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Molecular Oncology, Markers, Clinical Correlates |
Laboratory of Cancer Cell Biology and Genetics, Department of Human 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 and Laboratory Medicine [J. F. S.], Allegheny University of the Health Sciences, Allegheny General Hospital, Pittsburgh, Pennsylvania 15212
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ABSTRACT |
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INTRODUCTION |
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Numerous studies in human solid tumors, and in breast cancer in particular, have demonstrated relationships between p53 abnormalities (or loss of heterozygosity at 17p) and aneuploidy, between p53 abnormalities and Her-2/neu amplification/overexpression, between Her-2/neu amplification and/or overexpression 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 been found to be associated with distinctive forms of genetic instability that are manifested by the development of gross abnormalities in chromosome number/cell (aneuploidy) and by structural abnormalities involving individual chromosomes, such as chromosomal breakage, deletions of chromosomal material, and gene amplification (3 , 4) . The Her-2/neu gene is often amplified and overexpressed in human breast cancer (5 , 6) , particularly in aneuploid tumors (7, 8, 9, 10) . If Her-2/neu amplification were a direct or indirect consequence of p53-induced genetic instability, then one might anticipate that the development of p53 abnormalities might accompany or precede the development of Her-2/neu overexpression, aneuploidy, or both. The overexpression of ras protein has been found to be contingent on the loss of wild-type p53 function in a number of experimental systems (reviewed in Ref. 3 ). Thus, we hypothesized that p53 abnormalities might also precede the development of ras overexpression in human breast cancer.
Our methodological approach to extracting evolutionary sequence information from single tumor samples is based on the technical ability to perform multiple quantitative measurements on each cell by multiparameter flow cytometry. In previous studies, we found that breast cancer precursor populations generally persist in the background during and after the emergence of more advanced clones (those with larger numbers of genetic abnormalities/cell and/or higher levels of expression of individual oncogene proteins; Refs. 1 , 2 , and 11 ). Under such conditions, if there is a specific order in which different genetic abnormalities appear, the same early genetic changes among those measured would be found in the cells with the fewest abnormalities. In contrast, one might expect that abnormalities that consistently occur late would almost always be found to have accumulated in the same cells with abnormalities that had occurred early and had persisted.
In this study, multiparameter flow cytometric analyses were
supplemented by FISH3
studies
on the same tumors in which paired cell-by-cell measurements of a
locus-specific fluorescent probe for p53 and an 
satellite probe for
chromosome 17 were performed to further explore the relationship
between p53 abnormalities (manifested by allelic loss) and aneuploidy
in the same cells. We also performed paired cell-by-cell measurements
of a locus-specific probe for Her-2/neu and an satellite probe for
chromosome 17 in cells from the same tumors to examine the relationship
between Her-2/neu gene amplification and aneuploidy in the
same cells.
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PATIENTS AND METHODS |
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summarizes the clinical characteristics
of these patients. Four of these patients have had disease recurrences
to date.
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Immunofluorescence Staining.
Fluorescein-conjugated monoclonal antibody immunospecific for p53
protein (Clone DO-7) was purchased from Novocastra Laboratories, Ltd.
(Newcastle upon Tyne, United Kingdom). Rabbit polyclonal antibody to
c-erbB-2, purchased from Cambridge Research Biochemicals (Cambridge,
United Kingdom), was used for indirect staining.
Phycoerythrin-conjugated goat anti-rabbit IgG (1:20), purchased from
Vector (Burlingame, CA), was used as a secondary antibody. Rat
monoclonal antibody to human v-H-ras, which recognizes human c-H-ras,
K-ras, and N-ras, was purchased from Oncogene Science (Cambridge, MA).
This antibody was conjugated with Cy-5 (Amersham Life Sciences, Inc.,
Pittsburgh, PA) and used for direct staining as described previously
(2)
. JC 1939, a breast cancer cell line established in our
laboratory, was used as a positive staining control for p53, Her-2/neu,
and H-ras immunofluorescence and as a quantitative fluorescence
staining reference for p53 and Her-2/neu (see below). Lymphocytes from
healthy donors were used as low-level baseline immunofluorescence
staining controls and as relative reference standards for the ras
measurements.
DNA Staining and Flow Cytometry.
Cells were stained with 4',6-diamino-2-phenylindole (Sigma Chemical
Co., St. Louis, MO) at a final concentration of 0.1 µg/ml.
Multiparameter flow cytometry measurements were performed as described
previously (2)
.
Use of Lymphocytes as Reference Cells for Cell DNA Content and Cell
ras Content.
Methanol-fixed lymphocytes were used as diploid reference standards for
ploidy analysis of methanol-fixed tumor cells (12)
. The
criteria for tetraploidy and aneuploidy were as described previously
(2)
.
