high frequency of p53 gene mutation in adenocarcinomas of ...(52%) of 23 cases had p2! expression....

Vol. 5, 461-466, June 1996 Cancer Epidemiology, Biomarkers & Prevention 461

High Frequency of p53 Gene Mutation in Adenocarcinomas

of the Gallbladder’

Kiyomu Fujii, Hiroshi Yokozaki, Wataru Yasui,Hiroki Kuniyasu, Manabu Hirata, Goro Kajiyama,and Eiichi Tahara2

First Department of Pathology (K. F., H. Y., W. Y., H. K., E. T.) and First

Department of Intemal Medicine (K. F., M. H., G. K.) Hiroshima University

School of Medicine, 1-2-3 Kasumi, Minami-ku, Hiroshima 734, Japan

Abstract

p53 mutations in adenocarcinomas of the gallbladderwere analyzed by deoxynucleotide sequencing of the gene.of 23 cases, 16 (70%) harbored 18 missense mutations inexon 5, 6, or 8 of the p53 gene. The characteristics of the

p53 mutation spectrum observed in gallbladdercarcinomas were (a) frequent mutations at an A:T pair[10 (55%) of 18 mutations], (b) high transversionincidence [12 (66%) of 18 mutations], and (c) only onemutation at the CpG site. The immunohistochemicalstudy revealed that 36 (55%) of 65 cases showed an

abnormal accumulation of p53 hnmunoreactivity, and 12(52%) of 23 cases had p2! expression. No statisticalcorrelation was observed between p53 and p21immunoreactivity. These results suggest that p5.3mutations may confer the carcmogenesis of theadenocarcinoma of the gallbladder with the specificmutation spectrum of p53.

Introduction

It is known that the p.53 tumor suppressor gene product plays apivotal robe in normal cell growth and differentiation (1, 2).

Inactivation of the p53 gene by allelic loss and point mutation

have been found in a variety of human malignancies (3, 4). Wealso have demonstrated that allele loss and mutation of the p53gene are frequently associated with gastric cancer and occur inan early stage of human stomach carcinogenesis (5-7). Differ-

ences in the deoxynucleotide base-substitution spectrum of p53mutation have been clarified in several malignancies (8). Tran-sitions, especially at CpG dinucleotides, predominate in cob-

rectal carcinomas (9) and brain tumors (3), whereas G-�Ttransversions are frequently found in breast (3) and esophagealcarcinomas (10). Selective G-�T transversions at codon 249

are reported in hepatocellular carcinomas (1 1). Moreover, we

found that the characteristics of the p53 mutation spectrum

Received 1 1/30/95; revised 3/1/96; accepted 3/4/96.

The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement in

accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

I This work was supported in part by Grants-in-Aid for Cancer Research from the

Ministry of Education. Science, and Culture of Japan and from the Ministry ofHealth and Welfare of Japan.

2 To whom requests for reprints should be addressed. Phone: +81-82-257-5147;

Fax: +81-82-257-5149.

observed in primary gastric carcinomas were (a) frequent mu-

tation at an A:T pair and (b) high transversion incidence (6).The epidemiological significance of these accumulating muta-

tional spectra of the p53 gene is that specific mutagenic effects

of exogenous or endogenous carcinogens may be linked withthe risk factors of these malignancies (12).

Previous studies demonstrated that the incidence of gall-bladder carcinoma was more frequent in Japan than in the West(13). Although accumulations of abnormal p53 product havebeen reported to be seen often in gallbladder carcinomas

(14, 15), there is almost no analysis for p53 mutations per-formed directly by deoxynucleotide sequencing. The aims ofthis study are to examine p53 mutations in adenocarcinomas ofthe gallbladder and to determine whether p53 mutation or

accumulation correlates with the expression of p2 1 protein,

which is induced by wild-type p53.

Materials and Methods

Tissues. A total of 65 surgically resected adenocarcinomas ofthe gallbladder were used. Nineteen patients were men, and 46were women. The tissues were fixed routinely in 10% formalin

and embedded in paraffin wax. The definitions of histologicalclassification and stage grouping were made according to theWHO International Histological Typing of Tumors (16) and the

International Union against Cancer (17), respectively. Informedconsent was obtained from all subjects.

