[CANCER RESEARCH 52. 5291-5298, October 1. 1992]

p53 Mutations in Breast CancerChristopher Coles,1 Alison Condie, Udi Chetty, C. Michael Steel, H. John Evans, and Jane Prosser2

MRC Human Genetics Unit, Western General Hospital, Edinburgh EH4 2XU [C. C., A. C, C. M. S., H. }. E., 3. P.], and Department of Surgery, Royal Infirmary,Edinburgh EH3 9YU[U. CJ, Scotland

ABSTRACT

We have identified and analyzed 41 mutations in p53 in sporadicbreast tumors from 136 unselected breast cancer patients and estimatethat approximately 40% of such tumors contain p53 mutations. Thefrequency of G-T transversions and the incidence of guanosine muta

tions in the nontranscribed strand of the pSÃŒgene were found to behigher than expected, and we suggest, therefore, that exogenous carcinogens have an etiological role in sporadic breast cancers. Mutationswere recorded in 44 codons of the p53 gene, with no obvious mutationalhot-spots, although mutations at codons 175, 194, 273, and 280 accounted for 25% of the changes. One germ-line mutation was found in

136 patients and so we conclude that constitutional mutation ofp53 maybe an uncommon etiological factor in breast cancer.

INTRODUCTION

For female nonsmokers, cancer of the breast is the mostimportant malignancy in Western society, and by the age of 75approximately 10% of women in the United States will havedeveloped the disease. There is evidence for a genetic contribution to the risk of developing breast cancer, as well as an association with modern affluence (diet and alcohol consumption).In addition, the influence of reproductive factors supports ahormonal role in the etiology of the disease (1, 2). Breast canceris, however, a heterogeneous disease (3) and for this reasonlinkage studies to pinpoint a genetic locus or loci responsiblefor the inherited suceptibility have been beset with difficulties.Studies on LOH3 in sporadic breast tumors have shown loss of

genetic material at a number of loci including Iq, 3p, 6q, 16q,17p, and 18q in >50% and at 1p, 7q, 8q, 9q, 11q, 13q, 15q, 17q,and 22q in >30% of tumors (4-17), suggesting the possibleinvolvement of a number of tumor suppressor genes. In addition, several oncogenes (myc, neu/HER-2/c-erbB-2, and int2)have been implicated in the development of the disease (reviewed in Refs. 18 and 19). The etiology of breast cancer is,therefore, complicated both by disease heterogeneity and by thenumber and variety of genetic changes which appear to be important. However, one gene which is clearly involved in thedevelopment of both sporadic and some hereditary breast tumors is p53 (20-34), a gene which in its unmutated form behaves as a tumor suppressor gene but which in at least severalmutant forms acts as an oncogene (35).

Mutations in the p53 gene are the most common geneticalterations in all human cancers (35, 36) and have been found atnumerous sites in > 100 of the 393 amino acids that comprisethe protein (37, 38). Such a variety of mutations has permittedanalyses for significant differences in the mutational spectrabetween different cancers (37) and has led to the postulation ofetiological roles for particular mutagens, e.g., the role of afla-

Received 4/2/92; accepted 7/17/92.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.

1Supported by the British Breast Cancer Research Trust.2To whom requests for reprints should be addressed.3 The abbreviations used are: LOH, loss of heterozygosity; HOT, hydroxylamine

and osmium tetroxide; HA, hydroxylamine; PCR, polymerase chain reaction.

toxin Bl and the hepatitis B virus in the G-T transversion ofp53 codon 249 in hepatocellular carcinomas from southernAfrica and east Asia (39,40). The nature of molecular change isoften specific for a given mutagen, e.g., UV light predominantlycauses cyclobutane dimers (41) and /V-methylnitrosourea produces G-A transitions (42). The nature of the mutations in p53

may, therefore, point to the mutagen(s) involved. In skin cancers p53 mutations at dipyrimidine sites (43) were indicative ofthe role of UV light, and in lung and esophageal cancers basepair changes in p53 were characteristic of those produced bymutagens in tobacco and alcohol (44). The spectrum of mutations in radon-associated lung cancer in uranium miners is different from that usually seen in lung cancers and may reflect thegenotoxic effects of radon (45). p53 mutations in colon cancerare characterized by a very high incidence of C-T changes,mainly at CpG dinucleotides (37), although etiological reasonsfor this have not been advanced.

We have estimated the incidence of p53 mutations in twoseries of sporadic breast tumors, based on mutation detectionby the HOT detection technique (46). Using our own data on137 tumors and published results of others, we discuss theoverall incidence of p53 mutations in sporadic breast tumors,the nature of these mutations, and their possible etiology. Wehave compared the spectrum of p53 mutations in sporadicbreast cancer with the germ-line mutations found in the Li-Fraumeni syndrome and with similar analyses in colon andother tumors.

MATERIALS AND METHODS

Tumor samples were collected from patients undergoing Patey mastectomy or wide local excision. All patients had presented with palpablebreast lumps and had been referred by their general practitioners to thebreast clinic in the Royal Infirmary of Edinburgh. Patients with T4tumors or with distant métastasesat presentation were excluded, because they were usually treated by chemotherapy in the first instance.Two sets of tumors resulted from serial admissions collected by twosuccessive surgeons. All DNA preparations were made from wholeblood or tumor tissue by standard techniques. PCR amplification, sequencing of PCR templates, and the HOT technique (HA and osmiumtetroxide modifications) were carried out as previously described(21, 47). Scanning for mutations in exons 5 and 6 of the first 60-tumorset (mutations 1-8; Table 1) was carried out using HA modificationonly and labeled wild-type DNA; for mutations in exons 7, 8, and 9 ofthe 60-tumor set (mutations 9-16) both HA and OsO4 modificationsand labeled wild-type DNA were used. This was also done for exons 5and 6 of the second 77-tumor set (mutations 17-32). HA and OsO«modifications and both wild-type and potential mutant labeled DNAswere used for exons 7-9 in the 77-tumor set (mutations 33-41); therefore, these fragments were scanned twice to ensure that all mutationswere detected. The exact mutation in each instance was determined bydirect sequencing of independently amplified PCR fragments. The leukocyte DNAs from patients with tumors carrying pS3 mutations weresubsequently checked for germ-line mutations.

