[CANCER RESEARCH 55, 646-651, February 1, 1995]

p53 Abnormalities and Genomic Instability in Primary Human Breast Carcinomas1

Jorunn E. Eyfjörd,2Steinunn Thorlacius, Margret Steinarsdottir, Rut Valgardsdottir, Helga M. Ögmundsdottir, and

Kesara Anamthawat-Jonsson

Molecular and Cell Biology Research Laboratory, Icelandic Cancer Society, Skogarhlid 8, 105 Reykjavik [J. E. E., S. T., R. V., H. M. Ö.], and Cytogenetics Laboratory,Department of Pathology, University of Iceland. Reykjavik [M. S., K. A-J.J, Iceland

ABSTRACT

Abnormalities in the p53 tumor suppressor gene have been shown toaffect cell cycle control and lead to genetic instability in cell lines ofmurine and human origin. We have examined genetic instability in 183primary human breast carcinomas with and without p53 abnormalities.Mutation analysis was performed by constant dénaturant gel electro-

phoresis and DNA sequencing, and abnormal protein expression wasexamined by immunohistochemical staining methods. Genetic instabilitywas studied by detection of gene amplification, allelic loss, karyotypeanalysis, and fluorescent in situ hybridization. We found a significantassociation between /;5.? abnormalities and genetic instability detected bythese methods.

INTRODUCTION

Genomic instability is a common feature of cancer cells, but thereasons for this are still largely unknown. Alterations of the p53 tumorsuppressor gene have been reported in a spectrum of human cancers,suggesting that it plays a central role in the formation of tumors (1).

Proliferating cells have several mechanisms for repairing DNAdamage, and it has been shown that at least two stages in the cellcycle, the G,-S and G2-M transitions, are regulated in response to

such damage. These transitions serve as checkpoints to allow necessary repair to take place (2). A number of studies have implicated thep53 protein in the Gj-S arrest in response to DNA damage (3-5). The

content of p53 protein in the cell increases as a response to DNAdamage and recent studies have shown that p53 activates a MT21,000protein (Cipl/WAFl/SDI) that has been shown to inhibit the activityof cyclin-dependent kinases and thus induce either arrest in G, or

apoptosis (6). p53 has also been shown to be involved in the controlof DNA replication through interaction with PCNA3 (7). Cells with

abnormal p53 do not activate p21 and do not show the normal GÃŒarrest necessary for repair after exposure to DNA-damaging agents.

Normal cells in culture generally have a stable karyotype and do notundergo amplification, whereas most transformed and immortal cellsdo (8, 9). Cells that lack wild-type p53 can undergo gene amplifica

tion and show conversion to aneuploidy (10). Germ line mutations inthe p53 gene are found in families with the Li-Fraumeni cancersyndrome (11). Normal somatic cells from individuals with the Li-Fraumeni syndrome are heterozygous and wild-type/normal at the p53locus, whereas tumors that arise have no wild-type p53 alíele.Germ

line mutations have also been found in children with two differentmalignancies and in cancer-prone families that are not of the classicalLi-Fraumeni type, but such mutations are very rare in breast cancer

Received 8/29/94; accepted 11/29/94.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 with18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported by grants from The Icelandic Cancer Society Science Fund,The Nordic Cancer Union, The Icelandic Science Fund, the women's branch of the

Icelandic Red Cross, The National Hospital Science Fund, and The Memorial Fund ofBergthora Magnusdottir and Jakob J. Bjarnason.

2 To whom requests for reprints should be addressed, at Molecular and Cell Biology

Laboratory, Icelandic Cancer Society, P.O. Box 5420, 125 Reykjavik, Iceland.3 The abbreviations used are: PCNA, proliferating cell nuclear antigen; FISH, fluo

rescent in situ hybridization; AT, ataxia telangiectasia; CDGE, constant dénaturantgelelectrophoresis.

patients (12, 13). Studies of normal cells, non-neoplastic cells frompatients with the Li-Fraumeni syndrome that are heterozygous (wild-

type/mutant) at the p53 gene locus, murine embryonic fibroblasts withgerm line p53 mutation, and homozygous p53 mutant derivativesindicated that a loss or alteration of both copies of the p53 gene wasneeded for amplification to occur (10, 3). This p53-dependent path

way is not the only way amplification is regulated, however, sincethere are tumor cells with normal p53 that are able to undergo geneamplification (10).

