[CANCER RESEARCH 53,368-372. January 15, 1993]

Role and Mutational Heterogeneity of the p53 Gene in Hepatocellular Carcinoma I

N a o s h i N i s h i d a , Y o s h i h i r o F u k u d a , H i r o y u k i K o k u r y u , J u n y a T o g u c h i d a , D a v i d W. Yande l l , M i t u o I k e n e g a ,

H i r o o I m u r a , a n d K a n j i I s h i z a k i 2

The Second Department of Internal Medicine [N. N., Y F, H. K., H. L ] and Department of Orthopaedic Surgery [J. T. ], Faculty of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto; Radiation Biology Center IN. N., J. T., M. I., K. 1.], Kyoto University, Yoshida-konoecho, Sakyo-ku, Kyoto 606, Japan; and Howe Laboratory of Ophthalmology, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts 02114 [D. W. Y]

A B S T R A C T

The mutational spectrum of the p53 gene was analyzed in 53 hepato- cellular carcinomas. Somatic mutations of thep53 gene were detected in 17 cases (32%). Among these 17 mutations, 9 were missense mutations; the mutations in the other 8 cases were nonsense mutations, deletions, or mutations at the intron-exon junctions. These mutations were found in a wide region stretching from exon 4 to exon 10 without any single muta- tional hot spot. G:C to T:A transversions were predominant, suggesting the involvement of environmental mutagens in the mutagenesis of the p53 gene in a subset of the hepatocellular carcinoma cases. Mutations of the p53 gene occurred frequently in advanced tumors, although several tu- mors in the early stages also showed mutations. A deletion map of chro- mosome 17 was constructed by using 10 polymorphic probes and was compared with the p53 gene mutation in each case. Loss of heterozygosity (LOH) on chromosome 17p was observed in 49% of the cases (24 of 49), and two commonly deleted regions were detected (around the p53 locus and at 17p13.3 to the telomere). Sixteen of the 17 cases with p53 gene mutations showed LOH around the p53 locus, and mutations were rare in hepatocellular carcinomas without LOH. However, no mutations were detected in 8 cases with LOH on 17p, suggesting the possibility that an unidentified tumor suppressor gene(s) located on 17p may have also been involved in hepatocarcinogenesis.

I N T R O D U C T I O N

Increasing evidence has supported the fact that the p53 gene acts as

a tumor suppressor gene (1), and mutations of the p53 gene are

frequently found in a variety of cancers (2). Bressac et al. (3) reported an abnormal structure and expression of the p53 gene in HCC 3-

derived cell lines. In addition, a mutational hot spot at codon 249

of the p53 gene was found in patients in China and South Africa,

which was considered to be closely associated with dietary aflatoxin

B~ intake (4-6). In contrast, no such mutational hot spot has been reported thus far in Japanese HCC cases (7, 8), suggesting the in-

volvement of different etiological factor(s). Therefore, an intensive

analysis with a large number of samples is required to understand

the mutational mechanism underlying the p53 gene during hepato- carcinogenesis.

A study on colorectal carcinomas demonstrated that mutations of

the p53 gene were closely associated with LOH on 17p, and vice versa

(9). We have also reported that the LOH on 17p in HCCs was strongly

correlated with the aggressiveness of the tumor (10), suggesting a role

for p53 gene inactivation in the progression of HCCs. Several recent studies, however, have shown that the LOH on

chromosome 17p was not necessarily associated with mutations of the

p53 gene in tumors such as malignant gliomas, ovarian cancers, and neuroectodermal tumors (11-13). Allelotypic studies on HCCs and

Received 8/6/92; accepted 11/2/92. The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This work was supported by Grants-in-Aid from the Ministry of Education, Science and Culture, Japan.

2 To whom requests for reprints should be addressed. 3 The abbreviations used are: HCC, hepatocellular carcinoma; LOH, loss of heterozy-

gosity; RFLP, restriction fragment length polymorphism; PCR, polymerase chain reaction; SSCP, single strand conformation polymorphism.

breast cancers have also revealed a c o m m o n deleted region other than

the p53 locus, which suggests that an unidentified tumor suppressor

gene might be located on 17p (14, 15). Furthermore, other tumor

suppressor genes such as NF-1, nm23, and the prohibitin gene were

mapped to the proximal region of chromosome 17q (16-18). There-

fore, it may be interesting to compare mutations of the p53 gene with

the deletion map of chromosome 17 obtained from the same HCC

samples.

