p53 is frequently mutated in barrett’ s metaplasia of the ... · in be metaplasia. formalin-fixed...

Vol. 5, 559-565, July 1996 Cancer Epidemiology, Biomarkers & Prevention 559

p53 Is Frequently Mutated in Barrett’ s Metaplasia of the

Intestinal Type’

Paola Campomenosi, Massimo Conio, Massimo Bogliolo,Stefania Urbini, Paola Assereto, Anna Aprile,Paola Monti, Hugo Aste, Gabnella Lapertosa,Alberto Inga, Angelo Abbondandolo, andGilberto Fronza2

Centre for the Study of Tumors of Environmental Origins-Mutagenesis

Laboratory [P. C.. M. B.. S. U.. P. A., A. Ap.. P. M., A. I., A. Ab., G. F.l and

Gastroenterological Unit lM. C., H. Al. National Institute for Cancer Research(1ST) Largo R. Benzi 10. 16132 Genoa; and Chairs of Anatomical Pathology

1G. L.l and Genetics A. Ah.I, University of Genoa, Genoa. Italy

Abstract

Barrett’s Esophagus (BE) is a complication ofgastroesophageal reflux in which the normal squamousepithelium of the lower esophagus is replaced bymetaplastic tissue. The clinical significance of thiscondition is the associated predisposition toadenocarcinomas (ADCs). Three types of BE have beencharacterized: the gastric fundic (F) type, the gastriccardial (C) type, and the intestinal (I) type. The latter isthe most closely associated with the development ofADCs; the causes of this bias remain unknown. Todetermine whether p53 and/or K-ras gene alterations (a)are present in preneoplastic lesions and (b) are associatedwith a specific histotype, we performed PCR-baseddenaturing gradient gel electrophoresis (DGGE) analysisof exon 1 (codons 12-13) of K-ras gene and of exons 5-8

of the p53 gene in biopsies obtained from 30 patients withBE of the I type (9 patients), combined I type (I + C ± F;

10 patients) and non-I type (C, F, or C + F; 1 1 patients).None of the cases under study revealed K-ras mutations,whereas biopsies from 12 patients showed at least one p53DGGE variant. Four patients showed the exact same

variants in leukocytes also (polymorphisms), whereaseight cases revealed specific DGGE variants only inbiopsies. The molecular characterization of these variantsrevealed that four of them showed a single base pair

substitution, and four showed multiple mutations. Of 17somatic mutations, all but 1 were base pair substitutionslocated mainly in exons 7 and 8. The majority of thesemutations were GC targeted (13 of 16; 81%), 54% (7 of13) of which were transitions occurring at CpG sites. Allsomatic mutations were found in BE with at least one Icomponent. The association with the histotype was

Received 1/4/96; revised 4/3/96; accepted 4/8/96.

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 I 8 U.S.C. Section 1 734 solely to indicate this fact.

I This work was partially supported by Contract CHRX-CT94-058l from the

Commission of the European Communities and by the Italian Association for

Cancer Research (AIRC).2 To whom requests for reprints should be addressed.

statistically significant (P < 0.03; pure I type versus non-Itype; P < 0.04, combined I type versus non-I type;Fisher’s exact test). Loss of heterozygosity in the vicinityof the p53 locus was evaluated by PCR using a highlypolymorphic variable number of tandem repeats markeron 25 out of 30 cases. Ninety-two % of the cases analyzedwere informative, and none of them showed LOH. Inconclusion, we showed that p53 mutations are frequentlyobserved in specimens from BE patients of the I-type,whereas no involvement of K-ras (exon 1) mutationalactivation was observed. In light of the key roles that thep53 protein plays in controlling cell cycle and celldiploidy, this result may suggest why this type ofmetaplasia is the most closely associated to thedevelopment of ADCs.

Introduction

BE3 is a complication of GER in which the normal squamousepithelium of the lower esophagus is replaced by metaplastic

tissue. The clinical significance of this condition is the associ-ated predisposition to ADCs. Endoscopy reveals that 10% and

1% of patients with symptoms of chronic GER are affected byBE or ADCs, respectively. Individuals with BE have a risk ofdeveloping esophageal ADC 30-I 25 times greater than the

general population. Three types of BE have been characterized:the gastric fundic (F) type, the gastric cardial (C) type, and theintestinal (I) type. The latter is the most closely associated withthe development of ADCs (1-3), and the causes of this biasremain unknown.

