GYNECOLOGIC ONCOLOGY 21, 94-100 (1985)

Serum-Mediated Immunosuppression: A Possible Tumor Marker in

Patients with Ovarian Carcinoma
MATHIAS ONSRUD, M.D.
Department of Gynecologic Oncology, The Norwegian Radium Hospital, Oslo, Norway
Received May 10, 1984

Serum samples from 25 patients with ovarian carcinoma were tested for their suppressive
effects on in vitro response of normal lymphocytes. Patients examined prior to primary
surgery showed a significantly greater suppression than did patients examined after a radical
operation. Suppression was detected both in a natural killer cell assay and in an assay of
phytohemagglutinin-induced lymphoproliferation. Only the results of the later test showed
a correlation to the clinical course: Those patients who died during the first year of follow-
up presented the most marked suppression. Serial determinations performed in a few
patients indicated a certain correlation between immunosuppression and tumor burden. It
is concluded that this test may give additional prognostic information in patients with
ovarian carcinoma. 0 1985 Academic Press, Inc.

INTRODUCTION
Since the concept of “immunological tumor surveillance” was introduced by
Bumet [l] in 1970, various immune responses have been studied in cancer patients,
and the responses correlated to the clinical course and the treatment given.
Depressed immune responses are commonly seen in patients with advanced
cancers. The exact mechanism behind this immunosuppression is still unclear.
Substances released from the tumor cells or from host cells in response to the
tumor are possible mediators.
Serum from cancer patients has been shown to cause a nonspecific suppression
of various responses of normal lymphocytes [2]. When added to in vitro assays,
inhibition of both mitogen-induced T-lymphocyte proliferation 12-51 and of cytotoxic
activity of natural killer (NK) cells [61 have been described. In gastrointestinal
cancers a correlation between the level of circulating immunosuppressive factors
and the prognosis has been reported [4,5]. The purpose of this study was to
determine the suppressive effect of serum from ovarian cancer patients on normal
in vitro T-cell proliferation and on NK cell activity, and to correlate the results
to the clinical course of the disease.
MATERIALS AND METHODS
Patients. Twenty-five patients with epithelial ovarian cancers were studied.
The distribution according to stage (FIG0 classification) and histology is shown
in Table 1. The patients were referred to the Norwegian Radium Hospital either

94
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 SERUM-MEDIATED IMMUNOSUPPRESSION 95

TABLE 1
DISTRIBUTION ACCORDING TO STAGE AND HISTOLOGY OF OVARIAN TUMORS BEING EITHER
NONOPERATED OR RADICALLY OPERATED

Radically
Nonoperated operated
(14 patients) (11 patients)

Stage (FIGO)
I + II 3 9
III + IV 11 2
Histology
Serous 8 3
Mutinous - 4
Endometrioid 2 3
Clear-cell 2 1
Poorly differentiated 2 -

for primary operation (14 patients) or for adjunctive therapy after a radical
operation had been performed at the local hospital (11 patients). In the latter
group, the radicality of the primary operation was confirmed by a second-look
laparotomy or laparoscopy in our institution. The serum samples were taken
immediately before the primary operation or before the second-look operation.
Serial determinations were done in 4 patients who received combined treatment
with surgery, chemotherapy, and/or radiotherapy. All serum samples were stored
at -20°C until tested. One year after the first examination, follow-up reports
were obtained from the out-patient department of our hospital or from the local
doctor.
Immunologic assays. The methods used have been extensively described in
a previous publication [7]. Mononuclear cells of a healthy person were isolated
from defibrinated venous blood by Ficoll-Isopaque flotation. Consistent results
were obtained with cells from three different persons. The data presented here
were obtained in a single experiment, using cells from one donor only.
Mitogen-induced polyclonal T-cell activation was measured after stimulation
by phytohemagglutinin (PHA) at a suboptimal concentration (1:400 final con-
centration) for 72 hr. The incorporation of [3H]thymidine into DNA during the
final 6 hr was determined by liquid scintillation counting. Quadruplicate cultures
of 50,000 mononuclear cells were set up in 150 ~1 of medium (RPM1 1640 with
10% fetal bovine serum). Preliminary experiments showed that a dose-dependent
inhibition of the response occurred when lo-100 ~1 of patient serum was added
to the cultures. The following results were obtained using 50 ~1 of patient serum
or 50 ~1 of normal serum (pooled sera from 15 healthy, male blood donors). The
degree of suppression was defined as:

