p53 mutation and allelic loss of chromosome 3p, 9p of preneoplastic lesions in patients with...

p53 Mutation and Allelic Loss of Chromosome 3p, 9pof Preneoplastic Lesions in Patients with NonsmallCell Lung Carcinoma

Hiromasa Kohno, M.D.1

Kenzo Hiroshima, M.D.1

Tetsuya Toyozaki, M.D.1

Takehiko Fujisawa, M.D.2

Hidemi Ohwada, M.D.1

1 Department of Pathology, Institute of PulmonaryCancer Research, Chiba University School of Med-icine, Chiba, Japan.

2 Department of Surgery, Institute of PulmonaryCancer Research, Chiba University School of Med-icine, Chiba, Japan.

Address for reprints: Hiromasa Kohno, M.D., De-partment of Pathology, Institute of Pulmonary Can-cer Research, Chiba University School of Medicine,1–8-1, Inohana, Chuo-ku, Chiba 260–8670, Ja-pan.

Received April 21, 1998; revision received June16, 1998; accepted June 16, 1998.

BACKGROUND. An accumulation of mutations can result in carcinogenesis. Com-

paring genetic alterations in preneoplastic lesions with those seen in cancer in the

same patient may be helpful in the early diagnosis of lung carcinoma or preneo-

plastic lesions.

METHODS. To identify genetic alterations that may play a role in the development

of nonsmall cell lung carcinoma (NSCLC), the authors examined the p53 gene and

microsatellite markers on chromosome 3p (D3S643, D3S1317), 9p (D9S171, IFNA)

in 35 bronchial metaplastic lesions and 28 alveolar hyperplastic lesions from 61

patients.

RESULTS. A total of 8 metaplastic lesions (1 squamous metaplasia and 7 dysplasias)

and 3 alveolar hyperplastic lesions (with atypia) showed genetic alterations, in-

cluding loss of heterozygosity (LOH) of 3p, 9p and mutations of the p53 gene. In an

analysis of microsatellite markers, 5 of 35 cases of squamous cell carcinoma (SCC)

and 3 of 26 cases of adenocarcinoma (Ad) showed LOH in both preneoplastic

lesions and synchronous cancers. Nine patients (25.7%) with SCC and 6 patients

(23.1%) with Ad were shown to have mutations of the p53 gene by single-strand

conformation polymorphism. In 2 of these 9 patients with SCC, the same mutation

was observed in both dysplasia and SCC.

CONCLUSIONS. These findings suggest that several genetic alterations may occur in

preneoplastic lesions or the early stage of SCC of the lung, whereas the genetic

alterations examined appeared to occur relatively late in the pathogenesis of

pulmonary adenocarcinoma. Cancer 1999;85:341–7.

© 1999 American Cancer Society.

KEYWORDS: lung carcinoma, preneoplastic lesion, dysplasia, atypical adenomatoushyperplasia, p53 gene, microsatellite marker.

Carcinogenesis often occurs as a result of an accumulation ofmutations. Recent studies have revealed three different types of

genetic mechanisms in carcinogenesis: activation of oncogenes, in-activation of tumor suppressor genes, and alteration in microsatelliterepeat length.1 The involvement of known or presumed tumor sup-pressor genes has been suggested by the presence of chromosomaldeletions or losses and has been confirmed by restriction fragmentlength polymorphism (RFLP) analysis and analysis of microsatellitemarkers. In lung carcinoma, as in other human cancers, the inacti-vation of the p53 gene has been demonstrated by the loss of heterozy-gosity (LOH) and mutations in the remaining allele.2–5 Deletions inthe short arm of chromosome 3 and 9 have frequently been observedin lung carcinoma.1,6 –13

Early genetic alterations occur as a result of exposure of the

341

© 1999 American Cancer Society

bronchial epithelial cells to environmental carcino-gens, which leads to altered cellular morphology. His-tologic changes associated with the development ofsquamous cell carcinoma, such as hyperplasia, squa-mous metaplasia, dysplasia, and carcinoma in situ ofthe lung, have been documented. Dysplasia of thebronchial epithelium is a preneoplastic lesion thatmay develop into lung carcinoma, particularly squa-mous cell carcinoma.