In this present study, we have investigated the mutational profile of

thep53 gene in 53 Japanese HCC cases by PCR-SSCP analysis and by

direct genomic sequencing over the entire coding region, and com-

pared these mutations with several clinical parameters. We also dele-

tion mapped chromosome 17 by using 10 RFLP probes in the same

tumor samples, and analyzed the relationship between mutations of

the p53 gene and LOH.

M A T E R I A L S AND M E T H O D S

Samples. We used 53 HCC patients, with their informed consent, in this study. The patients underwent surgery at Kyoto University Hospital, and the tumors and surrounding noncancerous tissues were frozen immediately after surgical removal and stored at -80~ until the DNA isolation. The clinical stage of each patient was determined according to the classification scheme in the General Rules for the Clinical and Pathological Study of Primary Liver Cancer (19). Histological studies were performed at the Clinical Pathology Department of the hospital, and histological grades were assigned according to Edmondson's grading system (20). Among the cases analyzed, 40 were males and 13 were females. Furthermore, 7 patients were positive for serum hepatitis B virus surface antigen, and 46 were negative. Eight cases were at clinical stage 1, 7 were at stage II, 15 were at stage III, and 23 cases were at stage IV.

DNA Isolation and Southern Blot Analysis. High molecular weight DNA was isolated from the tumor and surrounding noncancerous tissues as previ- ously described (10). Restriction endonuclease digestion, agarose gel electro- pholesis, Southern blot hybridization, probe labeling by nick-translation, and autoradiography were also all performed as previously described (10).

RFLP Probes. The polymorphic probes used in this study are shown in Table 1. Probes pYNH37.3, pYNZ22, pMCT35.1, pHF12-1, p10.5, pAl0-41, pHHHI52, pCMM86, and pTHH59 were a gift from Dr. Y. Nakamura. Probe pR4-2 was a gift from Dr. C. W. Miller. Further details of these probes can be found in Human Gene Mapping !1 (21).

PCR-RFLP Analysis. In addition to BglII RFLP detected by Southern blot analysis with the pR4-2 probe, we also analyzed the BstU1 (AcclI) polymor- phism at codon 72, which can be detected by PCR-RFLP analysis (22). The fourth exon of the p53 gene was amplified by using a pair of primers which were the same as those used in the PCR-SSCP analysis. The amplified DNA fragments were then digested with 5 units of BstU 1 for 4 h and electrophoresed on 2% agarose gels.

PCR-SSCP and Direct Sequencing. Each sample was screened for muta- tions of the p53 gene by the PCR-SSCP method, as described by Toguchida et al. (23). For SSCP, each pair of primers was designed to cover the entire coding region and the intron-exon junctions. One hundred ng of genomic DNA was amplified in a buffer containing 0.1 lal [a-32p]dCTP ( 10 mCi/ml). Thirty cycles of PCR were performed, with 75 s of denaturation at 94~ 90 s of annealing at 52 or 60~ and 120 s of polymerization at 71~ The PCR products were diluted 8- to 10-fold with 0.1% sodium dodecyl sulfate and 10 rnM EDTA, and then mixed with the same volume of dye solution (95% formamide; 20 mM

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LOH ON CHROMOSOME 17 AND MUTATION OF p53 GENE IN HCC

Table 1 Frequency of LOH on each locus of chromosome 17

Chromosomal Cases of LOH/no. of Probes Locus symbol location Restriction enzyme informative cases (%)

pYNH37.3 D17S28 17p13.3 BamHI 1/3 (33) pYNZ22 D17S5 17p 13.3 TaqI 15/31 (48) pMCT35.1 D17S31 17p13.1-11.2 M~pI 12/21 (57) pR4-2 or TP53 17p 13.1 BstUI or 14/32 (44)