Paraformaldehyde/methanol-fixed lymphocytes were used as reference cells for the multiparameter flow cytometry studies. Tumor cell ras levels were expressed as multiples of the baseline normal lymphocyte reference (by analogy to the DNA index). In this study, for convenience of graphical comparison of ras levels to levels of Her-2/neu in the same cells (see below), the normal lymphocyte reference was assigned a nominal reference value of 10,000 units. Thus, for example, 5 x 104 units/cell would correspond to a 5-fold increase in ras content above that of normal lymphocytes, and 1 x 105 ras units/cell would correspond 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 77,000
molecules/cell of HER-2/neu, quantitated by ELISA assay (Oncogene
Science; Ref. 2
), and a mean of 7,500 p53 molecules/cell, also
quantitated by ELISA assay (Oncogene Research Products, Cambridge, MA).
On the basis of this reference value, the mean p53 content in 23
samples of normal lymphocytes was 6600 molecules/cell, in general
agreement with values reported by others for normal blood leukocytes
(13
, 14) .
Data Analysis.
Using a computer program developed by one of the authors (S. E. S.),
the logarithmic data for all three of the oncogene measurements per
cell were scaled to a common origin. A subtractive correction for
nonspecific antibody binding was applied to each measurement on a
cell-by-cell basis, as described previously (2)
. In all
data figures shown in this paper, nonspecific labeling has already been
subtracted from each measurement on a cell-by-cell basis.
FISH.
Tumor cells in single-cell suspension freshly fixed in methanol:glacial
acetic acid (3:1) were applied to slides and processed in accordance
with protocols developed by Vysis, Inc. (Downer’s Grove, IL) using the
HYBrite hybridization apparatus (Vysis). Combinations of gene
locus-specific and centromeric 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
4',6-diamino-2-phenylindole (50 µg/ml), and slides were viewed on an
Axiophot-II microscope (Carl Ziess, Inc). Standardized criteria for
spot counting were used as recommended by Vysis. At least 50 cells on
each slide were counted by each of two independent observers. Staining
quality assurance controls consisting of normal lymphocytes were
included in every staining batch for each probe set. Each bivariate
data set was corrected for staining background using a bivariate
template derived from at least five slides of normal lymphocytes, where
at least 100 normal cells were counted on each slide for each probe
set.
Criteria for Protein Overexpression: p53.
Brotheric et al. (13)
estimated the staining
intensity threshold for overexpression of p53 to be in excess of 9,000
molecules/cell for antibody DO-7 by flow cytometry. In this study, we
used a staining intensity threshold for p53 overexpression of 10,000
molecules/cell.
Criteria for Oncogene Overexpression: Her-2/neu.
Individual cells that overexpress Her-2/neu are generally detectable
immunohistochemically when intracellular levels are in the range of
200,000–500,000 molecules/cell or higher (3)
. We adopted
a mean level of Her-2/neu overexpression of >300,000 molecules/cell as
a staining intensity threshold for overexpression to relate our studies
to immunohistochemical studies. However, it was also apparent that flow
cytometric measurements were capable of detecting Her-2/neu levels that
were well below 300,000 molecules/cell but which were clearly higher
than normal (
50,000 molecules/cell). Therefore, in this study, we
used both a >150,000 mean molecules/cell threshold and a >300,000
mean molecules/cell threshold. We view the former threshold as more
inclusive for true overexpression and the latter as more appropriate
for comparisons with immunohistochemical studies.
Criteria for Oncogene Overexpression: ras.
A relative mean staining intensity per cell at least four times higher
than that of normal lymphocytes (i.e., 4 x 104 units) was used as a threshold level for
overexpression. This value exceeded the mean for normal lymphocytes
(relative to a fluorescent bead reference) by more than 2 SDs.
Criteria for Allelic Loss or Gene Amplification by FISH.
For the FISH studies, allelic loss was defined by the presence of fewer
p53 alleles/cell than chromosome 17 centromeres in the same
cells, with a frequency threshold requirement that at least 15% of
cells exhibit allelic loss, based on the studies of Kibbalaar et
al. (15)
. True Her-2/neu gene
amplification was defined by the presence of an excess of
Her-2/neu gene loci over the number of chromosome 17
centromeres in the same cells. Absolute increases in the number of
Her-2/neu loci/cell that were matched or exceeded by the number of
chromosome 17 centromeres in the same cells may have been attributable
solely to aneusomy and were treated separately. Frequency threshold
requirements were similar to those for p53 allelic loss.
Analytic Approach to Genetic Evolutionary Sequencing.
