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Clinical Investigations |
School of Biological Sciences [P. S. R., A. P-H., L. R., R. B.], Cancer Tissue Bank Research Centre [P. S. R., C. R.], and Department of Public Health [C. R. W.], University of Liverpool and Breast Unit, Royal Liverpool University Hospital [J. H. R. W.], Liverpool L69 3BX, United Kingdom
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1 woman in 12 developing cancer and
approximately half of these dying from this disease (1)
.
The major cause of death is the metastatic spread of the disease from
the primary tumor to distant sites in the body (2
, 3)
, and
therefore, the need for prognostic factors to indicate which patients
are more likely to die of metastatic disease is important. Research
into breast cancer has highlighted the prognostic significance of a
number of pathological factors (3)
; these include the size
of the primary tumor, the histological grade (4)
, and most
importantly, the involvement of the draining lymph nodes of the tumor
(5)
. Gene products involved in controlling cell
proliferation, e.g., c-erbB-2 (6
, 7)
, c-erbB-3
(8
, 9)
, particularly those concerned with estrogen action
[e.g.,
ER4
(10, 11, 12)
, pS2 (13)
, and PgR (14
, 15)
], cell death [e.g., p53 (16
, 17)
], and invasion [e.g., cathepsin D (14
, 18)
] in tissue cultured systems have been of more limited value
in predicting patient death from metastatic disease (19)
.
This may be due in part to the fact that very few of these gene
products have been shown to be capable of causing metastasis directly
in experimental systems. However, one gene product, p9Ka, has recently
been described with this property (20
, 21)
. p9Ka, now renamed S100A4, is a member of the S100 family of calcium-binding proteins (22) . S100A4 or its mRNA is found at higher levels in metastatic relative to nonmetastatic rat (23) and mouse (24) tumor cell lines and benign relative to malignant human breast tumors (25) . Elevation of the levels of rat (20) or human (26) S100A4 in benign rat mammary tumor cells by DNA transfection results in the induction of metastatic capability in some of the cells when they are injected into the mammary fat pads of syngeneic rats. In independent transgenic mouse models of breast cancer overexpression of S100A4 in neu oncogene-induced (27) or with mouse mammary tumor virus-induced (28) benign mammary tumors yields metastatic tumors. Moreover, in pilot studies on human colorectal adenocarcinoma specimens, elevated levels of immunocytochemically detected S100A4 are associated with the more malignant carcinomatous regions of the primary tumors and with liver metastases (29) . We now investigate, using immunocytochemical techniques, the presence of S100A4 in specimens of primary breast carcinomas from a comparatively large group of patients with sufficient follow-up time to assess whether its presence at time of diagnosis is significantly associated with patient death from metastatic disease.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The rabbit polyclonal antiserum to rat rS100A4 protein was prepared and purified by immunoaffinity chromatography on a column of rS100A4-Sepharose (30) . Two-dimensional gel electrophoresis of proteins from the Rama 29 cell line identified the natural rat S100A4 as a protein of Mr 9000 and an isoelectric point of 5.5, and only the polypeptide in this position on the gel corresponded to that for the Western blot with the antibody (30) .
Immunocytochemistry.
Histological sections on 3-aminopropyltriethoxysilane-coated
slides (31)
were cut from the paraffin-embedded sections
(32)
, endogenous peroxidase activity was blocked
(33)
, and indirect immunocytochemistry was carried out
using a commercially available antibody complex containing horseradish
peroxidase (34)
. The anti-S100A4 serum was diluted to
1/500, the sections were incubated at room temperature overnight, and
bound antibody was detected with biotinylated donkey antirabbit
immunoglobulin (Amersham, Bucks, United Kingdom), followed by ABComplex
(Dako Ltd). The bound complex was visualized with 3,
3'-diaminobenzidine (Sigma, Dorset), 0.003% (v/v)
H2O2 (35)
. The
sections were then counterstained in Mayers’ hemalum and mounted in
DPX (Merck Ltd, Dorset, UK).
