(CANCERRESEARCH 52, 6092-6097, NovemberI. 19921

Advances in Brief

p53 Mutation and Protein Accumulation during Multistage Human

Esophageal Carcinogenesis

William P. Bennett, Monica C. Holistein, Robert A. Metcalf, Judith A. Welsh, A. He, Si-mm Zhu, Inca Kusters,James H. Resau, Benjamin F. Trump, David P. Lane, and Curtis C. Harris'

Laboratory of Human Carcinogenesis, National Cancer institute, Bethesda, Maryland 20892 (W. P. B., R. A. M., J. A. W., C. C. H.]; International Agency forResearch on Cancer, Lyon Cedex 2, France fM C. H., I. K.]; Cancer Research Institute, China Medical University, Shenyang, Liaoling, People's Republic of China(A. H.]; Sun Vat-Sen University ofMedical Sciences, Guangzhou, People's Republic of China (S-m. Z.J; University ofMaryland, School ofMedicine, Baltimore,Maryland 21201 (1. H. R., B. F. Tj; and Cancer Research Campaign Laboratory, University ofDundee, Dundee, Scotland DDJ 4HN (D. P. L.J

Abstract

Preinvasive lesions of squamous cell carcinoma are well defined mor

phologically and provide a model for multistage carcinogenesis. Sincealterations in thep53 tumor suppressor gene occur frequently in invasiveesophageal squamous cell carcinoma, we examined a set of preinvasivelesions to investigate the timing of p53 mutation. Surgically resectedtissues from nine patients with esophageal squamous cell carcinomacontained precursor lesions which had not yet invaded normal tissues.Immunohistochemistry showed high levels of p53 protein in both preinvasive lesions and invasive carcinomas in six cases sequence analysisof all invasive tumors identified p53 missense mutations in two cases.Preinvasive lesions from both tumors with mutations plus one wild-typetumor were microdissected and sequenced. In one patient there weredifferent mutations in the invasive carcinoma (codon 282, CGG@@ >TGGirP) and a preinvasive lesion (codon 272, GTG@ > T/GTGIC/vdl).In a second case, an invasive carcinoma had a mutation in codon 175(CGCRrS> CAChIC),and adjacent preinvasive lesions contained a wildtype sequence. A carcinoma and preinvasive lesion from the third casecontained high levels of protein and a wild-type DNA sequence.Therefore, p53 mutation may precede invasion in esophageal carcinogenesis,

and multifocal esophageal neoplasms may arise from independentclones of transformed cells. The timing of p53 protein accumulation isfavorable for an intermediate biomarker in multistage esophageal carcinogenesis.

Introduction

Mutation in the p53 tumor suppressor gene is the most common genetic alteration in human cancer (reviewed in Ref. 1).Mutation occurs in all of the common cancers, and within anyone type, one-half or more of the individual tumors may beaffected. Increased levels of p53 protein are also found in 29—80% of malignant tumors but only rarely in benign tumors andnormal tissues (2). Molecular mechanisms leading to excessprotein accumulation include mutation with conformationalchange (3), complex formation with viral oncoproteins (4—7),and possibly aberrant expression of the p53 gene by cellulartranscriptional regulators (8). The frequency and specificity ofp53 protein accumulation suggest that elevated protein levelsmay be useful as an intermediate biomarker. This possibilityhas been the subject of recent debate among groups analyzinghuman tissues for evidence of malignancy (9, 10).

Received 7/27/92; accepted 9/15/92.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.

I To whom requests for reprints should be addressed, at Laboratory of Human

Carcinogenesis, National Cancer Institute, NIH, Building 37, Room 2C01, Bethesda, MD 20892.