When estimating the frequency ofp53 mutations in breast tumors, aproblem arises regarding the counting of multiple tumors from a single

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p53 MUTATIONS IN BREAST CANCER

Table 1 p53 mutations found in 137 sporadic breast tumors, with LOH results for the same samples

Mutationno.1234567891011121314151617181920212223242526272829303132333435363738394041Tumorno.82132343643518548920334757808101118212534365764677172951131148126184889298101110Codon164136°187°175201179152157250265281307283280242267208194140179192186182178175213163141194213194175285276273273237255239245282NucleotidechangeAAG—

CAGCAA—GAAGGT-TGTCGC—

CTCTTG—TT-CAT-GATCCG—

TCGGTC-TTCCCC-GCCCTG—

CCGGAC-GGCGCA—

ACACGC—CCCAGA—GGATGC-TTCCGG-CAGGAC—

GTCCTT—CGTGAC—G-CCAT-TATCAG—

TAGGAT-TATTGC-TGACAC-CCCCGC-CACCGA—

TGATAC-AACTGC-TACCTT—

CGTCGA—TGACTT-CGTCGC-CACGAG—

AAGGCC-CCCCGT-CATCGT—

CTTATG-ATTATC-AAC—

GACGGC-GACCGC—

CTGProtein

changeLys—

GinGin—GluGly—CysArg—LeuFrameshiftHis—

AspPro—SerVal-PhePro—

AlaLeu—ProAsp—GlyAla—ThrArg—ProArg—GlyCys—PheArg—GinAsp—ValLeu—ArgFrameshiftHis—

TyrGin—StopAsp—TyrCys—StopHis—ProArg-HisArg—

StopTyr—AsnCys—TyrLeu—ArgArg—StopLeu—ArgArg-HisGlu—

LysAla—ProArg—LeuArg—LeuMet—IleIle—DelAsn—AspGly—AsnArg—

LeuBHp53ULLLULNNUNLUNLN———LL—UULNU—LU———————UNL—LOHMCT35UUUuNLLULULLULU———UL—LULUU—Uu———————_uu—YNZ22LNLULLULNLLLNLLL———LL—NLLL—L————————_L—" Thompson et al. (32) give incorrect sequence changes for mutations at codons 136 and 187. In addition, no mutations were found in tumors 65 and 81 (codons 194

and 67) of their list. L, loss; N, no loss; U, uninformative; —¿�,not done.

individual. If multiple tumors unequivocally represented multipleprimaries, then, for the purposes of estimating mutation frequency,each tumor would be considered independently. But differentiating between multiple independent primaries and multiple deposits of a singleprimary is difficult. We consequently made the following decision: sixof the 136 patients had multiple tumors (total of 14 tumors); in fiveindividuals thep5J mutation status of each of the multiple tumors wasidentical and these were, therefore, counted as five tumors in total; inone individual with two tumors, one tumor contained mutant p53 andone did not, and these were counted as two separate tumors.

RESULTS AND DISCUSSION

Incidence of p53 Mutations in Sporadic Breast Cancer. Wehave found 41 mutations in sporadic tumors from 136 unse-lected breast cancer patients (137 tumors, as detailed in"Materials and Methods"). The precise codon alterations are

listed in Table 1 and sequence data on a number of mutationsare shown in Fig. 1. Two tumors contained two independentmutations. In a separate study,4 it was determined that theHOT technique as used (see "Materials and Methods") detects

90% of all mutations. For the purpose of estimating mutationfrequency, we consider only the second 77-tumor set, in which25 tumors (31%) contained mutations in exons 5-9 of thep55

4 Condie, A., Eeles, R., B*rreseu, A-L., Coles, C, Cooper, C, and Prosser, J.Detection of point mutations in the p53 gene: comparison of SSCP, CDGE, andHOT, submitted for publication.

gene. An allowance for the incomplete detection of mutationsuggests a probable frequency closer to 36%.

Our study does not take into account the total number ofpossible mutations in the entire p53 gene. In collated publisheddata regarding alterations to p53 in a wide variety of cancers(38), 6% of all mutations (22 of 368) were recorded outsideexons 5-9, in exons 1-4, 10, and 11 and in introns 3, 4, 5, 6, 7,and 9, despite a heavy bias in the literature against analyzingthese regions. The 6% of mutations found outside exons 5-9could, therefore, be a notable underestimate. Although few suchstudies have been undertaken, the sequencing of several entirep53 cDNA copies in a variety of tumors and tumor cell lines didnot reveal mutations which lie outside this region (20, 48-50).In addition, although we have included introns 5,7, and 8 in thePCR fragments of our survey, we have found no splicing mutations in 137 tumors. If approximately 10% of all p53 mutations lie outside exons 5-9, then the incidence of mutation inthe entire p53 gene in sporadic breast tumors must be on theorder of 40%. This estimate is in accord with, and extends,published findings on smaller series using nonimmunohis-tochemical methods (single-strand conformation polymorphism, HOT, constant denaturing gel electrophoresis, etc.) formutation detection (see Table 2).

Published estimates of mutations detected by immunohis-tochemical techniques suggest that on the order of 60% ofsporadic breast tumors have mutations in the p53 gene (23, 27,

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p53 MUTATIONS IN BREAST CANCER

8GATC

•¿�*

61GATC

12GATC

18GATC

36GATC

57GATC

,

f «OG

67GATC

I84

GATC

S

•¿�GATC

101GATC

Fig. I. Sequencing dala for 10 tumors in which the relative contributions of normal and wild-type alíelescan be assessed. For example, in tumor 8 the amount ofwild-type DNA is greater than that of mutant DNA. in tumor 61 the amount of mutant DNA is greater than that of wild-type DNA. and in tumor 57 only mutant DNAis present.

Table 2 Proportion of sporadic breast tumors with mutations in the p53 gene (nonimmunohistochemical methods of detection)Using DNA-based methods of mutation detection [constant denaturing gel electrophoresis (CDGE), (single-strand conformational polymorphism (SSCP),

HOT, direct sequencing], various regions of the p53 gene in breast tumors have been examined. The incidence of mutations has been compared in exons 5-8. Inorder to compare these estimates with those achieved by antibody staining, we have estimated mutation incidence in the entire p53 gene (see text).

MethodHOT

HOTCDGESSCPSSCPPCRRNase protectionPCRTumors

examined77

60322459112659Tumors

withmutation25

16109

1043

21Exons

studied5-9

5-95,7,8

5-85-95-9

Entire gene2-11Reported

mutations(%)32

27313817361236Estimated

mutations inexons 5-8(%)36"

33»34CEstimated

mutations in allexons(%)40

37384219

36Ref.This

paper21,32

293031283031

" If 25 represents 90% of mutations present, then 28 mutations would be the probable actual incidence in these 77 tumors (36%) (see text).* Prosser et al. (21 ) used the HOT technique with HA modification and wild-type labeled DNA. This theoretically detects 50% of all possible mutations but in sporadic

breast tumors, where 74% of mutations occur in GC base pairs, approximately 70% are detected. Therefore, the 8 mutations found in exons 5 and 6 would represent 11probable mutations. The HOT technique with wild-type DNA labeled using both HA and OsO4 modifications detects 90% of mutations.4 Consequently, the 8 mutationsfound in exons 7, 8, and 9 (32) would represent 9 probable mutations, giving a total of 20 probable actual mutations in the 60-tumor set (33%).

c Exons 5, 7, and 8 were studied. Mutations in exon 6 account for 13% ofp53 mutations recorded between exons 5 and 8 in all cancers surveyed (38). This estimate

has, therefore, been adjusted to include potential exon 6 mutations.

51, 52). Immunohistochemical staining uses p5J-specific antibodies which recognize various epitopes on the p53 protein.The increased stability of many mutant p53 proteins permitstheir detection in tissue sections with antibodies such aspAb421, pAblSOl, and polyclonal CM-1. Antibody pAb240specifically recognizes an epitope characteristic of many mutantproteins (53).