Recently, atypical adenomatous hyperplasia (AAH)has been described as a preneoplastic lesion in lungcarcinomas that develop peripherally, such as adenocar-cinomas.14–16 Although morphometric, immunohisto-chemical, and molecular analyses have been performedfor AAH,6,15–19 the sequence for pulmonary adenocarci-noma has not been clearly established.

It is important to compare the genetic alterationsin preneoplastic lesions with those found in cancer inthe same patient. The identification of the geneticalterations associated with preneoplastic lesions maybe useful in the early diagnosis of lung carcinoma orpreneoplastic lesions.

In this study, we examined mutations of the p53gene and LOH of 3p, 9p in preneoplastic lesions andsynchronous lung carcinomas.

MATERIALS AND METHODSWe studied genetic alterations of preneoplastic lesionsand synchronous lung carcinomas separately in thesquamous cell carcinoma group (SCC group) and theadenocarcinoma group (Ad group). Informed consentwas obtained from the guardian of Institute of Pulmo-nary Cancer Research, Chiba University School ofMedicine. The clinical features are listed in Table 1.Both tumor and preneoplastic lesion samples wereobtained from each of 61 patients who were histolog-ically diagnosed with squamous cell carcinoma (SCC;n 5 35, 34 males and 1 female, ages 39 –77 years[mean, 65.9 years]) or adenocarcinoma (Ad; n 5 26, 11males and 15 females, ages 48 –79 years [mean, 63.0years]) of the lung. These patients underwent surgeryat Chiba University Hospital during the period 1987–1997. The surgical procedures they underwent con-sisted of 1 partial resection, 53 lobectomies, and 7pneumonectomies (31 patients with pathologic StageI, 12 with pathologic Stage II, 12 with pathologic StageIIIA, 6 with pathologic Stage IIIB, according to theInternational Union Against Cancer TNM classifica-tion20). Fifty-one of 61 patients consisted of 15 non-smokers and 36 smokers (Tables 1, 4). All of the 15nonsmokers belonged in the Ad group, and 29 (80.6%)of 36 smokers belonged in the SCC group.

Preneoplastic lesions were identified by retro-spective review of the resected paraffin embedded

specimens. We carefully chose preneoplastic lesionsthat were distant from the tumors. Sections of eachresected specimen were stained with hematoxylin andeosin and examined by three reference pathologists(H.K., K.H., and H.O.). Bronchial metaplastic lesions (atotal of 35 lesions) from 35 patients with SCC (1 lesionper patient) were classified as dysplasia (Dys, n 5 22)or squamous metaplasia (Sm, n 5 13). Alveolar hyper-plastic lesions (a total of 28 lesions) from 26 patientswith Ad (1 lesion per patient, but only 1 patient had 3lesions) were classified as atypical adenomatous hy-perplasia (AAH, n 5 20) and adenomatous hyperplasiawithout atypia (AH, n 5 8). Metaplastic lesions werenot found in the patients with Ad, and alveolar hyper-plastic lesions were not found in the patients withSCC.

DNA was extracted from formalin fixed, paraffinembedded specimens. Previously identified areas ofcancer and preneoplastic lesions on hematoxylin andeosin–stained sections were microdissected from par-allel 10 mm sections, followed by standard phenol/chloroform methods. Ciliated epithelium of the bron-chus or alveolar epithelium was also microdissected asa source of normal DNA from each patient.

As described previously,11,21–23 the primers wereused for amplification and sequencing and were bio-tinylated at the 59-end for nonisotopic detection. Hot-start polymerase chain reaction (PCR) was performed.After initial denaturation at 94°C for 5 minutes, 40

TABLE 1Summary of Clinical Features

FeaturesSCC group(n 5 35)

Ad group(n 5 26)

Age, yrs (mean/range) 65.9/39–77 63.0/48–79Gender (M/F) 34/1 11/15Preneoplastic lesion Dys: 22 AAH: 20a