For each tumor sample analyzed, the cells were grouped by the number of
abnormalities they contained. If these abnormalities had developed in
an orderly sequence of inherited clonal changes, and if the precursor
clonal subpopulations had persisted in the presence of more evolved
clones, then the earliest abnormal change(s) would be identified among
the cells that contain a single abnormality (Fig. 1A)
. Once the earliest
abnormality is identified, the next abnormal change in the sequence can
then be deduced from the patterns observed in cells with two
abnormalities, and the third abnormality in the sequence can be
deduced, in turn, from the patterns observed in cells with three
abnormalities. In the hypothetical example shown in Fig. 1A
,
almost all cells with a single abnormality contained abnormality B;
hence, among the abnormalities studied, B is likely to have occurred
first. In this hypothetical example, among cells that contained two and
only two of the abnormalities measured, almost all contained
abnormalities A and B in the same cells. Having established that B
occurred first, abnormality A must have occurred after abnormality B.
In this example, because cells containing abnormality C almost always
contained abnormalities B and A as well, the sequence of accumulation
of all of these abnormalities must have been B 
A C.
Illustrative examples based on actual data are given below in Figs. 3
and 4
and Tables 2
and 3
.
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If precursor subpopulations had not persisted in sufficient numbers to
be detected, then this approach to genetic evolutionary sequencing
would break down (Fig. 1B)
. However, in the present study,
among 153 subpopulations with two or more abnormalities/cell that were
detected among the 56 breast cancers (diploid and aneuploid cell
subpopulations considered separately), at least one potential immediate
precursor (i.e., a subpopulation with one abnormality fewer)
was also present in the same tumor in all but one instance.
The presence of more than one candidate precursor subpopulation (Fig. 1C)
is not only possible but was actually quite common.
Other variants included parallel pathways that converge after multiple
steps. However, if all potential precursor populations are
present in the same tumor, then this approach to genetic evolutionary
sequencing would provide no useful information (Fig. 1D)
.
When the event frequency threshold for identifying a potential
precursor populations was set at 
1% of all cells analyzed in a given
sample, well-defined evolutionary sequences could be discerned in all
but one of the 56 tumors analyzed, and in that one case, an event
frequency threshold of 1% of cells may have been too low.
Statistical Analysis.
Means of two groups were compared using Student’s t test.
Associations between variables were assessed by the 
2
test or by Fisher’s exact test, as appropriate.
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RESULTS |
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To reduce the potential effects of dilution by normal diploid cells,
mean p53 levels/cell were calculated separately for the 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 the normal range in the majority
of diploid tumors and in most of the diploid components of aneuploid
tumors. In every aneuploid tumor, the aneuploid component exhibited
higher mean levels of p53/cell than the diploid component, even when
neither component exhibited a mean p53 level that exceeded 10,000
molecules/cell. When the presence of a separate aneuploid component
with an elevated mean p53 level was considered in classifying tumors
with respect to p53 status, the total number of samples exhibiting p53
overexpression increased to 29 of 56, or 51.8% of cases.
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Among the 46 cases in which FISH studies involving the p53 locus were performed, 26 showed allelic loss of p53 by FISH (56.5%). Fifteen of these 26 tumors also showed p53 overexpression by flow cytometry. In 11 cases, allelic loss of p53 by FISH was the only p53 abnormality noted. Overall, 37 of 46, or 80.4%, of tumors studied by both flow cytometry and FISH exhibited p53 abnormalities by either or both techniques.
Nineteen of the 26 tumors with allelic loss by FISH contained cells that were either monosomic for chromosome 17 or were disomic for chromosome 17 but exhibited only one p53 allele. Among the 19 cases that contained cells that exhibited disomy and p53 allelic loss or monosomy for chromosome 17 in the same cells, 9 were diploid by flow cytometry. In six of these nine diploid tumors, p53 protein levels by flow cytometry were in the normal range, and allelic loss was the only p53 abnormality detected. Because hemizygosity for wild-type p53 has been reported to be associated with increased susceptibility to oncogenic transformation (27) , p53 abnormalities that first became apparent in diploid cells in the present study may have been of clinical consequence.
FISH studies were performed on 17 of 23 diploid tumors. Nine of these 17 diploid tumors exhibited allelic loss of p53 (52.9%). All nine contained cells that exhibited disomy and p53 allelic loss or monosomy for chromosome 17 in the same cells. Overall, p53 allelic loss, p53 protein overexpression, or both were documented in a total of 13 of 17, or 76.5%, of the diploid tumors studied by both techniques.