PCR-RFLP a Bgl II pHFI2-1 D17SI 17p13 MspI 2/11 (18) p10.5 MYH2 17p13.1 HindIII 2/5 (40) pAl0-41 D17S71 17p12-11.2 MspI 1/3 (33) pHHHI52 D17S32 17q BamHI 0/13 (0) pCMM86 DI 7S74 17q TaqI 2/12 (17) pTHH59 DI 7S4 17q23-q25.3 TaqI 2/8 (25)

LOH on the TP53 locus was examined by Southern blot analysis with the pR4-2 probe and/or PCR-RFLP analysis in exon 4, as described in "Materials and Methods."

EDTA; 0.05% xylenecyanol; 0.05% bromophenol blue). This was followed by denaturation at 80~ for 3 min, and then the samples were applied to 6% neutral polyacrylamide gels with or without 10% glycerol, and electrophoresed at 5 W for 12 to 16 h with cooling by a desk top fan.

Samples which showed altered mobility from the normal controls on the PCR-SSCP gels were further analyzed by direct sequencing. A single-strand DNA fragment was amplified by asymmetric PCR, purified by SUPREC-02 (Takara), and then sequenced directly by using a T7-Sequencing kit (Pharma- cia). The sequencing primer was one of a pair of PCR primers, and was radiolabeled at the 5' end by using a MEGALABEL kit (Takara).

RESULTS

Mutational Spectrum of p53 Gene in HCC and Corresponding Clinical Stage. By PCR-SSCP analysis and direct sequencing, 17 different mutations were detected in 53 tumors (32%). Fig. 1 shows representative examples of the PCR-SSCP analysis and direct se- quencing of the mutant p53 genes. A summary of the p53 mutations, with the clinical profile from each patient, is shown in Table 2. The following types of mutations were detected: 9 cases of missense mutations, 4 cases of nonsense mutations, 2 cases of mutations at the intron-exon junction, and 2 cases of deletions (one with a 1-base pair deletion and the other with a 58-base pair deletion) (Table 2). Five of the missensc mutations (cases 8, 19, 26, 28, and 53) were within the conserved domains, whereas 2 missense mutations (cases 5 and 48) were observed at the conserved amino residues within cxons 6 and 10. Except for these subtle mutations, we could not detect any structural alterations with Southern blot analysis, using the pR4-2 probe.

Among the 15 base substitutions, G:C to T:A transversions were the most common changes observed (7 cases, 47%; see Table 3). In 6 of these transversions, a guanine residue on the non-transcribed strand was mutated. No mutations were detected in the histologically normal tissues surrounding the tumors that exhibited mutations.

The frequencies of p53 mutations in HCCs of each clinical stage were as follows: 9 of 23 (39%) in stage IV, 5 of 15 (33%) in stage III, 2 of 7 (29%) in stage II, and 1 of 8 (13%) in stage I (Fig. 2). This suggests a relationship between p53 mutations and the progression of the HCCs. No relationship was found between the p53 mutations and the serum hepatitis B virus surface antigen status (Table 2).

Deletion Map of Chromosome 17 and Mutations of p53 Gene. Table 1 summarizes the results from the LOH analyses on chromo- some 17, and Fig. 3 shows the deletion map of chromosome 17. In some tumor samples, residual faint bands which might be caused by contamination of normal cells were observed. When it was difficult to determine whether there was loss, we performed densitometric anal- ysis and only the cases in which the density of fainter band was <50% of normal tissue were determined as allelic loss. Among the 53 cases of HCC, 50 cases were informative with respect to at least one locus on chromosome 17, and LOH was detected in 24 cases (48%).

LOH on the short arm was observed in 24 of 49 cases (49%), whereas LOH on the long arm was found only in 3 of 25 cases (12%).