Slides were read independently by two observers using light microscopy. The percentage of carcinoma cells with cytoplasmic staining was recorded from two sections of each specimen, 10 fields/section at 200x magnification. Staining was evaluated initially in three groups: positive (+), >5%; borderline (±), 1–5%; and negative (-), <1% of the carcinoma cells stained. The borderline and negative staining groups were usually combined. Photographs were recorded on a Reichart Polyvar microscope fitted with a Wratten 44 blue green filter (36) . Increasing the concentration of antibody 10-fold or using commercially supplied rabbit antihuman rS100A4 protein gave identical results. Immunocytochemical staining for ER (340 patients), PgR (330 patients), pS2 (344 patients), p53 (348 patients), and c-erbB-3 (335 patients) was accomplished by similar standard procedures (37) to those described previously for c-erbB-2 (342 patients) and cathepsin D (270 patients) (7 , 18) .
Protein Samples and Western Blotting.
Rat and human rS100A4 were produced in Escherichia coli
(38)
, and human rS100A1 and rS100A2 were gifts of Dr. G.
Wang (University of Liverpool, United Kingdom). Soluble extracts of
human breast tumor specimens were prepared by crushing in liquid
nitrogen and homogenizing with 1 mM phenylmethyl
sulfonyl fluoride. The extract was centrifuged at 4°C for 1 min in a
microfuge, and to the supernatant were added SDS, glycerol, bromophenol
blue, and 2-mercaptoethanol prior to boiling, sonication, and
electrophoresis on 15% (w/v) polyacrylamide gels (39)
.
Molecular weight markers were run alongside the samples for molecular
weight determination. Proteins were then transferred to Immobilon P
membranes (Millipore Corporation, Watford, United Kingdom), which were
blocked with "blocking buffer" containing 2% (w/v) Marvel and
incubated with anti-S100A4 diluted as in the figure legends. In some
experiments, 1 mg/ml rat rS100A4 was present to provide a blocked
antibody control. Filters were then incubated for 1 h with
peroxidase-conjugated swine antirabbit IgG (Sigma, St. Louis, MO), and
bound antibodies were detected with the Super Signal West Pico
Chemiluminescence System (Pierce and Warriner, Rockford, Illinois) and
exposing the filter against Fuji RX film.
Statistical Methods.
Follow-up information was obtained from the Merseyside Cancer Registry
for patients used in this study and was updated for patient survival to
August 31, 1995. The accuracy of this data was subsequently checked by
inspection of General Practitioner records to confirm whether patients
were alive, dead of cancer, or dead of other causes. The association of
immunocytochemical staining for S100A4 with other tumor variables was
assessed using a Fisher’s exact test (40)
. These
variables on the same group of patients included tumor size,
histological grade, nodal status, menopausal status, patient age
(12)
, and presence of c-erbB-2 (7)
, cathepsin
D (18)
, ER, PgR, pS2, p53, and c-erbB-3 (37)
in the primary tumor. The cutoff values between those groups of
patients designated negatively or positively immunocytochemically
stained for the marker proteins included the borderline staining group
with the unstained group, unless otherwise specified (7
, 18
, 37)
.
The association of the staining for S100A4 in breast cancers with
patient survival was evaluated using life tables constructed from
survival data with Kaplan Meier plots and analyzed using generalized
Wilcoxon (Gehan) statistics (7)
. Patients found to be dead
from causes other than cancer were excluded from the analyses. To
determine whether the association of patient survival with S100A4 was
independent of other potential prognostic factors shown to approach
significance in univariate analysis, a multivariate analysis was
performed using the Cox proportional hazards model (41)
.
Other potential prognostic factors measured on the same group of
patients included tumor size, histological grade, nodal status
(12)
, the presence of c-erbB-2 (7)
, cathepsin
D (18)
, ER, PgR, pS2, p53, and c-erbB-3 (37)
.
The degree of agreement between observers was assessed using the 
statistic; a value >0.61 was taken to be a satisfactory level of
agreement (40)
. Data processing and statistical analyses
were performed using Excel version 5.0 (Microsoft Corp.,
Washington, D.C.) and Statistical Package for the Social
Sciences, version 6.1.2 (SPSS Inc., Chicago, IL).
