(CANCER RESEARCH 40. 3627-3631. October 1980]
0008-5472/80/0040-0000/S02.00
Circulating Immune Complexes in Sera of Dogs with Benign and Malignant
Breast Disease1
Bruce R. Gordon, Scott Moroff, Arthur I. Hurvitz, Robert E. Matus, E. Gregory MacEwen, Robert A. Good, and
Noorbibi K. Day2
Sloan-Kettering Institute for Cancer Research. New York. New York 10021 IB fÃ-.G., R. A. G.. N. K. D.], and The Animal Medica/ Center
10021 ¡S.M., A. I. H., R. E. M., E. G. M.¡
ABSTRACT
The Raji cell radioimmunoassay was adapted to measure
circulating immune complexes (CIC's) in the sera of 74 dogs
with benign and malignant breast disease. In dogs with recently
diagnosed spontaneous breast cancer, 57% (29 of 51) had
elevated CIC's, with some levels as high as those found in the
sera of dogs with systemic lupus erythematosus. The sera of
10 of 23 dogs with benign breast disease also demonstrated
elevated levels of CIC's. Two weeks following mastectomy,
repeat CIC levels were obtained in 30 dogs. Elevated CIC levels
returned to normal in all dogs with benign breast disease but
in only 33% of dogs with breast cancer. Dogs with persistent
elevation of CIC's were at greater risk of developing metastatic
breast cancer. Serum total hemolytic complement was signifi
cantly higher (p < 0.05) in dogs with untreated breast cancer
than in healthy dogs but did not correlate with the level of CIC's
found. Two weeks after mastectomy, total hemolytic comple
ment levels had returned to normal. By sucrose density gra
dient ultracentrifugation,
the complexes were shown to sedi
ment at 19 S. These studies indicate that the dog may be a
good model for elucidating the significance of elevated CIC's
in breast cancer.
INTRODUCTION
CIC's3 have been found in a variety of human neoplasms
when measured by techniques such as the Raji cell radioim
munoassay, C1q binding test, and C1q deviation test (4, 13,
17, 18). Sera from human breast cancer patients frequently
contain elevated levels of CIC's. Hoffken et al. (4), using a C1 q
binding test, reported that all 22 sera of women tested with
breast cancer had elevated levels of CIC's prior to mastectomy.
Theofilopoulos et al. (18) studied 44 breast cancer patients
and found 15 of 44 to have elevated complexes by the Raji cell
radioimmunoassay. Rossen et al. (13), using a C1q binding
technique, found elevation of CIC's in 20 of 27 patients. In 91
patients reported by Teshima et al. (17), levels of CIC's were
3-fold higher in sera of breast cancer patients than in sera of
healthy individuals.
Despite these studies, the use of CIC's for monitoring the
course of malignant disease is uncertain. The long natural
history of human breast cancer with the inherent difficulties in
' Supported by Grant HL-07029 from the National Institute of Heart and Lung;
Grants CA-08748 and CA-26287-01 from the National Cancer Institute; American
Cancer Society Grants IM-185. A1-11843, and Special Project MSKCC; and the
Catherine Ingels Fund.
2 To whom requests for reprints should be addressed, at Memorial SloanKettering Cancer Center, 1275 York Avenue, New York, N. Y. 10021.
' The abbreviations used are; CIC. circulating immune complex; TCHso, total
hemolytic complement; MEM. minimal essential medium.
Received February 11.1980; accepted July 10. 1980.
New York New York
follow-up make an animal model attractive to clarify the rela
tionship between immune complexes and the course of the
disease. The similarities between human and canine breast
cancer make the dog a useful experimental model in the study
of this tumor (2, 14). Spontaneous breast cancer is the most
common cancer in female dogs with an incidence of 105
cases/100,000
dogs at risk (14). The pathology including
lymph node spread is similar to that of breast cancer in the
human. The disease often spreads systemically, with bone,
lung, and liver being frequent sites of métastases.An additional
advantage is that the natural course of the disease is usually
manifested within 2 years in the dog as compared to 5 years,
10 years, or longer in humans.