Barren’s associated ADCs develop by a multistep processthat can be recognized morphologically as a metaplasia -�

dysplasia -* cancer sequence (Ref. 4 and references citedtherein). Diagnosis of dysplasia, especially low-grade dyspla-sia, is far from being conclusive because of the subjective

nature of histological interpretation and low intra- and inter-observer agreement. The neoplastic progression has been de-scribed more objectively through the determination of molec-

ular markers. With DNA content flow cytometry, thehistological progression of BE has been shown to parallel the

changes in DNA content indicating a diploid/aneuploid Se-quence (4-6). Aneuploidy might be triggered by the functionalinactivation of the tumor suppressor gene p53. Its gene productis a 53-kDa nuclear phosphoprotein. which acts as a transcrip-tional activator of cell proliferation inhibitor genes (7). Several

studies suggest that p53 may be required for the maintenance ofdiploidy because loss or inactivation of p53 can be associatedwith tetraploidy or aneuploidy (8-10). Mutational inactivation

3 The abbreviations used are: BE, Barrett’s esophagus; GER. gastroesophageal

reflux; ADC, adenocarcinoma; F, gastric fundic; C. gastric cardial; I. intestinal;

DGGE, denaturing gradient gel electrophoresis; LOH. loss of heterozygosity;

VNTR, variable number of tandem repeats.

Association for Cancer Research. by guest on November 2, 2020. Copyright 1996 Americanhttps://bloodcancerdiscov.aacrjournals.orgDownloaded from

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560 p53 Mutations tn Barrett’s Esophagus

of the p53 gene, a frequent feature of many human neoplasms,occurring as either early or late event during specific carcino-

genic processes ( 1 1 ), has been reported for BE-associatedADCs. In BE. p53 mutations have been characterized especially

in dysplastic BE or Barrett’s ADCs (8, 9, 12, 13). Different

authors, using an immunohistochemical approach, observed anincreasing incidence of p53 protein accumulation along thedifferent stages of the carcinogenic process (14-18).

K-ras proto-oncogene activation by point mutation may be

another factor contributing to the loss of cell cycle control. Rasproteins are key transducers of extracellular stimuli from theplasma membrane to the nucleus ( 19, 20). Mutations in theK-nis gene are observed in many human cancers (21) and areinvolved in both early and late stages of tumorigenesis. Using

an immunohistochemical approach, Jankowski et a!. (22) foundsimilar incidences (20%) of ras overexpression in metaplasia

and ADC, suggesting that alterations at the ras loci could be

early events but are not particularly associated with advancedstages in the BE progression. To our knowledge, no direct

studies of ras gene alterations in BE metaplasia are present inthe literature.

In the present study, we addressed the questions ofwhether p53 and/or K-ras gene alterations (a) are present inpreneoplastic lesions and (b) are associated with a specifichistotype. For this purpose, we screened for K-ras and p53 genemutations endoscopic biopsies histologically confirmed as BEmetaplasia, obtained from 30 patients undergoing endoscopicsurveillance (23), by applying the highly sensitive PCR-basedDGGE and sequencing methods (24, 25).

Patients and Methods

Patients. Thirty patients (27 males and 3 females; ages 36-85

years: mean age, 6 1 ) undergoing upper gastrointestinal endos-copy for upper-digestive tract symptoms, were diagnosed to be

affected by histologically confirmed BE (23). Endoscopically,BE was defined as the presence of velvety red gastric-likemucosa lining the distal esophagus for at least 3 cm between thegastroesophageal junction and the proximal displaced squamo-columnar junction (Z-line). Four to six biopsies per patient,taken from the metaplastic epithelium, were immediately fro-zen in liquid nitrogen. From each patient, 10 ml of blood werewithdrawn as control tissue. The analysis at the molecular level

was usually performed on pooled biopsies. In a few cases,

however, biopsies were analyzed separately. In the latter situ-

ation, the majority of cases (but not all) were concordant. Thissuggests that p53 mutations may be heterogeneously distributedin BE metaplasia. Formalin-fixed and paraffin-embedded ma-terial was histologically evaluated to characterize columnarepithelium as gastric type (C or F) and/or I type as describedpreviously (23). A single type ofmetaplasia was observed in 17patients, whereas in the remaining cases, more than one type of

metaplasia was observed. Biopsies were then classified as pureI type. combined I type (I + C ± F), and non-I type (C and/or

F; Ref. 23). Dysplasia was classified according to Schmidt et a!.