Sm: 13 AH: 8Surgical procedure Pa: 0 Pa: 1

Lo: 29 Lo: 24Pn: 6 Pn: 1

Smoking statusNonsmokers 0 15Smokers 29 7

(BI ,400) (1) (1)(BI $400) (28) (6)

Pathologic stageb I:17 I:14II:10 II:2IIIa:6 IIIa:6IIIb:2 IIIb:4

SCC: squamous cell carcinoma; Ad: adenocarcinoma; Dys: dysplasia; Sm: squamous metaplasia; AAH:

atypical adenomatous hyperplasia; AH: adenomatous hyperplasia without atypia; Pa: partial resection;

Lo: lobectomy; Pn: pneumonectomy; BI: Brinkman Index (the no. of cigarettes smoked/day 3 yrs).a Twenty lesions from 18 patients (1 patient had 3 lesions).b According to the International Union Against Cancer TNM classification.

342 CANCER January 15, 1999 / Volume 85 / Number 2

cycles of PCR were performed, consisting of 1 minuteat 94°C, 2 minutes at the optimal annealing tempera-ture (55– 65°C for each primer pair), and extension at72°C for 3 minutes, followed by a terminal extension at72°C for 10 minutes.

Microsatellite polymorphism on chromosome 3p(D3S1317, D3S643), 9p (IFNA, D9S171) was examinedin tumor, preneoplastic lesion, and normal epitheliumDNA from each patient. PCR products were dilutedwith a gel-loading buffer. After the DNA fragmentswere heated to 94°C, electrophoresis was performedon 7% polyacrylamide gel containing 7 M urea at 60 Wfor 3– 4 hours. Following electrophoretic separation ofthe DNA fragments, the DNA was transferred from thegel onto a nylon membrane. The membrane was in-cubated with a streptavidin-alkaline phosphatase con-jugate and applied with a chemiluminescent substratefor alkaline phosphatase, followed by exposure of themembrane to X-ray film.

Exon 4 of the p53 gene contains a sequence poly-morphism at codon 72.24,25 To detect LOH of chromo-some 17p, analysis of restriction fragment length poly-morphism (RFLP) at exon 4 of p53 gene wasperformed. The restriction digestion with ACCII wascarried out, followed by electrophoresis on 4% agarosegel.

For analysis of the p53 gene mutation by single-strand conformation polymorphism (SSCP), p53 exons5– 8 were amplified in PCR as described above. PCRproducts diluted with a loading buffer were loaded on5% polyacrylamide gel containing 5% glycerol. Elec-trophoresis was performed at 18 –22°C for 3– 4 hours,

and detection was carried out as described above.Following DNA transfer to the nylon membrane, thegel was dried at 80°C and stored. DNA was eluted fromthe dried gel at the same location on X-ray film wherethe variant band was detected. The extracted DNAfragments were sequenced by the cycle sequencemethod.

Using Fisher’s exact probability test, a statisticalanalysis was performed, with P , 0.05 consideredsignificant.

RESULTSA molecular analysis was performed for 61 patientswith nonsmall cell lung carcinoma (NSCLC). Tumorsand preneoplastic lesions were microdissected fromformalin fixed, paraffin embedded specimens.

Extracted DNA was examined by LOH analysis formicrosatellite polymorphism in chromosome 3p and9p, and RFLP was performed at exon 4 of the p53 gene.Table 2 shows the results of genetic alterations in theSCC group and the Ad group. Fourteen (40.0%) of 35patients with SCC showed LOH for microsatellite poly-morphism. Nine of these 14 cases showed that SCChad LOH but metaplastic lesions did not, and theremaining 5 cases showed LOH in both metaplasticlesions and SCC (Table 3). On the other hand, 8(30.8%) of 26 Ads showed LOH for microsatellite poly-morphism. Three of these 8 cases showed LOH in bothAAH and synchronous Ad (Table 3).