(a)

(b)

case 14 N T

case 18 case 35 case 53

N T N T N T

|

case 14 case 18

N T 3' 3' N T 3' 3' A G C T A G C T -A T:.. A G C T A G C T .:~ h. i ~ C" ~ , ~ : : ~ / T o'., ' !'ii =/a e- ~ ....................... ~3~ / c . G '" ~ / G IG - ~ ' : , ~ : .-" C

I ' ' ~ / A G ~ A A I G~u ^ <'~ 198 A ! .... A

G ~'~ ..... G G I ~ G -~'i!~i " '~"~o ~" T G G IGly T .... :: :~ G - ~ \ G Gly IGA - ~~"~ A - -

A / ''-. A Aa ii \~ ~.' ..... .A c s, A- s' C 5'

5'

case 35 case 53 N T N T

5' ,3' A G C T A G C T 3 ' 5' A G C T A G C T 188 Leul T- ~ /T Leu G' , / I ", ...... I

T

' ~ ' Ic: 242 T A t ~ i '~ t ~:~ G 242

,,., - ..... ,..,..

c,," ~ ._,.~ r - I G/ " 5' 5' 3' 3'

Fig. I. Examples of PCR-SSCP analysis (a), and direct sequencing (b) in human HCCs. Arrows indicate bands with a shifted mobility detected in the SSCP analysis (a). Case 14 showed an abnormal band with PCR-SSCP analysis in exon 6, and a nonsense mutation by direct sequencing. Case 18 showed a deletion mutation in exon 8, case 35 showed a base substitution mutation at the intron-exon junction in intron 5, and case 53 showed a missense mutation in exon 7. N, normal tissue sample; T, HCC sample. Capital letters indicate base sequences in exons, and lower case letters indicate base sequences in introns.

Furthermore, all 3 of these cases also showed LOH on the short arm, suggesting the loss of the whole chromosome. Although most of the cases with LOH on 17p showed allelic losses at all informative loci on the short arm, 3 cases (cases 33, 45, and 54) showed LOH at the D17S5 locus yet retaining heterozygosity at the p53 locus. On the other hand, one case (case 22) showed LOH at the p53 locus without LOH on more distal loci (Fig. 3). These results indicate that two commonly deleted regions exist on 17p in HCCs: one around the p53 locus, and the other stretching from the D17S5 locus to the telomere.

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LOH ON CHROMOSOME 17 AND MUTATION OF p53 GENE IN HCC

Table 2 Mutations of p53 gene in HCC and clinical profiles of patients

Case no. Age Sex HBsAg ~ Clinical stage b Codon

Mutations of p53 gene

Nucleotide change Amino acid change

2 48 M - IV 110 5 62 M - Ill 337 8 63 M - Ill 173

19 67 M - II 270 26 65 M - IlI 177 28 60 F - III 132 48 64 M - IV 215 53 67 M - IV 242 55 76 M - II 35

14 48 M + IV 198 22 66 F + IV 196 37 61 M - III 298 43 76 F - IV 135

18 61 M - IV 294--295 30 58 F - IV 90-109

4 45 M + IV Exon 7-intron 7 35 62 F - I Intron 5--exon 6

CGT ~ CTT Arg ---) Leu CGC --* TGC Arg ---) Cys GTG ---) TFG Val ---) Leu "ITF --~ GTT Phe ---> Val CCC ---) CGC Pro ---) Arg AAG ---) AAT Lys ---> Ash AGT ---) AAT Ser ---> Asn TGC ~ AGC Cys ---) Ser TTG ---> Trc Leu ---> Phe

GAA ---) TAA Stop c CGA ~ TGA Stop GAG ---) TAG Stop TGC ---) TGA Stop

1 base pair deletion (frame shift) 58 base pair deletion (frame shift)

AGgtc ---> AGgtg a agGT ---> atGT

a HBsAg, hepatitis B virus surface antigen; +, presence of serum HBsAg; -, absence of serum HBsAg. b According to the General Rules for the Clinical and Pathological Study of Primary Liver Cancer (19). c Stop indicates nonsense mutation. d Capital letters, sequences in the exon; lower case letters, sequences in the intron.