FISH studies were performed on 28 of 33 aneuploid tumors. Among these 28 tumors, 20 exhibited p53 overexpression (71.4%), and 17 exhibited p53 allelic loss (60.7%). Five of these 28 tumors exhibited p53 allelic loss as the only p53 abnormality, and 3 of the 28 tumors had no demonstrable p53 abnormalities at all. Overall, 25 of 28 aneuploid tumors (89.3%) studied by both flow cytometry and FISH exhibited p53 abnormalities by either or both techniques. The difference in frequency of p53 abnormalities between diploid and aneuploid tumors was not statistically significant (P = 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 of Her-2/neu
protein/cell that were considered to be potentially detectable by
immunohistochemistry (>300,000 molecules/cell), a value that is within
the range of reported frequencies of Her-2/neu overexpression in breast
cancer (28)
. Overall mean levels of Her-2/neu/cell
exceeding 150,000 molecules/cell were observed in 26 of 56, or 46.4%,
of breast cancers. The higher apparent frequency reflects the greater
sensitivity of detection of moderate increases in Her-2/neu expression
by flow cytometry in comparison with immunohistochemical techniques.
Even this value is still likely to underestimate the proportion of
tumors with Her-2/neu overexpression because of the presence of normal
diploid cells that have low intracellular levels of Her-2/neu.
To minimize the effect attributable to the presence of normal diploid
cells in aneuploid tumors, mean Her-2/neu levels were calculated
separately for the diploid and aneuploid components. The results are
shown in Fig. 2B
, together with Her-2/neu levels in the
purely diploid tumors. When the presence of a separate aneuploid
component with an elevated Her-2/neu level was considered in
classifying tumors with respect to Her-2/neu status, the total number
of samples exhibiting Her-2/neu overexpression (>150,000
molecules/cell) increased to 31 of 56 tumors, or 55.4% of cases.
It is apparent from Fig. 2B
that high mean levels of
Her-2/neu/cell were observed both among diploid tumors and among the
aneuploid components of aneuploid tumors. The aneuploid component of
each aneuploid tumor invariably exhibited higher mean levels of
Her-2/neu/cell than the diploid component of the same tumor, even when
neither component exhibited a mean Her-2/neu level that exceeded
150,000 molecules/cell. The diploid components of aneuploid tumors
appear to fall into two easily separable groups, i.e., those
with mean levels of Her-2/neu that were <150,000 molecules/cell and
those with mean levels of Her-2/neu that were >150,000 molecules/cell,
suggesting that in aneuploid tumors, the development of Her-2/neu
overexpression can precede the development of aneuploidy.
True Her-2/neu gene amplification by FISH (where the number of gene loci/cell exceeded the number of chromosome 17 centromeres in each cell) was observed in 14 of 46 tumors in which Her-2/neu FISH studies were performed (30.4%). Concomitant Her-2/neu gene amplification and protein overexpression (>150,000 molecules/cell) were present in the same tumor in 9 of these 14 cases (64%). Six of these tumors exhibited mean levels of Her-2/neu/cell that exceeded 300,000 molecules/cell. Two of the tumors with Her-2/neu gene amplification by FISH but no protein overexpression had unequivocally low/normal mean levels of Her-2/neu protein/cell (mean, <50,000 molecules/cell). Twelve of the 14 tumors that exhibited true Her-2/neu amplification were aneuploid (85.7%), and 12 of these 14 tumors exhibited p53 allelic loss, p53 overexpression, or both.
An absolute increase in Her-2/neu gene copy number/cell that was matched or exceeded by chromosome 17 centromere copy number in the same cells was observed in 12 additional tumors. Concomitant Her-2/neu protein overexpression was present in 6 of these 12 tumors (50%). These 6 tumors, together with the 9 tumors with true Her-2/neu amplification and mean levels of Her-2/neu overexpression exceeding 150,000 molecules/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 regulatory mechanisms, rather than increased gene dosage per 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 protein overexpression.
Abnormal Mean ras Expression and Ploidy.
Elevated mean levels of ras/cell (mean, >40,000 units/cell) were
observed in 14 of 56, or 25%, of tumors, in keeping with our earlier
studies (2)
. This is likely to be an underestimate of the
true proportion of tumors with ras overexpression because of the
artifactual effects of normal diploid cells with low intracellular
levels of ras. Hence, for aneuploid tumors, mean ras levels were
calculated separately for the diploid and aneuploid components. The
results are shown in Fig. 2C
, together with mean ras levels
in the purely diploid tumors. When the presence of a separate aneuploid
component with an elevated mean ras level was considered, the total
number of samples exhibiting ras overexpression increased to 25 of 56
tumors, or 44.6% of cases. As Fig. 2C
shows, most of the
aneuploid components of aneuploid tumors exhibited high mean levels of
ras/cell, whereas most of the diploid components did not. However, a
statistically significant relationship between ras overexpression and
aneuploidy was not supported by 2 analysis
(P > 0.09).
Abnormal Mean p53 Expression and Mean ras Overexpression.