There is clearly a serious discrepancy in the estimates ofmutation based on the two sorts of technique, one an analysis atthe DNA level (~40%) and the other at the protein level(~60%). This might suggest that sampling bias in the DNAstudies may be greater than estimated and that >10% of mutations may occur outside exons 5-9. On the other hand, theassumption that detection of expression of p53 protein meansoverexpression of mutant as opposed to wild-type protein maybe open to question. Indeed, some tumorigenic cell lines over-

express wild-type p53 (54), and it is known that there are differences in p53 protein levels due to factors affecting differentiation (55) and cell cycle progression (54). Moreover, in fourundifferentiated neuroblastoma-derived cell lines high-level expression of a stable, apparently wild-type, protein was found(56). It is of course possible that a gene outside the p53 locus isinvolved in the increased half-life of some proteins. Milner andWatson (57) observed that the wild-type murine p53 proteincould be expressed in a form immunologically similar to amutant protein, within l h after addition of fresh growth medium. If their observations hold true in human material, it ispossible that not all the p53 which stains with mutant-specificantibody is mutant. Interestingly, the temperature-sensitive p53mutant (Val-135) fluctuates between normal and mutant protein conformation depending on the growth temperature (58).Overall, there would appear to be good reason to question the

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p53 MUTATIONS IN BREAST CANCER

assumption that elevated expression of p53 is synonymous withexpression of mutant p53.

Notwithstanding the qualifications given above, in studies of24 positively staining breast tumors and cell lines known tooverexpress mutant p53,22 were shown to possess mutations inthe gene (22, 24, 25, 27, 29), and similar findings have beenreported for tumors and cell lines of lung, colon, ovary, andesophagus (44, 59-62). In fact, there are a number of reasons tosuppose that some mutant p53 genes would be undetected byantibody staining, i.e., those with stop codons truncating thegene before the recognized epitope, those with deletions of bothalíeles,and those with protein degraded by the human papillo-mavirus E6 protein. Some investigators have, indeed, foundmutations in negatively staining material (29, 44, 60). Atpresent, therefore, in the absence of direct comparison within alarge series of mutations detected both at the DNA level and atthe protein level, it is unclear whether DNA studies underdetectmutations or antibody-staining techniques overdetect them.

Relationship between p53 Mutation and LOH on Chromosome 17p. The p53 gene is located at 17pl3, and data on p53mutation and LOH for this region can indicate a temporalorder for the two events in tumor development. Our resultsshow that, of 20 tumors which are both mutant at the p53 geneand informative for markers at BHp53 or MCT35 (loci nearp53), 75% show LOH (Table 1) and may conform to the sequence of events of mutation in p53 followed by loss of thenormal alíeleon the partner chromosome. This value is similarto those found in other breast cancer studies also using markersnear the p53 gene (24, 29, 30). Evidence for two independentloci showing significant LOH on the short arm of chromosome17, one at 17pl3.1, the site ofp53, and one more telomeric tothis at 17pl3.3, has been found in a number of cancers including breast (8, 11), liver (63), and kidney (64). Unless the patternof genetic loss on 17p has been shown to involve only one locus,as is the case with colon cancer (48), LOH over the p53 genecannot be assumed by loss at a more telomeric site (e.g., thatdefined by the probe YNZ22).

Several investigators have directly addressed the question ofthe presence or absence of a wild-type alíelein tumors containing a mutation in p53, using sequence data or single-strandconformation polymorphism autoradiographs. Since whole tumor DNAs from our breast tumor series were PCR amplifiedand sequenced with no consideration for the relative amounts oftumor and normal tissue in each of the samples, in many instances it was difficult to determine whether the mutations inp53 were accompanied by loss of the wild-type p53 alíele.In our77 iunior series, sequencing data showed that the mutant alíelewas at least twice the intensity of the wild-type alíelein 11 of the25 mutations, evidence that, in the tumor tissue, the mutantalíelewas accompanied by loss of the normal alíele.In theremaining 14 mutations, however, the wild-type alíelewas either equal to or greater than the mutant alíelein intensity. Inthe literature, a high proportion of homozygous or hemizygousmutations in breast tumors and cell lines has been found(24, 28, 30, 31), and a similar high proportion of homo/hemi-zygosity pertains to tumors of the ovary, brain, liver, lung,uterus, and gut, as well as leukemia (39, 65-71). In the twotumors containing two independent p53 mutations, no LOHdata are available; neither is it known if the mutations are onseparate alíeles.

While in a proportion of sporadic breast tumors, as in coloncancer (20, 48, 72), p53 mutation may occur before loss of the

wild-type alíele,there are a substantial number in which loss isnot accompanied by mutation. Of 34 tumors in our series withLOH near the p53 gene 56% apparently have only normal p53DNA, suggesting that in many tumors loss of the normal wild-type alíelemay occur without concomitant p53 mutation. Otherstudies with similar results (20, 24, 30, 48, 70) suggest severalpossible explanations for this: that loss on 17p may be a random or nonselective loss, that the screening technique used maynot have detected the corresponding p53 mutation, that p53mutation may occur outside the region screened, that there maybe a dosage effect such that 50% dosage ofp53 confers a selective growth advantage to the cell, or, finally, that the alíelelossmay involve a second tumor suppressor gene.

Mutation of one copy of p53 and loss of the wild-type alíeleconform to KHudson's two-hit hypothesis of tumor suppressor

genes, and our results support the role of p53 as a tumor suppressor gene in a number of breast tumors. Nevertheless, itshould be remarked that we, and others (25, 30), have foundevidence for a proportion of tumors with p53 mutation and noconcomitant loss of the remaining wild-type alíele(five in thisstudy with no LOH of adjacent 17p markers). It is known thatcertain mutant forms of p53 behave as oncogenes (73-75), andit would be interesting to address the question of whetherstrongly oncogenic mutations require no loss of the wild-typep53 alíelein order to effect a growth advantage, while mutationswhich are either weakly oncogenic or not oncogenic (simplyloss of function) need to be associated with loss of the normalalíele.The relative oncogenicity of only a few mutant p53 geneshas been determined. Hinds et al. (73) and Halevy et al. (74)have compared different mutant p53 genes for their ability totransform primary embryo rat cells in culture. Milner and Med-calf (75) have compared mutant p53 genes for their ability todrive wild-type p53 into the mutant conformation. From thesestudies, a number of mutations have been catergorized asstrongly oncogenic (Phe-132, Val-135, Ser-151, His-175, Ile-247, and Pro-273) or weakly oncogenic (Trp-248, Cys-270,His-273, and Gly-281). Only two samples from our breast tumor series contain mutations of tested oncogenicity. Tumors 25and 32 (Table 1) both have the 'strong' His-175 mutation and

so might be expected to have retained the wild-type p53 alíele,but tumor 25 shows LOH at BHp53 and MCT35.1 and sequencing data show complete and partial loss of the wild-typealíelein tumors 25 and 32, respectively.

