Correlations Among p53, Her-2/neu, and ras Overexpression ... · Human Oncology [R. J., T. J., D. H., N. W.], and Pathology and Laboratory Medicine [J. F. S.], Allegheny University

Correlations Among p53, Her-2/neu, and ras Overexpression andAneuploidy by Multiparameter Flow Cytometry in HumanBreast Cancer: Evidence for a Common PhenotypicEvolutionary Pattern in InfiltratingDuctal Carcinomas1

Charles A. Smith, Agnese A. Pollice,Ling-Ping Gu, Kathryn A. Brown,Sarita G. Singh, Laura E. Janocko,Ronald Johnson, Thomas Julian, David Hyams,Norman Wolmark, Lillian Sweeney,Jan F. Silverman, and Stanley E. Shackney2

Laboratory of Cancer Cell Biology and Genetics, Department ofHuman Oncology [C. A. S., A. A. P., L-P. G., K. A. B., S. G. S.,L. E. J., L. S., S. E. S.], Departments of Human Genetics [S. E. S.],Human Oncology [R. J., T. J., D. H., N. W.], and Pathology andLaboratory Medicine [J. F. S.], Allegheny University of the HealthSciences, Allegheny General Hospital, Pittsburgh, Pennsylvania15212

ABSTRACTHuman solid tumors develop multiple genetic abnor-

malities that accumulate progressively in individual cellsduring the course of tumor evolution. We sought to deter-mine whether there are specific sequences of occurrence ofthese progressive evolutionary changes in human breastcancers by performing correlated cell-by-cell measurementsof cell DNA content, p53 protein, Her-2/neu protein, and rasprotein by multiparameter flow cytometry in 56 primarytumor samples obtained at surgery. In addition,p53 allelicloss and Her-2/neugene amplification were determined byfluorescence in situ hybridization in cells from the samesamples. We reasoned that if there is a specific order inwhich genetic changes occur, the same early changes wouldbe found consistently in the cells with the fewest abnormal-ities. We reasoned further that late-developing abnormali-ties would not occur alone in individual cells but wouldalmost always be found together with the early changesinherited by the same cells. By these criteria, abnormalitiesinvolving p53 generally occurred early in the course of

development of invasive breast cancers, whereas ras proteinoverexpression was found to be a late-occurring phenome-non. Within individual tumors, cellular p53 overexpressionwas often observed alone in individual cells, whereas rasprotein overexpression was rarely observed in the absence ofp53 overexpression and/or Her-2/neu overexpression in thesame cells. Furthermore, the intracellular level of each ab-normally expressed protein was found to increase progres-sively as new abnormalities were acquired. Infiltrating duc-tal carcinomas exhibited characteristic phenotypic patternsin which p53 allelic loss and/or p53 protein overexpression,Her-2/neu amplification and/or overexpression, aneuploidy,and ras overexpression accumulated within individual cells.However, this pattern was not a prominent feature of lobu-lar breast cancers. All six lobular breast cancers studiedwere diploid. p53 allelic loss and/or early p53 overexpres-sion, and late ras cooverexpression in the same cells wereless common in lobular breast cancers than in infiltratingductal carcinomas. Although Her-2/neu overexpression wasa common finding in lobular breast cancers, Her-2/neu am-plification was not observed in these tumors.

INTRODUCTIONHuman solid tumors undergo multiple genetic changes as

they progress from a near-normal state to aggressive malig-nancy. Some of these changes may occur in distinctive patterns,and there is reason to believe that some may develop in apreferred sequence. In a previous study, we performed multiplecorrelated cell-by-cell measurements by multiparameter flowcytometry on primary tumors from patients with breast cancerand found that the presence within a tumor of aneuploid cellsthat also overexpressed Her-2/neu and ras in the same cells wasan adverse prognostic feature (1). We sought to determinewhether this pattern of intracellular oncogene protein overex-pression was the result of a specific sequence of evolutionarychanges. In a subsequent study, we found that the overexpres-sion of EGF receptor and/or the overexpression of Her-2/neualmost always preceded the development of ras overexpression(2). The present study was undertaken to examine the relation-ship between p53 abnormalities and previously studied intracel-lular patterns of Her-2/neu overexpression, ras overexpression,and aneuploidy in individual breast cancers.

Numerous studies in human solid tumors, and in breastcancer in particular, have demonstrated relationships betweenp53 abnormalities (or loss of heterozygosity at 17p) and aneu-ploidy, between p53 abnormalities and Her-2/neu amplification/overexpression, between Her-2/neu amplification and/or over-

Received 5/11/99; revised 10/25/99; accepted 10/27/99.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisementin accordance with 18 U.S.C. Section 1734 solely toindicate this fact.1 This work was supported by Grant CA 55230 from the Department ofHealth and Human Services.2 To whom requests for reprints should be addressed, at AlleghenyUniversity of the Health Sciences, Allegheny Campus, 320 East NorthAvenue, Pittsburgh, PA 15212. Phone: (412) 359-4306; Fax: (412)359-3238.

112 Vol. 6, 112–126, January 2000 Clinical Cancer Research

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expression and aneuploidy, and between Her-2/neu amplification/overexpression and ras overexpression within individual tumors(reviewed in Ref. 3). Loss of wild type p53 function has beenfound to be associated with distinctive forms of genetic insta-bility that are manifested by the development of gross abnor-malities in chromosome number/cell (aneuploidy) and by struc-tural abnormalities involving individual chromosomes, such aschromosomal breakage, deletions of chromosomal material, andgene amplification (3, 4). The Her-2/neugene is often amplifiedand overexpressed in human breast cancer (5, 6), particularly inaneuploid tumors (7–10). If Her-2/neu amplification were adirect or indirect consequence of p53-induced genetic instabil-ity, then one might anticipate that the development of p53abnormalities might accompany or precede the development ofHer-2/neu overexpression, aneuploidy, or both. The overexpres-sion of ras protein has been found to be contingent on the lossof wild-type p53 function in a number of experimental systems(reviewed in Ref. 3). Thus, we hypothesized that p53 abnormal-ities might also precede the development of ras overexpressionin human breast cancer.

Our methodological approach to extracting evolutionarysequence information from single tumor samples is based on thetechnical ability to perform multiple quantitative measurementson each cell by multiparameter flow cytometry. In previousstudies, we found that breast cancer precursor populations gen-erally persist in the background during and after the emergenceof more advanced clones (those with larger numbers of geneticabnormalities/cell and/or higher levels of expression of individ-ual oncogene proteins; Refs. 1, 2, and 11). Under such condi-tions, if there is a specific order in which different geneticabnormalities appear, the same early genetic changes amongthose measured would be found in the cells with the fewestabnormalities. In contrast, one might expect that abnormalitiesthat consistently occur late would almost always be found tohave accumulated in the same cells with abnormalities that hadoccurred early and had persisted.

In this study, multiparameter flow cytometric analyseswere supplemented by FISH3 studies on the same tumors inwhich paired cell-by-cell measurements of a locus-specific flu-orescent probe for p53 and ana satellite probe for chromosome17 were performed to further explore the relationship betweenp53 abnormalities (manifested by allelic loss) and aneuploidy inthe same cells. We also performed paired cell-by-cell measure-ments of a locus-specific probe for Her-2/neu and ana satelliteprobe for chromosome 17 in cells from the same tumors toexamine the relationship between Her-2/neugene amplificationand aneuploidy in the same cells.

PATIENTS AND METHODSPatient Population. This study was approved by the

Institutional Review Board of Allegheny General Hospital(Pittsburgh, PA). Fresh tumor samples were obtained with in-formed consent from 56 primary breast cancers of patients whounderwent surgery at Allegheny General Hospital and other

Pittsburgh area hospitals between November 1, 1995 and Janu-ary 1, 1998. Table 1 summarizes the clinical characteristics ofthese patients. Four of these patients have had disease recur-rences to date.