There was an association between mean p53 level/cell and mean ras
level/cell in the same tumor. (In aneuploid tumors, the aneuploid
component was used in the analysis.) Among 29 tumors with elevated mean
p53 levels/cell, 19 tumors also had elevated mean ras levels/cell
(>40,000 units/cell). Among 27 tumors in which mean p53 levels were
not elevated, only 7 had mean ras levels/cell that exceeded 40,000
units/cell. This association was highly significant by 2
analysis (P = 0.007).
Abnormal Mean Her-2/neu Expression and Mean ras Overexpression.
There was a strong association between mean Her-2/neu level/cell and
mean ras level/cell in the same tumor (in aneuploid tumors the
aneuploid component was used in 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
not elevated, only 4 had mean ras levels/cell that exceeded 40,000
units/cell. The strength of this association was supported by
2 analysis (P < 0.0002).
Patterns of Intracellular Coexpression of p53, Her-2/neu, and ras
within Individual Tumors: Reconstruction of Evolutionary Pathways.
The foregoing analyses, which treated mean p53 overexpression, mean
Her-2/neu overexpression, and mean ras overexpression as uncorrelated
measurements, showed that there was an intricate pattern of
interrelationships among them. To examine these interrelationships in
greater detail, we took advantage of the fact that all of these
measurements were made simultaneously on each of a large number of
cells in each tumor and were, therefore, correlated on a cell-by-cell
basis. Our approach was designed to explore the possibility that there
may be preferred sequences in which these abnormalities developed in
individual tumors, based on the premises that early changes can appear
alone in individual cells, whereas late changes are likely to be
accompanied by persistent early changes in the same cells.
Subpopulations of cells that simultaneously overexpressed p53,
Her-2/neu, and ras in the same cells and represented 1% or more of the
cells in a given tumor were detected in 45 of 56, or 80.4%, of the
breast cancers studied. In most of these 45 tumors, the triple-positive
cell subpopulations represented at least 5% of the 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 46 tumors, 10–19.9% of the
cells in 13 of 46 tumors, and 20% of the cells in 5 of 46 tumors).
Triple-positive subpopulations were common among both diploid and
aneuploid tumors (19 of 23, or 82.6%, and 26 of 33, or 78.8%,
respectively).
Among the 45 tumors that contained triple protein-overexpressing cell
subpopulations, 31 contained precursor populations that supported
evolutionary sequences in which p53 overexpression was potentially the
first abnormality to occur among those studied. In 13 of 31 cases,
sequences in which p53 overexpression was the first detectable
abnormality were the only sequences supported by the data. Data from
one such tumor are presented in Fig. 3
and Table 2
.
Among the 45 tumors that contained triple protein-overexpressing cell
subpopulations, 36 contained precursor populations that supported
genetic evolutionary sequences in which Her-2/neu overexpression was
potentially the first abnormality to occur among those studied. In 16
of 36 cases, sequences in which Her-2/neu overexpression was the first
detectable abnormality were the only sequences supported by the flow
cytometry data. However, among these 16 tumors, there were 10 for which
p53 FISH data were available, and 4 of the 10 exhibited p53
allelic loss in cells that were monosomic or disomic for chromosome 17.
This suggests that p53 abnormalities were present in what may have been
diploid cells, and that these abnormalities actually may have preceded,
or occurred concomitantly with, Her-2/neu overexpression in at least
40% of tumors in which Her-2/neu overexpression might otherwise appear
to have occurred first. The example shown in Fig. 4
and Table 3
illustrates a tumor in
which p53 overexpression appears to have played a minor role in the
early stages of its genetic evolution but in which early p53
allelic loss was a prominent feature.
The overexpression of ras in more than 1% of the tumor cells was
observed in 51 of 56 tumors. In 38 of these 51 tumors, ras
overexpression was always accompanied by the overexpression of at least
one other protein in the same cells. In 13 of these 38 tumors, ras
overexpression first appeared in cells with p53 overexpression, in 18
of 38 tumors ras overexpression first appeared in cells with Her-2/neu
overexpression, and in 7 of 38 tumors, ras overexpression was always
accompanied by both p53 and Her-2/neu overexpression in the same cells.
Only 13 of 51 tumors contained precursor populations that supported
genetic evolutionary sequences in which ras overexpression was
potentially the first detectable abnormality among those studied. Among
the 13 tumors in which ras overexpression was one of the first
detectable abnormalities, there were only four tumors in which
sequences with ras first were the only sequences supported by the data;
p53 FISH data were available in three of these four cases, and in two
of the three tumors, p53 allelic loss was present. Taken
together, these findings suggest that ras overexpression generally
occurs late in the evolutionary sequence. 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 of Tumor
Evolution.