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RESULTS |
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, although some areas showed staining of both ducts (Fig. 1B)
and ductules. In contrast, individual invasive breast
carcinomas presented a more uniform staining pattern. Of the 349
invasive breast carcinomas evaluated, 152 (44%) were unstained (Fig. 1C)
, 53 (15%) possessed borderline staining (Fig. 1D)
, and 144 (41%) were strongly stained (Fig. 1E)
by antiserum to S100A4. The staining was confined mainly
to the cytoplasm (Fig. 1F)
and could be abolished by prior
incubation of the antiserum with rS100A4 (Fig. 1G)
. The
assessment was made only on the malignant cells; however, positive
staining was also present on normal blood vessels and certain reactive
stromal cells (Fig. 1H)
and lymphocytes (Fig. 1I)
present in the carcinomas. The borderline group was defined as
carcinomas possessing 1–5% of the malignant cells stained for S100A4
(see "Materials and Methods"). For the purposes of most analyses,
the borderline staining carcinomas were combined with the unstained
carcinomas into one group of negatively stained carcinomas, leaving the
clearly positive staining carcinomas as the other group.
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value of 0.79,
which represents a good degree of consistency between observers. In
addition, intratumor heterogeneity was assessed by comparing the
category of staining allocated when two well-separated sections from
the same tumor were analyzed independently. In 7% of all histological
sections studied, intratumor heterogeneity was sufficiently high to
affect whether a section was regarded as negatively or positively
stained, and then recourse was made to staining and analyzing two
further sections to obtain a consensus result.
The antibody to rat rS100A4 recognized both rat (not shown) and human
rS100A4 on Western blots, but did not cross-react with the closely
related human rS100A1 and rS100A2 proteins at the same loading on the
gel (Fig. 2A).
Western blots of extracts of selected carcinoma specimens confirmed
that the antibody detected a Mr 9000
protein in extracts of specimens that exhibited high levels of
immunocytochemically detectable S100A4 (Fig. 2B)
but not in
extracts of specimens that displayed no staining (not shown). The
binding of antibody to the Mr 9000
protein was blocked when human rS100A4 (at a concentration of 1 mg/ml)
was present during incubation of the antibodies to S100A4 with the
filter (Fig. 2C)
. In extracts of some, but not all tumor
specimens tested, a high molecular weight band of immunoreactivity was
also present at about Mr
60,000–65,000. Although this band corresponded approximately to the
molecular weight of serum albumin, there was no cross-reaction of the
antibody onto purified serum albumin (Fig. 2A)
. It is
possible that this immunoreactivity consisted of higher molecular
weight aggregates of S100A4 because essentially similar results were
obtained with a second commercially produced anti-S100A4 (not shown).
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. Of the pathological factors, only the presence of carcinoma in the
axillary lymph nodes showed a borderline association with
immunocytochemical staining for S100A4 in the primary tumor; 54% of
S100A4-positive tumors had tumor in the associated axillary lymph nodes
compared to 42% of those that were classified as S100A4-negative
(Fisher’s exact test, P = 0.058). There was
a statistically significant association of carcinoma staining for
S100A4 with positive staining for the potential molecular markers
c-erbB-3 (P = 0.002), cathepsin D
(P = 0.024), and c-erbB-2
(P = 0.048); (Table 1)
. There was also a
tendency for more p53-positive carcinomas to be positive for S100A4
when compared to the p53-negative carcinomas, but this association did
not achieve statistical significance (P = 0.095) (Table 1)
. The remaining tumor variables measured, tumor size,
histological grade, menopausal status, and staining for ER, PgR, and
pS2, showed no significant association with positive staining for
S100A4 in this group of patients, and there was no obvious age or other
demographic difference between patients with S100A4-negative and
S100A4-positive carcinomas (not shown).
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. Of 205 patients who were classified as S100A4-negative, about 80%
were alive at the census date, in comparison with about 11% of the 144
patients classified as S100A4-positive. The median survival of patients
whose tumors were classified as S100A4-negative was >228 months in
comparison with that of patients with a tumor classified as positive of
47 months. The data show that over the time period of 19 years, the
survival of patients with S100A4-positive carcinomas was highly
significantly worse than those patients with carcinomas classified as
S100A4-negative (Wilcoxon test, P < 0.0001;
Fig. 3
). This difference in survival of patients between the two groups
became statistically significant after 6 months of follow-up (Wilcoxon
test, 
2 = 4.39, 1 d.f.,
P = 0.036). Thereafter, increasing follow-up
times increased the significance of the difference (e.g.
for 1 year, 2 = 7.90, 1 d.f.,
P = 0.005; for 2 years,
2 = 22.14, 1 d.f.,
P < 0.0001; for 3 years,
2 = 47.63; for 4 years,
2 = 70.55; for 19 years,
2 = 131.5). When the results are
expressed in terms of the RR of a patient surviving, women with
S100A4-negative and -borderline carcinomas had an unadjusted RR for
survival of 8.7 (95% CI, 6.7–12.7) compared to the S100A4-positive
group.
