions HCC hepatocellular carcinoma

Introduction

Hepatocellular carcinoma (HCC) is one of the com- monest cancers in Asia and Africa (Simonetti et al. 1991). The resection rate of HCC has increased with the increasing discovery of early HCC; however, the recur-

This work was supported partially by China Medical Board Grant 93-583

B. He . Z.-Y. Tang (N~) . K.-D. Liu �9 Ge Zhou Liver Cancer Institute, Shanghai Medical University, Shanghai 200032, China. Tel: 0086 21 64037181; Fax: 0086 21 64037181

fence of HCC remains the major problem affecting the survival of the patients after resection. Although resec- tion remains the treatment of choice for HCC, the 5-year survival rate after resection was low. According to our previous study, even after radical resection, the 1-, 3- and 5-year recurrence rates were as high as 17.1%, 32.5% and 61.5% respectively (Tang et al. 1984).

HCC has been thought to be of multicentric origin because many patients showed multiple patterns; the recurrence rate after resection was high and multiple nodules were found in almost all of the chemically induced hepatic cancers in animals. On the other hand, in recent years long-term survivors have been observed after resection of small HCC, therefore there may also be a unicentric origin. Several methods have been used to investigate the cellular origins of HCC, such as hepatitis virus B (HBV) DNA integration, loss of het- erozygosity of the p53 gene and loss of heterozygosity of chromosome 16 (Chen et al. 1989; Hsu et al. 1994). The previous work of our institute using HBV DNA integration also demonstrated that both multicentric and unicentric origins existed (Liang et al. 1991). In this study we sought to use a new approach to differentiate the cellular origins of HCC and to investigate the relations between p53 mutation and recurrence of HCC.

Materials and methods

Patients

During 199~1992, 11 patients with resected primary HCC had disease pathologically proven at the Liver Cancer Institute of Shanghai Medical University. From these 11 patients, 29 paraffin- embedded samples of primary and recurrent tumours were selected for the mutation analysis at codon 249 of the p53 gene. The age of the patients ranged from 28 to 64 years. Serum HBsAg was found in 8 patients, and was absent in the remaining 3 patients. The other clinical data are listed in Table 1.

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Extraction of DNA

The paraffin samples were cut into pieces of 25 ~am. Thirty pieces were put into the microtubes and xylene was used twice to remove paraffin at 65~ The samples were washed twice with ethanol and dried under vacuum. TRIS/EDTA buffer (pH 7.4; 0.5 ml) and pro- teinase K (2 mg/ml) were added to each microtube. The samples were extracted by an equal volume of TRIS-buffer-saturated phenol and an equal volume of chloroform. The DNA was precipitated by ethanol. The pellets were dissolved in 30 gl TRIS/EDTA buffer (pH 7.4).

200bp-- 110bp--

1 2 3 4 5 6

---75bp

PCR

A pair of primers, synthesized in our institute, were selected to amplify the seventh exon of the p53 gene. The amplified fragment was 110 base pairs. The primer sequences were 5 ' -CCTGCCACAG GTCTCCCCAA-3' and 5 ' -AGGGGTCAGCGGCAAGCAGA-Y. The polymerase chain reaction (PCR) was carried out on the 5 gl extracted materials using 50pmol each primer and 2.5 U Taq polymerase (Perkin Elmer) in 1 x Taq polymerase buffer (Perkin Elmer) in a total volume of 50 gl for 40 cycles at 94~ for 1 min, 52~ for 1 min and 72~ for 1 min 30 s.

Detection of mutat ion

A 7-gl aliquot of PCR product was used to analyse the mutat ion of the p53 gene. The products of PCR were digested for 1 h at 37~ with 10 U HaeII I (Promega) in a total volume of 10 lal, then elec- trophoresed on 2% agarose gels. The wild-type p53 gene would result in cleavage by HaeIII at codon 249, giving products of 75 and 35 base pairs with the primers indicated above. Any changes at codon 249 at position 2 or 3 would result in destruction of the HaeII I site and an uncleaved product of 110 base pairs (Heyward and Walker 1991) (Fig. 1).

Results

Incidence of p53 mutation in different HCC samples

p53 mutation rates were 69.0% (20/29) of primary and recurrent tumors, 58.8 % (10/17)of tumors with a single nodule and 83.3% (10/12) of tumors with multiple nodules.

Fig. 1 Electrophoretogram of fragments by the HaeIII-restricted endonuclease. Lane 1 Marker, lanes 2, 4, 5 Mutant codon of p53 gene, lanes 3, 6 normal samples

Phenotypes of ~-fetoprotein-producing, p53 genotypes and cell origins

Eight cases (nos. 1, 3, 6, 7, 8, 9, 10, 11) were of the same c~-fetoprotein(AFP)-producing phenotypes and the other 3 cases (nos. 2, 4, 5) were of different AFP-produ- cing phenotypes. Among 11 pairs of primary and recur- rent tumors, 6 pairs showed different p53 genotypes (nos. 1, 3, 4, 5, 7, 9) and 5 pairs the same genotypes (nos. 2, 6, 8, 10, 11). The same genotypes were demonstrated in all of the 5 cases (nos. 2, 6, 8, 9, 11) with multiple nodules (Table 1). Seven recurrences (nos. 1, 2, 3, 4, 5, 7, 9) were of multicentric origin and 4 (nos. 6, 8, 10, 11) were of unicentric origin.

Discussion

The problem of the multicentric and unicentric origin of HCC is important, because it is a major issue in the control of the HCC. There are two possible origins of any recurrence: micrometastasis from the primary lesion and a new lesion from a multicentric origin.