Sample Preparation. Freshly obtained breast tumorsamples were finely scissor-minced in HBSS (Life Technolo-gies, Inc., Gaithersburg, MD), filtered through 64mm nylonmesh (Small Parts, Miami, FL), and washed once with HBSS. Asmall aliquot of cells was fixed in methanol;glacial acetic acid(3;1) for FISH studies, and the remaining cells were then fixedeither in methanol for DNA analysis or in paraformaldehydeplus methanol for multiparameter analysis, as described previ-ously (12). A small aliquot of paraformaldehyde/methanol-fixedcells was set aside for cytological confirmation of the presenceof tumor cells in each sample to provide assurance that diploid3 The abbreviation used is: FISH, fluorescencein situ hybridization.

Table 1 Clinicopathological features of the 56 breast cancersincluded in the present studya

Histopathological typeInfiltrating ductal carcinoma 43Infiltrating lobular carcinoma 6Other 7

Pure mucinous carcinoma 2Mucinous and infiltrating ductal carcinoma 1Lobular and infiltrating ductal carcinoma 1Invasive tubular carcinoma 1Tubulobular carcinoma 1Metaplastic squamous cell carcinoma 1

Tumor sizeT1 28T2 23T3 3T4 2

Nodal statusN0 30N1 19N2 1Nx 60 positive nodes 301–3 positive nodes 14.4 positive nodes 6Nodal status unknown 6

ER/PRb statusER2/PR2 17ER1/PR1 28ER1/PR2 11ER2/PR1 0

TreatmentUnknown 6None 3Radiation therapy only 4Chemotherapy only 16Tamoxifen only 4Chemotherapy1 Tamoxifen 4Chemotherapy1 radiation 7Chemotherapy1 Tamoxifen1 radiation 1Tamoxifen1 radiation 11

Total receiving adjuvant therapy 47Race

Caucasian 49African American 3Unknown 4

a Total number of patients, 56; mean patient age, 59 years (range,25–92 years).

b ER, estrogen receptor; PR, progesterone receptor.

113Clinical Cancer Research



cell samples that did not exhibit p53, Her-2/neu, or ras overex-pression did, in fact, contain monodispersed tumor cells.

Immunofluorescence Staining. Fluorescein-conjugatedmonoclonal antibody immunospecific for p53 protein (CloneDO-7) was purchased from Novocastra Laboratories, Ltd.(Newcastle upon Tyne, United Kingdom). Rabbit polyclonalantibody to c-erbB-2, purchased from Cambridge Research Bio-chemicals (Cambridge, United Kingdom), was used for indirectstaining. Phycoerythrin-conjugated goat anti-rabbit IgG (1:20),purchased from Vector (Burlingame, CA), was used as a sec-ondary antibody. Rat monoclonal antibody to human v-H-ras,which recognizes human c-H-ras, K-ras, and N-ras, was pur-chased from Oncogene Science (Cambridge, MA). This anti-body was conjugated with Cy-5 (Amersham Life Sciences, Inc.,Pittsburgh, PA) and used for direct staining as described previ-ously (2). JC 1939, a breast cancer cell line established in ourlaboratory, was used as a positive staining control for p53,Her-2/neu, and H-ras immunofluorescence and as a quantitativefluorescence staining reference for p53 and Her-2/neu (see be-low). Lymphocytes from healthy donors were used as low-levelbaseline immunofluorescence staining controls and as relativereference standards for the ras measurements.

DNA Staining and Flow Cytometry. Cells were stainedwith 49,6-diamino-2-phenylindole (Sigma Chemical Co., St.Louis, MO) at a final concentration of 0.1mg/ml. Multiparam-eter flow cytometry measurements were performed as describedpreviously (2).

Use of Lymphocytes as Reference Cells for Cell DNAContent and Cell ras Content. Methanol-fixed lymphocyteswere used as diploid reference standards for ploidy analysis ofmethanol-fixed tumor cells (12). The criteria for tetraploidy andaneuploidy were as described previously (2).

Paraformaldehyde/methanol-fixed lymphocytes were usedas reference cells for the multiparameter flow cytometry studies.Tumor cell ras levels were expressed as multiples of the baselinenormal lymphocyte reference (by analogy to the DNA index). Inthis study, for convenience of graphical comparison of ras levelsto levels of Her-2/neu in the same cells (see below), the normallymphocyte reference was assigned a nominal reference value of10,000 units. Thus, for example, 53 104 units/cell wouldcorrespond to a 5-fold increase in ras content above that ofnormal lymphocytes, and 13 105 ras units/cell would corre-spond to a 10-fold increase.

Quantitation of Her-2/neu and p53 in Molecules/Cell.Passage 39 of cell line JC 1939 was found to contain 77,000molecules/cell of HER-2/neu, quantitated by ELISA assay (On-cogene Science; Ref. 2), and a mean of 7,500 p53 molecules/cell, also quantitated by ELISA assay (Oncogene ResearchProducts, Cambridge, MA). On the basis of this reference value,the mean p53 content in 23 samples of normal lymphocytes was6600 molecules/cell, in general agreement with values reportedby others for normal blood leukocytes (13, 14).

Data Analysis. Using a computer program developed byone of the authors (S. E. S.), the logarithmic data for all three ofthe oncogene measurements per cell were scaled to a commonorigin. A subtractive correction for nonspecific antibody bindingwas applied to each measurement on a cell-by-cell basis, asdescribed previously (2). In all data figures shown in this paper,

nonspecific labeling has already been subtracted from eachmeasurement on a cell-by-cell basis.

FISH. Tumor cells in single-cell suspension freshly fixedin methanol:glacial acetic acid (3:1) were applied to slides andprocessed in accordance with protocols developed by Vysis, Inc.(Downer’s Grove, IL) using the HYBrite hybridization appara-tus (Vysis). Combinations of gene locus-specific and centro-meric enumeration probes used in this study were Her-2/neu/chromosome 17 and p53/chromosome 17, obtained from Vysis.After hybridization, cells were counterstained with 49,6-dia-mino-2-phenylindole (50mg/ml), and slides were viewed on anAxiophot-II microscope (Carl Ziess, Inc). Standardized criteriafor spot counting were used as recommended by Vysis. At least50 cells on each slide were counted by each of two independentobservers. Staining quality assurance controls consisting of nor-mal lymphocytes were included in every staining batch for eachprobe set. Each bivariate data set was corrected for stainingbackground using a bivariate template derived from at least fiveslides of normal lymphocytes, where at least 100 normal cellswere counted on each slide for each probe set.

Criteria for Protein Overexpression: p53. Brothericetal. (13) estimated the staining intensity threshold for overex-pression of p53 to be in excess of 9,000 molecules/cell forantibody DO-7 by flow cytometry. In this study, we used astaining intensity threshold for p53 overexpression of 10,000molecules/cell.

Criteria for Oncogene Overexpression: Her-2/neu. In-dividual cells that overexpress Her-2/neu are generally detecta-ble immunohistochemically when intracellular levels are in therange of 200,000–500,000 molecules/cell or higher (3). Weadopted a mean level of Her-2/neu overexpression of.300,000molecules/cell as a staining intensity threshold for overexpres-sion to relate our studies to immunohistochemical studies. How-ever, it was also apparent that flow cytometric measurementswere capable of detecting Her-2/neu levels that were well below300,000 molecules/cell but which were clearly higher than nor-mal (;50,000 molecules/cell). Therefore, in this study, we usedboth a .150,000 mean molecules/cell threshold and a.300,000 mean molecules/cell threshold. We view the formerthreshold as more inclusive for true overexpression and thelatter as more appropriate for comparisons with immunohisto-chemical studies.