Precursor lesions of SCC2 are recognized in several differenttissues including the esophagus, oropharynx, bronchus, cervix,and skin (1 1). Multiple stages are defined clinically and histologically in accordance with the multistage model of carcinogenesis. In the usual terminology, mild dysplasia is an earlylesion which may regress to a normal state, and moderate dysplasia is an unpredictable stage which may either regress orprogress to invasive cancer. Severe dysplasia and carcinomain situ are considered premalignant lesions which usuallyprogress to invasion and metastasis unless treated. The multiplestages of dysplasia have been called by several terms, includingincipient neoplasia and preinvasive, precursor, progenitor, orin situ lesions. In general, these terms are used interchangeablyto denote a neoplastic lesion which has not yet invaded thesurrounding tissues. We undertook this study to characterizep53 alterations in preinvasive squamous lesions of humanesophagus.

Materials and Methods

Esophageal tissues fixed in ethanol were collected from Shenyang

and Guangzhou in the People's Republic of China. The samples wereembedded in paraffin, and immunohistochemical analysis was performed using conventional peroxidase methods as previously described(12). The primary antibody was a 1:1000 dilution ofa polyclonal rabbitantiserum, CM-i, raised against wild-type human p53 protein whichwas produced in a bacterial expression system (13). The chromogen wasdiaminobenzidine (final concentration, 0.05 mg/mI) osmicated withnickel chloride (final concentration, 0.03%). Intense nuclear stainingwas the criterion for a positive reaction.

After identification of suitable precursor lesions and invasive tumors, paraffin sections were dewaxed for 10 mm in two changesof xylene and air dried. Using a dissecting microscope, selected areasof tissue were rehydrated with sterile water, excess water was blottedwith tissue paper, and specific lesions were dissected from theglass slides using a needle and syringe (Fig. 3C). The dissected tissuefragments were placed into 50—I®@clof deionized water, and DNAwas released by boiling in a water bath for 5 mm. The tissue waspelleted, and an aliquot of supernatant was taken for polymerasechain reaction. Exons 5—8of p53 were examined for both tumor andprecursor lesions in cases HE9O-72 and HE9O-27. For the invasivetumor in case HE9O-43, exons 5—8were examined, and a mutationwas found in exon 5; for the adjacent precursor lesions, only exon 5was examined. Direct sequencing of polymerase chain reaction product was performed essentially as previously described (14). Protocolmodifications included the use of nested primers in sequential rounds

2 The abbreviation used is: 5CC, squamous cell carcinoma.

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p53 MUTATION AND PROTEIN ACCUMULATION DURING CARCINOGENESIS

of amplification (15). In cases with identified mutations, the germline sequence of the affected exon was analyzed from nontumortissues.

Results and Discussion

This study began when squamous precursor lesions werenoted to contain elevated levels of p53 protein. In nine sets ofpreinvasive lesions and invasive tumors, the staining patternswere concordant; that is, in six pairs, both preinvasive lesionsand tumors contained high levels of p53 protein, and in threepairs, both were negative. These findings suggested that p53protein accumulation and/or mutation may occur before thedevelopment of invasion in esophageal SCC. Most of the progenitor lesions were next to invasive cancers, but in one instance, the tumor was distinct from the dysplastic area whichappeared to be an independent neoplasm (HE9O-72). This ohservation suggested that dietary or environmental agents causedwidespread genetic damage (i.e., a “fielddefect―)leading tomultiple independent neoplasms. Alternatively, a monoclonalproliferation may have spread through the mucosa or lymphatics to form multiple neoplasms derived from a common progenitor cell. To test these hypotheses, all six immunostainpositive, invasive tumors were sequenced in exons 5—8;missense mutations were found in two. Since a point mutationprovides a molecular marker, microdissection and sequenceanalysis were conducted on the precursor lesions from thosetwo tumors. An advanced third lesion (i.e., carcinoma in situfrom case HE9O-27) was also sequenced, although the adjacenttumor contained a wild-type sequence.