We found LOH of 3p and/or 9p in 8 preneoplasticlesions, consisting of 5 metaplastic lesions and 3 alve-

TABLE 2Results of Genetic Alterations in the SCC Group and the Ad Group

LOH (%)

p53 mutation (%)c Any alteration (%)3pa 9pa 3p and 9pa 17pb

SCC groupSCC (n 5 35) 9 (25.7) 2 (5.7) 3 (8.6) 4 (36.4)d 9 (25.7) 17 (48.6)Dys (n 5 22) 2 (9.1) 1 (4.5) 1 (4.5) 4 (36.4)d 2 (9.1) 7 (31.8)Sm (n 5 13) 1 (7.7) 0 (0.0) 0 (0.0) 0 (0.0)d 0 (0.0) 1 (7.7)

Ad groupAd (n 5 26) 3 (11.5) 5 (19.2) 0 (0.0) 2 (16.7)e 6 (23.1) 13 (50.0)f

AAH (n 5 20) 2 (10.0) 1 (5.0) 0 (0.0) 0 (0.0)e 1 (5.0) 3 (15.0)f

AH (n 5 8) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0)e 0 (0.0) 0 (0.0)

SCC: squamous cell carcinoma; Ad: adenocarcinoma; LOH: loss of heterozygosity; Dys: dysplasia; Sm: squamous metaplasia; AAH: atypical adenomatous hyperplasia; AH: adenomatous hyperplasia without atypia.a Examined by microsatellite analysis.b Examined by restriction fragment length polymorphism.c Examined by single strand conformation polymorphism.d Of 11 informative cases.e Of 12 informative cases.f The difference between Ad and AAH was statistically significant (P 5 0.01).

Mutation of Preneoplastic Lesions of the Lung/Kohno et al. 343

olar hyperplastic lesions (Table 3). Seven (87.5%) ofthese 8 lesions showed loss of the same allele.

Mutations of the p53 gene in SCCs were detectedby PCR-SSCP in 9 (25.7%) of 35 SCCs (Table 2). Incontrast to squamous metaplasia, in which we foundno mutations of the p53 gene, mutations in dysplasiawere detected by PCR-SSCP in 2 (9.1%) of 22 lesions(Tables 2, 3). In these 2 cases, the same mutation andLOH of 17p were observed in both SCC and dysplasia.One case (No. 3, listed in Table 3) showed 1 basedeletion of exon 8, and the other case (No. 7, listed inTable 3) showed 1 base insertion of exon 6 (Figs. 1–3).Of 26 adenocarcinomas, 6 cases (23.1%) demonstratedaberrant bands by the SSCP method (Table 2). In 1 ofthese 6 cases, an aberrant band with the same shiftedmobility was detected in both AAH and synchronousAd by SSCP (No.10, listed in Table 3).

We did not find any genetic alterations from pre-neoplastic lesions when the tumor had no geneticchange.

Fifty-one of 61 patients consisted of 15 non-smokers and 36 smokers (Tables 1, 4). Eight (53.3%)

of 15 nonsmokers and 15 (41.7%) of 36 smokers hadgenetic alterations. There was no significant differ-ence between nonsmokers and smokers. This find-ing may have resulted from the biased distribution,in which all the nonsmokers were the patients withAd. In addition, no significant association was ob-served between genetic alterations and pathologicstage.

In conclusion, we found genetic alterations, in-cluding LOH of chromosome 3p, 9p and/or alterationsof the p53 gene, in metaplastic lesions of 8 cases (1lesion of squamous metaplasia and 7 lesions of dys-plasia) and in alveolar hyperplastic lesions of 3 cases(all AAHs) (Table 3). There was no significant differ-ence between SCC (48.6%, 17 of 35 cases) and dyspla-sia (31.8%, 7 of 22 cases) or between dysplasia andsquamous metaplasia (7.7%, 1 of 13 cases) (P 5 0.06),however, dysplasia tends to accumulate more geneticalterations than squamous metaplasia. In contrast, thedifference between Ad (50.0%, 13 of 26 cases) and AAH(15.0%, 3 of 20 cases) was statistically significant (P 50.01) (Table 2).