Table 3 Nucleotide changes in p53 mutations found in Japanese HCCs

No. of No. of Transversion Cases (%) Transition Cases (%)

G:C to T:A 7 (47) G:C to A:T 3 (20) G:C to C:G 3 (20) A:T to G:C 0 (0) A:T to T:A 1 (7) A:T to C:G 1 (7)

50 (%)

, _

~ 4 0 E ~" 30

o 20 E

, .--10 o

0

J ! ! ! i

I II III IV

Fig. 2. Relationship between frequency of p53 gene mutation and clinical stage. Tu- mors were classified according to their clinical stage, and the percentage of tumors with mutations was determined in each group.

The relationship be tween L O H and the mutational status o f the p53

gene further supports the presence of two important regions on 17p.

A m o n g the 24 cases with L O H on 17p, 16 cases, including 1 case with

L O H only at the p53 locus (case 22), showed mutations in the residual

p53 allele (16 of 24, 67%; see Table 4). In the other 8 cases, there was

L O H on 17p but no alteration of the p53 gene. These 8 cases included

3 with L O H only at loci distal to the p53 locus.

Only one o f the 25 cases without L O H on 17p showed a mutation

of the p53 gene (1 of 25, 4%; see Table 4), where the mutat ion was

heterozygous.

DISCUSSION

Mutat ions of the p53 gene have been demonstra ted to be a c o m m o n

event in various types of human malignancies. The mutational profile

o f each type of tumor, however , seems to be different f rom other

tumors (24). Even within a single type of tumor, the mutational

spectrum might be affected by different genetic and/or environmental

factors, and thus a comparat ive study may reveal important etiological

factors behind the deve lopment of each tumor. In this paper, we have

characterized mutat ions o f the p53 gene in Japanese H C C patients,

with special attention to the L O H on 17p.

The overall f requency of p53 gene mutat ion in the H C C s in this

s tudy was 32% (17 o f 53), which is inconsistent with previously

reported values in Japanese patients (7, 8). Kress et al. (25) also

showed a lower f requency (15%) in H C C from Germany, and the

f requency is consistently higher (50%) in H C C patients from groups

where the codon 249 hot spot predominates (4, 5). This difference of

f requency is probably due to different et iologies of hepatocarcinogen-

esis in different areas. In contrast to the hot spot observed in H C C s in

China and south Africa, which we discuss later, no mutational hot spot

was observed, and mutat ions were generally found in a wide region

from exons 4 to 10. Mos t o f the missense mutat ions were observed at

residues within evolutionari ly conserved domains, or at scattered but

conserved amino residues, as reported in other types of cancers (24).

Unlike other cancers such as colorectal carcinomas, where missense

mutat ions accounted for 94% of the cases (9), almost one-half o f the

mutations (8 of 17) in H C C s were not missense mutations. Among

these 8 mutations, 4 were nonsense mutations and 2 were base sub-

stitutions at the intron-exon junctions, which had not been reported in

previous studies using HCCs (4, 5, 7, 8). We do not have any clear

explanation for this difference. However , different patient populat ions

or higher sensitivity of our P C R - S S C P method might give rise these

different results.

As ment ioned above, we did not detect any mutat ions at codon 249

which was previously reported as a mutational hot spot in H C C s in

China and South Africa (4-6) . This mutational hot spot was consid-

ered to be closely associated with aflatoxin B~, and therefore our

results suggest the involvement of mutagens other than aflatoxin B ~ in

the deve lopment of HCCs in the Japanese population.

G:C to T:A changes were predominant among the base substi tutions

observed, including 6 G to T transversions on the non-transcribed

strand, which may be attributable to preferential repair o f the tran-

scribed strand (26). This type of transversion in the p53 gene was

frequently observed in cancers in both small cell and non-small cell

lung cancers, which was considered to be associated with cigarette

smoke (24, 27). Recent reports have shown that a variety of mutagenic

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Fig. 3. Deletion map of chromosome 17 in HCCs. Among the 53 cases studied, 50 were infor- mative and 24 showed LOH with respect to at least one locus on chromosome 17. Mutations of the p53 gene were detected in 16 cases with LOH, but in only one cases without LOH on 17p. In case 22, only the TP53 locus showed LOH and the other 4 informative loci retained heterozygosity. In cases 33, 45, and 54, the TP53 locus retained heterozy- gosity but the D17S5 locus lost heterozygosity. Blank spaces indicate uninformative loci. +, pres- ence of a p53 mutation; - , absence of a p53 muta- tion.