Criteria for Oncogene Overexpression: ras. A relativemean staining intensity per cell at least four times higher thanthat of normal lymphocytes (i.e.,4 3 104 units) was used as athreshold level for overexpression. This value exceeded themean for normal lymphocytes (relative to a fluorescent beadreference) by more than 2 SDs.

Criteria for Allelic Loss or Gene Amplification byFISH. For the FISH studies, allelic loss was defined by thepresence of fewerp53 alleles/cell than chromosome 17 centro-meres in the same cells, with a frequency threshold requirementthat at least 15% of cells exhibit allelic loss, based on the studiesof Kibbalaaret al. (15). True Her-2/neugene amplification wasdefined by the presence of an excess of Her-2/neugene loci overthe number of chromosome 17 centromeres in the same cells.Absolute increases in the number of Her-2/neu loci/cell thatwere matched or exceeded by the number of chromosome 17centromeres in the same cells may have been attributable solely

114 p53, Genetic Instability, and ras Overexpression



to aneusomy and were treated separately. Frequency thresholdrequirements were similar to those forp53 allelic loss.

Analytic Approach to Genetic Evolutionary Sequenc-ing. For each tumor sample analyzed, the cells were groupedby the number of abnormalities they contained. If these abnor-malities had developed in an orderly sequence of inheritedclonal changes, and if the precursor clonal subpopulations hadpersisted in the presence of more evolved clones, then theearliest abnormal change(s) would be identified among the cellsthat contain a single abnormality (Fig. 1A). Once the earliestabnormality is identified, the next abnormal change in the se-quence can then be deduced from the patterns observed in cellswith two abnormalities, and the third abnormality in the se-quence can be deduced, in turn, from the patterns observed incells with three abnormalities. In the hypothetical exampleshown in Fig. 1A, almost all cells with a single abnormalitycontained abnormality B; hence, among the abnormalities stud-ied, B is likely to have occurred first. In this hypotheticalexample, among cells that contained two and only two of theabnormalities measured, almost all contained abnormalities Aand B in the same cells. Having established that B occurred first,abnormality A must have occurred after abnormality B. In thisexample, because cells containing abnormality C almost alwayscontained abnormalities B and A as well, the sequence of

accumulation of all of these abnormalities must have been B3A 3 C. Illustrative examples based on actual data are givenbelow in Figs. 3 and 4 and Tables 2 and 3.

It should be noted that the establishment of a regularsequence of occurrence for a group of measured abnormalitiesdoes not necessarily imply direct causal relationships among themembers of the sequence, nor does it preclude the occurrence ofother, unmeasured abnormalities within the sequence.

If precursor subpopulations had not persisted in sufficientnumbers to be detected, then this approach to genetic evolution-ary sequencing would break down (Fig. 1B). However, in thepresent study, among 153 subpopulations with two or moreabnormalities/cell that were detected among the 56 breast can-cers (diploid and aneuploid cell subpopulations considered sep-arately), at least one potential immediate precursor (i.e., a sub-population with one abnormality fewer) was also present in thesame tumor in all but one instance.

The presence of more than one candidate precursor sub-population (Fig. 1C) is not only possible but was actually quitecommon. Other variants included parallel pathways that con-verge after multiple steps. However, if all potential precursorpopulations are present in the same tumor, then this approach togenetic evolutionary sequencing would provide no useful infor-mation (Fig. 1D). When the event frequency threshold for

Fig. 1. Schematic representation of the approach to genetic evolutionary sequencing based on the measurement of multiple correlated measurements/cell. A, cells are grouped into classes based on the number of abnormalities/cell. Cell subpopulations with only one abnormality/cell are taken torepresent potential precursors to subpopulations of cells that have accumulated two abnormalities/cell. If a subpopulation with a particular abnormalitypredominates, this subpopulation is taken to represent the most likely precursor to subpopulations that contain this abnormality plus one other per cell.B, if precursor populations did not persist in the presence of more advanced cell subpopulations, this approach to genetic evolutionary sequencingwould break down. In practice, potential precursor populations were almost always present.C, the presence of multiple potential precursor populationswas a common occurrence.D, if all possible potential precursors were present, this approach to genetic evolutionary sequencing would break down.In practice, this was observed in only one of 56 tumors.




identifying a potential precursor populations was set at$1% ofall cells analyzed in a given sample, well-defined evolutionarysequences could be discerned in all but one of the 56 tumorsanalyzed, and in that one case, an event frequency threshold of1% of cells may have been too low.

Statistical Analysis. Means of two groups were com-pared using Student’st test. Associations between variableswere assessed by thex2 test or by Fisher’s exact test, asappropriate.

RESULTSp53 Abnormalities and Ploidy. An elevated overall

sample mean level of p53 protein/cell (mean,.10,000 mole-cules/cell) was observed in 21 of 56 or 37.5% of samples, whichis within the range reported in most studies using immunohis-tochemical techniques (6, 16–26). This is likely to be an under-estimate of the proportion of tumors with p53 overexpression,because the presence of variable proportions of normal diploidcells would tend to lower the overall mean level of p53/cell ineach sample.

To reduce the potential effects of dilution by normal dip-loid cells, mean p53 levels/cell were calculated separately forthe diploid and aneuploid components of the aneuploid tumors.The results are shown in Fig. 2A, together with p53 levels/cell in

the purely diploid tumors. Mean p53 levels/cell were in thenormal range in the majority of diploid tumors and in most ofthe diploid components of aneuploid tumors. In every aneuploidtumor, the aneuploid component exhibited higher mean levels ofp53/cell than the diploid component, even when neither com-ponent exhibited a mean p53 level that exceeded 10,000 mole-cules/cell. When the presence of a separate aneuploid compo-nent with an elevated mean p53 level was considered inclassifying tumors with respect to p53 status, the total number ofsamples exhibiting p53 overexpression increased to 29 of 56, or51.8% of cases.

Increased mean p53 levels/cell were observed in somediploid tumors and in the diploid components of some aneuploidtumors, suggesting that although the transition to aneuploidymay be associated with an increase in intracellular p53 protein,the development of p53 abnormalities can occur prior to, and/orindependently of, the development of aneuploidy. Because dip-loid tumors were not subdivided further in this study, true meanp53 levels/cell in diploid tumors are likely to have been higherthan those observed because of the dilutional effect of normaldiploid cells.

Among the 46 cases in which FISH studies involving thep53 locus were performed, 26 showed allelic loss of p53 byFISH (56.5%). Fifteen of these 26 tumors also showed p53

Table 2 Quantitative sequence analysis of the data from the same tumor shown in Fig. 3 The overall frequencies of the various subpopulationspresent in the diploid and aneuploid components of the tumor are separated into those with one, two, or three overexpressed proteins/cell. The

proportional representations of each protein-overexpressing cell subpopulation have thresholds at the 1% frequency level and at the 5% level. Themean levels of overexpression of each of the proteins measured are also given for each subpopulation. The diploid cell subpopulation

overexpressing p53 protein alone represents 9.3% of the cells present and is much more abundant than the cell subpopulation overexpressingHer-2/neu alone, which comprises 1.2% of all cells. Cells overexpressing ras alone are not present in the sample.