(26). No low- or high-grade dysplasia was found in any biopsy,but four cases showed indefinite dysplasia. In a follow-upranging from I to 3 years, none of the 30 cases progressed tohigh-grade dysplastic BE.

PCR Conditions. High molecular weight DNA was prepared

from biopsy and blood sample (control tissue) as describedpreviously (24). The region encompassing exons 5-8 of the p53

gene was selectively amplified using five pairs of meltingdomain specific primers. Primers and PCR conditions were asdescribed previously (24). The first exon of the K-ras gene was

amplified according to Pellegata et a!. (25). Briefly, for K-ras,PCR amplifications were performed using about 200 ng ofDNA, 15 pmol of each primer, 250 �M each dNTP, 50 mrvi KC1,10 msi Tris-HC1 (pH 8.3), 1 .5 m� MgCl2, and 1 .5 units of Taq

Polymerase (Promega, Madison, WI) in a final volume of 50;.tl. PCRs were performed in a MJPT-l00 thermal cycler (MJResearch, Inc., Watertown, MA) for 35 cycles. Each cycle

consisted of 1 mm at 95#{176}C,1 .5 mm at 55#{176}C,and 2 mm at 72#{176}C.Positive controls for K-ras analysis, consisting of DNA sam-ples extracted from four cell lines bearing known K-ras muta-

tions [SW 480 (codon 12: gGt-�gTt), SW 837 (codon 12Ggt-*Tgt), DLD-l (codon 13: gGc-’-�gAc), NIH�3T3* (NIH3T3 transformed with human mutated K-ras; codon 12:

Ggt-�Cgt)] were a generous gift from Dr. W. Giaretti (NationalInstitute for Research on Cancer, 1ST, Genoa, Italy).

DGGE Conditions. The PCR products obtained with the GC-

clamped ampliprimers were analyzed by DGGE. The DGGE

apparatus was from CBS Scientific Co. (Del Mar, CA). Paralleldenaturing gradient gels were prepared with 8% acrylamide-bisacrylamide (37: 1) in TAE [40 mrvi Tris acetate-l mrvi EDTA

(pH 8.0)1 and by varying denaturant concentrations (100%denaturant corresponds to 7 M urea and 40% formamide). Therange of the denaturing gradient was different and specific foreach amplified fragment (24, 25). Gels were run in TAE at60#{176}Cand 50 V for 16-20 h. After electrophoresis, the gels were

stained in ethidium bromide and photographed using Polaroidtype 55 films. DNA samples showing PCR-amplified fragmentswith altered DGGE profile were reanalyzed to confirm their

abnormal behavior, thus avoiding misinterpretation of possiblePCR artifacts.

DNA Sequencing. PCR products for sequencing were ob-tamed starting from either genomic DNA or eluted DGGEmutant homoduplex bands. The PCR products were cloned into

the plasmid pGEM-T (Promega, Madison, WI) according to themanufacturer’s instructions. The inserts of a series of independ-ent clones were characterized by PCR-DGGE analysis. Three tofive independent clones showing the same mutant homoduplexband pattern as the one obtained starting from genomic DNAwere then sequenced as previously described (24).

LOH. LOH in proximity to the p53 locus was evaluated byPCR using the highly polymorphic (VNTR) marker YNZ22(band Vlpl3.3) on 25 of 30 cases. Primers and PCR conditionswere those described by Batanian et a!. (27). For each case, 300

ng of DNA extracted from biopsies and blood sample (controltissue) were separately used as template for VNTR-specific

amplifications. PCR products were separated on a 2% agarosegel, stained with 0.5 p�g/ml ethidium bromide, and visualizedusing a UV transilluminator. LOH was considered to haveoccurred when one of the two alleles present in PCR productsobtained with leukocyte DNA was missing from PCR productsobtained from the biopsy DNA.