LOH ON CHROMOSOME 17 AND MUTATION OF p53 GENE IN HCC

c a s e 22 2 4 5 8 26 30 35 37 48 53 14 18 ;:>8 19 43 33 45 54 24 49 13 29 39 p 5 3 m u t a t i o n + + + + + + + + + + + + + + + + . .

p - te l

D17S28 ~._

! o . . o, s31 l �9 �9 �9 l O O o l �9 o o �9 O l O �9

D17S1 �9 0 MYH2 �9 D17S71 /

D17S32 ~ D17S74 ~ 0 0 0 8 0 0 0 0 8 D17S4 / �9 I _ 8 _ O

q-tel �9 allelic loss O retained

Table 4 Correlation between LOH on 17p and mutations in p53 gene

Among the 53 cases studied, 24 showed LOH, 25 retained heterozygosity, and 4 were not informative on the short arm of chromosome 17.

Mutation in p53 gene

LOH on 17p Presence Absence Total

Presence 16 (67%)~ 8 24 | P < 0.01 a

Absence 1 (4)- 24 25 NI b 0 (0) 4 4

Total 17 (32) 36 53

a p value by the X 2 test. b NI, not informative.

heterocyclic amines, which are commonly found in cooked food, can produce DNA adducts in the liver cells (28). Furthermore, endogenous mutagens caused by oxyradical damage can also produce G:C to T:A transversions (29). These mutagens may therefore induce transversion mutations of the p53 gene, and contribute to the progression of the cancer in some human HCCs, as discussed in the next section.

Previous reports on HCCs have emphasized the association of p53 gene mutations with tumors in advanced stages (7). Accordingly, the p53 gene mutations observed in this study were found in advanced tumors with a higher frequency (39 and 33% for stages IV and III, respectively) than in less advanced tumors. However, we found that several tumors in the early stages also carried mutations, although the frequency was lower (29 and 13% for stages II and I, respectively). Although these results provide no definite answer concerning the stage of the HCC when the p53 gene mutations first occur, the gradual increase in frequency of p53 gene mutations may represent a growth advantage for tumors with these mutations, as reported in brain tumors (30).

Among the 17 cases with p53 gene mutations, LOH was observed on 17p in 16 cases (94%); only one case retained constitutional heterozygosity. This indicates a close association of p53 gene muta- tions with LOH on 17p, as is observed in some types of cancers such as colorectal carcinomas (9) and lung cancers (27). Although it is supposed that some mutant p53 genes may act dominant negatively (31), we detected only one case of p53 mutation without LOH on 17p. All the other p53 mutations were homozygous or hemizygous, sug- gesting that once a mutation occurred in the p53 gene, it was rapidly followed by a loss of the residual normal allele, since this might confer an increased growth advantage in hepatocarcinogenesis.

On the other hand, LOH on 17p was not always found with p53 gene mutations in some HCCs. No mutations of the p53 gene were detected in 8 cases with LOH on 17p. It is possible that we did not detect mutations in these cases because of the limit of sensitivity of our method, or because the mutations were in regions of the gene beyond our study, such as in the promoter region. The results of the

371

deletion mapping, however, support the idea that another tumor sup- pressor gene located on the short arm of chromosome 17 might be involved in the development of some HCCs. The fact that three cases with LOH on the distal portion of 17p still retaining heterozygosity at the p53 locus and showed no detectable abnormalities in the p53 gene, and the fact that one case with LOH only at the p53 locus showed a p53 gene mutation further support the presence of two tumor suppres- sor genes on 17p, both of which may play a critical role in hepato- carcinogenesis.

ACKNOWLEDGMENTS

We thank Dr. Y. Nakamura and Dr. C. W. Miller for supplying the RFLP probes.

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1993;53:368-372. Cancer Res   Naoshi Nishida, Yoshihiro Fukuda, Hiroyuki Kokuryu, et al.   Hepatocellular Carcinoma

Gene inp53Role and Mutational Heterogeneity of the

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