DNA Preparation. Genomic DNA was prepared from formalin-fixed and paraffin-embedded tissue by the proteinase K-phenol-

chloroform extraction method (18) with some modifications.Briefly, paraffin blocks were sectioned at 10 �m in thickness and

attached to glass slides, and then tumor parts were excised fromeach specimen under a microscope. Paraffin was eliminated bythree extractions with xylene, tissues were then immersed in eth-anol and incubated in 500 p1 of lysis buffer [50 most Tris-HCI (pH9.0) containing proteinase K to a final concentration of 0.5

mg/mb] at 55#{176}Cfor 8 h. After phenol-chloroform extraction,genomic DNA was precipitated with ethanol. All possibleprecautions were taken to avoid contamination.

DNA Sequencing. Genomic DNA was dissolved in a totalvolume of 50 �l of solution containing 1 X PCR buffer, 0.2 mMof each deoxynucbeotide tniphosphate, I ,.LM of each primer, and

2.5 units of TaqI polymerase. Templates were denatured for 5

mm at 94#{176}Cfollowed by 35 cycles of PCR with incubations of

2 mm at 55#{176}C,2 mm at 72#{176}C,and 1 mm at 94#{176}C.Primerscovering conserved portions of exons 5-8 of the human p53

gene for the PCR amplification of the p53 gene used in thisstudy were as follows: exon 5, 5’-TCCTCTFCCTACAG-TACTC-3’ and 5’-CAGCTGCTCACCATCGCTA-3’; exon 6,

5’-CACTGArfGCTCTFAGGTCT-3’ and 5’-AGTFGCAA-ACCAGACCTCAG-3’; exon 7, 5’-CTFGCCACAGGTCTC-CCCAA-3’ and 5’-AGGGGTCAGCGGCAAGCAGA-3’; and

exon 8, 5’-TfGGGAGTAGATGGAGCCTG-3’ and 5’-CT-GCTFGCTFACCTCGCTFA-3’. All amplifications included

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462 p53 Mutations in Gallbladder Cancer

Ta ble I Mutations of the p53 gene detected by sequencing analysis in 23 hu man adenocarc inomas of the gallbladder

Immunohistochemieal

Case Age SexHistological

typeStage”

Existence of

gallstonestudy

p53 p21

Affected

codon

Nucleotide

substitution

Amino acid

changeLOH

I 63 F Pap’ III - - + 190 CCT-aCCC Pro-aPro -

201

277

TFG-STGG

TGT-s1TI’

Cys-sTrp

Cys-aArg

2 4 1 M Pap I - + + + - I 37 CTG-aCTI’ Phe-sPhe -

145

146

148

CTG-*CTC

TGG-*TCG

GAT-+AAT

Leu-sLeu

Trp-�Ser

Asp-aAsn

3 55 F Pap III - + + + - I 66

190

GTC-SGAC

CCT-aCCG

Val-aAsp

Pro-sPro

-

4 74 M Pap II - - + No point mutation -

5 54 F Pap III - + + + 201 11’G-*TCG Trp-sSer -

6 75 M Pap I - + ++ 270 ‘ITI’-sTI’G Phe-.Leu -

7 74 F Pap I - + + - 190 CCT-sACT Pro-sThr -

8 56 F Pap II - + + + - 163 TAC-*CAC Try-sArg -

9 55 F Pap II + + + + - 202 CGT-aCAT Arg-sHis +

10 74 F Pap III + - - 276 GCC-SGT1’ Ala-�Val -

I I 76 F Pap III + - - No point mutation -

12 53 F Pap III + - - - No point mutation -

13 71 F Well IV + +++ + 140 ACC-’CCC Thy-aPro -

14 64 F Well IV - + + + + 145

306

CTG-aGTG

CGA-*AGG

Leu-sVal

Arg-aArg

X”

IS 59 F Well I - - - 268 GAA--sGTA Glu-’Val -

16 61 F Well III + - + + No point mutation -

17

I 8

77

76

F

F

Well

Poor

I

IV

+

-

+ -

+ + +

147

277

304

GTI’-*GGT

TGT-STGC

ACT-aCCT

Val-aGly

Cys-aCys

Thr-sPro

-

-

19 74 F Poor I + - + + No point mutation X

20 65 F Poor I + - - 278 CCT-SGCT Pro-sAla +

21 65 M Poor IV - - + - No point mutation X

22 70 M Poor IV - - + - No point mutation -

23 64 F Poor IV + + + + + 193 CAT-*CGT His-’Arg -

a Histological typing was done according to Ref. 16.