We compared the levels of overexpression of p53 protein in cells that
overexpressed p53 alone with the levels of p53 protein overexpression
in cell subpopulations from the same tumor that overexpressed p53 plus
Her-2/neu, ras, or both. We reasoned that if intracellular p53
accumulation conferred no selective evolutionary advantage, then there
would be no basis for expecting systematic differences in mean p53
levels/cell between cells that overexpressed p53 alone and cells from
the same tumor that were further along in their evolutionary
development. The data are shown in Fig. 5
.
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Among the 31 tumors that contained cells with p53 overexpression alone, 16 also contained more advanced cell subpopulations that overexpressed p53 plus Her-2/neu in the same cells. In 12 of these 16 tumors, the mean number of p53 molecules/cell was higher in cells that overexpressed p53 plus Her-2/neu than in cells that overexpressed p53 alone in the same tumor. More striking increases in the mean number of p53 molecules/cell were seen in cells that overexpressed p53, Her-2/neu, and ras than in cells that overexpressed only p53 and Her-2/neu. Among the 14 of 16 tumors that contained cells that overexpressed all three proteins, the mean intracellular levels of p53 were higher among triple protein-overexpressing cells than among cells overexpressing only p53 and Her-2/neu in the same tumor.
The progressive increase in mean p53 levels/cell in cells that acquire increasing numbers of additional oncogene abnormalities, and particularly in cells that develop ras protein overexpression, suggests that the cells with high p53 protein levels may be favored with a survival advantage during the course of tumor evolution. Similarly, mean levels of Her-2/neu/cell increased progressively as Her-2/neu-overexpressing cells acquired increasing numbers of additional abnormalities (data not shown).
Histopathological Correlations.
The majority of tumors in this study (43 of 56, or 76.8%) were pure
infiltrating ductal cancers. Of these, 30 of 43 were aneuploid (70%).
Thirty-one tumors (72.1%) exhibited early p53 abnormalities
(p53 allelic loss in cells disomic or monosomic for
chromosome 17 and/or overexpression of p53 protein alone). Thirty-six
infiltrating ductal tumors (83.7%) contained subpopulations of cells
that simultaneously overexpressed all three oncogene proteins. In 23 of
43 cases (53.5%), the triple-overexpressing cells represented >5% of
the cells in the tumor.
Six tumors were classified as pure infiltrating lobular cancers. This
group of tumors appears to have followed genetic evolutionary pathways
that may have differed from those of most infiltrating ductal
carcinomas. All six lobular breast cancers were diploid. All were
positive for estrogen receptor, as compared with 27 of 43 (62.8%) pure
infiltrating ductal carcinomas. Although this difference is not
statistically significant (P = 0.069 by
2 analysis), the number of lobular breast cancers
analyzed was small. Three of the six lobular tumors contained cells
that simultaneously overexpressed all three proteins; but in two of the
three tumors, the triple-overexpressing subpopulation represented <5%
of the cells in each tumor. In view of numerous reports that Her-2/neu
overexpression is infrequent in lobular breast cancers, it is somewhat
surprising that Her-2/neu overexpression was so common in the lobular
tumors studied here. The greater sensitivity of flow cytometry over
immunohistochemistry in detecting Her-2/neu overexpression may account
in part for this discrepancy. Her-2/neu FISH data were available in
four of the six pure lobular tumors, and none of the four exhibited
Her-2/neu gene amplification. p53 FISH data were available
for five of the six pure lobular tumors, and only one of the five
exhibited p53 allelic loss.
Two tumors contained lobular breast cancer together with other histopathological elements. In both tumors, the patterns of ploidy and oncogene overexpression appeared to reflect the features of the nonlobular component. There were two pure mucinous (colloid) breast cancers among the tumors studied here. Both exhibited aneuploidy, early p53 abnormalities, and a subpopulation of triple protein-overexpressing cells that represented >5% of the cells in each tumor, features that were also characteristic of infiltrating ductal breast cancers. An additional tumor that contained both mucinous breast cancer and infiltrating ductal carcinoma exhibited similar features. A tubular breast cancer and a metaplastic carcinoma with squamous cell features were both diploid, and each contained a small triple protein-overexpressing cell subpopulation.
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DISCUSSION |
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Supplementary FISH studies showed that tumors often contain cell subpopulations that exhibit p53 allelic loss by FISH in the absence of p53 overexpression, and that allelic loss of p53 without p53 protein overexpression often accompanies or may precede the development of Her-2/neu abnormalities. A comparison of FISH studies with flow cytometric findings in the same tumors also indicates that overexpression of Her-2/neu can occur in the absence of demonstrable Her-2/neu gene amplification. Taken together, these findings suggest that there is a common molecular phenotypic evolutionary pattern that is associated with many infiltrating ductal carcinomas of the breast, and that this pattern can develop by a variety of molecular genetic mechanisms that have equivalent or overlapping phenotypic consequences.