After repeated testing of the same serum, the range of variation was normally
within 20%. The degree of suppression was unchanged after heating the sera at
96 MATHIAS ONSRUD

56°C for 30 min; and the sera showed no cytotoxicity against normal lymphocytes,
as determined by the trypan blue exclusion test.
To determine the effect of patient serum on the NK cell activity, mononuclear
blood cells from the same healthy donor were first incubated for 1 hr at 37°C
in tissue culture flasks. The nonadherent cells (lymphocytes) were cocultured
with “Cr-labeled K562 cell line targets for 4 hr and the specific release of radiolabel
from quadruplicate cultures was measured. Preliminary experiments showed that
patient serum caused a dose-dependent suppression of K562 cell lysis at various
effecter/target cell ratios. The results given here, were obtained at an effector/
target ratio of IO/l in 150 ~1 of medium with 50 ~1 of patient serum or 50 ~1 of
normal serum added. The degree of suppression was calculated as above. Addition
of patient serum to the target cells had no influence on the spontaneous release
of radiolabel, which was below 10% of maximum release.
The differences in immunosuppression between the patient groups were sta-
tistically evaluated by Wilcoxon’s rank sum test.
RESULTS
Suppression In the ovarian cancer patients examined prior
of NK cell activity.
to operation, the serum-mediated suppression of NK cell activity varied from
-2 to 58% (median 27%) (Fig. 1). In the patients who showed no tumor left at
the second-look operation, the suppression varied from - 6 to 42% (median 9%).
The difference in suppression between the two patient groups is of borderline
significance (P = 0.04). No correlation between suppression of NK cell activity
and prognosis could be seen.
Suppression of PHA-induced lyrnphoproliferation. The patients examined prior
to the primary operation showed a median suppression of PHA-induced lym-
phoproliferation of 61% (range 6-99%), whereas the median suppression seen in
the radically operated group was 21% (range -2O-48%) (Fig. 2). The difference
between the groups is significant (P < 0.005). In the first group four patients
died from cancer within 1 year. The sera from these patients were the ones that
showed the highest degree of suppression in their group (71, 72, 88, and 99%).
In addition, there was one death from intercurrent disease (cardiac infarction)

Non- Radically
operated operated
-10 r

f lo” +- fb-
5 20
0 p-o.04
g 30 I
E 40 .
% .
g 50
$ 60 ZT
.- 70 r-
60 asalive
90 ocdead

FIG. 1. The suppression of natural killer (NK) cell activity of sera from nonoperated ovarian
cancer patients and from radically operated patients with relation to the l-year survival.
 SERUM-MEDIATED IMMUNOSUPPRESSION 97

Non- Radically
operated operated
-20 - 8
-10 . .

8 1:. .
‘0 20. ;
‘2 30. .
z 40. +. p<o.o05 ;
2 50. :
(jj 60. t
70 _ 8
80 _ l = alive
90 _ 0 0. dead
100 0
FIG. 2. The suppression of phytohemagglutinin-induced lymphoproliferation of sera from nonoperated
ovarian cancer patients and from radically operated patients with relation to the l-year survival.
The asterisk indicates a patient who died from intercurrent disease.

1 month after operation. In the patients who had no tumor left after primary
operation, only one recurrence and death during the first year was observed.
This patient was the one who presented the highest degree of immunosuppression
in her group (Fig. 2).
Irnmunosuppression and tumor burden. In the patients examined before the
primary operation the degree of immunosuppression was related to the extent
of the disease as found during laparotomy (Table 2). No definite correlation
between immunosuppression and stage of the disease (FIGO) was seen. Similarly,
the presence or the quantity of ascites did not have any clearcut influence on
the PHA response. For disseminated tumors (stages III and IV), a correlation

TABLE 2
CORRELATION BETWEEN THE DEGREE OF IMMUNOSUPPRESSION AND STAGE, QUANTITY OF ASCITES,
AND TUMOR WEIGHT IN 14 PATIENTS OPERATED FOR OVARIAN CARCINOMA

Suppression of Stage Ascites Tumor

Patient PHA-response (%) (FIGO) (ml) weight (g)