TABLE 3Results of LOH of Chromosome 3p, 9p, 17p and Mutations of p53 gene of the Cases in Which Preneoplastic Lesions Showed Genetic Alterations

Case no. Age/gender StageaHistologictype

LOH Mutation of p53 gened

3pb 9pb 17pc Exon Codon

1. 73/M IIIa SCC (1) (2) NI (2)Sm (1) (2) NI (2)

2. 76/M I SCC (1) (1) NI (2)Dys (1) (1) NI (2)

3. 77/M I SCC (1) (2) (1) 8 266 GGA3GADys (2) (2) (1) 8 266 GGA3GA

4. 70/M IIIb SCC (2) (2) (1) (2)Dys (2) (2) (1) (2)

5. 64/M I SCC (2) (2) (1) (2)Dys (2) (2) (1) (2)

6. 57/M II SCC (2) (1) NI (2)Dys (2) (1) NI (2)

7. 76/M II SCC (1) (2) (1) 6 190 CCT3ACCTDys (1) (2) (1) 6 190 CCT3ACCT

8. U I SCC (1) (2) NI (2)Dys (1) (2) NI (2)

9. 75/F IIIb Ad (1) (2) NI (2)AAH (1) (2) NI (2)

10. 79/F I Ad (2) (1) NI 8 N.D.AAH (2) (1) NI 8 N.D.

11. 64/M I Ad (1) (2) (2) (2)AAH (1) (2) (2) (2)

SCC: squamous cell carcinoma; Sm: squamous metaplasia; Dys: dysplasia; Ad: adenocarcinoma; AAH: atypical adenomatous hyperplasia; U: unknown.a According to International Union Against Cancer TNM classification.b Examined by microsatellite analysis.c Determined by restriction fragment length polymorphism.d Determined by single strand conformation polymorphism.


DISCUSSIONCarcinogenesis is a multistep process that results froman accumulation of genetic alterations. SCC of thelung is associated with squamous metaplasia of thebronchial epithelium. Recent studies have shown thatAAH is a potential precursor to pulmonary adenocar-cinoma.14 –16 In this study, we examined LOH usingmicrosatellite polymorphism on chromosomes 3p, 9pand the p53 gene mutations in squamous metaplasia,adenomatous hyperplasia, and synchronous NSCLCfor individual cases.

Previous studies have demonstrated that frequentLOH is observed on chromosome 3p and 9p in NSCLC.It has been reported that the frequency of allelic loss is49 – 86% on chromosome 3p6 –9,12,13 and 13–71% onchromosome 9p.1,8 –13 Some investigators have foundthat LOH on 3p and 9p is more frequent in SCC thanin Ad.1,8 We observed that LOH was present on 3p in12 SCCs (34.3%) and 3 Ads(11.5%), and on 9p in 5SCCs (14.3%) and 5 Ads (19.2%). All these numbers arelow compared with others from previous studies.1,6 –13

This finding may have resulted from the difference inour method of detection from the methods used inother studies.

It is reported that the same allele is lost in widelyseparated areas of the respiratory epithelium.1,6 Wefound LOH of 3p and/or 9p in 8 preneoplastic lesions,consisting of 5 metaplastic lesions and 3 alveolar hy-perplastic lesions. Seven (87.5%) of these 8 lesionsshowed loss of the same allele. This is consistent withthe findings of previous studies.1,6

LOH of chromosome 3p and/or 9p was observedin 1 of 13 squamous metaplasias and in 4 of 22 dys-plasias, and LOH of 17p was present in 4 of 11 infor-mative cases (Table 2). In all of 4 cases, metaplasticlesions were dysplasias. The incidence was low incomparison with SCC; however, these genetic alter-ations occurred more frequently in dysplasia than insquamous metaplasia. Furthermore, in 2 patients withSCC, the same mutation of the p53 gene was observedin both dysplasia and SCC. It may be significant thatdysplasia tends to accumulate genetic alterations.These findings suggest that LOH of chromosome 3p,9p and the mutations of the p53 gene are commonlyacquired before malignant transformation and dyspla-sias with genetic alterations may be considered trulypreneoplastic lesions. Researchers have described the

FIGURE 1. Histologic findings in Case No. 7 (cited in Table 3) are depicted

here. (a) Moderately differentiated squamous cell carcinoma is shown (H & E,

original magnification 3100). (b) Dysplasia is shown (H & E, original magni-

fication 3100).