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). These overall differences became significant after 1 year (Wilcoxon
test, 2 = 13.01, 2 d.f.,
P = 0.0015). The completely negatively
stained group of 152 patients showed a very high level of cumulative
survival of 98% and a median survival of >228 months. The group of 53
patients with borderline staining showed a level of survival of 29%
and a median survival of 82 months, which was significantly different
from either the completely negatively (P < 0.0001) or positively stained group of patients (P = 0.017). The former difference became statistically significant
after 1 year (2 = 11.73, 1 d.f., P = 0.0006) and the latter difference
after 5 years (2 = 4.22, 1 d.f., P = 0.04). The same positively stained
group of 144 patients as above showed a level of survival of 11% and a
median survival of 47 months as before (Fig. 4)
. Women with
S100A4-negative carcinomas had an unadjusted RR for survival of 53.4
(95% CI, 16.4–174) compared to those with S100A4-borderline
carcinomas and of 91.6 (95% CI, 29.0–289) compared to those with
S100A4-positive carcinomas. The comparisons with the group of patients
with unstained carcinomas, however, may be open to question because
there were only three deaths and therefore 149 censored observations in
this group (Fig. 4)
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2 = 18.00, 1 d.f.,
P < 0.0001), tumor size
(2 = 13.6, 3 d.f.,
P = 0.0035), histological grade
(2 = 8.66, 2 d.f.,
P = 0.013), staining for c-erbB-2
(2 = 10.03, 1 d.f.,
P = 0.0015), ER
(2 = 4.83, 1 d.f.,
P = 0.028), and p53
(2 = 4.61, 1 d.f.,
P = 0.032). The association of staining for
cathepsin D, PgR, pS2, and c-erbB-3 with survival time failed to reach
statistical significance (not shown). The association of patients with
carcinomas staining for S100A4 and their survival in subgroups defined
by the different tumor variables described above was analyzed. The
borderline cases of carcinoma cell staining for S100A4 were included
once again in the unstained carcinoma cell group of patients to ensure
sufficient numbers in the subgroups for statistical validity. In all of
these subgroups of patients, staining for S100A4 was associated with
poorer survival, including that for patients with involved lymph nodes
(Fig. 5)
. There were only three tumor variables which, when they occurred with
S100A4, showed a statistically significant reduction in patients’
survival time over that obtained with S100A4 alone: involved lymph
nodes (Fig. 5)
, fixed tumors (T4), and staining
for c-erbB-2 (Fig. 6)
. The other tumor variables, including tumor sizes
T1-T3 and histological
grades II and III, showed no such effect (not shown). Thus, the 51
patients who were positive for S100A4 and negative for involved lymph
nodes exhibited longer survival times than the 60 patients who were
positive for S100A4 and also positive for involved lymph nodes
(P < 0.0001). The differences were 24% and
3% in cumulative proportion surviving and 85 months and 36 months in
median survival times, respectively (Fig. 5)
. Similarly, 129 patients
positive for S100A4 without fixed tumors (T1,
T2, T3) had longer survival
times than the modest 10 patients positive for S100A4 and possessing
fixed tumors (T4)(2 =
5.93, 1 d.f., P = 0.015; not shown). In
this case, the differences were 81% and 48% in cumulative proportion
surviving and >228 months and 106 months in median survival times,
respectively. Although only at borderline significance, the 102
patients who were positive for S100A4 and negative for c-erbB-2 had
longer survival times than the 39 patients who were positive for S100A4
and also positive for c-erbB-2 (P = 0.050;
Fig. 6
). In this case, the differences were 11% and 6% in cumulative
proportion surviving and 49 months and 35 months in median survival
times, respectively. Neither the presence of involved lymph nodes nor
staining for c-erbB-2 in the S100A4-negative group of patients showed
any correlation with poorer prognosis (Figs. 5
and 6)
. It is possible
that the statistical validity may, however, have been influenced by the
numbers of patients in the subgroups analyzed.