The diploid cell subpopulation overexpressing p53 alone is a potential precursor to a large aneuploid cell subpopulation (13.3% of all cells) thatalso overexpresses p53 alone at somewhat higher levels (mean p53 levels of 2.73 104 molecules/cell in the diploid component increasing to 3.43 104

molecules/cell in the aneuploid component). The small diploid cell subpopulation overexpressing Her-2/neu alone has no existing aneuploid progeny,but it is a potential precursor to another small diploid cell subpopulation (1.7% of all cells) that exhibits Her-2/neu overexpression at comparable levels(mean, 2.5–3.03 105 molecules/cell) and has also developed overexpression of p53 protein in the same cells.

Among the aneuploid cells overexpressing two proteins/cell, the subpopulation overexpressing both p53 and Her-2/neu is the most prominent(17.1% of all cells). Both the aneuploid cell subpopulation overexpressing p53 alone and the diploid cell subpopulation overexpressing both p53 andHer-2/neu could be precursors to this cell subpopulation, but the aneuploid cell subpopulation overexpressing p53 alone seems more likely, based onits greater abundance. This cell subpopulation could also serve as a precursor to a minor aneuploid cell subpopulation (1.9% of all cells) thatoverexpresses both p53 and ras.

There is an abundant aneuploid cell subpopulation (17.4% of all cells) in which all three proteins are overexpressed in the same cells, which couldhave arisen either from an aneuploid cell subpopulation overexpressing both p53 and Her-2/neu or from an aneuploid cell subpopulationoverexpressing both p53 and ras. Again, the former appears to be the more likely precursor, because of its greater abundance (17.1%versus1.9%).There is also a small diploid cell subpopulation (2.4% of all cells) that overexpresses all three proteins, for which the diploid subpopulationoverexpressing p53 and Her-2/neu is the most likely precursor.

Overall, the relative abundances of the various precursor populations suggest that the predominant evolutionary pathway in this tumor involvedthe progressive, stepwise development of p53 overexpression in diploid cells, to p53 overexpression in aneuploid cells, to p53 overexpression plusHer-2/neu overexpression in aneuploid cells, to p53 overexpression, Her-2/neu overexpression, and ras overexpression in aneuploid cells.

Diploid% (p53,a Her-2/neu,a rasb)

Aneuploid% (p53,a Her-2/neu,a rasb)

p53 alone 9.3% (2.73 104, - - -, - - -)c 13.3% (3.43 104, - - -, - - -)c

Her-2/neu alone 1.2% (- - -, 2.53 105, - - -)d 0.0%ras alone 0.0% 0.0%p531 Her-2/neu 1.7% (2.33 104, 3.03 105, - - -)d 17.1% (4.23 104, 3.13 105, - - -)c

p53 1 ras 0.8% 1.9% (1.43 105, - - -, 7.0 3104)Her-2/neu1 ras 0.0% 0.0%p531 Her-2/neu1 ras 2.4% (5.13 104, 8.33 105, 5.13 104)d 17.4% (1.13 104, 1.03 106, 1.13 105)c

a Mean molecules/cell.b Mean units/cell.c Threshold at the 5% level. - - -, no data.d Threshold at the 1% level. - - -, no data.




overexpression by flow cytometry. In 11 cases, allelic loss ofp53 by FISH was the only p53 abnormality noted. Overall, 37 of46, or 80.4%, of tumors studied by both flow cytometry andFISH exhibited p53 abnormalities by either or both techniques.

Nineteen of the 26 tumors with allelic loss by FISH con-tained cells that were either monosomic for chromosome 17 orwere disomic for chromosome 17 but exhibited only onep53allele. Among the 19 cases that contained cells that exhibiteddisomy andp53allelic loss or monosomy for chromosome 17 inthe same cells, 9 were diploid by flow cytometry. In six of thesenine diploid tumors, p53 protein levels by flow cytometry werein the normal range, and allelic loss was the onlyp53 abnor-mality detected. Because hemizygosity for wild-type p53 hasbeen reported to be associated with increased susceptibility tooncogenic transformation (27), p53 abnormalities that first be-came apparent in diploid cells in the present study may havebeen of clinical consequence.

FISH studies were performed on 17 of 23 diploid tumors.Nine of these 17 diploid tumors exhibited allelic loss ofp53(52.9%). All nine contained cells that exhibited disomy andp53allelic loss or monosomy for chromosome 17 in the same cells.Overall, p53 allelic loss, p53 protein overexpression, or bothwere documented in a total of 13 of 17, or 76.5%, of the diploidtumors studied by both techniques.

FISH studies were performed on 28 of 33 aneuploid tu-mors. Among these 28 tumors, 20 exhibited p53 overexpression(71.4%), and 17 exhibitedp53allelic loss (60.7%). Five of these28 tumors exhibitedp53allelic loss as the only p53 abnormality,and 3 of the 28 tumors had no demonstrable p53 abnormalitiesat all. Overall, 25 of 28 aneuploid tumors (89.3%) studied byboth flow cytometry and FISH exhibited p53 abnormalities byeither or both techniques. The difference in frequency of p53

abnormalities between diploid and aneuploid tumors was notstatistically significant (P5 0.399 by Fisher’s exact test).

Abnormal Mean Her-2/neu Expression and Ploidy.Nineteen of 56 tumors (33.9%) had mean overall levels ofHer-2/neu protein/cell that were considered to be potentiallydetectable by immunohistochemistry (.300,000 molecules/cell), a value that is within the range of reported frequencies ofHer-2/neu overexpression in breast cancer (28). Overall meanlevels of Her-2/neu/cell exceeding 150,000 molecules/cell wereobserved in 26 of 56, or 46.4%, of breast cancers. The higherapparent frequency reflects the greater sensitivity of detection ofmoderate increases in Her-2/neu expression by flow cytometryin comparison with immunohistochemical techniques. Even thisvalue is still likely to underestimate the proportion of tumorswith Her-2/neu overexpression because of the presence of nor-mal diploid cells that have low intracellular levels of Her-2/neu.

To minimize the effect attributable to the presence ofnormal diploid cells in aneuploid tumors, mean Her-2/neu levelswere calculated separately for the diploid and aneuploid com-ponents. The results are shown in Fig. 2B, together with Her-2/neu levels in the purely diploid tumors. When the presence ofa separate aneuploid component with an elevated Her-2/neulevel was considered in classifying tumors with respect to Her-2/neu status, the total number of samples exhibiting Her-2/neuoverexpression (.150,000 molecules/cell) increased to 31 of 56tumors, or 55.4% of cases.

It is apparent from Fig. 2Bthat high mean levels of Her-2/neu/cell were observed both among diploid tumors and amongthe aneuploid components of aneuploid tumors. The aneuploidcomponent of each aneuploid tumor invariably exhibited highermean levels of Her-2/neu/cell than the diploid component of thesame tumor, even when neither component exhibited a mean

Table 3 Quantitative sequence analysis of the data from the same tumor shown in Fig. 4The overall frequencies of the various subpopulations present in the diploid and aneuploid components of the tumor are separated in to those

with one, two, or three overexpressed proteins/cell. The proportional representations of each protein-overexpressing cell subpopulation havethresholds at the 1% frequency level and at the 5% level. The mean levels of overexpression of each of the proteins measured are also given for eachsubpopulation. The diploid cell subpopulation overexpressing p53 protein alone represents only 0.2% of all cells and falls below the 1% abundancecriterion for overexpression. The presence of a small aneuploid subpopulation (1.3% of all cells) that overexpresses p53 alone raises the possibilityof a minor evolutionary pathway in which p53 overexpression occurs early. Cells overexpressing ras alone are virtually absent from the sample. Therelative abundance of diploid and aneuploid subpopulations overexpressing Her-2/neu alone suggests the presence of a major evolutionary pathwayin which Her-2/neu overexpression occurs early. However, the presence of substantial allelic loss in a substantial proportion of both the diploid andaneuploid cells by FISH (see Fig. 4C) suggests that the cells overexpressing Her-2/neu alone might also have contained p53 abnormalities that werenot accompanied by p53 protein overexpression. The abundance of diploid and aneuploid cell subpopulations in which both Her-2/neu and ras areoverexpressed in the same cells suggests that ras overexpression occurred later than Her-2/neu overexpression. Again, the FISH data suggest that thesecells might also have contained p53 abnormalities that were not accompanied by p53 protein overexpression. Finally, the abundance of aneuploid cellsoverexpressing all three proteins suggests that p53 overexpression may have been a late effect that accompanied the earlier changes.