Analysis of the pS3 Mutational Spectrum in Sporadic BreastTumors. A total of 84 mutations in sporadic breast tumorsfrom our series and from the literature are listed in Table 3.When the specific types of mutations are analyzed (Table 4),there are some notable observations. There is an excess of pointmutations at G/C base pairs (75%). (The overall content ofGC in ihep53 gene is 56%.5) It is possible that 50% (42 of 84)

of all breast tumor mutations are point mutations occurring atguanosines in the noncoding strand. This bias is seen in GC-CG, GC-AT, and GC-TA mutation events, although it is mostpronounced for GC-TA transversions (14 of 16 or 88%). Elevenof 14 GC-AT transitions at CpG dinucleotides may be morerealistically viewed as C-T alterations in the coding strand, butthe remaining 31 of 84 mutations (37%) which could haveoccurred at guanosines in the noncoding strand are in excess ofexpectation. This is significant in view of the fact that thenucleoside guanosine is a preferential target for most chemical

5 P. M. Chumakov, EMBL Accession Number is X54156 for HSP53G.

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fS3 MUTATIONS IN BREAST CANCER

Table 3 Codon number and specific alteration for breast tumor mutations(mutations from this paper and from the literature)

Single nucleotidesubstitutionsCodon

number128128132134136141151152157157163163164175175175175175175178179179182186187192193194194194194196208213213234237237237238238239242245245245248248248249250254265266267273273273273276278280280280280281281282282282283285285285307CodonchangeCCT-CCGCCT-TCTAAG—

CAGTTT—CTTCAA—GAATGC—TACCCC-TCCCCG—

TCGGTC-TTCGTC-TTCTAC—

AACTAC-TGCAAG—

CAGCGC—CACCGC-CACCGC—

CACCGC—CACCGC-CTCCGC—

CACCAC-CCCCAT—

TATCAT-GATTGC—

TGAGAT-TATGGT-TGTCAG—

TAGCAT-CCTCTT-CGTCTT-CGTCTT-CGTCTT-TTTCGA-CCAGAC-GTCCGA-TGACGA-TGATAC-TAAATG-ATTATG—

AAGATG—ATATGT-TTTTGT—

TTTAAC—GACTGC-TTCGGC-GACGGC-GTCCGC—

GACCGC—TGGCGG-CAGCGC—

CAGAGG-AGCCCC—

GCCATC—AACCTG-CCGGGA—

GTACGG-CAGCGT-CATCGT-CTTCGT-CATCGT-CATGCC-CCCCCT—

GCTAGA—ACAAGA—ACAAGA—GGAAGA-AAAGAC-GGCGAC-GGCCGG-CTGCGG-CCGCGG-CTGCGC-CCCGAG—

AAGGAG—AAGGAG—AAGGCA—

ACANucleotide

changeT-GC-TA—

CT-CC-GG—

AC-TC-TG-TG-TT—

AA-GA—

CG—AG-AG—

AG—AG-TG—

AA-CC—

TC-GC-AG-TG-TC—

TA-CT-GT-GT-GC-TG-CA-TC-TC-TC-AG-TT-AG-AG-TG—

TA-GG-TG—

AG—TG-AC-TG-AG—

AG-CC-GT—

AT-CG-TG-AG-AG-TG—

AG—AG-CG-CG-CG-CA—

GG-AA-GA-GG-TG-CG-TG-CG-AG-AG—

AG-ACpGdinucleotideNonNonNonNonCpGCpGCpGCpGCpGNonNonNonCpGCpGNonNonNonCpGCpGCpGCpGCpGCpGCpGNonNonNonNonNonSourceTumorTumorCell

lineTumorTumorTumorTumorTumorTumorCell

lineTumorTumorTumorTumorTumorTumorTumorTumorCell

lineTumorTumorTumorTumorTumorTumorTumorTumorTumorTumorTumorCell

lineTumorTumorTumorTumorCell

lineTumorTumorTumorTumorTumorTumorTumorTumorTumorTumorTumorTumorTumorCell

lineTumorTumorTumorTumorTumorCell

lineTumorTumorTumorTumorTumorTumorTumorTumorCell

lineTumorTumorTumorTumorTumorTumorTumorTumorCell

lineTumorRef.2929222921This

paper26212128This

paper2421This

paperThispaper27292128This

paperThispaper21This

paperThispaper21This

paperaThis

paperThispaperThispaper20aThis

paperThispaperThispaper31This

paper29243029This

paper322429This

paper29242522322532253220This

paperThispaper29This

paper24302832223229This

paper272432This

paper302232

Table 3—ContinuedDeletionsCodon140167172175-180201235-239255329Exon

11Codon

changeGAC—

G-CCAG-CA-GTT-GTDeletion

of 6codonsTTG—TT-FrameshiftdeletionATCdeletionACC—AC-Deletion

of30basepairsDeletionAT

basepairGCbasepairTAbasepair1

8 basepairsGCbasepair14

basepairs3basepairsCG

basepair30base pairsSourceTumorTumorTumorTumorTumorTumorTumorTumorTumorRef.This

paper2729242128This

paper2830

1P. Devilee. unpublished observations.

carcinogens (76) and that the nontranscribed strand is morecommonly the site of damage (possibly because of a bias in therate of DNA strand repair such that the coding strand is preferentially repaired over the noncoding strand) (77).

Two categories of mutation are, therefore, noticeably prevalent in sporadic breast tumors: CG-TA transitions and GC-TAtransversions. The increased incidence of CG-TA changes oc

curs both at CpG dinucleotides (19%) and at cytidines andguanosines not contained in this sequence conformation (20%).Although CG-TA changes at CpG dinucleotides are common,

they are not as frequent as in colon cancer, where they accountfor 67% of the total changes (37). With GC-TA transversions,

it is apparent that they are more frequent in sporadic breastcancer than expected. Hollstein et al. (37) found that GC-TAchanges occur at high frequency in lung cancer (non-small celllung cancer, 57%), liver cancer (74%), and esophageal cancers(24%), where a number of specific mutagenic factors are believed to be important. Aflatoxin Bl is a potent liver carcinogenwhich induces G-T transversions, and carcinogens in tobacco(for example, benzo[a]pyrene) are also known to elicit G-Ttransversions. However, in other solid tumors (colon, bladder,ovary, sarcomas, and brain) GC-TA transversions constitute asmall proportion of all mutations (5-13%). Of 75 point mutations in breast tumors in our series, 16 (21%) are GC-TAtransversions. The increased frequency of GC-TA transversionsand the high incidence of mutation of guanosines in the non-coding strand of the gene might, therefore, imply that externalcarcinogens have a role in the development of sporadic breasttumors.

The germ-line mutations to p53 found in the Li-Fraumenifamilial cancer syndrome (which includes breast tumor) have amutational spectrum with a preponderance of CG-TA transitions and few GC-TA transversions (Table 4). Forty-four % ofthe germ-line mutations are at CpG dinucleotides, a change

that is frequent in mammalian genomes (78) and may be attributed to spontaneous deamination of cytosine. It, therefore, appears that different factors predispose cells to the somatic p53mutations found in a large proportion of sporadic breast tumorsand to the germ-line p53 mutations which occur as part of theLi-Fraumeni inherited cancer syndrome.