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. On controlling the data for T4, nodal status,
grade III, and S100A4, there was no significant association between
tumor sizes T1-T3, grades
I-II, c-erbB-2, p53, ER, and patient survival (Cox analysis, residual
2 = 3.53, 6 d.f.,
P = 0.74). Consistent with this result, the
univariate analysis for tumor size gave the most significant and
shortest survival for patients when groups with fixed tumors
(T4) were compared with the rest
(T1-T3) (Wilcoxon test,
2 = 11.72, 1 d.f.,
P = 0.0006) and for histological grade when
groups with high grade (III) tumors were compared with the rest (I-II)
(Wilcoxon test, 2 = 6.44, 1 d.f., P = 0.011). None of the possible
pairwise or higher order interaction terms in the multivariate analysis
was statistically significant, implying that the effect of staining for
S100A4 on patient survival is similar over the various prognostic
groups defined by nodal status, tumor fixation, and high-grade tumors.
The adjusted RR for survival of patients with S100A4-negative and
-borderline carcinomas compared to those with S100A4-positive
carcinomas was second highest at 7.5 (95% CI, 4.2–13.5) compared to
the highest of 11.6 (95% CI, 13.1–42.8) for patients with nonadherent
carcinomas compared to those few with carcinomas adherent to the chest
wall (Table 2)
. If all of the other molecular tumor variables and tumor
sizes were not entered into the Cox proportional hazards model to
generate a slightly higher number of 145 patients with complete data
sets for nodal status, histological grade and S100A4 status, S100A4
with an RR of 7.4 (95% CI, 4.2–13), and involved nodes with an RR of
2.7 (95% CI, 1.6–4.4) were still independent predictive variables of
patient death (2 = 79.6, 2 d.f., P < 0.0001) with similar RRs to those
in Table 2
, but high histological grade (III) was no longer a
significant independent variable (2 = 1.99, 1 d.f., P = 0.16).
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DISCUSSION |
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In this and previous studies with this group of patients, the tumor
variables that show a significant association with survival time of the
patients are nodal status (P < 0.0001),
tumor size (P = 0.0035), histological grade
(P = 0.013), staining for c-erbB-2
(P = 0.0015), ER (P = 0.028), and p53 (P = 0.032) for the
full follow-up period of 19 years. Previous publications on this group
of patients have reported that the presence of ER showed only a trend
with improved prognosis for the patients after 14 years when measured
biochemically (P = 0.09) (12)
.
This difference is probably due to the method of detection used because
after the full follow-up period of 19 years, no association of patient
survival with ER measured biochemically is detected
(2 = 0.45, 1 d.f.,
P = 0.5), whereas a strong association is
detected using immunocytochemical methods (P = 0.028) (37)
. Also, cathepsin D has been shown
previously to be associated with a poorer prognosis for this group of
patients (P = 0.025) after 14 years of
follow-up (18)
, whereas after the full follow-up of 19
years, only a trend (P = 0.093) is observed
(37)
. This difference may reflect the comparatively few
patients dying of cancer with cathepsin D-positive carcinomas between
the two census dates. Although previous reports have suggested
associations between patient survival and the presence of PgR
(14
, 15)
, pS2 (13)
, and c-erbB-3 (8
, 9)
in breast carcinomas, these associations represent only
trends in the present group of 349 patients and are not statistically
significant [2 = 3.6, 1 d.f.,
P = 0.058; 2 = 1.3, 1 d.f., P = 0.25; and
2 = 0.37, 1 d.f.,
P = 0.54, respectively (Ref.
37
)].
Of all of the above tumor variables that represent potential prognostic markers for patient outcome, only the presence of carcinoma in the lymph nodes at borderline statistical levels and immunocytochemical staining for c-erbB-3, cathepsin D, and c-erbB-2 show a statistically significant association with staining for S100A4 in the primary carcinomas, and a trend is established for p53. These results may suggest that the same underlying change(s) is responsible for the altered expression of those tumor variables, involved lymph nodes, c-erbB-3, cathepsin D, c-erbB-2, and possibly p53, which show some correlation with S100A4. However, the fact that there is a borderline correlation between the presence of S100A4 and the major pathological tumor variable associated with poor patient prognosis, that of involved lymph nodes, but not with the other two variables of tumor size and histological grade, may reflect the characteristics of the tumors in this group of patients and the size of sample. In this study, a minority of 18% and 6%, respectively of the tumors are classified as T3 and T4; these are small fractions in comparison with other groups of patients (6) . Moreover, histological grading has been undertaken on only 51% of the tumors in this study, with only 27% of these classified as grade III. These smaller numbers may make tests for associations less meaningful.