Diploid% (p53,a Her-2/neu,a rasb)

Aneuploid% (p53,a Her-2/neu,a rasb)

p53 alone 0.2% 1.3% (1.73 104, - - -, - - -)c

Her-2/neu alone 13.2% (- - -, 3.43 104, - - -)d 5.4% (- - -, 3.03 104, - - -)d

ras alone 0.3% 0.3%p53 1 Her-2/neu 0.4% 1.2% (1.83 104, 3.2 3 105, - - -)c

p53 1 ras 0.0% 0.4%Her-2/neu1 ras 16.0% (- - -, 1.13 106, 1.1 3 105)d 8.1% (- - -, 8.33 105, 1.0 3 105)d

p53 1 Her-2/neu1 ras 2.7% (2.73 104, 2.2 3 106, 2.8 3 105)c 17.9% (3.03 104, 2.0 3 106, 2.2 3 105)d

a Mean molecules/cell.b Mean units/cell.c Threshold at the 1% frequency level. - - -, no data.d Threshold at the 5% frequency level. - - -, no data.




Her-2/neu level that exceeded 150,000 molecules/cell. The dip-loid components of aneuploid tumors appear to fall into twoeasily separable groups,i.e., those with mean levels of Her-2/neu that were,150,000 molecules/cell and those with meanlevels of Her-2/neu that were.150,000 molecules/cell, suggest-ing that in aneuploid tumors, the development of Her-2/neuoverexpression can precede the development of aneuploidy.

True Her-2/neugene amplification by FISH (where thenumber of gene loci/cell exceeded the number of chromosome17 centromeres in each cell) was observed in 14 of 46 tumors inwhich Her-2/neu FISH studies were performed (30.4%). Con-comitant Her-2/neugene amplification and protein overexpres-sion (.150,000 molecules/cell) were present in the same tumorin 9 of these 14 cases (64%). Six of these tumors exhibited meanlevels of Her-2/neu/cell that exceeded 300,000 molecules/cell.Two of the tumors with Her-2/neugene amplification by FISHbut no protein overexpression had unequivocally low/normalmean levels of Her-2/neu protein/cell (mean,,50,000 mole-cules/cell). Twelve of the 14 tumors that exhibited true Her-2/neu amplification were aneuploid (85.7%), and 12 of these 14tumors exhibitedp53 allelic loss, p53 overexpression, or both.

An absolute increase in Her-2/neugene copy number/cellthat was matched or exceeded by chromosome 17 centromerecopy number in the same cells was observed in 12 additionaltumors. Concomitant Her-2/neu protein overexpression waspresent in 6 of these 12 tumors (50%). These 6 tumors, togetherwith the 9 tumors with true Her-2/neu amplification and meanlevels of Her-2/neu overexpression exceeding 150,000 mole-cules/cell, accounted for only 65% of the 23 tumors with Her-2/neu overexpression in which FISH studies were performed,suggesting that transcriptional and/or posttranscriptional regu-latory mechanisms, rather than increased gene dosageper se,may have played a role in increasing the intracellular levels of

Her-2/neu in at least some of the tumors with Her-2/neu proteinoverexpression.

Abnormal Mean ras Expression and Ploidy. Elevatedmean levels of ras/cell (mean,.40,000 units/cell) were ob-served in 14 of 56, or 25%, of tumors, in keeping with ourearlier studies (2). This is likely to be an underestimate of thetrue proportion of tumors with ras overexpression because of theartifactual effects of normal diploid cells with low intracellularlevels of ras. Hence, for aneuploid tumors, mean ras levels werecalculated separately for the diploid and aneuploid components.The results are shown in Fig. 2C, together with mean ras levelsin the purely diploid tumors. When the presence of a separateaneuploid component with an elevated mean ras level wasconsidered, the total number of samples exhibiting ras overex-pression increased to 25 of 56 tumors, or 44.6% of cases. AsFig. 2Cshows, most of the aneuploid components of aneuploidtumors exhibited high mean levels of ras/cell, whereas most ofthe diploid components did not. However, a statistically signif-icant relationship between ras overexpression and aneuploidywas not supported byx2 analysis (P. 0.09).

Abnormal Mean p53 Expression and Mean ras Over-expression. There was an association between mean p53level/cell and mean ras level/cell in the same tumor. (In aneu-ploid tumors, the aneuploid component was used in the analy-sis.) Among 29 tumors with elevated mean p53 levels/cell, 19tumors also had elevated mean ras levels/cell (.40,000 units/cell). Among 27 tumors in which mean p53 levels were notelevated, only 7 had mean ras levels/cell that exceeded 40,000units/cell. This association was highly significant byx2 analysis(P 5 0.007).

Abnormal Mean Her-2/neu Expression and Mean rasOverexpression. There was a strong association betweenmean Her-2/neu level/cell and mean ras level/cell in the same

Fig. 2. A, patterns of mean levels of expression/cell of p53 protein in diploid tumors and in the diploid and aneuploid components of aneuploid tumors.Each tumor is plotted separately, and the mean p53 levels of the diploid and aneuploid components of each aneuploid tumor are connected byverticallines. B, patterns of mean levels of expression/cell of Her-2/neu in diploid tumors and in the diploid and aneuploid components of aneuploid tumors,plotted as inA. C, patterns of mean levels of expression/cell of ras in diploid tumors and in the diploid and aneuploid components of aneuploid tumors,plotted as inA. High levels of all three proteins could be observed in some diploid tumors, as well as in substantial proportions of the aneuploidcomponents of aneuploid tumors. For further discussion, see text.




tumor (in aneuploid tumors the aneuploid component was usedin the analysis). Among 31 tumors with elevated mean Her-2/neu levels/cell, 21 tumors also had elevated mean ras levels/cell;among 25 tumors in which mean Her-2/neu levels were notelevated, only 4 had mean ras levels/cell that exceeded 40,000units/cell. The strength of this association was supported byx2

analysis (P, 0.0002).Patterns of Intracellular Coexpression of p53, Her-2/

neu, and ras within Individual Tumors: Reconstruction ofEvolutionary Pathways. The foregoing analyses, whichtreated mean p53 overexpression, mean Her-2/neu overexpres-sion, and mean ras overexpression as uncorrelated measure-ments, showed that there was an intricate pattern of interrela-tionships among them. To examine these interrelationships ingreater detail, we took advantage of the fact that all of thesemeasurements were made simultaneously on each of a largenumber of cells in each tumor and were, therefore, correlated ona cell-by-cell basis. Our approach was designed to explore thepossibility that there may be preferred sequences in which theseabnormalities developed in individual tumors, based on thepremises that early changes can appear alone in individual cells,whereas late changes are likely to be accompanied by persistentearly changes in the same cells.

Subpopulations of cells that simultaneously overexpressedp53, Her-2/neu, and ras in the same cells and represented 1% ormore of the cells in a given tumor were detected in 45 of 56, or80.4%, of the breast cancers studied. In most of these 45 tumors,the triple-positive cell subpopulations represented at least 5% ofthe cells present (1–1.9% of the cells in 6 of 45 tumors, 2–4.9%of the cells in 8 of 46 tumors, 5–9.9% of the cells in 13 of 46tumors, 10–19.9% of the cells in 13 of 46 tumors, and$20% ofthe cells in 5 of 46 tumors). Triple-positive subpopulations werecommon among both diploid and aneuploid tumors (19 of 23, or82.6%, and 26 of 33, or 78.8%, respectively).