When the mutational spectra between breast cancer and colon cancer are compared, there are obvious differences. A highproportion (67%) of all p53 mutations in colon cancer areCG-TA transitions at CpG dinucleotides. There are no recorded GC-TA transversions (37, 38) and three codons areindisputably hot-spots for mutation: codons 175, 248, and 273

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p5ìMUTATIONS IN BREAST CANCER

Table 4 Frequency of specific mutations in breast tumors, in Li-Fraumeni patients, and in cancers other than breast

Published breast mutations except thosefromthislaboratory (20, 22,24-31)"Breastmutations from this laboratory (thispaper;Refs.

21 and32)Totalbreast tumormutationsPercentage

of specific mutations in breasttumorsGerm-linep53 mutations (mainlyLi-Fraumeni)Percentageof specific germ-linemutationsPercentageof specific p53 mutations in allcancersother

than breast(38)Percentageof specific p53 mutations in allcancersother

than breast, liver, and lung (38)GC-CG6511150098GC-TA7916212102111GC-AT

(CpG,non)16

(8,8)13

(6,7)29(14,

15)39(19,20)15

(10,5)71(47,24)48(36,12)56

(42, 14)AT-GC34793141113AT-CG358111556AT-TA22450055Total37387510021100100100

" P. Devilee, unpublished observations.

(accounting for >50% of all the alterations). In contrast, inbreast cancer the picture is very different; of the 75 point mutations analyzed, only 19% occur at CpG dinucleotides, 21%are GC-TA transversions, and only 24% of breast cancer mutations occur at four codons: 175 (six), 194 (four), 273 (four),and 280 (four). Overall, CG-TA transitions at CpG are lessfrequent, GC-TA transversions are more frequent, and there arenot such pronounced hot-spots for mutation as found in coloncancer.

Analysis of the Particular Mutations and Codons Involved.Data from a number of studies show that the most frequent typeof change occurring in the p53 gene is a single-base missensesubstitution which alters a single amino acid in the protein (e.g.,Refs. 20, 37, and 38), and this is true in breast tumors also,where 89% (75 of 84) are single-nucleotide substitutions (84%result in amino acid substitutions and 5% in stop codons). Theremaining 11% (9 of 84) are deletions. The locations of all pointmutations in sporadic breast cancer published to date (Refs.20-32, 37, and 38 and this paper) have been in conservedcodons of the gene; 68% are in conserved domains II to V, 8%in domain II, 12% in domain III, 23% in domain IV, and 25%in domain V. Of the remaining mutations, 17 of 24 occur atcodons conserved from Xenopus to mammals and the rest arefound in codons conserved among mammals. A number of neutral mutations and polymorphisms have been reported for p53in the literature. In 76 patients we have found two samples withthe CGA-CGG neutral mutation constitutionally present atcodon 213 (79, 80).

One of the 41 mutations in 136 patients was found to be aconstitutional change, CGG-CAG at codon 267, giving an incidence of constitutional mutations in p53 in sporadic breastcancer of 0.7% (81). The patient carrying this constitutionalmutation was subsequently found to have come from a cancer-prone family, although not a Li-Fraumeni family. We havereanalyzed the five breast cancer-prone families previously reported (47) and confirm our inability to find constitutional p53mutations in these individuals. In our 77-tumor set we found norelationship between p53 mutation and tumor size, diseasestage, or menopausa! status. This was also true for the 60-tumor

set (32).Conclusions, (a) From our study of 136 breast cancer pa

tients we estimate that 40% of sporadic breast tumors carrymutations in thep53 gene. A review of the data on incidence ofp53 mutations in sporadic breast cancer shows that the estimates of mutation frequency are significantly different depending upon whether the measurements are made by DNA analysis(~40%) or by immunohistochemical methods (~60%). Thereare theoretical reasons for both techniques to underestimate the

actual incidence, but there is good evidence to suggest thatimmunohistochemical staining may well result in an overesti-mation. Until a body of material selected on the basis of immunohistochemical screening is extensively analyzed by non-immunohistochemical methods and/or sequencing, it will bedifficult to account for the observed differences.

(b) In sporadic breast tumors, the increased frequency ofGC-TA transversions, together with a very high incidence ofguanosine mutations in the nontranscribed strand of the p53gene, leads to the conclusion that exogenous carcinogens mayhave an etiological role in these tumors. Information was notavailable on what proportion of the breast cancer patientswere smokers and whether this was relevant in accounting forsome G-T transversions. Carcinogenic hormones are generallyinefficient in the production of point mutations at the genelevel (82), but increased estrogen levels are known to promote cell growth and may indirectly increase the incidence ofmutation.

(c) Germ-line p53 mutations in the Li-Fraumeni family cancer syndrome (which includes breast cancer) are mainly CG-TAtransitions at CpG dinucleotides. These may be naturally occurring endogenous events.

(d) Somatic mutations in breast tumors have been found in 44codons of the p53 gene between exons 5 and 9. A preponderance of mutations was found at codons 175, 194, 273, and 280,but no particular mutational hot-spot was identified.

(e) Analysis of the data for LOH and mutation in p53 insporadic breast tumors suggests that in a proportion of tumorsmutation may occur before alíeleloss but this sequence ofevents is not followed in a substantial number of tumors.

(/) In breast cancer patients an autosomal dominant mode ofinheritance accounts for approximately 5-10% of cases (83).Based on our finding of one germ-line mutation in sporadicbreast tumors from 136 patients (81) and on our finding of noapparent p53 mutation in five families with breast cancer (47),we conclude that mutation in p53 is a rare etiological factor inhereditary breast cancer.

ACKNOWLEDGMENTS

The authors thank Wendy Bickmore and Nick Hastie for discussionand critical reading of the manuscript and Peter Devilee for permittingus to use unpublished data.

REFERENCES1. Wille«,W. The search for the causes of breast and colon cancer. Nature

(Lond.), 338: 389-394, 1987.2. Henderson, B. E., Ross, R. K., and Pike, M. C. Toward the primary preven

tion of cancer. Science (Washington DC). 254: 1131-1137, 1991.

5296

on May 13, 2021. © 1992 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

pS3 MUTATIONS IN BREAST CANCER

3. Devilee, P., and Cornelisse, C. J. Genetics of human breast cancer. Cancer 28.Surveys, 9:605-630, 1991.

4. Lundberg, C.. Skoog, L.. Cavenee, W. K., and Nordenskjold, M. Loss ofheterozygosity in human ductal breast tumours indicates a recessive mutationon chromosome 13. Proc. Nati. Acad. Sci. USA, 84: 2372-2376, 1987. 29.

5. Devilee, P., van der Broek, M., Kuipers-Dijkshoorn, N., Kolluri. R., Khan, P.M., Pearson, P. L., and Cornelisse, C. J. At least four different chromosomalregions are involved in loss of heterozygosity in human breast carcinoma.Genomics, 5: 554-560, 1989. 30.

6. Genuardi, M., Tsihira, H., Anderson, D. E.. and Saunders. G. F. Distaldeletion of chromosome Ip in ductal carcinoma of the breast. Am. J. Hum.Genet., «.-73-82, 1989.

