[CANCER RESEARCIt 53, 5745 -574t), December t, 1993]

TP53 Gene Mutation Profile in Esophageal Squamous Cell Carcinomas 1

M. P. Audr6zet, M. Robaszkiewicz, B. Mercier, J. B. Nousbaum, J. P. Bail, E. Hardy, A. Volant, P. Lozac'h, J. F. Charles, H. Gou6rou, and C. F6rec Centre de Biogdndtique, C.D.T.S., BP 454, 29275, Brest Cedex, France [M. P. A., B. M, E. H., C. El; and Service d'Hdpato-Gastro-Entdrologie [M. R., J. B. N., H. G.], Service de Chirurgie G6ndrate et Digestive [J. P. B.. P. L., J. F. C.], and Service d'Anatomie Pathologique, C.H.U., Brest, France [A. V.]

A B S T R A C T

Esophageal squamous cell carcinoma is a form of cancer occurring most commonly in males, particularly those living in some areas of Asia, Africa, and western Europe. In some of these tumors, a sequence alter- ation has been identified in the coding region of the TP53 gene which is known to inactivate the tumor suppressor function of its product. Using a GC clamp (i.e., a GC rich domain) denaturing gradient gel electrophoresis assay we have been able to identify sequence modifications in 27 of the 32 tumor samples analyzed (84%). Most of the mutations occur in exon 6, a region of the gene which has not previously been reported as being a hot spot for the mutations of other cancers. Twelve of the mutations reported here have not been described in other types of tumors and these consist mostly of frameshift or splice mutations.

The distribution of mutations [transitions (45%), transversions (34%), and frameshift (21%)] suggests that the etiological contribution of geno- toxic factors might be complex and might associate different exogenous and endogenous mutagen exposures.

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

The T P 5 3 gene is probably the most commonly mutated gene in human cancers (1, 2). One of the several lines of evidence of T P 5 3

involvement in human malignancies is the loss of heterozygosity in 17p associated with mutations in the other T P 5 3 allele (3). The normal allele of this gene encodes a 375 amino acid nuclear phosphoprotein involved in the regulation of cell proliferation (4). It has been pro- posed also that the p 5 3 function can be inactived in a single allele, i.e., with a dominant negative effect (5). T P 5 3 genes contain five highly conserved domains which are the most frequent sites of mutations in

many human tumors (6, 7). Extensive analysis has shown about 1600 molecular defects in more than 35 cancer types (1, 2). Most of these mutations are somatic, but germline mutations have been described in the familial Li Fraumeni syndrome (8, 9). Mutated alleles of T P 5 3

have been found in solid tumors of the colon (10), lung (11), breast (12), ovary (13), brain (14), liver (15, 16), and in hematopoietic tissues (17). These various mutant alleles code for proteins that have altered growth-regulatory properties (3, 5). Interestingly, the pattern of point mutations in T P 5 3 gene varies in different human cancers and

each pattern appears to associate with the cancer of a particular organ (1, 2). It has also been observed that specific etiological agents can produce specific mutations. For example, the G --> T transversion in codon 249 of hepatocellular carcinomas can be the result of the

interaction of aflatoxin B with DNA in certain areas (18). Other chemical carcinogens such as nitrosamine are also able to produce a high incidence of organ-specific tumors each with a particular pattern of mutations in the T P 5 3 gene (19). Cancer of the esophagus occurs with a high frequency in certain regions of the world such as China, Iran, South Africa, and France (20). In western Europe and North America, the classical etiological factors of esophageal cancer are tobacco and alcohol consumption. Because in the western part of France, Brittany, the incidence rate of esophageal cancers is high

(27/100,000) (21), we have undertaken a systematic screening of point

Received 6/9/93; accepted 9/30/93. 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.

1 This work was supported by the Ligue Nationale contre le Cancer, Comit6 du Finist~re.

mutations in the T P 5 3 gene in a series of 32 esophageal squamous cell carcinomas in patients of a same ethnic origin. For this study, we have

used a GC clamp denaturing gradient gel electrophoresis assay to analyze the conserved domains of the gene and we have determined

the spectrum of mutations in the T P 5 3 gene. The particular profile described in this work might reflect the genotoxic effects of different

environmental factors in this type of cancer.