The determination of CIC levels in dog serum has been
reported previously (12, 15). Terman ef al. (15), using the C1 q
binding test, showed that 4 of 4 sera of dogs with breast cancer
had elevated levels of CIC s. Poskitt and Poskitt (12), using the
binding of CIC's to the Fc receptor of the L1210 murine
leukemia cell, studied 20 normal dogs and 16 dogs with sys
temic lupus erythematosus. None of the sera from normal dogs
exhibited binding, while 10 of 16 sera from dogs with systemic
lupus erythematosus demonstrated binding to the L1210 cell.
In the present study, the Raji cell radioimmunoassay was
adapted to measure CIC's before and after mastectomy in the
sera of dogs with benign and malignant breast disease.
MATERIALS AND METHODS
Patient Population
Fifty-one dogs with spontaneous breast cancer and 23 dogs
with benign breast disease were studied prior to mastectomy
at the Animal Medical Center of New York. Follow-up studies
were done 2 weeks after mastectomy in 30 dogs and 3 months
after mastectomy in 22 dogs. The average age of the dogs with
breast cancer was 9.5 years (range, 7 to 13 years). The
average age of the dogs with benign breast disease was 10
years (range, 7 to 13 years). Twenty healthy female dogs with
an average age of 9 years (range, 6 to 13 years) were used as
controls. These dogs were randomly selected without regard
to breed. Two dogs with systemic lupus erythematosus were
also studied. The criteria for the diagnosis of canine systemic
lupus erythematosus was polysystemic disease with an ele
vated titer of anti-nuclear antibodies.
Serum Samples
The blood was allowed to clot at room temperature for 1 hr
and was centrifuged at 200 x g at 4°for 10 min in a refriger
ated centrifuge (Damon PR-600 IEC centrifuge, Fisher Scien
tific Co., Springfield, N. J.). Serum samples were aliquoted and
stored at -70° and were used only once.
OCTOBER 1980
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3627
B. R. Gordon
et al.
Measurement of TCH.„,
50 n\ MEM) were incubated (37°, 45 min) with 50 jul of pooled
normal dog serum or heat-inactivated (56°,20 min) normal dog
sowas titrated according to a modified method of Mayer
(7) using sensitized sheep erythrocytes as follows. Serial dilu
tions of dog serum in 0.5 ml of 30 mM sodium chloride:1 5 HIM
sodium barbital buffer (pH 7.5): 1.0 HIM MgCI2:0.1 5 mM CaCI2,
to which 25 g dextrose and 0.5 g gelatin have been added,
were incubated at 37°for 1 hr with 0.5 ml of sensitized sheep
erythrocytes (1x1 08/ml) and 2 ml of 30 mM sodium chloride:
serum. The cells were washed 3 times in MEM and incubated
with 100 fil of fluorescein isothiocyanate-conjugated
goat anticanine C3 for 30 min at 4°.The cells were again washed in
15 mM sodium barbital buffer (pH 7.5):1 .0 mM MgCI2:0.1 5 mM
CaCI?, to which 25 g dextrose and 0.5 g gelatin have been
added. Next, 4 ml of 0.9 M NaCI were added to each tube, and
the release of hemoglobin was determined spectrophotometrically at 41 2 nm.
Measurement of CIC's
CIC's were measured
by a modification
of the Raji cell
radioimmunoassay (20) as follows.
Preparation of Aggregated Dog IgG. Dog globulin from
Cohn Fraction II (Miles Laboratories, Inc., Elkhart, Ind.) was
dissolved in 0.01 M sodium phosphate buffer (pH 7.5). This
was chromatographically
purified on a DEAE-cellulose 52 col
umn. The protein in the first peak concentrated to 6 mg/ml was
shown to be IgG by immunoelectrophoresis using monospecific
rabbit anti-dog IgG (Cappel Laboratories, Downingtown, Pa.).