‘, Staging was done according to Ref. 17.

, Pap. papillary; Well, well-differentiated; Poor, poorly differentiated.

,I Not an informative case.

negative controls featuring the amplification mixture and

deionized water added in place of template DNA.The amplified PCR products were separated by electro-

phoresis with low-melting-point agarose and purified usingWizard PCR Prep (Promega, Madison, WI). Identification ofmutation in the p53 gene was determined by direct sequencingof DNAs, using a Circum Vent Thermal Cycle Dideoxy DNA

Sequencing Kit (New England Biolabs, Beverly, MA). TheDNA preparation, PCR, and sequencing reaction were repeated

at least three times to confirm the results.

PCR-RFLP.3 Allelic boss was examined using the PCR-RFLPtechnique with two different polymorphisms within p53. One

marker was AccII (19), and the other was BamHI4 polymor-phism within the p.53 gene. Primers for this study were thefollowing: Acdll, 5’-TCTGGGAAGGGACAGAAGATGAC-3’

and 5’-TfGCCGTCCCAAGCAATGGATGA-3’; BamHl, 5’-

GAAGAGCCFCGGUATGGGTATACA-3’ and 5’-TCAGA-

AGGAAGTAGGAAGGACTCAG-3’.

Immunohistochemistry. p53 and p21 proteins were detectedimmunohistochemically by means of the avidin-biotin-peroxi-

dase complex method using 10% formaldehyde-fixed, paraffin-

embedded sections. Several blocks from each case were used

for im.munohistochemical staining. A mouse monoclonal anti-body against human p53 protein (DO-7, Novocastra Laborato-

ries, Newcastle, UK) and a mouse monoclonal antibody againstp21 protein (187, Santa Cruz Biotechnology, Inc., Santa Cruz,CA) were used as the primary antibodies at the 1 : 100 and 1:300dilutions, respectively. The immunoreactivity was graded as -

to + + + according to the number of cells stained and intensityof the reaction in individual cells. Grades were defined asfollows: -, almost no positive cells; +, 10-30% of tumor cellsshowed positive immunoreactivity; + +, 30-60% of tumor

cells showed moderate immunoreactivity and/or 10-30% of

tumor cells showed strong immunoreactivity; + + + , more than60% of tumor cells showed strong immunoreactivity.

Statistics. Statistical analysis was performed using the Kendalltau b correlation analysis, the signed rank test, the generalizedWilcoxon test, the Kaplan-Meier model, and the Cox propor-tional hazards model. A probability of P < 0.05 was considered

statistically significant.

3 The abbreviations used are: RFLP. restriction fragment length polymorphism;

LOH, loss of heterozygosity.4 This information is deposited as G00-029-684 with Genome Data Base (1991);

H. Saito, personal communication.

Results

DNA Sequencing Analysis. Of 23 cases, 16 (70%) harbored

I 8 missense mutations in exon 5, 6, or 8 of the p53 gene, asshown in Table 1 . All cases with alterations in p53 gene had



Wild type Case No. 10Fig. 2. In case 2. missense mutations at codon 146 (TGG-. TCG. Trp-�St’r)

and codon 148 (GAT-”AAT. Asp-sAsn) were found to be close to silent mutation

at codon /45 (CTG-’ CTC. Leu--sLeu).

codon276

� 0 I GCC-GTF. � I (Ala-’Val)

�1

Table 2 Spectrum of p53 mutations in adenocarcinomas of the gallbladder

Substitutions No. of mutations

G:C-�A:T

G:C-’T:A

G:C-aC:G

A:T-sT:A

A:T-’G:C

A:T-”C:G

3 ( I 7�)

2(113)

3(l7’7f)

15’/)

3(l7e%)

6 (33%)

Total 18(100%)

b

Fig. I. Examples of sequencing reactions demonstrating p53 gene mutation inadenocarcinomas of the gallbladder. a, in case 17, there was a missense mutation

at codon 147 (G7T-”GGT, Val-sGlv). b, in case 10, missense mutation by

substitution of two bases was detected at codon 276 (GCC-sGl7’. Ala-�Val). 220bp134bp-

75bp- -

-�)0bp-S9bp-3 lbp

Cancer Epidemiology, Biomarkers & Prevention 463

a

Wildtype Case No.17ACGT ACGT

codon 147

�GU-.GGT(val-’c;Iy)