The flow cytometric methodology used in this study did not permit direct correlations between the cell-based measurements that were performed and the morphological appearance of the cells that were studied. Instead, the observed intracellular patterns of the abnormalities themselves were used to characterize the various cell subpopulations present in each tumor sample. In diploid samples in which none of the cells exhibited overexpression of the oncogenes studied, the presence of tumor cells in the monodispersed cell suspensions was confirmed by independent cytological examination. Direct correlations between morphological appearance and quantitative phenotypic measurements on a cell-by-cell basis must await the application of quantitative multiparameter fluorescence imaging technologies.
A relationship between the overexpression of p53, Her-2/neu, and ras
proteins and the development of gross genetic abnormalities in the same
cells is established by the finding that the levels of expression of
each of these proteins were invariably higher in the aneuploid
components of aneuploid tumors than in the diploid components of the
same tumors (Fig. 2)
. However, the finding that levels of each protein
increased progressively with the acquisition of new abnormalities (Fig. 5
and associated discussion) suggests that this progressive
overexpression was the result of multiple events, some of which may
have been attributable to effects of genes coding directly for the
specific proteins involved, and others of which may have resulted from
indirect effects, including the possibility of perpetuated epigenetic
changes.
The relationship between p53 protein overexpression and the presence of genetic abnormalities that might affect p53 function directly or indirectly is complex. Wild-type p53 protein has a short half-life and does not ordinarily accumulate in normal cells in the absence of DNA damage. In experimental cell systems, sustained overexpression of the wild-type p53 protein commonly leads to cell cycle arrest, apoptosis, and/or inhibition or reversion of the transformed phenotype. In human tumors, p53 overexpression is commonly associated with missense mutations (29) . The accumulation of mutant p53 protein in the cell nucleus is thought to be attributable to the failure of the mutant protein to induce MDM2 (30 , 31) , a regulatory protein that normally binds p53, inactivates it, and targets it for ubi- quitination and subsequent destruction.
p53 protein overexpression is not always associated with p53 gene mutations. Wild-type p53 protein can also accumulate in human tumors and, like mutant p53 protein, is detected by the DO-7 antibody to p53 used in this study. When wild-type p53 protein is overexpressed in human tumors, it is thought to be maintained in a functionally inactive state by any of several possible mechanisms. In up to one-third of human breast cancers, wild-type p53 is excluded from the nucleus and accumulates in the cytoplasm (32) , where it is transcriptionally inactive. Cytoplasmic p53 overexpression is associated with an adverse clinical outcome in breast cancer, even in the absence of nuclear overexpression (33) . In the nuclear compartment, the level of MDM2 and the level of wild-type p53 are normally regulated mutually and reciprocally (30 , 34 , 35) . However, the MDM2/p53 regulatory loop is modulated by additional factors, including the states of phosphorylation of the MDM2 and p53 proteins, and the level of p14ARF, which regulates the level of MDM2, in turn (35) . Simultaneous overexpression of both MDM2 and wild-type p53 proteins has been documented in a variety of human solid tumors, including a small proportion of breast cancers (36) , which may reflect simultaneous inactivation of wild-type p53 by MDM2 binding and impairment of the ubiquitin pathway that leads to p53 degradation (30) . Thus, direct or indirect interference with normal p53 transcriptional activity would appear to be a common underlying phenotypic link among the several known genetic mechanisms that can lead to wild-type or mutant p53 protein overexpression.
Abrogation of wild-type p53 function is not always accompanied by
mutant or wild-type p53 protein overexpression. Frame shift and stop
codon mutations, which represent 15% of all p53 mutations, often
result in the absence or near absence of intracellular p53 protein. In
the present study, 50% of all tumors were found to overexpress p53
protein, using sensitive flow cytometric techniques to detect p53
overexpression. With the addition of FISH studies to detect
p53 allelic loss, p53 abnormalities were identified in
80.4% of all tumors studied by both techniques, suggesting that a
substantial number of p53 abnormalities might be missed if one were to
rely on p53 overexpression alone.
With regard to the molecular mechanisms underlying Her-2/neu
overexpression, our FISH studies indicated that gene dosage effects
attributable to gene amplification and/or increased chromosome copy
number/cell could account for overexpression in 65% of cases.