7. Borresen, A. L., Ottestad, L., Gaustad, A., Anderson, T. I., Heikkila, R.. 31.Jahnsen, T., Tveit, K. M., and Nesland, J. M. Amplification and proteinover-expression of the neu/HER-2/c-erbB-2 proto-oncogene in human breastcarcinomas: relationship to loss of gene sequences on chromosome 17. family 32.history and prognosis. Br. J. Cancer, 62: 585-590, 1990.

8. Coles, C., Thompson, A. M., Elder, P. A., Cohen, B. B., Mackenzie, 1. M.,Cranston, G., Chetty, U.. Mackay, J., Macdonald, M., Nakamura, Y., Hoy-heim, B., and Steel, C. M. Evidence implicating at least two genes on chro- 33.mosome I7p in breast carcinogenesis. Lancet, 336 (2): 761-763, 1990.

9. Cropp, C. S., Lidereau, R., Campbell, G.. Champene, M. H.. and Callahan,R. Loss of heterozygosity on chromosomes 17 and 18: two additional regionsidentified. Proc. Nati. Acad. Sci. USA, 87: 7737-7741, 1990.

10. Larsson, C., Bystrom, C., Skoog, L., Rotstein, S., and Nordenskjold, M. 34.Genomic alterations in human breast carcinomas. Genes Chrom. Cancer, 2:191-197, 1990.

11. Sato, T., Tanigami, A., Yamakawa, K., Akiyama, F., Karsumi, F.. Sakamoto. 35.G., and Nakamura, Y. Allelotype of breast cancer: cumulative alíelelossespromote progression in primary breast cancer. Cancer Res., 50: 7184-7189, 36.1990. 37.

12. Thompson, A. M., Steel, C. M.. Chetty, U., Hawkins, R. A., Miller, W. R.,Carter, D. C., Forrest, A. P. M., and Evans, H. J.pS3 gene mRNA expression 38.and chromosome 17p alíeleloss in breast cancer. Br. J. Cancer, 61: 74-78,1990.

13. Devilee, P., van de Broek, M., Mannens, M., Slater, R., Cornelisse. C. J., 39.Westerveld, A., and Khan, P. M. Differences in patterns of alíeleloss betweentwo common types of adult cancer, breast and colon carcinoma and Wilms'tumour of childhood. Int. J. Cancer, 47: 817-821, 1991. 40.

14. Devilee, P., van Vliet, M., Kuipers-Dijkshoorn, N., Pearson, P. L., andCornelisse, C. J. Somatic genetic charges on chromosome 18 in breast carcinomas: is the DCC gene involved? Oncogene, 6: 311 -315, 1991. 41.

15. Devilee, P., van Vliet, M., van Sloun, P., Dijkshoorn, N. K.. Hermans, J.,Pearson, P. L., and Cornelisse, C. J. Allelotype of human breast carcinoma:a second major site for loss of heterozygosity is on chromosome 6q. Onco- 42.gene, 6: 1705-1711, 1991. 43.

16. Sato, T., Akiyama, F., Sakamoto, G., Kasumi, F., and Nakamura, Y. Accumulation of genetic alterations and progression of primary breast cancer.Cancer Res., 51: 5794-5799, 1991.

17. Bieche, I., Chámpeme, M. H., Matifas, F., Hacene, K., Callahan. R., and 44.Lidereau, R. Loss of heterozygosity on chromosome 7q and aggressive primary breast cancer. Lancet. 339 (I): 139-143, 1992.

18. Callahan, R., and Campbell, G. Mutations in human breast cancer: an overview. J. Nati. Cancer. Inst., 81: 1780-1786, 1989. 45.

19. van de Vijer, M. J., and Nüsse,R. The molecular biology of breast cancer.Biochim. Biophys. Acta, 1072: 33-50, 1991.

20. Nigro, J. M., Baker, S. J., Preisinger, A. C.. Jessup, J. M., Hosteller, R.. 46.Cleary, K., Bigner, S. H., Davidson, N., Baylin, S., Devilee, P.. Glover, T..Collins, F. S., Weston, A., Modali. R., Harris. C. C., and Vogelstein, B.Mutations in the p53 gene occur in diverse human tumour types. Nature(Lond.), 342: 705-708, 1989. 47.

21. Prosser, J., Thompson, A. M., Cranston, G., and Evans, H. J. Evidence thatp53 behaves as a tumour suppressor gene in sporadic breast tumours. Oncogene, 5: 1573-1579, 1990. 48.

22. Bartek, J.. Iggo, R., Gannon, J., and Lane, D. P. Genetic and immunochem-ical analysis of mutant pS3 in human breast cancer cell lines. Oncogene, 5:893-899, 1990.

23. Bartek, J., Bartkova, J., Vojtesek, B., Staskova, Z., Rejthar, A., Kovarik, J.,and Lane, D. P. Patterns of expression of the p53 tumour suppressor in 49.human breast tissues and tumours in situ and in vitro. Int. J. Cancer, 46:839-844, 1990.

24. Davidoff. A. M., Humphrey, P. A., Iglehart. J. D., and Marks, J. R. Geneticbasis for p53 over-expression in human breast cancer. Proc. Nati. Acad. Sci.USA, ««.-5006-5010,1991. 50.

25. Davidoff, A. M., Kerns, B-J. M., Iglehart, J. D., and Marks. J. R. Maintenance of p53 alterations throughout breast cancer progression. Cancer Res.,51: 2605-2610, 1991.

26. Chen, L-C, Neubauer, A., Kurisu, W., Waldman, F. M., Ljung, B-M., Good-son, W., Goldman, E. S., Moore, D., Balazs, M., Liu. E.. Mayall, B. H., and 51.Smith. H. S. Loss of heterozygosity on the short arm of chromosome 17 isassociated with high proliferative capacity and DNA aneuploidy in primaryhuman breast cancer. Proc. Nati. Acad. Sei. USA, Ä«:3847-3851, 1991.

27. Varley. J. M.. Brammar, W. J., Lane. D. P., Swallow, J. E., Dolan, C., and 52.Walker, R. A. Loss of chromosome 17pl3 sequences and mutation ofp53 inhuman breast carcinomas. Oncogene. 6:413-421, 1991.