Results

We performed PCR-based DGGE analysis of exon 1 (codons12-13) of the K-ras gene and of exons 5 to 8 of the p53 genein biopsies obtained from 30 patients with BE of the pure I type(9 patients), combined I-type (I + C ± F; 10 patients) and non-I

type (C and/or F; 1 1 patients). Metaplastic tissue was observedin all samples, and no high-grade dysplasia was observed. Four

cases showed indefinite dysplasia. The screening allowed us toidentify DGGE variants, which were molecularly characterized

by sequencing.A typical result of PCR-based DGGE analysis on exon 1

of K-ras is shown in Fig. 1 . DNA extracted from four cell lines



Cancer Epidemiology, Biomarkers & Prevention 561

Fig. I. PCR-hased DGGE analysis on K-ras exon I from positive controls and

BE biopsies. The first four lanes on the left show DGGE variants of positive

controls (NIH 3T3, DLDI. StV837, and SW480 cell line DNA). Because SW480

atsd NIH-3T3’ are homozygous for the K-ras mutation, PCR products were

titixed. denatured, atsd renatured in presence of wild-type PCR products to induce

the fortoation of heteroduplexes. before DGGE analysis. Numbered lanes show

the wild-type DGGE pattem of biopsies DNA from nine BE patients (cases I , 3,5. 6. 10. 1 1. 12. 20. and 21 (. Homod. wt and mutant homoduplexes; heterod,

heteroduplexes.

[three human: SW480, SW837, and DLD-l, and a murine cellline (NIH 3T3) transformed with human mutated K-ras, re-

ferred as NIH 3T3*] carrying known mutations in either codon12 or codon I 3 of the human K-ras gene, were used as positivecontrols. No biopsy from BE patients showed any DGGEvariant (Table 1 ), whereas DGGE patterns of the positive con-

trol DNAs (Fig. I ) were consistent with the mutation reportedfor each of them. This result suggests that mutational activation

of K-nis oncogene, at least in codons 12 and 13, plays amarginal role (if any) in BE metaplasia.

Biopsies from 12 patients showed at least one p53 DGGE

variant (Table I ). All samples bearing a mutated p53 alleleshowed a DGGE band pattern that also contained the wild-type

allele. This may indicate either that cells with mutated p53 wereheterozygous for the observed mutation or that normal cells

carrying wild-type alleles were present in sample tissues fromwhich DNA was extracted. The relative intensity of mutantversus wild-type bands suggests that the mutated allele was

present in a considerable fraction of cells in the specimen(>25-35%). Four patients showed exactly the same variantsalso in leukocytes (control tissue), and the remaining eightcases revealed specific DGGE variants only in the biopsies. Fig.2 shows a typical result of PCR-based DGGE analysis on p53

exon 6 (A) or exon 7 (B). All somatic mutations were found inBE with at least one I component. The association with the

histotype was statistically significant (P < 0.03 for pure I-typeversus non-I type; P < 0.04 for combined I-type versus non-I

type: Fisher’s exact test).DGGE variants at the p53 locus were further characterized

at the molecular level (Table 2). Sequence analysis confirmedthe presence of a known polymorphism (Ref. 28; AT-�GCtransition at codon 213, resulting in no amino acid substitution)in three out of four cases in which the same variant was present

both in blood and biopsy (cases 3, 29. and 30). Case 28 showedan AT-SGC transition located in intron 6, 3 1 bp downstreamfrom the 3’ end of the exon 6. By applying a method able to

Table I Molecular characterization of biopsies from 30 BE patients for the

presence of K-ras and p53 mutations and LOH at the l7pl3 region

DGGEc variants” LOH I 7p I . I 3Case Sex/age Histotype -�-----� - number

ras p53 (alleles)”

Pure I (9 patients, 4 p53 mutations

1 M/46 I Mut NI’

2 M/75 I ND

3 M/SS I” Pol 2(lO.ll(

4 M/53 I” ND

S F/56 I Mut 2(1.3)

6 M/42 I 2(4.12

7 M/SS I Mut 2(3.6)

8 M/67 I Mut ND

9 M/36 I” 23,12

Combined (10 patients, 4 p53 mutations)

10 M/62 C+I 2(1.2)

11 MflO C+F+I” 2(4,11)

12 Mfll C+l Mut ND

13 M/73 C+I 2(5,11)

14 M/54 C+F+I 2(3.11)

15 M/68 C+I Mut 2(2.10)

16 M/67 C+F+I Mut NI

17 M/42 C+I Mut 22.3

18 M/77 C+I 24.9

19 M/63 C+I 24.12)

Non-I ( I I patients. no p53 mutations)