Case No.2Wild typeACGT ACGT

‘.‘� �

� 4 � �uI.aI GAT-’AAT (Asp-’Aan)

codoiil48

4.ul�!� a- codonl46� � ITOO-’TCG (Trp-Scr), i codonl45�. � CTG-CTC (�-.�)

-�

.� I_m� �

ACGT

#{149}d� �

A COT

missense mutations, resulting in amino acid substitutions (Fig.1). Missense mutations were detected in 10 (83%) of 12 pap-illary adenocarcinomas, in 3 (60%) of 5 webb-differentiatedadenocarcinomas, and in 3 (50%) of six poorly differentiatedadenocarcinomas, respectively. Two papillary adenocarcino-

mas each showed two missense mutations (cases 1 and 2). Inaddition, six of the base substitutions were without amino acid

substitution (silent mutation). Silent mutations were found to be

near missense mutational points (Fig. 2, case 2). Silent mutationitself does not affect p53 protein. But it was accompanied withmissense mutation in this study; therefore, its occurrence maybe related to genetic instability. Statistically, there was nocorrelation between p53 mutations and clinicopathological fea-tures, including histology and clinical stage (data not shown).

The p53 mutational spectrum of gallbladder carcinoma

observed in this study is summarized in Table 2. One (5%) wasA:T-�T:A transversion, three (17%) were A:T-’G:C transi-

tions, and six (33%) were A:T-*C:G transversions; therefore,10 (55%) of 18 mutations were detected at an A:T pair. In

addition, transversions were found frequently in gallbladderadenocarcinomas studied [12 (66%) of 18 mutations: G:C toT:A, 11%; G:C to C:G, 17%; A:T to T:A, 5%; A:T to C:G,33%]. Mutation at CpG dinucleotide was observed in only one

Cascl8 Cascl9 Casc2()Marker N T N T N T

Fig. 3. PCR-RFLP analysis of 3’ untranslated region of the p53 gene. (‘ase 18

showed three bands in both the normal (N) and tumor (1) regions; therefore, this

was a negative case. ease 19 showed only one band (90 bp). and this case was

not informative. In aise 20, LOH was detected because of deletion of two bands

(59 and 31 bp) in only tumor region. The lowest bands at all lanes were from PCR

primer.

case (case 9) at codon 202. No CpG-�TpG transition at codons

175, 245, 248, 273, and 282 was found. Seven (39%) missensemutations were found in exon 5, five (28%) in exon 6, andsix (33%) in exon 8. In contrast, there was no mutation inexon 7. Of 10 cases complicated with gallstones, 6 showed

p53 mutation.

PCR-RFLP. We performed PCR-RFLP analysis for LOH de-tection at the p53 locus. LOH was detected in 2 (10%) of 20informative cases by BamHI polymorphism (Fig. 3), but there

was no LOH by AccII polymorphism. This frequency of LOHwas lower than that of other malignant tumors (5). Two cases



.-

#{149}e�.,,.J . .

l�-%� � .,

� �.

‘!�s �.

�,. SW

a � �, � .,� -‘� #{149}� I� �

I � � ,. ,

.�!

464 p53 Mutations in Gallbladder Cancer

Fig. 4. Immunostaining of p53 protein in human gallbladder carcinoma. Well-

differentiated tubular adenocarcinoma at X 00 (case 13). Many tumor cells were

positive l�r p53 protein within the nuclei.

Ta/’le .1 Immunohistochemical detection of p53 protein in human

adenocarcinomas of the gallbladder

Histological type’ Positive casesp53 inimunoreactivity

+ ++ +++

Pap” 9/21 (43%) 4 2 3

Well 14/25(56%) 2 I 11

Mod 4/5 (80%) 2 2 0

Poor 9/14(64%) 3 4 2

Total )P = 0.3626’ ) 36/65 (56%) 8 9 16

‘, Histological typing was done according to Ref. 16.

‘, Pap. papillary. Well. mod, poor; well-. moderately. and poorly differentiated.

respectively.

, Significant correlation was not detected.

(cases 9 and 20) with LOH at p53 locus had a missensemutation of the gene.

Immunohistochemical Analysis. Of 65 adenocarcinomas ofthe gallbladder, 36 (55%) showed positive immunoreactivity

for p53 protein, regardless of histological type and staging. Inall positive cases, p53 immunoreactivities were confined to thenuclei of the tumor cells (Fig. 4).