5297

Kovach, J. S., McGovern. R. M., Cassady, J. D., Swanson, S. K., Wold, L. E.,Vogelstein, B., and Sommer, S. S. Direct sequencing from touch preparationsof human carcinomas: analysis of p53 mutations in breast carcinomas. J.Nati. Cancer. Inst., 83: 1004-1009, 1991.Borresen, A-L., Hovig, E., Smith-Sorensen, B.. Malkin, D., Lystad, S.,Andersen, T. I., Nesland, J. M., Isselbacher, K. J., and Friend, S. H. Constantdénaturantgel electrophoresis as a rapid screening technique for p53 mutation. Proc. Nati. Acad. Sci. USA, ««:8405-8409, 1991.Osborne, R. J.. Merlo, G. R., Mitsudomi, T., Venesio, T.. Liscia, D. S.,Cappa, A. P. M., Chiba, L, Takahashi, T., Ñau, M. M., Callahan, R., andMinna. J. D. Mutations in the p53 gene in primary human breast cancers.Cancer Res., 51: 6194-6198. 1991.Runnebaum, I. B., Nagarajan, M.. Bowman, M., Soto, D., and Sukumar, S.Mutations in p53 as potential molecular markers for human breast cancer.Proc. Nati. Acad. Sci. USA, ««:10657-10661, 1991.Thompson. A. M., Anderson, T. J.. Condie, A., Prosser, J., Chetty, U.,Carter, D. C.. Evans, H. J., and Steel, C. M. p53 alíelelosses, mutations andexpression in breast cancer and their relationship to clinico-pathologicalparameters. Int. J. Cancer, 50: 528-532, 1991.Malkin, D., Li, F. P., Strong, L. C., Fraumeni, J. F., Nelson, C. E., Kim, D.H., Kassel, J., Gryka, M. A., Bischoff, F. Z., Tainsky, M. A., and Friend, S.H. Germ lim- />•><mutations in-a familial syndrome of breast cancer, sarcomas, and other neoplasms. Science (Washington DC), 250: 1233-1238,1990.Srivastava, S., Zou, Z., Pirollo. K.. Blattner. W., and Chang, E. H. Germ-linetransmission of a mutated p53 gene in a cancer-prone family with Li-Frau-meni syndrome. Nature (Lond.), 348: 747-749, 1990.Lane, D. P., and Benchimol, S. p53: oncogene or antioncogene? Genes &Dev., 4: 1-8, 1990.Vogelstein, B. A deadly inheritance. Nature (Lond.), 348: 681-682. 1990.Hollstein, M., Sidransky, D., Vogelstcin, B., and Harris, C. C. p53 mutationsin human cancers. Science (Washington DC), 253: 49-53, 1991.Carón de Fromentel, C., and Soussi, T. TP53 tumour suppressor gene: amodel for investigating human mutagenesis. Genes Chrom. Cancer, 4:1-15,1992.Hsu, I. C., Metcalf, R. A.. Sun, T., Welsh, J. A., Wang. N. J., and Harris, C.C. Mutational hotspot in the p53 gene in human hepatocellular carcinomas.Nature (Lond.), 350: 427-428, 1991.Bressac, B., Kew, M., Wands, J., and Ozturk, M. Selective G to T mutationsof the p53 gene in hepatocellular carcinoma from southern Africa. Nature(Lond.), 550:429-431, 1991.Glickman, B. Mutational specificity of UV light in E. coli. In: C. W.Lawrence (ed.). Induced Mutagenesis, pp. 135-177. New York: PlenumPress, 1983.Barbacid, M. ras genes. Annu. Rev. Biochem., 56: 779-827, 1987.Brash, D. E.. Rudolph. J. A.. Simon, J. A., Lin, A., McKenna, G. J., Baden,H. P., Halperin, A. J.. and Ponten, J. A role for sunlight in skin cancer:UV-induced p53 mutations in squamous cell carcinoma. Proc. Nati. Acad.Sci. USA, 88: 10124-10128, 1991.Bennct, W. P., Hollstein, M. C, He, A.. Zhu, S. M., Resau, J. H., Trump, B.F., Metcalf, R. A., Welsh, J. A., Midgley, C, Lane, D. P., and Harris, C. C.Archival analysis of p53 genetic and protein alterations in Chinese esoph-ageal cancer. Oncogene, 6: 1779-1784, 1991.Vahakangas, K. H., Samet, J. M., Metcalf, R. A., Welsh, J. A., Bennett, W.P., Lane, D. P., and Harris, C. C. Mutations of p53 and ras genes in radonassociated lung cancer from uranium miners. Lancet, 339 (1): 576-579, 1992.Cotton, R. G. H., Rodrigues, N. R., and Campbell, R. D. Reactivity ofcytosine and thymine in single-base-pair mismatches with hydroxylamine andosmium tetroxide and its application to the study of mutations. Proc. Nati.Acad. Sci. USA, «5:4397-4401, 1988.Prosser, J., Elder, P. A., Condie. A.. Macfayden, I., Steel, C. M., and Evans,H. J. Mutations in p53 do not account for heritable breast cancer: a study infive affected families. Br. J. Cancer, 63: 181-184, 1991.Baker, S. J., Fearon, E. R., Nigro, J. M., Hamilton, S. R., Preisinger, A. C.,Jessup, J. M., van Tuinen, P., Ledbetter. D. H., Barker, D. F., Nakamura, Y.,White, R., and Vogelstein, B. Chromosome 17 deletions and p53 gene mutations in colorectal carcinomas. Science (Washington DC), 244: 217-221,1989.Chiba, I., Takahashi, T., Ñau, M. M., D'Amico, D., Curici, D. T., Mitsudomi, T.. Buchhagen, D. L., Carbone. D., Piantadosi, S., Koga, H., Reiss-man, P. T., Slamon. D. J., Holmes, E. C., and Minna, J. D. Mutations in thep53 gene are frequently in primary, resected non-small cell lung cancer.Oncogene, 5: 1603-1610, 1990.Lehman, T. A., Bennet, W. P., Metcalf, R. A., Reddel, R.. Welsh, J. A.,Ecker, J., Modali. R. V., Ullrich, S.. Ramano, J. W., Appella, E.. Testa, J. R.,Gerwin, B. I., and Harris, C. C. p53 mutations, ras mutations, and p53-heatshock 70 protein complexes in human lung carcinoma cell lines. Cancer Res.,57:4090-4096, 1991.Bartek, J., Bartkova, J., Vojtesek, B.. Staskova, Z., Lukas. J., Rejthar, A.,Kovarik. J., Midgeley, C. A., Gannon, J. V., and Lane, D. P. Aberrantexpression of the p53 oncoprotein is a common feature of a wide spectrum ofhuman malignancies. Oncogene, 6: 1699-1703, 1991.Horack, E., Smith, K.. Bromley. L., Lejeune, S., Greenall, M., Lane, D., andHarris. A. L. Mutant p53. EGF receptor and c-erl>B-2 expression in humanbreast cancer. Oncogene. 6: 2277-2284, 1991.

on May 13, 2021. © 1992 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

pS3 MUTATIONS IN BREAST CANCER

53. Gannon, J. V., Greaves, R., Iggo, R., and Lane, D. P. Activating mutationsin p53 produce a common «informational effect. A monoclonal antibodyspecific for the mutant form. EMBO J., 9: 1595-1602, 1990.

54. Reich, N. C., and Levine, A. J. Growth regulation of a cellular tumourantigen, p53, in nontransformed cells. Nature (Lond.), 308: 199-201, 1984.

55. Reich, N. C., Oren, M., and Levine, A. J. Two distinct mechanisms regulatethe levels of a cellular tumour antigen, p53. Mol. Cell. Biol., 3: 2143-2150,1983.

56. Davidoff. A. M., Pence, J. C., Shorter, N. A., Iglehart, J. D., and Marks, J.R. Expression of p53 in human neuroblastoma- and neuroepithelioma-de-rived cell lines. Oncogene, 7: 127-133, 1992.

57. Milner, J., and Watson, J. V. Addition of fresh medium induces cell cycle andconformation changes in p53, a tumour suppressor protein. Oncogene, 5:1683-1690, 1990.