Interphase FISH techniques rely on the identification of multiple,
spatially separated gene loci to identify gene amplification. It is
conceivable that contiguous amplified loci, such as HSR regions,
might be missed in FISH studies of interphase cells, possibly resulting
in underestimation of gene amplification by this technique. The high
level of sensitivity of flow cytometric techniques might also result in
the categorization of tumors with moderately elevated levels of
Her-2/neu expression as overexpressors, which might have been
categorized otherwise in immunohistochemical studies. Apart from
technical considerations, levels of Her-2/neu expression are known to
be impacted by factors other than gene amplification, including
down-regulation by estrogen through estrogen receptor-mediated pathways
(37
, 38)
and down-regulation by Rb (39)
,
either or both of which can be inactivated or lost in breast cancer.
Overexpression of ras is common in breast cancer, but ras is rarely mutated in this disease (40, 41, 42) . Studies in experimental tumor systems have suggested that increased levels of the ras protein may be more closely associated with tumorigenicity, rapid growth rate, and acquisition of metastatic potential than the presence or absence of a mutated ras gene per se (reviewed in Ref. 3 ), suggesting that this phenotypic measurement may be a more reliable indicator of a hyperfunctional ras state.
There is substantial evidence linking the development of both gross numerical chromosomal changes and structural chromosomal abnormalities to the abrogation of wild-type p53 function (reviewed in Refs. 3 and 4 ). Although both types of chromosomal abnormalities often occur together, they probably arise by different p53-related mechanisms. Tetraploidy, an early step in the development of aneuploidy, has been attributed to the failure of p53-deficient or p53-inactivated cells to arrest at a mitotic checkpoint (43, 44, 45) and/or to the development of centrosome abnormalities (46) . An association between p53 abnormalities and the development of structural genetic abnormalities, particularly in aneuploid cells, has been observed in a number of experimental studies (47 , 48) . Of special relevance to the present study, loss of wild-type p53 function has been found to be associated with increased levels of gene amplification, which has often been used as a marker for genetic instability in the experimental setting, as well as the development of aneuploidy (49) . In the present study, p53 abnormalities commonly preceded or occurred simultaneously with Her-2/neu overexpression; p53 abnormalities were present in 12 of the 14 tumors that exhibited Her-2/neu amplification by FISH, and 12 of these tumors were aneuploid. These findings are consistent with experimental studies linking p53 abnormalities with genetic instability that may be manifested by gene amplification and/or aneuploidy in the clinical setting. However, one cannot conclude from the sequence information provided by our studies that p53 abnormalities are causally linked to Her-2/neu amplification, nor do our studies rule out other potential sources of genetic instability that might lead to Her-2/neu amplification and/or aneuploidy (50) .
The frequent association of p53 abnormalities with ras overexpression in infiltrating ductal carcinomas is of particular interest, in view of the well-documented cooperation between mutant p53 and ras abnormalities in the transformation of normal cells in experimental cell systems (51, 52, 53, 54) . Cooperativity might account for the statistically significant association between these two abnormalities, but it would not explain why ras overexpression is almost always accompanied by p53 overexpression in the same cells, whereas p53 overexpression can occur alone in individual cells in the same tumor. There is mounting experimental evidence that ras-mediated mitogenic signaling can be antagonized by p53-mediated apoptotic and growth-inhibitory responses (3) . It has been reported recently that oncogenic ras transfected into mouse embryo fibroblasts induces an increase in p53 protein expression and a reduction in cell proliferative activity by a p19ARF-mediated pathway that is separate and distinct from the DNA damage-induced p53 response pathway (which does not involve p19ARF; Ref. 55 ). Our findings support the premise that for a strategy for neoplastic transformation that relies on sustained mitogenic signaling to succeed, prior abrogation of wild-type p53 function (with or without p53 protein overexpression) might be required. The present study suggests that both elements of this strategy may be embraced by many, and perhaps most, infiltrating ductal carcinomas.
Lobular breast cancers appear to pursue a different course from that followed by infiltrating ductal carcinomas. Our findings that lobular carcinomas are generally diploid are in keeping with other published studies (56) , as are our findings that p53 abnormalities (16 , 57, 58, 59) , Her-2/neu amplification (60) , and loss of estrogen receptor (58 , 61) are not prominent features, even in advanced stages of disease. The present study and our previously published multiparameter study in human breast cancer (2) suggest that both epidermal growth factor receptor overexpression and normal to moderately elevated levels of Her-2/neu expression are more characteristic features of lobular breast tumors.
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FOOTNOTES |
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1 This work was supported by Grant CA 55230 from
the Department of Health and Human Services. 
2 To whom requests for reprints should be
addressed, at Allegheny University of the Health Sciences, Allegheny
Campus, 320 East North Avenue, Pittsburgh, PA 15212. Phone: (412)
359-4306; Fax: (412) 359-3238.
3 The abbreviation used is: FISH, fluorescence
in situ hybridization.
Received 5/11/99; revised 10/25/99; accepted 10/27/99.
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