The presence and structure of circulating immune
complexes in patients with renal cell cancer
Artykuł opublikowany w Urologii Polskiej 2008/61/3.
Ryszard Gołda, Wojciech Jóźwicki, Grzegorz Przybylski, Zbigniew Wolski, Jan Domaniewski, Małgorzata Wyszomirska, Jacek Michałkiewicz
- Department of Immunology, The Ludwik Rydygier Collegium Medicum in Bydgoszcz Nicolaus Copernicus University in Toruń
Department of Tumour Pathology, The F. Łukaszczyk Oncology Center in Bydgoszcz
Department of Respiratory Medicine and Tuberculosis, The Ludwik Rydygier Collegium Medicum in Bydgoszcz Nicolaus
Copernicus University in Toruń
Department of General, Oncologic and Pediatric Urology, Ludwik Rydygier Collegium Medicum in Bydgoszcz Nicolaus
Copernicus University in Toruń
The level and characteristics of circulating immune complexes
(CIC) present in the sera of tumour patients have shown both good,
and poor correlation with the disease progress [1,2,3,4,5,6,7,8,9,10].
As far as prognosis is concerned many authors have observed a variety
of findings, ranging from partial to very good correlation between
CIC levels and prognosis in the disease [1,2,11,12,13,14,15]. For
example, melanoma tumor-associated antigen (TAA) containing CIC
was detected in 75% of patients with recurrence of the disease, as
compared to their absence in 71% of the patients, who remained
disease-free . Mavligit and Stuckey have found the presence of
CEA in CIC isolated from the serum of colorectal patients . It
has been demonstrated that detection of antigen-specific CIC in the
sera of cancer patients may provide an approach for more precise
defining of the nature of tumour antigens that is important for
immune prognosis [11,18,19,20]. The detailed characterisation of
CIC should enable both detection as well as identification of their
antigenic components [18,19,21,22,23,24].
The aim of this study was to separate and determine the
antigenic components of CIC isolated from the sera from 52
patients with renal cell cancer.
Materials and methods
We assayed the serum samples originated from 18 patients
with renal cell carcinoma in differentiation G-1, 21 with G-2 and
13 with grade G-3. Mean age of 52 cancer patients was 61.5
±9.5 yr. Control group consisted of the sera samples taken from
45 healthy men aged between 25 and 69 ( 41.1 ±10.8 years).
CIC assays by PEG-test
Serum samples (2 ml) were diluted in 2 ml of 7% PEG-6000
solution in borate buffer (0.1 M, pH 8.4). The samples were incubated
at 4oC for 18 h and centrifuged at 15.000g for 30 min at
the same temperature. The supernatant was decanted and the
precipitate was washed with the 3.5% PEG-6000 solution in borate
buffer, suspended in 2ml of 0.1M NaOH and incubated at 25oC.
After 30 min, the optical density was read on a LKB spectrophotometer
at 280 nm (0.1 optical density unit was read as 0.07 g/
dm3 of CIC protein). The results were considered as positive when
optical density value was higher than 0.130 (0.112+0.018) (mean
value ±0.018 observed in 45 healthy men) [25-27].
The quantity of 0.5 ml of patient serum was incubated with 0.5
ml of 7.5% PEG in borate buffer (0.1M, pH 8.4) for 24 hour at 4oC.
The precipitate was then washed twice with 3.5% PEG in borate
buffer, centrifuged at 2500 rpm for 20 min. at 4oC and subsequently
resuspended in a volume of 0.5 ml PBS . The CIC suspension in PBS
was then diluted in the borate buffer and used for polyacrylamide gel
Gel electrophoresis and silver staining
The nature of the protein present in CIC was studied by SDS-PAGE
gel electrophoresis, according to the method of Laemmli . The CIC
sample in PBS was diluted 1:1 in the TRIS-HCl buffer of pH 6.8, (1%
glycerol, 6% SDS, 5% 2-mercaptoethanol, 0.05% bromophenol blue),
then heated at 100oC for 3 min. and subsequently applied to the
plate composed of a stacking gel (3% acrylamide) and resolving gel
(11% acrylamide). Approximate molecular weights were estimated by
comparison with standard protein markers (MW-SDS-200-SIGMA).
At the end of the electrophoretic run, the proteins were stained with
Coomassie brilliant blue R250 and silver .
Statistical analysis of data was done by Student’s t-test.