Noncancerous esophageal tissues from the first patient,HE9O-72, contained extensive dysplastic mucosa, but invasivecancer was not detected in serial sections through 255 @tmof theparaffin block. In the available cross-section, the abnormal cellsextended through a 0.9-cm-long segment of the mucosa. If thiswere the diameter of a round lesion, the area would be approximately 0.64 cm2. In about 75% of the abnormal mucosa, onlythe lower and middle levels were occupied by cells with enlarged, darkly stained, irregularly shaped nuclei; this morphology is typical of moderate dysplasia (Fig. IA). In the remainderof the affected mucosa, the irregular nuclei filled the mucosa ina manner typical of severe dysplasia; in some places, there wasan abrupt transition between normal and abnormal mucosa(Fig. 1C). Immunohistochemical analysis showed high levels ofp53 protein in the irregular nuclei, but the cells in the submucosa, superficial layers, and adjacent normal mucosa were unstained (Fig. 1, B and D). Microdissection and sequence analysis of two different areas, one moderately and one severelydysplastic, showed the same heterozygous G:C to T:A transversion in the first position of codon 272, GTGVaI > T/GTGleL@/vaI(Table 1 and Fig. 2). In contrast, analysis of tissues containingthe invasive tumor from this patient demonstrated a heterozygous C:G to T:A transition in the first position of codon 282,CGGarK > TGGt@@.Persistence of the wild-type allele indicatesthat either the tumor contains normal and mutated copies of thep53 gene or that nonneoplastic tissues associated with the tu

mor contributed the normal allele. Histological review showedtumor-infiltrating muscle bundles composing the esophagealwall plus extensive inflammatory infiltrates and fibrous tissue.While microdissection can eliminate gross contamination bynonneoplastic tissues, it is most likely in this case that thewild-type allele was contributed by nonneoplastic tissues intermingled with the tumor. Normal tissues had wild-type se

quences at both codons (Fig. 2). The finding of a p53 mutation

in the preinvasive lesions demonstrates that mutation may occur before invasion, and the expansion of the cell populationthrough a macroscopic region of the mucosa suggests that themutation confers a growth advantage. Whether deletion of theremaining wild-type allele ofp53 is sufficient to cause invasionis unknown. The timing ofp53 mutation in this single exampleis similar to the molecular agenda for colorectal cancer (I 6),although one could argue that mutation at the stage of moderate dysplasia (i.e., HE9O-72) is earlier than the late adenomastage in the colorectal model.

In a second case, HE9O-43, a homozygous sequence in exon5 at codon 175 (CGC@@5> CAChIS) was found in the invasivetumor, but no exon 5 mutation was found in the adjacent dysplastic mucosa. This base change indicated a G:C to A:T transition plus a deletion of the second allele in the invasive tumor.There were extensive precursor lesions including mild, moderate, and severe dysplasia, and both the invasive tumor and adjacent precursor lesions contained high levels of p53 protein.There are two main interpretations of these data: (a) The precursor lesions contain wild-type p53 sequence and the proteinaccumulated by a nonmutational mechanism (e.g., complex formation with viral or cellular oncoproteins); in this scenario, p53mutation would correlate with invasion. (b) The precursor lesions (some within microns of the invasive tumor) contain p53mutations in unexamined exons and may represent independent neoplasms.

In the third case (i.e., HE9O-27), both the invasive tumor andthe contiguous progenitor lesions contained high levels of p53protein and wild-type DNA sequence in exons 5—8.This caseillustrates a neoplastic pathway which is independent of p53mutation in the highly conserved sequences. Although accumulation ofp53 protein frequently reflects a nonconservative pointmutation, exceptions to this rule have been observed (12, 17—20). Often only the highly conserved sequences in exons 5—8areexamined, but in some human tumor cell lines expressing detectable levels of p53 protein, exons 2—11 have been sequencedwithout finding a mutation (21, 22). Possible nonmutationalmechanisms for p53 protein accumulation include (a) inactivation of an enzymatic pathway responsible for p53 protein degradation (23), (b) stabilization of wild-type protein throughcomplex formation with a DNA tumor virus protein (4—7)or acellular oncogene, (c) aberrant posttranscriptional modification

conferring extended protein half-life, and (d) altered expressionof the p53 gene by cellular transcriptional regulators (8). Regardless of the mechanism, it is notable that the excess proteinprovides a distinct marker ofboth the preinvasive lesion and theinvasive tumor (Fig. 3).