In this paper, we have shown that the overall survival for patients
with carcinomas expressing immunocytochemically detectable levels of
S100A4 is significantly worse than for those patients with carcinomas
considered negative for S100A4. The level of association at
P < 0.0001 using Wilcoxon statistics is more
significant than for other tumor variables in this group of patients
and is comparable with the best association shown thus far for involved
lymph nodes (P < 0.0001). When the
corresponding Kaplan Meier plots are analyzed using log-rank sums, a
similar level of significance is achieved
(2 = 178, 1 d.f.,
P < 0.0001) and the median survival times of
>231 and 46 (95% CI, 38–55) months (not shown) compare favorably
with >228 and 47 months using Wilcoxon statistics (Fig. 3)
. This
relationship for S100A4 achieved statistical significance after 6
months of follow-up and remained statistically significant for the full
19 years of follow-up of the patients, unlike some of the relationships
between other tumor variables and patient survival [e.g.
ER (12
, 44)
and cathepsin D (18
, 37)
].
Moreover, when the borderline cases (defined as 1–5% carcinoma cells
stained) of immunocytochemical staining for S100A4 are analyzed
separately, they are correlated with a level of patient deaths that is
intermediate between that for the completely unstained group and that
for the positively stained group for all follow-up times of 5 years and
beyond (Fig. 4)
. Once again, Kaplan Meier plots followed by analysis of
log-rank sums gave the same results [2 = 224, 2 d.f., P < 0.0001, median
survival times were >231, 78 (95% CI, 30–125), and 46 (95% CI,
38–55) months for patients with S100-negative carcinomas, with
borderline carcinomas, and with positive carcinomas, respectively (not
shown)]. This result suggests that not only the presence, but also the
levels of immunoreactive S100A4 may be correlated with the time of
demise of the patients. It should be noted, however, that only 3 deaths
in 152 cases are observed in the group of patients with completely
unstained carcinomas, and this very low percentage may cast some doubt
on the validity of the overall statistical test for significance.
Moreover, the fact that in our study a large number of patients is
required to obtain a statistically significant result may mean that
small fluctuations in data can alter considerably the significance of
the results. Thus, when the positively stained group is separated into
patients with 5–25%, 25–50%, and 50–75% of stained carcinoma
cells in their tumors, there are too few highly stained carcinomas to
verify this effect statistically using 5% confidence limits.
Nevertheless, the fact that the presence of immunoreactive S100A4 in
the carcinoma cells is so highly correlated with early demise of this
group of patients may reflect that this change is more closely
associated with their cause of death than some of the other tumor
variables studied. Because S100A4 was first discovered as a
metastasis-inducing protein in rodent models of breast cancer
(20
, 21)
, and metastasis is the major event responsible
for death of patients from human breast cancer (3)
, it is
possible that S100A4 is causing premature deaths by its ability to
induce metastasis in humans as well.