There was no significant correlation between the expres-

sion of p53 protein and histological type (Table 3). No obviouscorrelation was observed between p53 immunoreactivity andother clinical factors determined according to TNM classifica-

tion (data not shown). In addition, there was no statistical

relationship between survival rate of the gallbladder carcinomapatients and expression of p53 protein (data not shown). Fourof 16 cases in which mutation could be detected by the DNAsequencing technique did not show p53 immunoreactivity

(Table 1).We next examined whether p21 expression is correlated

with p53 expression. p21 (20), the expression of which is

directly induced by wild-type p53, is an important mediator ofp53-dependent tumor growth suppression (2 1 ). Twelve (52%)

of 23 cases showed p2 1 immunoreactivity, which was seen

mainly in the nuclei of the tumor cells. The expression of p21immunoreactivity displayed heterogeneity of that level withinthe same tumor. p21 immunoreactivity was not related to anyclinicopathological factors (data not shown).

-‘ - .. � . . -.�.

, ,.‘- ‘5,

,�&, S �� . . .. . .-...-.�. ..-. .... . ,�‘.4�!b .

..;�z�:1;�’-’�-’ S �. �� .�! � #{149}�‘�v

��:‘ � � *u�,t.S� .

. � . . � . .

,,‘

)� � � ., -

‘‘)-l (�5 t:4,�(

e$� �<p�’��0 �s p� ,

a � :: � � � � d: z� � � � � �

b

‘.5 4 i:t;�,� �

, � ,,. ‘ . ..�

. � %..., .4,,.

#{163}�l�4� #{149} 2 . . � �%� . . - ..

I’4�� ‘� � ... -..‘�

. is �1. � � � �‘ ‘5- 4� �b-%..-. � �� :. ‘*�,�‘:.

: � ... / -a’- � � � ‘k��#{149}

#{149}:� �:� �i!z�r(�! &:.. � ‘ � �414�ti�l� ..�. #{149}�. . . .. (7.”, . -S.”

S � �‘‘ � .‘.,. . w’ . -

. S �‘ � �

Fig. 5. Immunostaining of p2 1 and p53 protein in human gallbladder carcinoma

(case 14, well-differentiated adenocarcinoma). p21 immunoreactivity (a) and p53

immunoreactivity (h) were detected mainly within the nuclei of tumor cells at the

same lesion. X 1(8).

Recently, it was reported that there is a strong negativecorrelation between the presence of p53 mutations and p21

expression (22). We expected that there would be a negativecorrelation between p21 and p53 expression of the gallbladder

carcinomas. However, six cases with p21 expression showedp53 immunoreactivity, whereas five cases did not show either

p21 or p53 immunoreactivity. In some cases, expressions ofboth were observed in the same area (Fig. 5). In this study, it

was not clear whether there was any relationship between p21and p53 expression.

Discussion

In gallbladder carcinoma, little is known about the molecularmechanism existing behind the tumor progression and metas-

tasis. Recently, we reported that decreased expression of nm23may play an important role in local invasion of this malignancy

(23). Some investigators have suggested that p53 mutation was

detected in high frequency with the use of immunohistochem-icab methods (70-90%, Refs. 14 and 15). However, there has

been only one report (24) on the p.53 mutation in gallbladdercarcinoma by the deoxynucleotide sequencing method. To our



Cancer Epidemiology, Biomarkers & Prevention 465

knowledge, this is the first detailed report of the analysis of p53mutations in adenocarcinomas of the gallbladder by de-

oxynucleotide sequencing.It has been described that overexpression of p53 protein

correlates with the existence of missense mutations of the gene(25). From this point of view, the frequency of p53 missense

mutations in gallbladder carcinomas reported previously byTakagi et a!. (24) was quite bow in comparison with the fre-

quency of p53 overexpression by immunohistochemistry asreported previously (70-90%; Refs. 14 and 15) as well as that

found in the present study (50%). On the other hand, the highfrequency of p53 missense mutations (70%) correlated wellwith the frequency of abnormal accumulation of p53 proteinfound in this study. However, 4 of 16 gallbladder adenocarci-

nomas that were found to have missense mutations by the DNA

sequencing technique were found negative for p53 protein

immunohistochemically. This might be due to the degradationof p53 protein irritated by bile. Therefore, DNA sequencing

should be performed for precise identification ofp53 mutations

in this malignancy.Many investigators have reported that most p53 mutations

have occurred in the regions of the gene that are highly con-served through evolution and presumably of functional impor-tance, primarily between exons 5 and 8 (codons 126-306) (3).In our study, all 18 mutations were found in exons 5-8. Afrequent mutational point, a so-cabled “hot spot,” has beenreported in various tumors (4, 26). However, this study did not

indicate the existence of a hot spot in gallbladder adenocarci-noma.