Table 1 The clinical data and p53 mutation of 11 patients. P primary liver cancer, R recurrent liver cancer, A F P e-fetoprotein

No. AFP of 1st operation (ng/~l)

AFP of Interval between Nodules (n) p53 mutation 2nd operation operations (ng/pl) (months) P R P R

1 2 3 4 5 6 7 8 9 l0 11

518 < 20 1260 < 20 < 20

172 381

1145 332

< 20 1172

154 12 1 1 - + 77 18 1 2 -

1712 14 1 1 + - 342 8 1 1 - + 410 8 1 1 + - 300 6 1 2 + + , + 145 7 1 1 + -

1260 7 1 2 + + , + 1145 14 1 2 - + , + < 20 3 1 1 + + 1545 12 4 1 4 + +

765

As mentioned above, approaches to the study of the cellular origin of HCC included integration of HBV DNA, the loss of heterozygosity of the p53 gene and the loss of heterozygosity of chromosome 16. The close relationship between hepatitis B virus and HCC has been recognized. Integration of HBV DNA is fre- quently found in cells of HCC. The locus, the fragments and the quantity of integration are random. The inte- grated HBV DNA is a good parameter for the study of the clonal origin of HCC; however, it is limited in patients without HBV infection. Therefore, the loss of heterozygosity of the p53 gene and the loss of hetero- zygosity of chromosome 16 will be additional ap- proaches for the study of multicentric origins.

The tumor-suppressor gene for p53 is located on chromosome 17p13 (Levein et al. 1991). It has 11 exons and participates in the cell-cycle regulation. Mutations of the p53 gene have been found to be a common change in human cancer (Hollstein et al. 1991). In hepatocellular carcinoma, a mutational "hot spot" at codon 249 has been reported in a substantial propor- tion of tumors from areas of aflatoxin exposure, and this contrasts with the less specific pattern of mutations reported in other tumors (Ozturk et al. 1991). More than 90% of the mutation of the p53 gene in HCC occurs at codon 249 (Bressac et al. 1991; Unsal et al. 1994). In this study the p53 gene is chosen as a para- meter to study the clonal origin of HCC because of the higher mutation rate of this gene at codon 249 demon- strated in our institute (Qin et al. 1995).

In this study, different p53 genotypes were demon- strated in 6 pairs of samples and the same p53 geno- types in 5 pairs of samples. The tumors of multiple nodules in the same patient had the same genotypes. The cellular origins of HCC were analysed by p53 genotype. The phenotype of AFP-producing and time to recurrence were also used to help the diagnosis. In 6 pairs of different genotypes, three pairs (nos. 3, 5, 7 in Table 1) were of multicentric origin because of a muta- tion at codon 249 in the primary lesions and no muta- tion in the recurrent lesions. One pair (no. 4) was of multicentric origin because there were different geno- types, a short recurrence time (2 months) and different phenotypes of AFP-production in the primary and recurrent tumors. Two pairs (nos. 1, 9) were more possibly of multicentric origin considering the different genotypes and the longer recurrence times (more than 12 months). In 5 pairs of the same genotype, 1 pair (no. 2) was of multicentric origin on the basis of the non-mutant genotype, a long recurrence time (18 months) and different AFP-producing phenotypes in the primary and the recurrent lesions. Three pairs (nos. 6, 8, 10) were of unicentric origin because they had mutant genotypes, similar AFP-producing phenotypes and short recurrence times (less than 8 months). One pair (no. 11) may be of unicentric origin because of mutant genotypes and multiple nodules in the primary lesion. Our previous observations had suggested that

HCC can develop multicentrically and unicentrically (Chen et al. 1989; Hsu et al. 1994).

Mutation of the p53 gene is frequently found in hepatocellular carcinoma. Hsu et al. (1991) reported that the mutation at codon 249 is a hot spot in HCC. In this study, the p53 mutation rates of different surgical specimens were analysed. In a total of 29 samples the p53 mutation rate was 69.0%; p53 mutation rates were 58.8% (10/17) in tumors with a single nodule and 83.3% (10/12) in tumors with multiple nodules. Oda et al. (1992) reported that the incidence of p53 mutations was significantly associated with cancer cell differenti- ation. In poorly differentiated HCC the mutation was detected in more than half of the cases, whereas in well-differentiated HCC, the mutation rate was low. Our data showed that codon 249 is a hot spot for mutation in HCC in China and the tumors of recurrent cases may be poorly differentiated.

Carcinogens can induce mutations by adduct-driven mutagenesis or by triggering increased cellular turn- over, which increases the mutation rate indirectly (Vol- gelstein et al. 1992). It has been reported that G to T transversion at the third base of codon 249 is a spe- cific feature of aflatoxin-related endemic HCC (Ozturk et al. 1991; Bressac et al. 1991). Moreover, mutation at codon 249 has also been preferentially observed in HCC from HBV-infected patients in areas of high ex- posure to aflatoxin (Scorsone et al. 1992). Aflatoxin exposure may be also the reason for the mutation at codon 249 of HCC in China. A relationship between p53 mutation and advanced tumor stage has been noted for cancer of the liver (Murakami et al. 1991; Nishida et al. 1993) indicating that, for HCC, p53 mutations may be a late event contributing to tumor progression. In our study, one recurrent tumor of multicentric origin had a p53 mutation and short recur- rence time (8 months), p53 mutation may also occur at an early stage.

Because there are both unicentric and multicentric origins of HCC, two lines of approach should be taken: early resection and preventive therapy to reduce para- operative micrometastasis, and the prevention of HBV infection, aflatoxin contamination, etc. to decrease the recurrence rate of HCC.

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