Purified IgG was heated in a water bath at 63°for 20 min and
then centrifuged (Sorvall RC2-B; Ivan Sorvall, Inc., Norwalk,
Conn.) at 1000 x g for 90 min in order to remove large
aggregates. The protein content off the top half of the super
natant was determined (6) and was adjusted to 4 mg/ml.
Radioiodination of Rabbit Anti-Dog IgG. Rabbit anti-dog
IgG was radioiodinated with 12bl(New England Nuclear, Boston,
Mass.) by the chloramine-T method (8). The labeled anti-dog
IgG was extensively dialyzed for 36 hr against 0.01 M sodium
phosphate buffer (pH 7.5). The specific activity of the 1?5Irabbit
anti-dog IgG was 0.05 to 0.2 /iCi/jug of protein. The protein
concentration was 0.3 mg/ml.
Assay Procedure. Raji cells were used after a 48-hr culture
in MEM. The cells were washed 3 times in MEM, and viability
was determined by the trypan blue exclusion test. An aliquot of
4 x 106 Raji cells was incubated (37°, 45 min) with 25 ¡i\of
test sera at 1:4 dilution in MEM. After 3 washes in MEM, the
samples were incubated (4°,45 min) with 100 /¿I
of '"I rabbit
anti-dog IgG. The cells were again washed 3 times, and the
radioactivity in the pellet was counted in a Packard Instruments
Auto-Gamma counter. Results were compared against a stan
dard curve of aggregated dog IgG. Serial dilutions of aggre
gated dog IgG from 0 ng in 10 ¡i\of MEM to 40,000 ng in 10
¿tiof MEM were incubated (37°, 30 min) with pooled normal
dog serum or human serum as a complement source. Since
dog serum and human serum yielded similar standard curves,
only pooled normal dog serum was subsequently used. Levels
of CIC's were expressed as fig equivalents of aggregated dog
IgG. For simplicity, we will refer to this as jug/ml. A standard
curve using heat-inactivated dog serum (56°,30 min) was also
tested.
Immunofluorescence
Studies
Fluorescein isothiocyanate-conjugated
was obtained from Cappel Laboratories.
3628
goat anti-canine C3
Raji cells (2 x 106 in
MEM. Wet preparations were read with a Leitz Dialux fluores
cent microscope equipped with a 100-watt mercury light
source. Slides were evaluated for percentage of stained cells
and intensity of staining.
Isolation of Complexes by Sucrose Density Gradient Ultracentrifugation
Sera from 2 dogs with breast cancer, one dog with benign
breast disease, and 2 healthy dogs were used. A 200-/il sample
of each serum was subjected to sucrose density gradient
ultracentrifugation (10 to 40%, w/w) at 100,000 x g for 15 hr
(Beckman Model L5-65; Beckman Instruments, Inc., Fullerton,
Calif.). 125IC1 q (11 S), IgG (7 S), and IgM (19 S) were used as
markers. Twenty fractions, each containing 15 drops, were
collected. In another experiment, the above sera were dialyzed
against glycine-HCI buffer (pH 2.5) and subjected to sucrose
gradient ultracentrifugation. Every second fraction was tested
for the presence of IgG by the Ouchterlony double immunodiffusion method. Immune complexes were determined by the
Raji cell method following the addition of normal dog serum (1 :
4 dilution) as a complement source. The 125IC1 q (11 S) marker
was determined in a gamma counter. Each aliquot was checked
for sucrose concentration by a refractometer to ensure com
parability among the different samples.
Biostatistical Methods
A multiple comparison test (3) was used to compare the
mean CIC and TCH;,o values of the dogs with breast cancer
and benign breast disease with those of the normal dogs. For
the CIC values, a logarithmic transformation was first applied
to the data. The Wilcoxon paired-sample test (21) was used to
compare the pre- and postmastectomy CIC and TCHbo values.