FIGURE 2. (a) Restriction fragment length polymorphism analysis in Case

No.7 (cited in Table 3) is depicted here. The heterozygous pattern disappeared

in squamous cell carcinoma and dysplasia. (b) Single-strand conformation

polymorphism analysis of the p53 gene at exon 6 of the same case is shown.

Aberrant bands (black arrows) with shifted mobility were observed in squamous

cell carcinoma and dysplasia. N: normal epithelium; D: dysplasia; C: squamous

cell carcinoma; white arrows: normal bands.


mutation of the p53 gene as a late event associatedwith tumor progression in other human tumors, suchas colorectal,26 ovarian,3 thyroid,4 and prostate carci-nomas.27 In lung carcinoma, particularly squamouscell carcinoma, these changes may occur relativelyearly in preneoplastic lesions of the bronchial epithe-lium.

In contrast, we found genetic alterations in only 3(10.7%) of 28 alveolar hyperplastic lesions. All of 3lesions showed atypia, so-called AAH. This may indi-cate that the genetic alterations examined in this studyoccur relatively late in the pathogenesis of pulmonaryadenocarcinoma and may be associated with the de-velopment of the malignant phenotype. It was re-ported that LOH on chromosome 3p and 9p occurredvery frequently in preneoplastic lesions, including hy-perplasia, dysplasia, and carcinoma in situ.6,11,28 Al-though standardized pathologic criteria for AAH hasnot been clearly established and it is therefore diag-nosed subjectively, this may explain why our resultswere not consistent those of with previous studies.

In summary, we found genetic alterations, includ-ing LOH of chromosome 3p, 9p and mutations of thep53 gene, in only 3 (15.0%) of 20 AAHs. Although theseresults indicate that the genetic alterations we exam-ined occur relatively late in the pathogenesis of pul-monary adenocarcinoma, it is necessary to examineother genetic alterations associated with preneoplasticprocesses. On the other hand, we found genetic alter-ations in 7 (31.8%) of 22 dysplasias and 1 (7.7%) of 13squamous metaplasias. From these observations, wesuggest that bronchial squamous metaplasia withoutatypia is rather stable genetically because few geneticalterations were detected. In contrast, several geneticalterations accumulated in some dysplasias, and thesame mutation of the p53 gene was observed in bothdysplasia and SCC in 2 cases, suggesting that thesealterations may occur in early stages of SCC of the lungor in preneoplastic lesions. These findings may pointto the neoplastic nature of dysplasia and may be use-ful in the early diagnosis of preneoplastic lesions ofbronchial epithelium, not only by pathologic studybut also by molecular analysis.

TABLE 4Association of Genetic Alterations with Smoking Status

LOH (%)

p53 mutation (%)c Any alteration (%)3pa 9pa 17pb

Nonsmokers (n 5 15) 2 (13.3) 4 (26.6) 1 (12.5)d 3 (20.0) 8 (53.3)Smokers (n 5 36) 10 (27.8) 5 (13.9) 4 (36.4)e 10 (27.8) 15 (41.7)

BI ,400 (n 5 2) 1 0 0 1 1BI $400 (n 5 34) 9 5 4 9 14

Not described (n 5 10) 3 (30.0) 1 (10.0) 0 (0.0) 5 (50.0) 7 (70.0)

LOH: loss of heterozygosity; BI: Brinkman Index (the no. of cigarettes smoked/day 3 yrs).a Examined by microsatellite analysis.b Examined by restriction fragment length polymorphism.c Examined by single strand conformation polymorphism.d Of 8 informative cases.e Of 11 informative cases.

FIGURE 3. (a) Sequencing analysis of the p53 gene exon 6 of squamous cell

carcinoma and (b) dysplasia in Case No. 7 (cited in Table 3) is depicted here.

The same mutation was shown at codon 190 of the p53 gene. (c) Microsatellite

analysis of chromosome 3p locus D3S643 of the same case is depicted.

Squamous cell carcinoma and dysplasia showed loss of heterozygosity. N:

normal epithelium; D: dysplasia; C: squamous cell carcinoma.


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p53 mutation and allelic loss of chromosome 3p, 9p of preneoplastic lesions in patients with...

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