Nonrandom alterations of chromosomes are well known in leuke-mias and lymphomas, and more recently in some solid tumors (14,15). In some cases these changes have been found to involve particular oncogenes or tumor suppressor genes (16). Random changes orcytogenetic noise also occur in cancer cells, especially at later stagesof the tumor progression. All these changes reflect the chromosomalinstability of tumor cells. Relatively little is known about specificchromosomal changes in breast carcinomas, but a number of chromosomes have been shown to be affected (17). Chromosomal changescan be detected by karyotype analysis based on G-banding, but more

recently FISH has become widely used. FISH provides additionalinformation, e.g., about translocations, amplifications, and alterationsin interphase cells (18).

Human cancer prone syndromes, like Fanconi's anaemia, Bloom's

syndrome, and AT, that are characterized by chromosomal instability,provide evidence that genetic instability can be heritable and that thisis associated with a predisposition to cancer (19, 2). The AT gene(s)has been implicated in the same signal transduction pathway as p53that controls cell cycle arrest following DNA damage (4). Cells frompatients with the AT syndrome lack the increase in p53 protein levelsthat is induced by ionizing radiation in normal cells.

The recent findings concerning the role of the p21 protein suggesta mechanism by which p53 could influence and perhaps coordinatecell cycle progression, DNA synthesis, and, possibly, DNA repair.This may explain the apparent importance of p53 abnormalities incancer. If p53 abnormalities cause genetic instability, this may giverise to genetic changes that occasionally result in neoplasia.

Do p53 abnormalities lead to increased genomic instability inprimary tumor cells? We have examined genetic instability in primaryhuman breast tumor samples with and without p53 abnormalities,using four different approaches: (a) detection of gene amplification;(b) detection of allelic loss; (c) karyotype analysis; and (d) FISH.

MATERIALS AND METHODS

Materials. Tumor specimens were obtained from 183 patients with primary, invasive breast cancer. Peripheral blood was collected from the samepatients on the day of surgery. The tumor tissue was finely minced, half of itprocessed for tissue culture and direct harvest for chromosomal analysis, andthe other half was kept at -80°C for DNA analysis. The mean age of patients

was 58.0 years (range, 29-94) compared to a mean age of 59.4 years for

Icelandic breast cancer patients over a period of 30 years (20). Information wascollected on tumor type, tumor size, axillary lymph node involvement, estrogen and progesterone receptor status, and clinical outcome of the disease (datanot presented).

DNA Analysis. DNA was extracted from breast tumor samples and bloodsamples by conventional phenol/chloroform extraction. p53 mutation analysis

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was carried out by PCR amplification and CDGE. Mutants were confirmed bydirect DNA sequencing. PCR conditions, CDGE, and sequencing conditionswere as described in Thorlacius et ai. (21) and Börresen el al. (22). All thesamples were screened for mutations in exons 5-8 of the p53 gene. Allelic loss

was examined by standard Southern blotting and hybridization techniques with7 different chromosome 17 probes and 7 different PCR polymorphisms. Theprobes used were: pl44D6 (D17S34); pYNZ22 (DI7S5); pYNH37.3(DÌ7S28);pTHH59 (DI7S4); pBHP53 (TP53); pCMM86 (DI7S74); andpRMU3 (D17S24). Three of the DNA markers map to 17pl3.3 (pl44D6,pYNH37.3 and pYNZ22), one maps to the p53 gene region (TP53), one mapsclose to the BRCA-I gene (pCMM86), and two of the probes map to 17q23-25

(pTHH59 and pRMU3). PCR polymorphisms used were: Accll polymorphismwithin the p53 gene (23); two different nucleotide repeats at the p53 locus(24);" three microsatellite markers, Mfdl88 (D17S579), THRA-1 (25), and

MfdlS (DÌ7S250),close to the BRCA-l gene; and a marker close to the end

of 17q, DI7S802.Amplification of the erbB2 oncogene was detected by Southern blotting

with the probe ERB-B2 (26) and comparative PCR amplification (27). Am

plification was measured by relative comparison of the signal strength betweentumor sample and a normal control. A control probe for a different marker, NJ4.1, was used to compare the amount of DNA loaded on the gel in the standardSouthern blotting method.