When smaller subgroups of patients are analyzed for their survival
times, small fluctuations in data may have an even more dramatic effect
on the significance of the results than when analyzed as a whole and/or
patient numbers may be too small to observe a significant effect. The
magnitude of both interobserver error and intratumor heterogeneity in
this study could conceivably result in such a situation. Nevertheless,
when subgroups of patients with carcinomas classified as positive or
negative for S100A4 and for another tumor variable were examined, those
subdivided by lymph node status or by c-erbB-2 are of particular
interest. Results of statistical analyses were virtually identical if
Wilcoxon (Figs. 5
and 6)
or log-rank tests (not shown) were used. There
was virtually no difference in patient survival in the S100A4-negative
group of patients with or without involved lymph nodes, but a
significantly more rapid demise was observed for patients in the lymph
node-negative group with rather than without S100A4 (Fig. 5)
. These
results may suggest that the presence of S100A4 in the tumor is the
more dominant factor at predicting patient outcome than that of
involved lymph nodes. Moreover, once S100A4 is detected in the primary
tumor, then patients with involved lymph nodes die more quickly than
those without involved lymph nodes (Fig. 5)
. These results are
consistent with those obtained in a Cox multivariate regression
analysis model where the presence of S100A4 is found to be the most
significant predictor of patient death, but nodal status is itself a
significant independent predictive variable (Table 2)
. The other
independent predictive variables in this proportional hazards model are
small subsets of the remaining two pathological variables, tumor fixed
to the chest wall (T4), and high histological
grade (III) (Table 2)
. When slightly larger data sets were analyzed for
only lymph node status, histological grade, and S100A4 status, S100A4
status was retained as the most significant predictor of patient death,
but high histological grade (III) was eliminated as an independent
predictive variable. Similarly, there was virtually no difference in
patient survival in the S100A4-negative group of patients with or
without c-erbB-2, but a significantly more rapid demise was noted for
patients in the c-erbB-2-negative group with, rather than without,
S100A4 (Fig. 6)
. Moreover, once S100A4 was detected in the primary
tumor, then patients with c-erbB-2-positive tumors died more quickly
than those with c-erbB-2-negative tumors (Fig. 6)
. These results
suggest once again that the presence of S100A4 in the tumor is the more
dominant factor at predicting patient outcome than that of c-erbB-2,
but that c-erbB-2 can synergize with S100A4 in accelerating the demise
of patients. The fact that c-erbB-2 was rejected as an independent
prognostic factor in the Cox multivariate regression analysis (Table 2)
may suggest that c-erbB-2 was confounded with one or more of the
independent pathological prognostic variables in the proportional
hazards model. That c-erbB-2 can synergize with S100A4 in producing
accelerated patient demise is consistent with one of the mouse models
for breast cancer in which transgenic mice require both the expression
of the mutated form of c-erbB-2, neu, and S100A4 to induce
metastasis (27)
.
How S100A4 may be overexpressed and its role in human breast cancer are not clear. In rodent model systems, its expression is normally under the control of both positive and negative regulatory factors (45, 46, 47) , and multiple copies of the rodent and human genes have been introduced into rodent and human cells to cause metastasis in rodents (20 , 21 , 26, 27, 28) . In humans, the gene for S100A4 occurs in a cluster of 13 S100 genes on chromosome 1 (48) , a region of the human genome, which is also often amplified in breast cancer and which contains jumping elements (49) . In the rodent model systems, however, elevated levels of S100A4 can only synergize with growth-promoting oncogenic products like c-erbB-2 (27) or be expressed in already benign neoplasms before metastasis can be induced (20 , 21 , 28) . By itself, it has no neoplastic or metastatic effect in normal rodent cells (50) . Because the majority of invasive human breast carcinomas do not contain c-erbB-2 (7) , interaction of S100A4 with other growth-promoting oncogenic products may also occur in those human breast carcinomas that fail to express c-erbB-2. In the rodent model systems, S100A4 is thought to interact with components of the cytoskeleton (20 , 51, 52, 53) , thereby enhancing the motile properties of cells (54 , 55) . But motility per se is unlikely to be the sole property required to accomplish the metastatic cascade in rodent models, let alone in human breast cancer (56 , 57) . However, it is plausible that one step in the overall progression from an invasive breast carcinoma to a growing metastasis may be more or less rate-limiting and that, under appropriate conditions, S100A4 or similar molecules may accelerate that step. In conclusion, our results show that the presence of the calcium-binding protein S100A4, which can cause metastasis in rodent models, is now associated with a poor prognosis for one group of breast cancer patients. It remains to be determined how widespread this association will prove to be, not only in breast, but in other metastatic carcinomas.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by the Cancer and Polio Research Fund,
the North West Cancer Research Fund, and the Cancer Tissue Bank
Research Centre. 
2 To whom requests for reprints should be
addressed, at School of Biological Sciences, Life Sciences Building,
University of Liverpool, P. O. Box 147, Liverpool L69 3BX, United
Kingdom.
3 Present address: Department of Surgery, North
Manchester General Hospital, Manchester M8 5RB, United
Kingdom.
4 The abbreviations used are: ER, estrogen
receptor; d.f., degrees of freedom; PgR, progesterone receptor;
rS100A4, recombinant S100A4 protein; RR, relative risk; CI, confidence
interval.
Received 7/19/99. Accepted 1/17/00.
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