RESULTS
Pathology. Table 1 lists the pathology at diagnosis in dogs
with malignant and benign breast disease. A total of 74 dogs
were investigated. Adenocarcinoma limited to the breast was
the most frequent finding (35 dogs). In 4 dogs, there was lymph
node spread, and 5 dogs presented initially with widespread
metastatic disease. Adenoma was by far the most frequent
benign pathological diagnosis (18 dogs).
Levels. Using the one-sided Dunnett's test, the serum
levels of dogs with untreated breast cancer were signif
icantly elevated (p < 0.05) when compared with serum levels
of healthy dogs (Chart 1; Table 2). The mean ± S.D. of 51
Table 1
Pathology at diagnosis in 74 dogs with benign and malignant breast disease
Diagnosis
No. of dogs
Mastitis or hyperplasia
Adenoma
Adenocarcinoma in situ
Localized
Positive lymph nodes
Métastasespresent
Total
5
18
7
35
4
5
74
CANCER
RESEARCH
VOL. 40
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C/C's in Dogs with Breast Disease
dogs with breast cancer was 110 ± 33 compia ,-1 with 20
230
healthy dogs at 93 ±32. TCH50 decreased from TI ±34 to
90 ± 21 in 22 dogs where follow-up sera were obtained 2
IR
O
ÃŽ3 170
weeks after mastectomy (Chart 2). In the 23 dogs with benign
breast tumors, serum TCH60 prior to mastectomy was 100 ±
25. This was not significantly different from values for the 20
healthy dogs.
Levels of CIC's in Sera of Dogs with Benign and Malignant
150
Breast Disease. A typical standard Raji cell radioimmunoassay
curve using normal dog serum and increasing concentrations
of aggregated y-globulin is shown in Chart 3. When normal dog
serum is heat inactivated at 56° for 30 min, only a slight
increase in 125Ianti-dog IgG binding to the Raji cells occurred.
.y loo
50
The addition of monomeric IgG from 0 to 4 mg/ml resulted in
minimal 125Ianti-dog IgG binding to the Raji cells (data not
shown).
The CIC values for dogs with benign and malignant breast
disease were both significantly higher (p < 0.01 and p < 0.05,
respectively) than those for sera of healthy dogs (Chart 4;
Table 2). When the cutoff value for CIC levels is set at 25 jug/
ml, then only 10% of the 20 normal dogs had CIC values above
this as compared to 43% of the 23 dogs with benign breast
disease and 57% of the 51 dogs with breast cancer. Seven of
9 dogs with either lymph node or systemic métastases had
elevated levels of CIC's.
Pre
Post
Surgery
Surgery
Benign BreastDisease
Pré
Post
Surgery
Surgery
Breast Ca
Chart 2. TCH^o levels in the sera of 22 dogs with breast cancer (Ca) and 8
dogs with benign breast disease before and 2 weeks following mastectomy.
Bars, mean
NormalPooled
DogSera
t
B
'2
Repeat CIC levels were determined in 22 dogs with breast
cancer (Chart 5). Twelve dogs (54%) had increased CIC levels
preoperatively, with elevated CIC's present 2 weeks after mas
HeatInactivated
DogSera
tectomy in 8 dogs. Metastatic breast cancer developed within
3 months in 5 of 8 dogs with persistently elevated CIC levels,
O
25
50
100
500
1000
5000
10.000
40.000
Nanograms
of Aggregated
DogIgG
240
Chart 3 Raji cell uptake of 12SIanti-dog IgG after incubation with increasing
amounts of aggregated dog y-globulin in fresh or heated (56°, 30 min) pooled
dog sera.
% ISO
-c lopoo
I
••:*:
••••
1,000
.1
0
60
E
100
Dogs with
Breast Ca
Oogs with
Benign Breast
Disease
Normal
Dogs
Chart 1. ICH«, levels in the sera of 5 dogs with breast cancer (Ca) and 23
dogs with benign breast disease. Serum TCHâ„¢levels for 20 normal dogs shown
for comparison. Bars. mean.