Several authors have proposed that p53 mutations are

associated with the progression of various cancers (26). Point

mutations of the p53 gene are observed frequently at the CpGdinucleotide site (4). Conversely, p53 mutation at the CpG

dinucleotide site is a rare event of esophageal cancer (8). Takentogether, the pattern of p53 mutations in tumor differs depend-ing on cancer type, suggesting that mutational type may reflect

the mutagen, which may cause specific base changes, some-times of specific p53 codons (12). For example, benzo(a)pyreneis a mutagen in cigarette smoke that causes G-�T transversionsin bronchogenic carcinoma (27). In contrast, in the gastriccarcinomas and adenomas, there were no specific changes in

the p53 gene, but mutations at the A:T pair occurred frequently

(6, 28).The present study also revealed that a high incidence

(55%) of mutation at A:T pair was observed in adenocarcino-mas of the gallbladder. Especially, of 18 mutations, 3 (17%)

were A:T-+G:C transitions, and 6 (33%) were A:T-�C:G

transversions. Mutation at the CpG dinucbeotide site was de-tected in one case at codon 202, which is not a hot spot. As

discussed for esophageal cancer by Holbstein et ab. (8, 10), ahigh incidence ofp53 mutation at an A:T pair occurred by DNA

depurination caused by irritants to mucosa, including ethanol orurethan contained in alcoholic beverages. In the case of gall-bladder carcinoma, environmental factors or chemical carcin-ogens have not yet been determined. In this context, our resultsdo suggest the involvement of some specific agent in bile, e.g.,

deoxycholic acid, lithocholic acid, and cholesterol, which in-tates the mucosa of the gallbladder. However, to date, there hasbeen no report that any component of bile itself is directly

implicated in carcinogenesis. In addition, we investigatedwhether the existence of gallstones is rebated to p53 mutation,but there was no significant statistical relationship between p53

mutation and complications with gallstones.In contrast to a high p53 mutation rate, frequency of LOH

at p53 was very low (10%). In most malignant tumors, LOH at

p53 occurs more frequently. We reported that LOH on I 7pl 3.1was observed in more than 60% of gastric carcinomas, regard-

less of histological type (5). At least, as each case showingLOH at the p53 locus had a missense mutation, the mechanism

ofp53 inactivation may be related to both mutation and LOH.p21 (sdil/WAF1/CJPI), whose product binds to cyclin-

cyclin-dependent kinase complexes and inhibits the function ofcyclin-dependent kinases (29), encodes the wild-type p53-in-

ducibbe transcript in mammalian cells (21). Recently, it wasreported that overexpression of p2/ suppresses proliferation

and cell growth of tumors in vitro (30). Oz#{231}eliket a!. (22)suggested that p53 mutation may reduce the ability of p53 toinduce p21 in vivo. Taken together, we anticipated that gall-

bladder carcinomas would show no or lower levels of p21

expression. However, p21 expression was detected in 12 (52%)of 23 cases immunohistochemically [especially, + + positive

cases were 7 (31%); Table I]. In addition, expression of bothp21 and p53 was detected in six cases. Therefore, it was

difficult to estimate whether there is a significant correlationbetween the expression of p21 and p53. Additional study with

an increased number of cases on this issue would be required.

In conclusion, the data presented here indicate that p53

gene mutations with the specific p53 mutation spectrum isimplicated in the development of gallbladder carcinomas.

Acknowledgments

We thank Drs. M. Itoh and K. Hanada for comments on the manuscript. Dr. M.

Yamamoto and Dr. F. Shimamoto for histological interpretation. and Dr. M.

Hiraoka for statistical analysis.

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1996;5:461-466. Cancer Epidemiol Biomarkers Prev K Fujii, H Yokozaki, W Yasui, et al. the gallbladder.High frequency of p53 gene mutation in adenocarcinomas of

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