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

Patients. Thirty-two patients with an esophageal squamous cell carcinoma were included in this study. Most of them had history of smoking and excessive alcohol consumption (Table 1). For each patient, biopsy specimens were taken from the tumor, and whole blood samples were collected. DNA were prepared from peripherical leukocytes and from biopsy specimens after proteinase K digestion and phenol-chloroform extraction as described elsewhere.

All the patients were seen at the time of diagnosis and none had chemo- therapy or radiation therapy prior to the time the biopsies were taken from the tumor.

The tumors were staged according to the new TNM classification by the International Union Against Cancer (22, 23) (Table 1).

Primers. Eight couples of primers have been used to study exons 2-8 containing the 5 domains highly conserved through evolution, as follows:

exon 2, 2i 5GC (GC) - CT TGC AGC AGC CAG ACT GCC T 2i3 CGG GGT GGT GGC CTG CCC "IT (this PCR product explores codon 1 to 25);

Exons 2--4 (partial), 2i5 CAG GTG ACC CAG GGT TGG AAG 4e3GC (GC) - GGA AGG GAC AGA AGA TGA CAG (this PCR product explores codon 26--84);

Exon 4 (partial), 4i5GC (GC) - CC AGC AGC TCC TAC ACC GGC 4i3 GAA TCC CAA AGT TCC AAA CA (this PCR product explores codon 84-125);

Exon 5 (partial), 5i5GC (GC) - CT TTC AAC TCT GTC TCC 'ITC 5i3 CCT GGG CAA CCA GCC CTG TC (this PCR product explores codon 126-148)

5i5 TGG CCA AGA CCT GCC CTG TG 5i3GC (GC) - CC TGG GCA ACC AGC CCT GTC GT (this PCR product explores codon 148-186);

Exon 6, 6i5 CCC CAG GCC TCT AGT TCC TC 6i3GC (GC) - TG CTC ACC CGG AGG GCC ACT GA (this PCR product explores codon 187-224);

Exon 7, 7i5GC (GC) -T TGC CAC AGG TCT CCC CAA G 7i3 GGG CAC AGC AGG CCA GTG TG (this PCR product explores codon 225 to 261);

Exon 8, 8i5GC (GC) - GGA CAG GTA GGA CCT GAT TTC 8i3 AAT CTG AGG CAT AAC TGC AC (this PCR product explores codon 262-306).

The presence of (GC) in a primer indicates the position of a GC clamp.

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TP53 GENE MUTATIONS 1N ESOPHAGEAL SQUAMOUS CELL CARCINOMAS

Table 1 p53 mutations in our series of human esophageal carcinomas

Patient Sex Age

Drinking >80 g

alcohol/day

Tumor Extension

Smoking TNM Stage Exon Nucleic acid Event Mutation

(amino acid no.)

1 F 57 - 2 M 53 + 3 M 73 + 4 M 60 + 5 M 67 - 6 M 63 - 7 M 55 + 8 M 77 - 9 M 56 -

10 M 71 + i1 M 62 + 12 M 74 - 13 M 62 - 14 M 68 + 15 M 76 + 16 M 62 - 17 M 58 + 18 M 59 + 19 M 64 + 20 M 41 - 21 M 61 unknown 22 M 70 - 23 M 80 - 24 M 79 + 25 M 62 + 26 M 52 + 27 M 66 + 28 M 63 + 29 M 29 +

30 M 64 - 31 M 47 + 32 M 58 -

+ TnN1Mo III + TzN1M o liB + T3NoM o IIA 8 GAG --~ AAG + T1NoM o I 8 CCT --> TCT - T3N1M o III 6 CAT --, CGT + T4NoM o III 5 del GTCA + T3NoM o IIA 5 dupli ACATGACG + T4NaMo III + T3NoMo IIA 8 CCT --> TCT

8 CCT ---> ACT + TINoM o I intron 7 -2A ---, T + TINoM o I 8 CGT --> TGT + T3NoM o IIA intron 5 -2A ---> T + T3NoM o IIA 6 ATC ---> "I-TC + TaNoMo I + TaNoM0 Ill + T3NoMo IIA 5 CAT --> CTI" + T3NoM o IIA 6 del 16 NT + T3N1Mo III 5 CAT --> CIT - TzNoM o IIA 6 TAT --~ TGT + T2NIMo liB 8 CGT --~ TGT

unknown T2N1Mo liB 5 del C + T2NoMo IIA 6 ATC --, AGC + TINoM o I 8 CCT -o CGT + T3NIM o III 5 ins CCCTI" + T3NoM o IIA 6 AGA --> TGA + T3N1Mo III 6 TAT ~ TGT + T3N1M o III 6 TAT ---> TGT + TINoM o I 6 TAT ---> TGT + T3N1M o III 6 TAT --~ TGT