20 F/37 F 23,4

21 M/72 C 2(4.12)

22 M/6l C 2(4.10)

23 M/64 C+F 2(2.4)

24 M/6l C+F ND

25 M/70 F 2(4,9)

26 M185 C 24.ll)

27 F/46 C+F 2(2.11)

28 M/60 C Pol 2(3.5)

29 M/42 C Pol 2(4.12)

30 M/68 C Pol 2(3,10)

“ DGGE variant present in the DNA extracted from biopsy or from blood sample.

When biopsy and blood sample showed identical DGGE variant(s) the mutationwas considered to be of germinal origin (Pol, polymorphism) when the variant(s)

were present exclusively in the biopsy the mutation was of somatic origin (Mut).

b Number of alleles present in the samples analyzed; in parenthesis. number of

repeats of each allele.‘- NI, not informative; ND. not done.

‘I Indefinite dysplasia.

identify splice sites with a high degree of accuracy (29), weverified that this mutation was predicted not to create an alter-native splice site, suggesting that this gene alteration has no

consequence for p53 mRNA splicing and may represent a newpolymorphism.

The molecular characterization of the p53 DGGE variantspresent only in the biopsies from eight patients revealed that

four of them (cases 5, 7, 12, and 16) showed a single base pairsubstitution, whereas the remaining four showed multiple (2-5)mutations (Table 2). Of 17 somatic mutations, all but 1 ( + IT,

case 16) were base pair substitutions located mainly (82%) in

exons 7 and 8. The majority of these mutations were GC

targeted (13 of 16; 81%), 54% (7 of 13) of which were tran-sitions occurring at CpG sites. DNA sequencing results of p53DGGE variants from sample 12 (exon 6) and 15 (exons 7 and

8) are shown in Fig. 3.

Inactivation of the p53 gene is usually a two-hit phenom-

enon: point mutation in one allele and deletion in the other or.alternatively, point mutations in both alleles. LOH at the highlypolymorphic (VNTR) marker YNZ22 was evaluated in 25 out



ex6

12 812 Cl 13

h�emd[

H:

A

ex7

C2 c3 17 817 8 B8 7 B7

� � � -� _

B

562 p53 Mutations in Barrett’s Esophagus

All p53 mutations were in the sequence coding for the

Fig. 2. PCR-based DGGE analy-sis on p53 exon 6 (A) and exon 7

(B) from positive controls (Cl. C2.

and C3) and a series of biopsies

(cases 12 and 13 for exon 6; cases

17, 8, and 7 for exon 7) along with

their matching blood samples (B12.

817, B8, and B7). A, the DGGE

variant present in the biopsies of

case 12 is clearly absent in DNA

heIsrod extracted from blood (B12). Case

13 shows a wild-type DGGE pat-tern in the biopsy. CI, positive con-

trol (cGa > cAa. Arg2t3 > Gln). B,

similarly. DGGE variants present in

biopsies from patients /7, 8, and 7

are absent in the matching blood

samples (B17. 88. and B7). C2,

positive control (eGg > cAG,

Arg24#{176}> GIn); C3, positive control

(tAc > tGc, Tyr�34 > Cys). Ho-

mod, wt and mutant homoduplexes;

heterod, heteroduplexes.

Table 2 Molecu lar characterization o f p53 DGGE variants observed in BE biopsies

Case Sex/age Histotype Exon Base change Amino acid change CpG site

Polymorphysm (4 cases)

3 M/SS I” 6 cgA-’cgG Arg213-”Arg

28 M/60 C 6 ggA-sggG l6:+31”

29 M/42 C 6 cgA-*cgG 21

30 M/68 C 6 cgA-scgG Arg2tS��sArg

Mutations (8 cases. 17 mutational events observed)