Among the 45 tumors that contained triple protein-overex-pressing cell subpopulations, 31 contained precursor popula-tions that supported evolutionary sequences in which p53 over-expression was potentially the first abnormality to occur amongthose studied. In 13 of 31 cases, sequences in which p53overexpression was the first detectable abnormality were theonly sequences supported by the data. Data from one such tumorare presented in Fig. 3 and Table 2.

Among the 45 tumors that contained triple protein-overex-pressing cell subpopulations, 36 contained precursor popula-tions that supported genetic evolutionary sequences in whichHer-2/neu overexpression was potentially the first abnormalityto occur among those studied. In 16 of 36 cases, sequences inwhich Her-2/neu overexpression was the first detectable abnor-mality were the only sequences supported by the flow cytometrydata. However, among these 16 tumors, there were 10 for whichp53 FISH data were available, and 4 of the 10 exhibitedp53allelic loss in cells that were monosomic or disomic for chro-mosome 17. This suggests that p53 abnormalities were presentin what may have been diploid cells, and that these abnormal-ities actually may have preceded, or occurred concomitantlywith, Her-2/neu overexpression in at least 40% of tumors inwhich Her-2/neu overexpression might otherwise appear to haveoccurred first. The example shown in Fig. 4 and Table 3 illus-trates a tumor in which p53 overexpression appears to have

played a minor role in the early stages of its genetic evolutionbut in which earlyp53 allelic loss was a prominent feature.

The overexpression of ras in more than 1% of the tumorcells was observed in 51 of 56 tumors. In 38 of these 51 tumors,ras overexpression was always accompanied by the overexpres-sion of at least one other protein in the same cells. In 13 of these38 tumors, ras overexpression first appeared in cells with p53overexpression, in 18 of 38 tumors ras overexpression firstappeared in cells with Her-2/neu overexpression, and in 7 of 38tumors, ras overexpression was always accompanied by bothp53 and Her-2/neu overexpression in the same cells. Only 13 of51 tumors contained precursor populations that supported ge-netic evolutionary sequences in which ras overexpression waspotentially the first detectable abnormality among those studied.Among the 13 tumors in which ras overexpression was one ofthe first detectable abnormalities, there were only four tumors inwhich sequences with ras first were the only sequences sup-ported by the data; p53 FISH data were available in three ofthese four cases, and in two of the three tumors,p53allelic losswas present. Taken together, these findings suggest that rasoverexpression generally occurs late in the evolutionary se-quence. This is readily apparent in the examples shown in Figs.3 and 4 and Tables 2 and 3.

Levels of Protein Overexpression during the Course ofTumor Evolution. We compared the levels of overexpressionof p53 protein in cells that overexpressed p53 alone with thelevels of p53 protein overexpression in cell subpopulations fromthe same tumor that overexpressed p53 plus Her-2/neu, ras, orboth. We reasoned that if intracellular p53 accumulation con-ferred no selective evolutionary advantage, then there would beno basis for expecting systematic differences in mean p53levels/cell between cells that overexpressed p53 alone and cellsfrom the same tumor that were further along in their evolution-ary development. The data are shown in Fig. 5.

Among 31 tumors that contained cells with p53 overex-pression alone, 15 also contained cell subpopulations that over-expressed p53 plus ras in the same cells. In all cases, the meannumber of p53 molecules/cell was higher in cells that overex-pressed p53 plus ras than in cells that overexpressed p53 alonein the same tumor. Furthermore, in 13 of these 15 tumors, themean number of p53 molecules/cell was higher in cells thatoverexpressed p53, ras, and Her-2/neu than in cells that over-expressed only p53 and ras in the same tumor.

Among the 31 tumors that contained cells with p53 over-expression alone, 16 also contained more advanced cell sub-populations that overexpressed p53 plus Her-2/neu in the samecells. In 12 of these 16 tumors, the mean number of p53molecules/cell was higher in cells that overexpressed p53 plusHer-2/neu than in cells that overexpressed p53 alone in the sametumor. More striking increases in the mean number of p53molecules/cell were seen in cells that overexpressed p53, Her-2/neu, and ras than in cells that overexpressed only p53 andHer-2/neu. Among the 14 of 16 tumors that contained cells thatoverexpressed all three proteins, the mean intracellular levels ofp53 were higher among triple protein-overexpressing cells thanamong cells overexpressing only p53 and Her-2/neu in the sametumor.

The progressive increase in mean p53 levels/cell in cellsthat acquire increasing numbers of additional oncogene abnor-




Fig. 3. Data from a breast cancer illustrating the early appearance of p53 overexpression in diploid cells, followed by aneuploidy, Her-2/neuoverexpression, and ras overexpression.Heavy reference linesin A (diploid cell component) andB (aneuploid cell component) indicate the thresholdfor p53 overexpression (vertical reference line) and the threshold for Her-2/neu overexpression (horizontal reference line).Red dots, the coordinatesof individual cells that express more than 40,000 units of ras/cell. In this example, the diploid cell component included almost all of the cells thatdid not overexpress any of the oncogene protein products studied here (A, left lower quadrant). This nonoverexpressing cell subpopulation wouldinclude admixed normal epithelial, stromal, hematopoietic, and lymphoid cells. In contrast, the aneuploid cell component is almost entirely devoidof cells that did not overexpress any of the oncogene protein products studied (B, left lower quadrant). This tumor contains both diploid and aneuploidcells that overexpress p53 alone (AandB, right lower quadrants). However, there are very few cells present that overexpress Her-2/neu alone, eitheramong the diploid or the aneuploid cells (AandB, left upper quadrants). Most cells in this tumor simultaneously overexpress both p53 and Her-2/neuprotein, and the majority of these are aneuploid (compareA andB, right upper quadrants). Almost all of the ras-overexpressing cells (red dots) areconfined to theright upper quadrantsof A and B, indicating that they overexpress p53 and Her-2/neu simultaneously; most of these triple-overexpressing cells are aneuploid. FISH data for this case are shown inC (paired measurements ofp53 gene copy number and chromosome 17centromere copy number/cell) andD (paired measurements of Her-2/neugene copy number and chromosome 17 copy number/cell).C, there isconcordance betweenp53gene copy number and chromosome 17 copy number/cell in most cells.D, over half of the cells exhibit.10 copies of theHer-2/neugene/cell but only two or three copies of chromosome 17 in the same cells (dark columns), indicating the presence of true geneamplification.