8 GAG ~ GAA + T3N1M o III 5 del TGA + TaNIM 0 III 6 CAT --~ CTr - T3N1Mo III 6 CGA -~ TGA

Transition E285K (glutamic acid --, lysine) a Transition P278S (proline ~ serine) a Transition H214R (histidine ~ arginine) ~ Frameshift 165 Frameshift 168

Transition P278S (proline ~ serine) a Transversion P278T (proline --, threonine) b Transversion Acceptor splice site b Transition R273C (arginine ~ cysteine) a Transversion Acceptor splice site b Transversion I195F (isoleucine --> phenylalanine) b

Transversion H179L (histidine --> leucine) a Frameshift 215 o Transversion H179L (histidine --> leucine) a Transition Y205C (tyrosine --> cysteine) a Transition R273C (arginine ~ cysteine)" Frameshift 159 b Transversion I195S (isoleucine --> serine) c Transversion P278R (proline ~ arginine) a Frameshift 143 b Transversion R209X (arginine --> stop codon) c Transition Y205C (tyrosine ---> cysteine) a Transition Y220C (tyrosine --> cysteine) a Transition Y220C (tyrosine ~ cysteine) a Transition Y220C (tyrosine ~ cysteine) a Transition E271E Polymorphism b Frameshift 169 b Transversion H193L (histidine --> leucine) ~ Transition R196X (arginine ---> stop codon) a

a Previously described mutations (Refs. 13, 32, 33, 44-47). b Novel mutations found in this work. c Personal communication.

A m p l i f i c a t i o n . Approximately 300 ng of genomic DNA were amplified in

a 100-p,1 reaction mixture containing 10 mM Tris-HC1 (pH 8.3), 50 mM KCI, 1.5

mM MgClz, 200 /XM of each deoxynucleotide triphosphate, 50 pmol of each

primer, and 2 units of Taq polymerase. Thirty-five cycles were performed in a

Perkin Elmer Cetus DNA thermocycler 9600 (2 min at 94~ followed by 5

cycles of 30 s at 94~ 30 s at 54~ 30 s at 72~ and 30 cycles of 30 s at 94~

30 s at 50~ 30 s at 72~ After these cycles, the samples were denaturated

for 2 min at 94~ and hybridized for 15 min at 70~ to create heteroduplexes.

Eleetrophoresis . A 20-/.d aliquot of the PCR product was electrophoresed

in a 6,5% polyacrylamide gel (37, 5: 1) with a linearly increasing denaturing

gradient from 50-100% (100% denaturating: 7 M urea and 40% formamide

V/V) at 75 V for 7-9 h at constant 60~ temperature, depending on the melting

map of the amplified DNA.

Computer analyses were performed using Melt 87, Melt Map Programs,

generously provided by Lerman. The optimal times of migration for electro-

phoresis were determined for each PCR product.

Following electrophoresis the gels were stained with ethidium bromide and

photographed under UV transillumination.

D N A S e q u e n c i n g . PCR products displaying an altered mobility in the gel

were sequenced. Single-strand DNA was obtained in the asymmetric PCR.

Each single-strand product was concentrated using Centricon 100 (Amicon,

Danvers, MA) and the reaction products were used for sequencing by the

dideoxy nucleotide chain termination method.

The nomenclature used to design the mutations is detailed by Beaudet et al.

(24). Controls . Different types of controls have been carried out in order to

verify that the mutated alleles that we have found are not artefacts of the sequencing assay or due to the theoretical possibility of error rates of the Taq

polymerase. (a) The DGGE z analyses have been performed twice each time a

modified pattern of migration of the PCR product was detected. (b) All the

sequencing data were obtained by sequencing from another aliquot of the tumor DNA, and this means that all these mutations were characterized on a

different sample that the first one in which the mutation was initially identified.

2 The abbreviations used are: DGGE, denaturing gradient gel electrophoresis.

All sequencing data were controled and confirmed twice. (c) Each time the

mutated allele creates or destroys a restriction site a confirmation of its pres-

ence was performed by restriction analysis of the PCR product and character-

ization of the novel restriction pattern. For each mutated allele we have looked

for the creation or the desctruction of a restriction site on the mutated sequence.