Immunohistochemistry. Five-|xm-thick sections from fixed, paraffin-em

bedded tumors were stained with two different p53 antibodies, a monoclonal(DO-1) (28) detected by a streptavidin-biotin complex and immunoperoxidaselabeling and a rabbit polyclonal antibody (CM-1) (29) detected with

immunoperoxidase.Cell Culture and Chromosome Preparation. Tissue samples were

minced in a Petri dish, digested with collagenase, and cultured in a specialserum-free medium (30) at 37°Cfor 3—6days. The cells were arrested in

mitosis by colchicine treatment and chromosomes were harvested by standardmethods as described by Pandis et al. (31). In addition a small fraction of theminced tissue was used for direct harvesting of chromosomes.

Karyotype Analysis. Chromosomes were G-banded with Wright's stain.

Karyotype analysis was performed according to an International System forHuman Cytogenetic Nomenclature (32). The number of cells analyzedranged from 5 to 300. A sample was defined as having a clonal abnormalityif it contained at least two cells with the same structural abnormality or thesame additional chromosome, or at least three cells lacking the samechromosome. Single structural changes or up to four numerical alterationswere defined as simple changes. All other aberrations were called complex.

FISH. PCR-amplified whole chromosome probes for chromosomes 1, 3,

16, and 17 were used for in situ hybridization. These are the chromosomesmost frequently involved in changes in the breast carcinomas studied here.5

Labeled DNA probes were obtained from Cambio (Cambridge, England) andin situ hybridization was performed according to the company's protocol.

Probes for chromosomes 1 and 3 were biotin labeled, detected with Avidin-Texas Red, and amplified over anti-Avidin. Probes for chromosomes 16 and 17

were directly labeled with fluorochrome FITC and the detection was amplifiedwith anti-FITC. Each chromosome preparation was hybridized with probes for

chromosomes 1 and 16 simultaneously, or with probes for chromosomes 3 and17. Chromosomes were counterstained with 4',6-diamidino-2-phenylindole-

dihydrochloride hydrate (DAPI).Statistical Analysis. When comparing two proportions, ic test with

Yates's correction was applied for testing the significance, or Fisher's exact

test, when appropriate.

RESULTS

Abnormalities in the pS3 Gene. 183 primary breast tumor samples were screened for mutations in exons 5-8 of the p53 gene by

CDGE. Suspected mutants were confirmed by DNA sequencing. Inour material p53 mutations were found in 17.6% of cases. The

4 M. Santibanez-Koref, personal communication.5 Margret Steinarsdottir, Steinunn Snorradoltir, Ingibjorg Pelursdoltir. Kesara

Anamathawat-Jonsson, Jorunn E. Eyfjörd, and Helga M. Ögmundsdottir. Differentkaryolypic profiles detected in breast carcinomas by direct harvesting and short termculture, submitted for publication.

majority of changes were single base substitutions leading to an aminoacid change (Fig. 1). The sites most frequently affected, codons 248and 273, are within the proposed DNA-binding area (33).

Tumor samples were also studied with immunohistochemical methods, and nuclear p53 staining was detected in 33.7% of cases. Nuclearp53 staining was detected in all the cases that had a mutation leadingto an amino acid change but not in tumors with nonsense or frameshiftmutations. In addition there are p53 mutation-negative tumors that

show nuclear staining. When taken together, abnormalities, p53 mutations, and/or nuclear p53 staining were detected in 36% of theprimary breast tumor samples.

Allelic Loss. Allelic loss was studied on chromosome 17 usingfour different probes and three different PCR polymorphisms for the

Fig. 1. CDGE of PCR fragments in exon 8 (codons 265-301 ). The CDGE technique is

based on the melting behavior of DNA strands. It allows maximal separation betweenmutant and wild-type DNA fragments in a specific dénaturantconcentration. The samplesare then sequenced to confirm the nature of Ihe mutation. Lane I. wild-type; Lane 2, Argto Cys mutation in codon 273; Lane 3, Arg to Trp mutation in codon 282; Lane •/,6-basedeletion in codons 279-281 (Arg and Asp deleted).