I M/46 I 7

7

8

tAc-”tGc

aaC-#{176}aaT

Cgt-.Tgt

Tyr�34-’Cys

Asn235-’Ser

Arg273-sCys

-

-

+

S F/56 I 7 gGc-sgAc G1y245-sAsp -

7 M/SS I 7 Atc-”Ttc 11e255-+Phe -

8 M/67 I S

6

7

7

8

cGc-’cAc

Cga-sTga

gGc-sgAc

cGg-�cAg

Cgt-”Tgt

Arg’55-*His

Arg2t3-*Stop

G1y245-+Asp

Arg248-#{176}Gln

Arg273-#{176}Cys

+

+

-

+

+

12 M/7l C + I 6 Cag-+Tag Gln#{176}’2-�Stop -

IS M/68 C + I 7

7

8

tAc-’tGc

aaC-�aaT

Cgt-+Tgt

Tyr�34-’Cys

Asn235-’Ser

Arg273-*Cys

-

-

+

I 6 M/67 C + F + I 7 gac-*Tgac Asp259-*Stop -

I 7 M/42 C + I 7

8

gGc-sgAc

Cgt-+Tgt

Gly245-Asp

Arg273-’Cys

-

+

“ Indefinite dysplasia.

S Mutation located in intron 6, 3 1 bp downstreans from the 3 ‘ end of exon 6. This mutation was not predicted to create an alternative splice site. Thus, this gene alteration

may represent a new polymorphism.

of 30 cases. Batanian et a!. (27) described 12 types of alleles,characterized by different numbers of repeats. In the 25 cases

analyzed, we observed all these types of alleles but two (7 and8 repeats). LOH was considered to have occurred when one of

the two alleles present in PCR products obtained with leuko-

cytes was missing from PCR products obtained from the match-ing biopsy. With this criterion, 23 of 25 cases analyzed (92%)were informative, and none of them showed LOH (Table 1). Atypical result of this analysis is shown in Fig. 4.

Discussion

BE may be regarded as a model of an in viva multistep carci-nogenic process, in which the replacement of the normal squa-

mous epithelium of the lower esophagus with metaplastic tissue

represents its first step. In the hypothetical cascade of genetic

alterations leading to the formation and progression of BE to

ADCs, aneuploidy and p53 mutations seem to be early events

(3), both strongly associated with the appearance of dysplasia

(4, 8, 9, 18). In the present study, we determined that p53

mutations occurred in 27% (8 of 30) of patients with BE of the

I type in absence of dysplasia. Other authors, using mainly

immunohistochemical approaches, reported that in the absence

of dysplasia and/or in the presence of indefinite dysplasia, the

incidences of BE positive for p53 accumulation ranged from 0

to 25% (14-16, 18, 30, 31).



ag ct

casel5 casel5

exon 7 exon 8

agct agct

*c > I � G > A *

‘p � #{149}*c>TA>G* �

B CA

�‘�2 � � � �1


Fig. 3. Sequence analysis of

DGGE variants observed in biop-

sies from eases 12 and 15. A, C T

transition (Cag > Tag. sense

strand) at codon 192 resulting in

the substitution of a glutammine

with a stop codon (case 12). B.

double transition observed in

((iSP’ /.5. es-on 7 A > G (tAc > tGc

sense strand. Tyr�34 > Cys) and

C > T (aaC > aaT sense strand;

Asn’5 > Ser). C. third mutationobserved in case 15. exon 8

(Cgt > Tgt. antisense strand.

Arg273 > Cys).

case 12

exon 6

csJ

Fig. 4. Analysis of the LOH at the YNZ22 marker in biopsies (samples 16, 29,

22. and 30) and in the corresponding control tissue (JOB, 29B, 22B, and 30B). A

100-hp ladder was used as a DNA size marker. The kind of alleles present in each

sample are indicated in parentheses beside each pair of samples.

highly conserved domains (or amino acids) of the protein (32);

thus, they might all alter the biological functions of the p53protein. The presence of p53 mutations does not imply, how-

ever, that p53 functions were completely abolished, although itis known that some mutant p53 proteins, when present in aheterozygous state, behave in vivo in a dominant, negativefashion (33, 34). The features of the DGGE variants (three orfour bands) we observed, suggest the presence of both thewild-type and the mutated p53 alleles in the DNA extractedfrom the biopsy. Analysis of LOH at the highly polymorphiclocus YNZ22 (telomeric with respect to the p53 locus) revealedthat none of the informative cases showed LOH. Becausenormal cells are expected to be present in the biopsy, our

approach may have underestimated l7p LOH. However, this isin keeping with an early finding by Meltzer et a!. (35), who

found no l7p LOH in Barrett’s metaplasia. These results sug-

gest that p53 mutations may precede complete loss of p53functions in absence of dominant negative phenotype of the

mutant protein. Complete p53 inactivation might, nevertheless,

be hypothesized in half of the BE patients, in whom multiple

somatic p53 mutations in different exons were observed, ifthese mutations involved different alleles.