Fig. 4. Data illustrating a breast cancer in which Her-2/neu overexpression appears to have preceded the development of p53 overexpression and rasoverexpression but in whichp53allelic loss may have been present in diploid cells.Reference linesin A (diploid cell component) andB (aneuploidcell component) indicate the threshold for p53 overexpression (vertical reference line) and the threshold for Her-2/neu overexpression (horizontalreference line).Red dots, the coordinates of individual cells that express.40,000 units of ras/cell. There are very few cells in this tumor thatoverexpress neither p53 nor Her-2/neu (AandB, left lower quadrants). This tumor contains substantial proportions of cells that overexpress Her-2/neualone (Aand B, left upper quadrant) but has very few cells that overexpress p53 alone (Aand B, right lower quadrants). Most of the cells thatoverexpress both Her-2/neu and p53 (Aand B, right upper quadrants) also overexpress ras. However, ras overexpression is not confined totriple-overexpressing cells and is present in both diploid and aneuploid cells that overexpress Her-2/neu alone (A andB, left upper quadrant). Althoughthe flow cytometry data did not demonstrate the early appearance of p53 overexpression in this tumor, the FISH data from the same tumor suggestedthat early abrogation of wild-type p53 function may have occurred even in the absence of overexpression of the protein.C shows that there wasextensive allelic loss of p53 in this tumor, particularly in cells that were disomic for chromosome 17, and presumably diploid. Although levels ofHer-2/neu overexpression in this tumor were in a range comparable with that in the example shown in Fig. 3, this tumor did not exhibit Her-2/neugene amplification (D).




malities, and particularly in cells that develop ras protein over-expression, suggests that the cells with high p53 protein levelsmay be favored with a survival advantage during the course oftumor evolution. Similarly, mean levels of Her-2/neu/cell in-creased progressively as Her-2/neu-overexpressing cells ac-quired increasing numbers of additional abnormalities (data notshown).

Histopathological Correlations. The majority of tumorsin this study (43 of 56, or 76.8%) were pure infiltrating ductalcancers. Of these, 30 of 43 were aneuploid (70%). Thirty-onetumors (72.1%) exhibited early p53 abnormalities (p53allelicloss in cells disomic or monosomic for chromosome 17 and/oroverexpression of p53 protein alone). Thirty-six infiltrating duc-tal tumors (83.7%) contained subpopulations of cells that simul-taneously overexpressed all three oncogene proteins. In 23 of 43cases (53.5%), the triple-overexpressing cells represented.5%of the cells in the tumor.

Six tumors were classified as pure infiltrating lobular can-cers. This group of tumors appears to have followed geneticevolutionary pathways that may have differed from those ofmost infiltrating ductal carcinomas. All six lobular breast can-cers were diploid. All were positive for estrogen receptor, ascompared with 27 of 43 (62.8%) pure infiltrating ductal carci-nomas. Although this difference is not statistically significant(P 5 0.069 byx2 analysis), the number of lobular breast cancersanalyzed was small. Three of the six lobular tumors containedcells that simultaneously overexpressed all three proteins; but intwo of the three tumors, the triple-overexpressing subpopulationrepresented,5% of the cells in each tumor. In view of numer-ous reports that Her-2/neu overexpression is infrequent in lob-ular breast cancers, it is somewhat surprising that Her-2/neuoverexpression was so common in the lobular tumors studiedhere. The greater sensitivity of flow cytometry over immuno-histochemistry in detecting Her-2/neu overexpression may ac-

count in part for this discrepancy. Her-2/neu FISH data wereavailable in four of the six pure lobular tumors, and none of thefour exhibited Her-2/neugene amplification. p53 FISH datawere available for five of the six pure lobular tumors, and onlyone of the five exhibited p53 allelic loss.

Two tumors contained lobular breast cancer together withother histopathological elements. In both tumors, the patterns ofploidy and oncogene overexpression appeared to reflect thefeatures of the nonlobular component. There were two puremucinous (colloid) breast cancers among the tumors studiedhere. Both exhibited aneuploidy, early p53 abnormalities, and asubpopulation of triple protein-overexpressing cells that repre-sented.5% of the cells in each tumor, features that were alsocharacteristic of infiltrating ductal breast cancers. An additionaltumor that contained both mucinous breast cancer and infiltrat-ing ductal carcinoma exhibited similar features. A tubular breastcancer and a metaplastic carcinoma with squamous cell featureswere both diploid, and each contained a small triple protein-overexpressing cell subpopulation.

DISCUSSIONWe have shown that p53 protein overexpression, Her-2/neu

overexpression, and ras overexpression often occur together inthe same tumor cells in various combinations in most breastcancers. Multiparameter flow cytometry studies demonstratedthat it is uncommon for ras protein overexpression to occur incells that do not contain p53 abnormalities and/or overexpressHer-2/neu protein. In contrast, simultaneous p53 protein andHer-2/neu protein overexpression occurs frequently in the ab-sence of ras overexpression in the same cells, suggesting thatthey are likely to have developed earlier than the ras overex-pression that is present in the triple-positive cells in sametumors. Overexpression of all three proteins, together with

Fig. 5. Levels of p53 overexpression per cellincrease progressively with the acquisition of newabnormalities. Each group of three points showsthe levels of p53 overexpression in cell subpopu-lations within a given tumor that overexpress one,two, or three proteins/cell, respectively.Boldpoints and lines represent the overall means forcorresponding cell subpopulations averaged for alltumors. A, in tumors where p53 overexpressionwas the predominant abnormality in cells withonly one abnormality and in which p53 overex-pression and ras overexpression were the predom-inant pair of abnormalities in cells with only twoabnormalities, the rise in p53 levels/cell was mostpronounced when ras overexpression was present.B, in tumors where p53 overexpression was thepredominant abnormality in cells with only oneabnormality and in which ras overexpression oc-curred predominantly as the third abnormality incells that also overexpressed both p53 and Her-2/neu, again, the rise in p53 levels/cell was mostpronounced when ras overexpression was present.




aneuploidy, were found to be prominent features of infiltratingductal carcinomas but not of lobular breast cancers.

Supplementary FISH studies showed that tumors oftencontain cell subpopulations that exhibit p53 allelic loss by FISHin the absence of p53 overexpression, and that allelic loss of p53without p53 protein overexpression often accompanies or mayprecede the development of Her-2/neu abnormalities. A com-parison of FISH studies with flow cytometric findings in thesame tumors also indicates that overexpression of Her-2/neu canoccur in the absence of demonstrable Her-2/neugene amplifi-cation. Taken together, these findings suggest that there is acommon molecular phenotypic evolutionary pattern that is as-sociated with many infiltrating ductal carcinomas of the breast,and that this pattern can develop by a variety of moleculargenetic mechanisms that have equivalent or overlapping pheno-typic consequences.

The flow cytometric methodology used in this study didnot permit direct correlations between the cell-based measure-ments that were performed and the morphological appearance ofthe cells that were studied. Instead, the observed intracellularpatterns of the abnormalities themselves were used to charac-terize the various cell subpopulations present in each tumorsample. In diploid samples in which none of the cells exhibitedoverexpression of the oncogenes studied, the presence of tumorcells in the monodispersed cell suspensions was confirmed byindependent cytological examination. Direct correlations be-tween morphological appearance and quantitative phenotypicmeasurements on a cell-by-cell basis must await the applicationof quantitative multiparameter fluorescence imaging technolo-gies.

A relationship between the overexpression of p53, Her-2/neu, and ras proteins and the development of gross geneticabnormalities in the same cells is established by the finding thatthe levels of expression of each of these proteins were invariablyhigher in the aneuploid components of aneuploid tumors than inthe diploid components of the same tumors (Fig. 2). However,the finding that levels of each protein increased progressivelywith the acquisition of new abnormalities (Fig. 5 and associateddiscussion) suggests that this progressive overexpression wasthe result of multiple events, some of which may have beenattributable to effects of genes coding directly for the specificproteins involved, and others of which may have resulted fromindirect effects, including the possibility of perpetuated epige-netic changes.