58. Michalovitz. D., Halevy, O., and Oren, M. Conditional inhibition of transformation and of cell proliferation by a temperature-sensitive mutant ofp53.Cell, 62:671-680, 1990.

59. Iggo, R., Gatter, K., Bartek, J., Lane, D., and Harris, A. L. Increased expression of mutant forms of p53 oncogene in primary lung cancer. Lancet, 335(J): 675-679, 1990.

60. Rodrigues, N. R., Rowan, A., Smith, M. E. F., Kerr, I. B., Bodmer, W. F.,Gannon, J. V., and Lane, D. P. p53 mutations in colorectal cancer. Proc.Nati. Acad. Sci. USA, 87: 7555-7559, 1990.

61. Marks, J. R., Davidoff, A. M., Kerns, B. J., Humphrey, P. A., Pence, J. C,Dodge, R. K., Clarke Pearson, D. L.. Igleheart, J. D., Bast, R. C., andBerchuck, A. Overexpression and mutation of pS3 in epithelial ovarian cancer. Cancer Res., 51: 2979-2984, 1991.

62. Gusterson, B. A., Anbazhagan, R., Warren, W., Midgely, C., Lane, D. P.,O'Hare, M., Stamps, A., Carter, R., and Jayatilake, H. Expression of p53 inpremalignant and malignant squamous epithelium. Oncogene, 6:1785-1789,1991.

63. Fujimori, M., Tokino, T., Hiño,O.. Kitagawa, T.. Imamura, T., Okamoto,E., Mitsunobu, M., Ishikawa, T., Nakagama, H., Harada, H., Yagura, M.,Matsubara, K., and Nakamura, Y. Allelotype study of primary hepatocellularcarcinoma. Cancer Res., 51: 89-91, 1991.

64. Morita, R., Ishikawa, J., Tsutsumi, M., Hikiji, K., Tsukado, Y., Kamidono,S., Maeda, S., and Nakamura, Y. Allelotype of renal cell carcinoma. CancerRes., 51: 820-823, 1991.

65. Gaidano, G., Ballerini, P., Gong, J. Z., Inghirami, G., Neri, A., Newcomb, E.W., Magrath, I. T., Knowles, D. M., and Dalla-Favera, R. p53 mutations inhuman lymphoid malignancies: association with Burkitt lymphoma andchronic lymphocytic leukemia. Proc. Nati. Acad. Sci. USA, 88: 5413-5417.1991.

66. Hensel, C. H.. Xiang, R. H., Sakaguchi, A. Y., and Naylor, S. L. Use of singlestrand conformation polymorphism technique and PCR to detect p53 genemutations in small cell lung cancer. Oncogene, 6: 1067-1071, 1991.

67. Mashiyama, S., Murakami, Y.. Yoshimoto, T., Sekiya. T., and Hayashi, K.Detection of p53 gene mutations in human brain tumors by single strandconformation polymorphism analysis of polymerase chain reaction products.Oncogene,«: 1313-1318, 1991.

68. Okamoto, A., Sameshima, Y., Yamada, Y., Teshima, S., Terashima, Y.,Terada, M., and Yokota, J. Allelic loss on chromosome 17p and p53 muta

tions in human endometrial carcinoma of the uterus. Cancer Res., SI: 5632-5636, 1991.

69. Okamoto, A., Sameshima, Y., Yokoyama, S., Terashima, Y., Sugimura, T.,Terada, M., and Yokota, J. Frequent allelic losses and mutations of ihe p53gene in human ovarian cancer. Cancer Res., 51: 5171-5176, 1991.

70. Shaw, P., Tardy, S., Benito, E., Obrador, A., and Costa, J. Occurence ofKi-ros and p53 mutations in primary colorectal tumours. Oncogene, 6:2121-2128, 1991.71. 'I amura. G., Kihana, T., Nomura, K., Terada, M., Sugimura, T., and Hiro-

hashi, S. Detection of frequent p53 gene mutations in primary gastric cancerby cell sorting and polymerase chain reaction single strand conformationpolymorphism analysis. Cancer Res., 51: 3056-3058, 1991.

72. Baker, S. J., Preisinger, A. C., Jessup, J. M., Paraskeva, C., Markowitz, S.,Willson, J. K. V.. Hamilton, S., and Vogelstein, B. p53 gene mutations occurin combination with 17p allelic deletions as late events in colorectal tumor¡genesis.Cancer Res., 50: 7717-7722, 1990.

73. Hinds, P. W., Finlay, C. A., Quartin, R. S., Baker, S. J., Fearon, E. R.,Vogelstein, B., and Levine, A. J. Mutant p53 DNA clones from human coloncarcinomas cooperate with ras in transforming primary rat cells: a comparison of the "hot spot" mutant phenotypes. Cell Growth & Differ., /: 571-580,

1990.74. Halevy, O., Michalovitz, D., and Oren, M. Different tumor derived p53

mutants exhibit distinct biological activities. Science (Washington DC), 250:113-116, 1990.

75. Milner, J., and Medcalf, E. A. Cotranslation of activated mutant p53 withwild-type drives the wild-type p53 protein into the mutant conformation.Cell, 65: 765-774, 1991.

76. Kriek, E., Engeise, L. D., Scherer, E., and Westra, J. G. Formation of DNAmodifications by chemical carcinogens. Identification, localization and quantification. Biochim. Biophys. Acta, 738: 181-201, 1984.

77. Mellon, I., Spivak, G., and Hanawalt, P. C. Selective removal of transcription-blocking DNA damage from the transcribed strand of mammalianDHFR gene. Cell, 51: 241-249, 1987.

78. Sved, J., and Bird, A. The expected equilibrium of the CpG dinucleotide invertebrate genomes under a mutational model. Proc. Nati. Acad. Sci. USA,«7:4692-4696, 1990.

79. Serra, A., Gaidano, G. L., Revello, D., Guerrasio, A., Ballerini, P., Pavera, R.D., and Saglio, G. A new Taq\ polymorphism in the p53 gene. Nucleic AcidsRes., 20: 928, 1992.

80. Carbone, D., Chiba, I., and Mitsudomi, T. Polymorphism at codon 213within thep53 gene. Oncogene, 6: 1691-1692, 1991.

81. Prosser, J., Porter, D., Coles, C., Condie, A., Thompson, A. M., Chetty, U.,Steel, C. M., and Evans, H. J. Constitutional p53 mutation in a non Li-Fraumeni cancer family. Br. J. Cancer, 65: 527-528, 1992.

82. IARC Monographs on the Evalution of Carcinogenic Risk to Humans, Vol.1-42, Suppl. 7, An Updating of IARC Monographs, pp. 272-310. Lyon,France: International Agency for Research on Cancer, 1987.

83. Lynch, H. T., Marcus, J. M., Watson, P., Conway, T., Fitzsimmons, M. L.,and Lynch, J. F. Genetic epidemiology of breast cancer. In: H. T. Lynch andT. Hirayama (eds.). Genetic Epidemiology of Cancer, pp. 289-332. BocaRaton, FL: CRC Press, Inc. 1989.

5298

on May 13, 2021. © 1992 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

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