These data indicate that p53 alterations may occur at different time points during the development of esophageal 5CC. Asillustrated by case HE9O-72, p53 mutation can occur in an earlystage of moderate dysplasia and may confer a growth advantageto the preinvasive cells. Loss of the remaining wild-type alleleand/or accumulation of additional mutations may produce aninvasive subclone within the original population. The secondand third cases show that excess p53 protein may accumulate inthe absence of either invasion or apparent p53 mutation. Further studies will be needed to define the predominant stage ofp53 alteration in the pathogenesis ofesophageal SCC; however,these findings suggest that the timing of p53 protein accumulation may be suitable for an intermediate biomarker.

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p53 MUTATION AND PROTEIN ACCUMULATION DURING CARCINOGENESIS

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Fig. 1. p53 immunohistochemical analysis in moderate to severe dysplasia (case HE9O-72). A and B, serial sections of moderately dysplastic squamous mucosa. Thelower and middle epithelial levels contain cells with enlarged, darkly stained, irregularly shaped, dysplastic nuclei (A; H&E). Immunohistochemical analysis shows

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Table 1p53 sequenceanalysisofpreinvasivelesioasand invasivetumors―Preinvasive

lesionInvasivetumorPatientHistologyCodonSequenceami@@0

acidHistologyCodonSequence―@―°acidWTb

MUTWTMUTHE9O-72

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> TGGtnCGC@ > CAChIS

p53 MUTATION AND PROTEIN ACCUMULATION DURING CARCINOGENESIS

aAllprecursorlesionsandinvasivetumorsselectedforanalysiscontainedhighlevelsofp53protein.b WT, wild type; MUT, mutant MSD, moderate to severe dysplasia CIS, carcinoma in situ.C Heterozygous mutation showing two bands in the first base position.

d The invasive tumor was previously reported as HE9O-15 in Ref. 12.

found in SCC but not in precursor papillomas (27). Immunohistochemical analyses have shown elevated levels of p53 protein in preinvasive lesions of the human testis (28) and breast(29, 30). These data suggest that in these tissues, p53 mutationsand/or protein accumulation frequently occur near the time

VAL that cells in precursor lesions become malignant and invade

surrounding tissues.However, the timing of p53 alteration may be different in

other tumors. For example, in bladder cancer, the incidence ofp53 mutation is greater in high-grade and invasive tumors thanin superficial and low-grade tumors. Similar observations havebeen made in low- and high-grade tumors of the brain and

ARG thyroid (31, 32). These findings suggest that, in some tissues,p53 mutation may contribute more to tumor progression thanthe conversion to malignancy. These data suggest that theagenda for p53 alteration may vary with tumor type and underscore the need for analysis of preinvasive lesions in a variety oftissues.

Multifocal Cancer. Esophageal cancer may develop in multiple sites either simultaneously or sequentially (33). Two theones have been advanced to explain these so-called synchronous and metachronous carcinomas. The monoclonal neoplasiatheory holds that progeny from a single transformed cell mayspread to produce multiple tumors (34—36),while the “fielddefect―model predicts that independent tumors develop fromthe genotoxic effects of carcinogens (37, 38). If a patient istreated for one 5CC and later develops a second, then there aretwo possibilities, (a) the second 5CC is a new, independentneoplasm (i.e., “fielddefect―model) or (b) the second 5CCdeveloped from an occult focus of the first cancer which survived the initial treatment (i.e., monoclonal neoplasia theory).The distinction between these mechanisms is important bothfor defining multistage carcinogenesis and for refining cancertreatment protocols. For example, if the first tumor recurs, thetreatment may have been ineffective, or progeny from the primary tumor may have spread through the mucosa or lymphaticsto produce an occult tumor which survived the treatment regimen. Alternatively, the genotoxic effects of tobacco, alcoholicbeverages, and certain dietary factors may have caused a “fielddefect―predisposing the entire esophageal mucosa to developmultiple independent cancers (39). In this setting, there mightbe multiple neoplasms in the early stages of development, andchemoprevention might be effective (see Comments in Ref. 40).The proof of these theories required demonstration of theclonal nature of cancer, which was not established until theadvent of molecular genetics (34—36).