Table 2
, ano CIC 's in dog serum
BenignBreast
Disease
Normal
SLE
Chart 4 Levels of CIC s in the sera of 51 dogs with breast cancer (Ca) and
23 dogs with benign breast disease. Sera from 20 healthy dogs and 2 dogs with
systemic lupus erythematosus (SLE) shown for comparison.
but in only one of 14 dogs without increased CIC's after surgery
ICH»Breast
of
dogs51
CIO
25ng/ml57
(logt.)5.4664.51a
cancer
1 99
4314
Benign tumors
100
25
2320Mean110a
3.34CICsS.D.2.35
0.30%of
93S.D.33 32Mean
Normal dogsNo.
3 p< 0.05. significantly elevated from normal one-sided Dunnett s test
6 p < 0.01. significantly elevated from normal one-sided Dunnett s test.
OCTOBER
Breast Ca
(p < 0.05). Eight dogs with benign breast disease were studied
prospectively (see Chart 5). All 3 dogs with initially increased
CIC levels were found to have normal levels 2 weeks after
mastectomy.
Several dogs had levels of CIC s in their sera comparable to
the elevated levels of CIC's found in the sera of 2 dogs with
systemic lupus erythematosus. Despite elevated levels of CIC s
in the sera of many dogs, blood urea nitrogen and creatinine
1980
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3629
S. R. Gordon et al.
elevated levels of CIC's has provoked many unanswered ques
tions. An animal model to study the implications of CIC's in
ir.ooo
cancer would be useful. The dog has been considered a good
model for human breast cancer because of the many similarities
between the tumors in both species. In this study, the Raji cell
radioimmunoassay was adapted to measure CIC's in dog sera.
Elevated levels of CIC's were demonstrated in the sera of dogs
1,000
s
J
with both benign and malignant breast disease. TCH50 was not
found to correlate with the CIC level. This is not surprising in
view of the individual variability in both rate of complement
synthesis and the ability of CIC's to bind complement. Kinetic
•s
•ë100
Pré
Mostectomy
Post
Mosteclomy
Breast Ça
Chart 5. Levels
8 dogs with benign
Pré
Mastectomy
Post
Mastectomy
Benign Breast Tumors
of CIC s in the sera of 22 dogs with breast cancer (Ca) and
breast disease before and 2 weeks following
mastectomy.
25ró-QKtÃ-—«Normal
Dog Sera
»
,
IgM ?,
Clg
IgG Markers
"p-ftV
-^
V /f «T f f T 1
/
/
•—•Dog
-ä—Serum
BreastCa
pH7.5
* —*Dog
\Serum
BreastCa
23äö"er^
\--IgG/
pH2.5
.\\'
with breast cancer and from the serum of one dog with benign
breast disease. The complexes sedimented at approximately
19 S and contained IgG. Fractions obtained from centrifugation
of normal dog serum under identical conditions or fractions of
serum of dogs with breast cancer using glycine buffer (pH 2.5)
did not contain CIC's, and IgG was restricted to the 7 S region.
The finding of elevated CIC's in dogs with benign breast
disease was of interest. Day" has also noted elevated CIC's in
\//
2lC\J-T31Ü1
\
\1t
\1
\1
\/
19oS
\
\.X^^^I^
studies of complement synthesis and catabolism would be
required to more fully define the relationship between comple
ment and CIC level.
Theofilopoulos ef al. (19), using human sera, have previously
demonstrated that the Raji cell binds immune complexes pre
dominantly through C3 and C3b receptors on the cell surface.
We have obtained several lines of evidence for this in the dog.
Using an immunofluorescence technique, C3 from normal dog
serum was shown to bind to Raji cells. Further, using the Raji
cell radioimmunoassay, aggregated dog IgG bound to the Raji
cells only minimally except in the presence of complement
which enhanced binding dramatically. By sucrose density gra
dient ultracentrifugation, we isolated CIC's from sera of 2 dogs
/
^^^^f^~-~--^—.