For P278T, the nucleotide change creates a RsaI site; for the H193L, the

mutation creates a AluI site. The confirmation of the presence of the mutated

sequence was obtained after amplification and enzyme digestion. Some frame-

shift mutations corresponding to 2 or 3 base pair deletion/insertion are detected

on a simple nondenaturing 8% polyacrylamide gel electrophoresis; this is due

to the creation of an heteroduplex between the wild type and the mutated allele.

All the deletions/insertions described here have been confirmed in such a way.

R E S U L T S

We have d e v e l o p e d the dena tu r i ng g rad ien t gel e l e c t r o p h o r e s i s

assay to de tec t any s ingle base c h a n g e that o c c u r s w i t h i n a D N A

f r agmen t , w h e r e b y such a change al ters that f r a g m e n t s m e l t i n g p rop -

ert ies. T h e m e l t i n g t e m p e r a t u r e o f a D N A f r a g m e n t is d e p e n d e n t on its

s e q u e n c e and f r a g m e n t s d i f f e r ing by on ly one n u c l e o t i d e wi l l usua l ly

exh ib i t d i f f e r ences in thei r me l t i ng behavior . In p rac t ice , me l t i ng is

a c h i e v e d by e l ec t ropho re s i s t h rough a p o l y a c r y l a m i d e gel con t a in ing

an inc reas ing c o n c e n t r a t i o n o f d e n a t u r i n g agents . I f two f r a g m e n t s

d i f fe r by a s ingle base pair, these f r a g m e n t s wil l me l t at d i f fe ren t

pos i t ions in the gel ( 2 5 - 2 7 ) . Th i s is i l lus t ra ted by the D G G E ana lys i s

o f e x o n 5 (Fig. 1). D u e to ex t ens ive repor t s on the func t iona l impor -

t ance o f h igh ly c o n s e r v e d d o m a i n s in gene p roduc t s and the h igh

f r e q u e n c y o f mu ta t i ons in the c o r r e s p o n d i n g reg ion o f the gene , we

have d e v e l o p e d an assay w h i c h a l lows c o m p l e t e ana lys i s o f these

reg ions , i.e., the f ive e x o n s (2, 4, 5, 7, and 8) o f the gene that e n c o d e

for these f ive c o n s e r v e d d o m a i n s and the e x o n s 3 and 6.

Our D G G E cond i t i ons have p e r m i t t e d the iden t i f i ca t ion o f an al-

t e red pa t te rn o f m i g r a t i o n in 27 s amp le s a m o n g 32 (84%) . Tab le 1

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TP53 GENE MUTATIONS 1N ESOPHAGEAL SQUAMOUS CELL CARCINOMAS

DISCUSSION

1 2 3 4 5 6 7 Fig. 1. DGGE analysis of exon 5. Lane 1, del GTCA, 165; Lane 2, dupli ACATGACG,

168; Lane 3, CAT ---> CTr, H179L; Lane 4, del C, 159; Lane 5, ins CCCTI', 143; Lane 6, del TGA, 169; Lane 7, normal. DGGE analysis of amplified PCR product of exon 5: Lanes 1--6 refer to different squamous cell carcinoma samples carrying different mutations. The control DNA (Lane 7) is from a genomic DNA. The PCR product was electrophoresed in a 50-100% gradient denaturing gel for 8 h. The two upper bands are heteroduplexes and the two lower bands are homoduplexes of normal and mutant DNA.

illustrates the molecular defects we have found. We have identified 10 transversions (34%), 13 transitions (45%), and 6 frameshift mutations (21%). Two missense mutations (P278T and P278S) were found in one sample as well as a polymorphism (E271E) and a mutation (Y220C) in another tumor sample. Of these mutations, 12 have not been reported to date: two of these were transversions which affect the splice site consensus sequence (IVS5-2, A---> T; IVS7, -2 A--> T) these nucleotide changes at position -2 of the acceptor site in Eucary- ote genes destroy the essential sequence for normal splicing (28, 29); 6 mutated alleles were clearly frameshift: 165 del GTCA; 168 dupli ACATGACG; 159 del C; 169 del TGA; 143 ins CCCTr in exon 5; 215 del 16 NT in exon 6. Four were missense: H193L; I195F; H214R; P278T (amino acid number). Sequencing results of some of these novel mutated alleles are shown in Fig. 2.