M. M. 6 IV No 20 b
A. L.O. 28 Ia No 700
S. M. 38 IV 1300 100
I. H. 42 III 2500 200
u. J. 42 III 2000 500
J. H. 46 III 1500 400
L. R. 61 III 4400 900
M. H. 61 Ic 4600 1400b
A. J. 62 Ia No 1200b
u. w. 71 III 1000 200
H. A. 72 III No 500
B. R. 72 III No 300
M. F. 88 III 2000 1200
H. H. 99 IV 5500 1500

’ Estimated from surgical and histopathological report

b Encapsulated cystic tumors.
98 MATHIAS ONSRUD

between estimated tumor weight and immunosuppression was observed. Of the

three patients with localized (stage I) disease, the two patients with large cystic
tumors suppressed the PHA-response more than did the patient with a smaller
cystic tumor.
Serial determinations. Serial determinations of the suppression of PHA-induced
lymphoproliferation were performed in four patients (Fig. 3). Patient S.M. had
an ovarian carcinoma stage III, which was nonradically operated. During post-
operative treatment with 6 cycles of cis-diaminodimethylplatinum (CDDP) 75
mg/m2, the degree of immunosuppression decreased. At second-look laparoscopy
there was no evidence of disease, and colloid 32P was installed intraperitoneally.
Six months later she developed ascites and abdominal tumor masses. The recurrence
was accompanied by increasing immunosuppression. Patient M.M. had a very
small ovarian tumor with spread to the pleural cavity (stage IV). During post-
operative treatment with CDDP the pleural fluid disappeared. Three months later
the pleural effusion reappeared together with an abdominal mass. In both these
cases the degree of immunosuppression reflected the clinical course fairly well.
The patients M.F. and U.W. had extensive stage III tumors that could only be
biopsied at the primary operation. Both patients showed no response to chem-
otherapy or radiotherapy and died within a few months. The immunosuppressive
effects of their sera remained high.
DISCUSSION
Depression of cellular immunity commonly accompanies a variety of malig-
nancies. The mechanisms involved are not known, but numerous reports indicate

FIG. 3. Serial determinations of serum-mediated suppression of the PHA response in four patients
who received combined treatment for ovarian cancer (see text). (1): no evidence of disease, (2):
disease progression.
 SERUM-MEDIATED IMMUNOSUPPRESSION 99

that noncytotoxic, nonspecific factors in malignant serum may play a role

[2-61. The cellular origin of these factors is unknown, but malignant ascites fluids
are abundant sources [3,6,8]. There are indications that the determination of
such factors may give adjunctive information about prognosis and tumor burden
[4,51.
A definite characterization of the immunosuppressive factor(s) has not yet
been done. The a-globulin fractions have been shown to possess immunosuppressive
properties and their elevated levels tend to correlate with metastatic spread
[9- 1 I]. A lymphocyte-inhibitory acid protein with a molecular weight of about
50,000 has been isolated from malignant ascites [6,81. This factor shows close
resemblance to the sheep erythrocyte receptor of human T cells [8]. Serum
inhibitory factors similar to those found in advanced cases of cancer have been
found also in normal individuals, although in much lower quantity. It has been
suggested that rather than being a product of tumor cells the immunosuppressive
factor(s) could be regulatory molecules released from activated T cells [8].
In ovarian cancer new tumor markers are warranted. The preliminary data
reported here show that the determination of serum-mediated immunosuppression
can be of clinical value. Sera from ovarian cancer patients suppress the in vitro
activity of both NK cells and T cells. A correlation between immunosuppression
and prognosis was found only for the T-cell proliferative (PHA) test. This test
also seems to be of value in monitoring the clinical course of the disease. The
material was too small to determine any correlation between immunosuppression
and histological type. It seemed, however, that the degree of immunosuppression
correlated better with the tumor burden than to the stage of the disease. The
tumor weight is difficult to assess when a radical operation is not performed,
and the relative content of cystic fluid may vary. The data presented must
therefore be looked at with caution, and more data are needed before firm
conclusions can be drawn. In future studies the immunosuppressive capacity of
peritoneal effusions from patients with ovarian carcinomas and from patients
with benign diseases will also be tested.
ACKNOWLEDGMENTS
The study was supported by grants from the Norwegian Cancer Society. The author thanks Dr.
E. Thorsby and the Tissue Typing Laboratory for the opportunity to perform the assays.

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