All somatic mutations were found in BE with at least oneI component, and the association with the histotype was statis-

tically significant (P < 0.03 for pure I type versus non-I-type;

P < 0.04 for combined I-type versus non-I type; Fisher’s exacttest). Reid et a!. (36) found that, in the absence of dysplasia, BE

of the I type but not BE of the gastric type showed the presenceof aneuploid cell populations. These results (Ref. 36; this study)

may give a molecular explanation to the clinical observationthat the I type metaplasia is the most closely associated withBE-associated tumor development. Hurlimann and Saraga (37),

studying the expression of p53 by immunohistochemistry, dem-

onstrated an association between p53 accumulation and I-typegastric cancers. More recently, Ranzani et a!. (38) elegantlyshowed that p53 gene mutations and protein nuclear accumu-

lation are early events in I-type gastric cancer but late events in

diffuse-type. All of these results (Refs. 36-38; this study) mayindicate that the intestinal metaplasia in the upper digestivetract is particularly prone to cancer-promoting gene alterations.

Why were p53 mutations only found in association withthe I-type metaplasia? It could be hypothesized that this meta-plasia is more exposed and/or less resistant to endogenous orexogenous mutagenic factor(s). BE results from long-lasting

GER and is accompanied by inflammatory processes. I-typemetaplasia is expected to be more sensitive than non-I type to

acid, an important component of GER. At the site of inflam-mation, a plethora of mutagenic leukocyte-derived oxidant free

radicals (39, 40) are delivered. Finally, some features of theintestinal metaplastic cells, such as high proliferation rates andthe ability to metabolically activate carcinogens (41), may also

contribute to the observed phenomenon.In light of the molecular epidemiology notion that a car-

cinogen leaves fingerprints on DNA (42), can we pinpoint apotential agent responsible for these mutations? The molecular

features of the p53 mutants observed in BE indicate that theputative mutagenic factor(s) specific for I-type metaplasia are



564 p53 Mutations in Barrett’s Esophagus

mainly GC-�AT inducer(s). The majority of these transitions

(7 of 13) occurred at CpG sites. A similar mutation profile hasbeen found in gastric ADC (43-45) and esophageal ADC (8, 9,13). It has been recently demonstrated that cytosines in the CpGdinucleotides present in p53 are methylated in vivo (5-methyl-cytosine; Ref. 46). It is known that 5-methylcytosine deamina-tion occurs frequently in the genome and that the repair of theresulting GrI’ mismatch is relatively inefficient (47). This may

suggest that some of the p53 mutations observed in BE had aspontaneous origin. The role of tobacco and alcohol in ADC on

BE is still debated (48-50). A high frequency of GC-�TA

transversions has been observed in squamous cell carcinomasof the esophagus (1 1) and in lung carcinomas, closely related tocigarette smoking (5 1 , I 1 ). The absence of such transversionsin Barrett’s metaplasia and in BE-associated ADC (Refs. 8, 9,

and 13; this work) gives a hint to tentatively exclude a muta-genic role for these two risk factors in the genesis of BE.

In conclusion, we showed that p53 mutations are fre-quently observed in biopsies from BE patients of the I type inthe absence of dysplasia, whereas no involvement of K-ras(exon 1) mutational activation was observed. In light of the keyroles that the p53 protein plays in controlling cell cycle and celldiploidy, this result suggests why this type of metaplasia is themost closely associated to the development of ADCs.

Acknowledgments

We thank Prof. A. J. Cameron for critical reading of the manuscript; and Drs. F.

Munizzi and M. Picasso (Gastrocnterology Unit, National Institute for Cancer

Research, Genoa, Italy), F. Morchi and F. Costa (Operative Unit of Gastroenter-ology. University of Pisa, Pisa, Italy), and P. Ravelli and R. Cestari (Chair of

Diagnostics and Endoscopic Surgery, University of Brescia, Brescia. Italy) for

having provided some of the clinical materials for this study.

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