If we consider the distribution of mutations in the gene, 7 molecular defects were located in exon 5 (25%), 13 were located in exon 6 (46%), and 8 were located in exon 8 (29%) (Fig. 3). These data clearly underline a cluster of mutations in exon 6 of the TP53 gene of these tumor samples. We note also that 5 frameshift mutations were situated in exon 5. Due to the frequent loss of heterozygosity in esophageal cancer cells (30, 31), all our samples have been mixed with the PCR product of wild-type p53 DNA in order to detect, by heteroduplex formation in denaturing gradient gels, the presence of a possible mutation on one allele of the tumor, the other allele having been lost. The mix of the PCR product of the samples has not shown any additional altered pattern in the gel. This could be explained by the fact that tumor samples do not contain exclusively tumoral cells.

In parallel, the genomic DNA of these patients was investigated in the same way. One missense mutation was found in the germline DNA (Y205C). Clinical and genetic inquiries will be perform in the siblings of this patient.

Acquired mutations in the TP53 gene have been detected in epi- thelial, mesenchymal, hematopoietic, lymphoid neoplasms, and tu- mors of the central nervous system (1, 2). Approximately one-half of the cancers of the colon, breast, lung, liver, and brain contain mutant TP53 gene and gene product. Recent studies have reported the pres- ence of alterations of TP53 gene in esophageal squamous cell carci- noma (32-34). Loss of heterozygosity affecting the TP53 locus has been detected in approximately one-half of esophageal cancers (30, 31) and mutations in the TP53 gene sequence have been identified in 10 to 47 p.100 of the cancers (32, 35, 36). In these studies, most of the tumor samples analyzed were of Chinese or Japanese origin or of mixed origin as recently reported by Y. Huang (34).

In this work, by screening the 7 exons (2-8) of the gene that code for the 5 conserved domains of the p53 protein, we succeeded in identifying mutations in 27 of 32 esophageal squamous cell carcino- mas. This corresponds to 84% of the cancers, and is, to our knowl- edge, the highest incidence of TP53 mutations reported in human malignancies.

Our results also suggest that somatic mutation in this type of tumor might be much more frequent than previously reported. This may be due to the fact we have used one of the most powerful assays for detection of mutations in a human gene. The denaturing gradient gel electrophoresis initially described by Lerman (25) and improved by Sheffield (26) by his addition of a GC clamp at one end of DNA fragment has already allowed the successful screening of other genes responsible for human diseases such as globin (37), rhodopsin (38), or cystic fibrosis transmembrane conductance regulator (39). However, in five tumors we have not been able to detect any sequence abnor- mality. To explain this lack of mutation, several potential explanations could be proposed: (a), we have screened only those exons which encode the conserved domains of the tumor suppressor gene, while exons 9, 10, and 11 have not been studied; (b) some mutations could be located in the intronic regions of the gene (1); (c) because tumor samples contain inflammatory and stromal cells with normal DNA, a mutation might be hidden by the wild type sequence of the TP53 gene. Despite these five tumors with undetected mutations, at this time, our data are highly suggestive that in esophageal cancer, mutation of the TP53 gene might occur with considerable frequency. This work also highlighted the location of a highly mutated region, exon 6 (codon 187-224) in which 45% of the mutations were found. These data would indicate that, although this region does not encode part of a conserved domain, it should be considered a hot spot in esophageal tumors.

Esophageal cancer is one of the six most common cancers world- wide with very significant variation in its geographical distribution. In western Europe and in the United States, alcohol and tobacco use are closely linked with esophageal carcinoma. Each of these is associated

Fig. 2. Sequencing data of four novel mutations.

c c T-- ->G A T T

C T A G

1195S

5747

C T A G

c T G G A--->T T T C T C

IVS5 (-2, A- ->T)

C T A G

A--->G C--->T~ G--->A A--->C C--->A A--->C C--->G T--->A G-->C A C G A A C /

del GTCA 165

C T A G

T--->G T

G--->T A--->T G G C--->A A--->G G T--->C A ~

del TGA 215

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TP53 GENE MUTATIONS IN ESOPHAGEAL SQUAMOUS CELL CARCINOMAS

14

Number of mutat ions

12

10

8

6

4

2

0 i i

Exon 5 Exon 6 Exon 7

Fig. 3. ~, frameshifi; D, transversions; [~, transitions.