^1
17•
4
7
10
13
FractionNumber
16
19
2l
Chart 6. Sera from dogs with breast disease and high levels of CIC s were
subjected
to sucrose density gradient
ultracentrifugation
at pH 7.5. Normal dog
sera and dog sera with elevated
levels of CIC's (pH 2.5) were also ultracentrifuged. Fractions
were
IgG by the Ouchterlony
tested for CIC s by the Raji cell radioimmunoassay
double immunodiffusion
method. Ca, cancer.
and for
values were always normal. No correlation was found between
the CIC level and the TCH50 value.
Isolation of CIC's by Sucrose Density Gradient Ultracen
trifugation. Sera of 2 dogs with breast cancer and one dog
with benign breast disease with elevated levels of CIC's were
fractionated according to size and buoyant density using su
crose density gradient ultracentrifugation at pH 7.5. Sera of 2
healthy dogs were used as controls. The fractions were tested
for the presence of CIC's by the Raji cell radioimmunoassay.
The sedimentation coefficient of CIC's from sera of dogs with
breast disease was 19 S (Chart 6). IgG was present in Fractions
7 to 16 from dogs with malignant and benign breast disease.
In contrast, IgG from normal sera was restricted to Fractions
13 to 16. When the above sera were subjected to sucrose
density gradient ultracentrifugation at pH 2.5, a pattern similar
to that of normal dog serum was obtained. No difference in
sedimentation profiles was found between sera containing
CIC's of dogs with benign or malignant breast disease.
several women with benign breast tumors. Women who have
had a biopsy for benign breast disease have about a 3-fold risk
of subsequently developing breast cancer (11). Fibrocystic
disease, fibroadenomas, and intraductal papillomas have all
been reported to have potential for malignant transformation
(5). The connection between benign breast disease and sub
sequent breast cancer in the dog has not been adequately
studied. Only further follow-up studies of dogs with benign
breast disease will determine whether the presence of elevated
CIC's is of prognostic importance.
The presence of elevated levels of CIC's in both benign and
malignant dog breast sera raises the question of whether a
similar mechanism underlies their formation. This is of partic
ular interest in view of several reports associating the mouse
mammary tumor virus and antibodies to this virus with breast
cancer in several species including humans (9, 10, 22). There
is no information concerning a possible relationship between
this virus and breast cancer in the dog.
Terman et al. (16) have recently reported necrosis of canine
mammary carcinoma after extracorporeal perfusion of plasma
over Staphylococcus aureus. The Cowans I strain of Staphylococcus aureus binds IgG and IgG-containing immune com
plexes. Our finding of elevated CIC levels in 57% of dogs with
breast cancer may be of significance with respect to the study
of Terman ef al..
The stage or extent of disease has been correlated with the
level of CIC's in several reports (1,4,16,18).
Seven of 9 dogs
DISCUSSION
The observation
3630
that sera of cancer patients often contain
•
N. K. Day,unpublishedobservations
CANCER
RESEARCH
VOL. 40
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CIC s in Dogs with Breast
in this study with lymph node or systemic métastases had
elevated CIC's. The dogs with persistent elevation of CIC levels
10.
appeared to be at greatest risk of developing metastatic breast
cancer during the 3-month period after mastectomy. The short
natural history of breast cancer in the dog should enable further
clarification of the relationship between immune complexes
and the clinical course of the disease.
11.
12.
13.
ACKNOWLEDGMENTS
14.
We wish to thank Regina Del Prete for her excellent technical assistance and
Dr Ying Wang for help in the statistical analysis.
15.
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3631
Circulating Immune Complexes in Sera of Dogs with Benign and
Malignant Breast Disease
Bruce R. Gordon, Scott Moroff, Arthur I. Hurvitz, et al.
Cancer Res 1980;40:3627-3631.
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