Table 1 Clonal chromosomal changes related to p53abnormalitiesCaseI23456789101112131415161718192(1212223Clonal

typeex/s"cxFISH

instCXCXcx/sCXsCXCXsCXcx/sssCXsp53

abnormalityExon

8Exon5Exon5Exon8Exon8Exon5Exon5Exon

5Pos.stainPos.stainPos.

stainNegNegNegNegNegNegNegNegNegNegNegNegCase2425262728293031323334353637383940414243444546Clonal

typeSCX/SCXCXCXCX/Sscx/sCXCXCXcx/sCXp53abnormalityNegNegNegNegNegNegNegNegNegNegNegNegNegNcgNegNegNegNegNegNegNegNegNeg

a CX, complex changes (>1 structural change and/or >4 numerical changes); S, simple

changes (1 structural change, or s4 numerical changes): Neg, negative; Pos, positive.

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Fig. 2. A, B, and C, two metaphases from tumor case 2 (left, normal; right, abnormal). A, 4',6-diamidino-2-phenylindole-dihydrochloride hydrate staining. B, Texas Red fluorescence

of chromosome 3. C, FITC green fluorescence of chromosome 17. Small arrowheads, normal chromosomes detected by FISH. This tumor contained three related clones with complexchromosome rearrangements, and the abnormal cells were from near-diploid to pentaploid. FISH revealed several translocations involving chromosomes 3 and 17. One pair of

translocated chromosomes (large arrowheads) contained segments from both chromosomes at opposite telomeric ends, indicating at least two translocation events prior to chromosomalduplication. More translocations and deletions can be seen. D and E, a metaphase from tumor case 4. D, 4',6-diamidino-2-phenylindole-dihydrochloride hydrate staining. E, red and

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short arm and three different probes and four different PCR polymorphisms for the long arm. On 17p alleile loss was detected in 70.8% ofthe tumors with p53 mutation as compared with 45% of the mutation-negative tumors, whereas on 17q loss was found in 65% of thesamples with p53 mutation and 32% of the mutation-negative samples. In both cases there is a statistically significant difference(P < 0.04; P < 0.007). When tumors rated abnormal on the basis ofprotein staining were included there was also a difference, i.e., loss ofheterozygosity for all sites was more frequent in the tumors with p53abnormalities, but this was not statistically significant.

Amplification of the erbB2 Oncogene. Amplification of theerbB2 oncogene was detected with the probe ERB-B2 and PCRcomparative amplification in 25% of the tumors. Amplification wasfound in 41% of the samples with p53 abnormalities as compared to15.9% of the samples without p53 abnormalities. This difference isstatistically significant (P < 0.008).

Cytogenetic Study. Fifty-six of the primarybreastcarcinomasamples screened for p53 abnormalities were put into culture to obtainmetaphase cells for cytogenetic analysis. In 46 cases successfullycultured, 30 showed clonal chromosomal abnormalities by karyotypeanalysis and FISH (Table 1). Although changes were found both insamples with and without p53 abnormalities, those with the p53abnormalities (samples 1-11) exhibited a significantly higher proportion of samples with chromosome aberrations than the sampleswithout p53 abnormalities (P < 0.04). Furthermore, the proportionwith complex clonal changes was much higher in the p53 abnormalgroup: 82% as compared to 40% in the p53 mutation-negative group(P < 0.008). No specific clonal changes could be associated with allsamples with p53 abnormalities.

Chromosome instability in breast tumor samples with p53 abnormalities included numerical and structural changes: loss and gain ofwhole or parts of chromosomes, as well as chromosome rearrangements. Chromosomes 1, 3, 16, and 17, which were most frequentlyinvolved in aberrations in our samples, were examined by the FISHtechnique, and some of the abnormalities are shown in Fig. 2. TheFISH method also identified changes undetected by other means andallowed examination of interphase chromosomes. The tumor case 3appeared normal when analyzed by G-banding, but FISH detectedhigh frequency of deletions and translocations of chromosomes 16and 17. DNA analysis showed allelic loss on 17p and amplification ofthe erbB2 oncogene on 17q in this tumor. Another tumor sample, case10, showed extensive DNA amplification on chromosome 17 whenexamined by FISH on both metaphase and interphase chromosomes.The pattern of the in situ hybridization signal suggested amplificationof dispersed repetitive DNA sequences in this tumor. Chromosome 17was also involved in translocation and duplication, while DNA analysis showed allelic loss on 17p and 17q and amplification of oncogeneerbB2.