Tumor Dysplasia Normal Wild TypeI I I II 11 1ACGT ACGTACGT

Fig. 2. pS3 sequence analysis of invasive tumor, preinvasive dysplasia, andnormal tissue (case HE90-72). The tumor contains a C:G to T:A transition in thefirst base position of codon 282; the wild-typeC may be contributed by normalstroma contaminating the tumor sample. Two microdissected samples from different sites within the dysplasticmucosacontained the same heterozygousG:C toT:A transversion in the first base position of codon 272; it is likely that thewild-type G represents an undeleted normal allele. The wild-type sequence ispresent in normal tissue from the same patient.

An intermediate biomarker should be present before or during the early stages of invasion to minimize the occurrence offalse negative results. The timing ofp53 mutation and proteinaccumulation has been studied best in colorectal adenocarcinoma. This cancer develops in several well-defined stages, andp53 mutation most frequently occurs between the stages of late

adenoma and invasive carcinoma (16, 24). This timing is favorable for a tumor marker because a positive result would signaleither a late adenoma or an invasive tumor, typically both aretreated by surgical resection.

In addition to the colorectal model, recent studies indicatethat alterations in the p53 gene and/or its protein product frequently occur in preinvasive lesions of the esophagus, skin,testis, and breast. In the esophagus, p53 mutations were foundcommonly in Barrett's epithelium, which is considered a precursor to adenocarcinoma of that site (25). In human skin cancer, elevated levels of p53 protein were seen in some squamousdysplastic lesions as well as in 16% ofcases of Bowen's disease,a preinvasive form of epidermal SCC (26). In a murine modelfor epidermal carcinogenesis, p53 deletion and mutation were

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staining in the dysplastic nuclei, but the submucosaand superficial layers are unstained (B; CM-l; no counterstain). C and D, serial sections of severelydysplasticsquamous mucosa (right) with abrupt transition to normal mucosa (left) (C; H&E). Immunostain shows p53 protein in the abnormal mucosa, but the normal mucosais unstained (1.@,CM-I; no counterstain). x200.

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p53 MUTATION AND PROTEIN ACCUMULATION DURING CARCINOGENESIS

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Fig. 3. Microdissection and p53 immunohistochemical analysis ofcarcinoma in situ (HE9O-27E). Serial sections show an abrupt transition between normal mucosa(left) and carcinoma in situ (right)(A; H&E). Immunostaining shows p53 protein confined to the carcinoma in situ (B@,CM-l; no counterstain). Microdissection followedby DNA isolation and amplification allows genetic analysis of histologically defined lesions (C; H&E). xIOO.

B

Recent analyses of multifocal hepatocellular carcinomas support the field defect model and the development of multiple,independent cancers within a single patient (41). The presenceof different p53 mutations in two esophageal neoplasms fromcase HE9O-72 also may be interpreted in this light, but thepossibility remains that progeny from a single transformed celllater developed independent p53 mutations. Conversely, thereis evidence supporting monoclonal neoplasia. For example, werecently examined a bronchial SCC which contained extensivedysplasia near the invasive cancer (42). Immunohistochemicalanalysis showed high levels of p53 protein in both dysplasticand invasive areas. Microdissection and DNA sequence analysis of three separate foci revealed the same p53 missense pointmutation in preinvasive, microinvasive, and fully invasive lesions. Similar evidence has been found in multifocal carcinomas of the bladder (43), renal pelvis (44), and liver (41 ). Insummary, these data support both the field defect and the monoclonal neoplasia models for carcinogenesis. Further studiesare needed to determine the frequency of each in esophagealSCC and in other cancers.

Acknowledgments

We thank Ricardo V. Dreyfuss for expert photomicrography andDorothea Dudek for editorial assistance.

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1992;52:6092-6097. Cancer Res   William P. Bennett, Monica C. Hollstein, Robert A. Metcalf, et al.   Human Esophageal Carcinogenesis

Mutation and Protein Accumulation during Multistagep53

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