:i : :::I::IH.:. I:H �84

iii!:!i:i i: :i ~ i;:~:

" r

~//r/ll/lllllll/li/lll~

. . . . . . . . . . . I

Exon 8

with an increased risk of cancer of the esophagus. Both factors have

a synergistic effect on carcinogenesis in the upper alimentary tract,

and their combined use is associated with even higher risk. Alcohol

can increase the susceptibility of the esophageal epithelium towards

carcinogens by several mechanisms. Ethanol can enhance the activa-

tion of procarcinogens to carcinogens through microsomal enzyme

induction. Ethanol can interfere with the cellular DNA repair mecha- nisms and the immune system. Alcohol by itself may increase the risk

of cancer by directly injuring the esophageal mucosa and leading to hyper regeneration. Finally, alcohol usage may be associated with

nutrition deficiencies that may also lead to an increased risk of cancer

(40). A wide variety of carcinogens have been found in cigarette

smoke and various alcoholic beverages. These include benzopyrene,

nitrosamines, polycyclic hydrocarbons, fermentation oils, and other

mutagenic compounds. Increased exposure to carcinogens by smoking

and excessive alcohol consumption contributes to mutagenic events

affecting DNA and cancer development. In our study, all the patients

originated from Brittany, an area of western Europe where epidemio-

logical studies have shown an increase in the risk of developing

esophageal cancer from alcohol consumption (41). Of particular importance also is the profile of mutations we have

found in the TP53 gene of these patients. The spectrum of mutations

observed in this series of 27 esophageal cancers of identical ethnic origin is quite particular with 45% of the DNA lesions being transi-

tions, 34% transversions, and 21% being composed of deletions,

duplications or small insertions. It is now well established that the

pattern of mutations vary greatly in human neoplasms and is in part

related to organ site. For example, G:C to A:T transitions predominate

in colon lymphoid or brain tumors, and most of these occur at CpG

dinucleotides (42). On the other hand, G:C to T:A transversions are the

most frequent substitutions observed in cancers of the lung and the

liver. These transversions are known to be induced by benzopyrene

and aflatoxin B associated with the etiology of lung and liver tumors,

respectively. These results have been confirmed in vitro showing that the TP53 nucleotides identified as mutational hot spots in lung and

liver cancers are targets for benzopyrene and aflatoxine B. It has been shown in rats (19) that esophageal tumors induced by

alkylating nitroso compounds contain 78% transitions clustered in

exon 6 of the gene. An article by K. H. V~ih~ikancas (43) has reported the mutational spectrum of TP53 mutations in lung cancer of under-

ground miners exposed to radon: 2 deletions were found in 7 patients,

although deletions have rarely been reported in lung cancers or other forms of cancer. In fact, less than 40 deletions-insertions have been

reported among the 400 TP53 DNA lesions so far published (1). The results we have obtained in this work in which we have been

able to identify 84% of samples carrying a mutated allele in the TP53

gene of esophageal tumors is of importance since this could indicate that such a mutational lesion in the encoded tumor suppressor protein

5748

is a very frequent, and perhaps obligate, step in the development of the

carcinogenesis of the esophagus. The particular profile of mutations

observed by us in the TP53 gene in part contradicts the pattern

previously reported by Hollstein (32) in which the occurrence of

transversions was reported, at least in the first tumors analyzed, as

being exceptionally frequent; however, in this series of 15 squamous

cell carcinoma samples from the western part of France, 3 were

carrying a G:C > T:A transition. This molecular analysis of TP53 in esophageal neoplasms could

suggest that these different profiles of mutations could be the result of

exposure to several genotoxic environmental factors. For example,

nitrosamine could cause transitions and benzopyrene transversions, and other genotoxic agents of esophageal cancer could be responsible

for deletional-insertional events. Since the definitive role and exact list of genotoxic agents capable

of inducing mutations have yet to be deduced, it is noteworthy that our

data are highly suggestive that esophageal tumors in this part of the

world (western Europe) could be due to the synergistic effect of

different carcinogens. These data may be helpful in defining the

chemicals agents capable of producing the spectrum of mutations we

have described as well as in understanding the etiology of human

esophageal cancer.

ACKNOWLEDGMENTS

We are indebted to Professor T. Soussi for providing us with the genomic sequences of the TP53 gene and for reading the manuscript. We thank Chantal Baclet for assistance in manuscript preparation and Arianne Le Menn for the mounting of photographs.

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Gene Mutation Profile in Esophageal Squamous CellTP53

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