DISCUSSION

Our results support earlier findings suggesting that p53 has a role incell cycle control and that p53 abnormalities may lead to geneticinstability. Moreover, the data indicate that this is true for primarycancer cells. Primary breast tumor cells with p53 abnormalities showsignificantly increased genetic instability measured as amplification

of the erbB2 oncogene, allelic loss on chromosome 17, and clonalchromosomal abnormalities involving several chromosomes.

Nuclear staining with p53 antibodies reflected the p53 mutations;all tumors with mutations leading to amino acid changes showednuclear staining, but no staining was seen in tumors with nonsense orframeshift mutations. In addition nuclear staining was detected insome tumors without proven p53 mutations. In these cases the proteinmight have been stabilized by complexing with other proteins or theymay harbor mutations in p53 exons not included in our analysis.

Cell line studies indicate that both copies of the p53 gene haveto be lost or inactivated for gene amplification to occur (3). In ourmaterial we detected alíeleloss on 17p in over 70% of the tumorswith a p53 mutation. This is in agreement with our earlier studiesand those of some others (21, 34-36), whereas other investigatorshave not found any correlation between alíeleloss on 17p and p53mutations in breast tumors (37). All but one of the tumors with ap53 mutation and erbB2 amplification that were informative (heterozygous) for one or more probes on 17p showed a loss ofheterozygosity at one or more sites. We cannot conclude from ourdata, however, whether the wild-type alíelehas been lost in thetumors with p53 mutations. The information on loss within the p53gene is too limited and the more informative probes are too distantfrom the gene for this to be possible. Deletions on 17p, large andsmall, are frequent in the tumors, however, and more so in the oneswith p53 mutations.

The association between p53 abnormalities and genetic instability such as oncogene amplification may indicate that p53 plays arole early in mammary tumor formation. Abnormal p53 proteinexpression has been detected in mammary carcinoma in situ,implicating p53 in mammary tumor evolution from in situ toinvasive disease. Interestingly, there was no association betweenp53 abnormalities and amplification of the erbB2 oncogene in thein situ lesions, whereas such association was found in invasivecarcinoma (38).

In this study we present only DNA data on allelic loss on chromosome 17, but both the chromosome data and our preliminary data onallelic imbalance on other chromosomes indicate that this is a moregeneral phenomenon involving several different chromosomes. This isin agreement with the view that p53 has a wide ranging monitoringfunction in the cell cycle. Specific chromosomes may be more frequently involved, however, in cytogenetically aberrant cells in thetumors. Results from a more extensive study of chromosome instability in breast carcinomas5 showed that chromosomes 1, 3, 16, and 17

had the highest frequency of alterations, and chromosomes 7 and 11were also relatively unstable. This is in fair agreement with thefindings of others (17, 39). Further studies will be required to verifythe importance of these chromosomes in breast cancer. The examination of interphase nuclei will also be important for screening ofchromosomal instability in tumors, especially for characterizingheterogeneous cell populations.

Our comparison of cytogenetic changes in breast tumors withand without p53 abnormalities revealed significant differences.Only 1 of the 11 tumor samples with p53 abnormalities that weresuccessfully analyzed by cytogenetic methods did not show clonalchromosomal abnormalities. More important, complex changeswere much more frequent in the samples with p53 abnormalities.

green fluorescence of chromosomes 1 and 16, respectively. No normal chromosomes 1 and 16 were found. In this near-diploid clone, a reciprocal translocation 1(1;16) was detected(large arrowheads). The karyotype analysis showed the breakpoints to be Iq21 and 16ql2. F, a partial metaphase from tumor case 5 showing red fluoresence of chromosome 3 andgreen fluorescence of chromosome 17. Small arrowheads, normal chromosomes. This tumor showed complex chromosomal changes. Abnormal cells were hypopentaploid and mostof the chromosomes were rearranged. When probed with chromosomes 3 and 17, two deleted chromosome 3 and three copies of normal chromosome 17, and 13 translocatedchromosomes were seen. Similarly complex results were obtained using chromosome 1 and 16 probes.

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ACKNOWLEDGMENTS

REFERENCES

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This high frequency of complex clonal changes, along with thediversity of chromosomal aberrations, is likely to reflect a malfunction of the cell division process.

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1995;55:646-651. Cancer Res   Jorunn E. Eyfjörd, Steinunn Thorlacius, Margret Steinarsdottir, et al.   Breast Carcinomas

Abnormalities and Genomic Instability in Primary Humanp53

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