The relationship between p53 protein overexpression andthe presence of genetic abnormalities that might affect p53function directly or indirectly is complex. Wild-type p53 proteinhas a short half-life and does not ordinarily accumulate innormal cells in the absence of DNA damage. In experimentalcell systems, sustained overexpression of the wild-type p53protein commonly leads to cell cycle arrest, apoptosis, and/orinhibition or reversion of the transformed phenotype. In humantumors, p53 overexpression is commonly associated with mis-sense mutations (29). The accumulation of mutant p53 protein inthe cell nucleus is thought to be attributable to the failure of themutant protein to induce MDM2 (30, 31), a regulatory proteinthat normally binds p53, inactivates it, and targets it for ubi-quitination and subsequent destruction.

p53 protein overexpression is not always associated with

p53gene mutations. Wild-type p53 protein can also accumulatein human tumors and, like mutant p53 protein, is detected by theDO-7 antibody to p53 used in this study. When wild-type p53protein is overexpressed in human tumors, it is thought to bemaintained in a functionally inactive state by any of severalpossible mechanisms. In up to one-third of human breast can-cers, wild-type p53 is excluded from the nucleus and accumu-lates in the cytoplasm (32), where it is transcriptionally inactive.Cytoplasmic p53 overexpression is associated with an adverseclinical outcome in breast cancer, even in the absence of nuclearoverexpression (33). In the nuclear compartment, the level ofMDM2 and the level of wild-type p53 are normally regulatedmutually and reciprocally (30, 34, 35). However, the MDM2/p53 regulatory loop is modulated by additional factors, includ-ing the states of phosphorylation of the MDM2 and p53 pro-teins, and the level of p14ARF, which regulates the level ofMDM2, in turn (35). Simultaneous overexpression of bothMDM2 and wild-type p53 proteins has been documented in avariety of human solid tumors, including a small proportion ofbreast cancers (36), which may reflect simultaneous inactivationof wild-type p53 by MDM2 binding and impairment of theubiquitin pathway that leads to p53 degradation (30). Thus,direct or indirect interference with normal p53 transcriptionalactivity would appear to be a common underlying phenotypiclink among the several known genetic mechanisms that can leadto wild-type or mutant p53 protein overexpression.

Abrogation of wild-type p53 function is not always accom-panied by mutant or wild-type p53 protein overexpression.Frame shift and stop codon mutations, which represent;15% ofall p53 mutations, often result in the absence or near absence ofintracellular p53 protein. In the present study, 50% of all tumorswere found to overexpress p53 protein, using sensitive flowcytometric techniques to detect p53 overexpression. With theaddition of FISH studies to detectp53 allelic loss, p53 abnor-malities were identified in 80.4% of all tumors studied by bothtechniques, suggesting that a substantial number of p53 abnor-malities might be missed if one were to rely on p53 overexpres-sion alone.

With regard to the molecular mechanisms underlying Her-2/neu overexpression, our FISH studies indicated that genedosage effects attributable to gene amplification and/or in-creased chromosome copy number/cell could account for over-expression in;65% of cases. Interphase FISH techniques relyon the identification of multiple, spatially separated gene loci toidentify gene amplification. It is conceivable that contiguousamplified loci, such as HSR regions, might be missed in FISHstudies of interphase cells, possibly resulting in underestimationof gene amplification by this technique. The high level ofsensitivity of flow cytometric techniques might also result in thecategorization of tumors with moderately elevated levels ofHer-2/neu expression as overexpressors, which might have beencategorized otherwise in immunohistochemical studies. Apartfrom technical considerations, levels of Her-2/neu expressionare known to be impacted by factors other than gene amplifi-cation, including down-regulation by estrogen through estrogenreceptor-mediated pathways (37, 38) and down-regulation byRb (39), either or both of which can be inactivated or lost inbreast cancer.

Overexpression of ras is common in breast cancer, but ras




is rarely mutated in this disease (40–42). Studies in experimen-tal tumor systems have suggested that increased levels of the rasprotein may be more closely associated with tumorigenicity,rapid growth rate, and acquisition of metastatic potential thanthe presence or absence of a mutatedras geneper se(reviewedin Ref. 3), suggesting that this phenotypic measurement may bea more reliable indicator of a hyperfunctional ras state.

There is substantial evidence linking the development ofboth gross numerical chromosomal changes and structural chro-mosomal abnormalities to the abrogation of wild-type p53 func-tion (reviewed in Refs. 3 and 4). Although both types of chro-mosomal abnormalities often occur together, they probably ariseby different p53-related mechanisms. Tetraploidy, an early stepin the development of aneuploidy, has been attributed to thefailure of p53-deficient or p53-inactivated cells to arrest at amitotic checkpoint (43–45) and/or to the development of cen-trosome abnormalities (46). An association between p53 abnor-malities and the development of structural genetic abnormali-ties, particularly in aneuploid cells, has been observed in anumber of experimental studies (47, 48). Of special relevance tothe present study, loss of wild-type p53 function has been foundto be associated with increased levels of gene amplification,which has often been used as a marker for genetic instability inthe experimental setting, as well as the development of aneu-ploidy (49). In the present study, p53 abnormalities commonlypreceded or occurred simultaneously with Her-2/neu overex-pression; p53 abnormalities were present in 12 of the 14 tumorsthat exhibited Her-2/neu amplification by FISH, and 12 of thesetumors were aneuploid. These findings are consistent with ex-perimental studies linking p53 abnormalities with genetic insta-bility that may be manifested by gene amplification and/oraneuploidy in the clinical setting. However, one cannot con-clude from the sequence information provided by our studiesthat p53 abnormalities are causally linked to Her-2/neu ampli-fication, nor do our studies rule out other potential sources ofgenetic instability that might lead to Her-2/neu amplificationand/or aneuploidy (50).

The frequent association of p53 abnormalities with rasoverexpression in infiltrating ductal carcinomas is of particularinterest, in view of the well-documented cooperation betweenmutant p53 and ras abnormalities in the transformation of nor-mal cells in experimental cell systems (51–54). Cooperativitymight account for the statistically significant association be-tween these two abnormalities, but it would not explain why rasoverexpression is almost always accompanied by p53 overex-pression in the same cells, whereas p53 overexpression canoccur alone in individual cells in the same tumor. There ismounting experimental evidence that ras-mediated mitogenicsignaling can be antagonized by p53-mediated apoptotic andgrowth-inhibitory responses (3). It has been reported recentlythat oncogenicras transfected into mouse embryo fibroblastsinduces an increase in p53 protein expression and a reduction incell proliferative activity by a p19ARF-mediated pathway that isseparate and distinct from the DNA damage-induced p53 re-sponse pathway (which does not involve p19ARF; Ref. 55). Ourfindings support the premise that for a strategy for neoplastictransformation that relies on sustained mitogenic signaling tosucceed, prior abrogation of wild-type p53 function (with orwithout p53 protein overexpression) might be required. The

present study suggests that both elements of this strategy may beembraced by many, and perhaps most, infiltrating ductal carci-nomas.

Lobular breast cancers appear to pursue a different coursefrom that followed by infiltrating ductal carcinomas. Our find-ings that lobular carcinomas are generally diploid are in keepingwith other published studies (56), as are our findings that p53abnormalities (16, 57–59), Her-2/neu amplification (60), andloss of estrogen receptor (58, 61) are not prominent features,even in advanced stages of disease. The present study and ourpreviously published multiparameter study in human breast can-cer (2) suggest that both epidermal growth factor receptor over-expression and normal to moderately elevated levels of Her-2/neu expression are more characteristic features of lobular breasttumors.

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2000;6:112-126. Clin Cancer Res Charles A. Smith, Agnese A. Pollice, Ling-Ping Gu, et al. Evolutionary Pattern in Infiltrating Ductal CarcinomasBreast Cancer: Evidence for a Common Phenotypicand Aneuploidy by Multiparameter Flow Cytometry in Human Correlations Among p53, Her-2/neu, and ras Overexpression

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Correlations Among p53, Her-2/neu, and ras Overexpression ... · Human Oncology [R. J., T. J., D. H., N. W.], and Pathology and Laboratory Medicine [J. F. S.], Allegheny University