PEG test from cancer and normal serum samples
The CIC level was studied in the sera of 52 patients and
45 healthy men by means of PEG test; CIC-protein level are
presented on Fig.2. In a normal serum mean optical density as
measured at 280 nm was 0.113 (0.091 g/l CIC protein). The
concentration of CIC in patients with renal cell cancer was significantly
higher. The analysis of 52 cancer serum samples divided
according to cancer differentiation grade showed the following
mean concentration values: 0.158 ±0.1 g/l (P<0.01) in stage
G-1; 0.19 ±0.086 g/l (P<0.01) in stage G-2 and 0.214 ±0.104
g/l (P<0.01) in stage G-3 (Fig.2). The percentage of CIC positive
patients in the G-1, G-2, and G-3 groups was as follows: 27.7%
in G-1 (elevated in 5 of 18 patients), 52.4% in G-2 (elevated
in 11 of 21 patients), and 53.8% in G-3 (elevated in 7 of 13
patients). In general, an increase in the level of CIC was observed
in 23 out of 52 patients with renal cell cancer (44.2%).
SDS-PAGE analysis of CIC originating from control
and cancer serum
The distribution and frequency of appearance of the protein
fractions in the CIC of control sera showed 21 bands of molecular
weight between 22 and 198 kDa (Table I). This mode of
the bands distribution was highly reproducible. In contrast, the
protein fractions of CIC obtained from sera of tumour patients
showed up to 28 bands of molecular weight between 20 and
231 kDa. The results, showing the distribution of atypical proteins
in relation to molecular weight are displayed in the Fig.3.
Their frequency is shown in the Table II.
The results presented in this paper indicated that 44.2%
of renal cell carcinoma patients under study had an increase
in the level of CIC in the serum. The values described here are
higher than those reported by other researchers. Sakai and Kato
have found considerably lower percentage (18.8%) of tumour
patients with increased level of CIC . However, they have
examined much smaller group of patients that makes quite
difficult to compare their results with those obtained by us.
Additionally, they indicated on the relation between the level of
CIC and the size of tumor, its invasive propensity, and the level
of its virulence . The results of this work are consistent with
our observations concerning both an increase in the level of CIC
in the patients with prostate cancers as well as the different
molecular weight of CIC proteins that in turn was depending
on the stage of cancer [11,12].
The origin of the antigens present in CIC originated from
cancer patients is unknown [11,14,18]. Perhaps the composition
of the antigens in CIC is related to the level of the cancer
progress . In 1991 Wiederkehr and Bueler  carried out
a comparative electrophoresis of immune complexes, isolated
from sera of healthy persons and persons with cancer. It turned
out that the protein profiles in electrophoretic sections of control
group were similar, but there were some small quantitative
differences. In the available literature we did not find any work
concerning the electrophoretic analysis of CIC present in the
sera of prostate cancer patients, so it is hard to compare our
results with the results of others.
We have found so far only one paper dealing with an analysis
of the protein fractions present in the CIC  originated
from cancer patients. The authors examined CIC isolated from
the sera of patients with colon cancer, pancreas cancer, stomach
cancer and melanoma. In the most cases the existence of a
few common proteins were found the CIC fractions: 1) for the
albumin (64-72 kDa), 2) for the heavy immunoglobulins chains
(52-61 kDa) and, 3) for the light immunoglobulins chains
(24-28 kDa). The results of this work are consistent with the
results previously described by others. Bartoloni and others
 indicated on the presence of atypical protein fractions with
molecular weight ranging from 29-33 kDa to 78-88 kDa found
in the serum of colon, pancreas and stomach cancer patients.
Additionally, a protein fraction of molecular mass of 99 kDa was
defined only in the sera of colon cancer patients. In the CIC originated
from the serum of melanoma patients the atypical protein
fractions with molecular weight of 30-49 kDa and 71-76
kDa were found . By comparison, our results dealing with
renal cancers indicated on the presence of 28 protein fractions
of molecular weight ranging from 20 kDa to 231 kDa. Thus, the
number of protein fractions obtained by us was much higher as
compared to the results of Bartoloni’s work. This may depend
on much more sensitive technique (silver method) used by us for
the detection of protein fraction present in CIC (sensitivity 1-10
ng). This technique is much better than the classical method
of Coomassie Brilliant Blue R-250, with sensitivity ranging from
1-10 μg . The electrophoretic analysis of isolated CIC originated
from serum of renal cell cancer patients indicated on the
presence of 10 atypical protein fractions. Molecular masses of
these fractions were as follow: 20,31,33,39, 82,92,95,102,124
and 231 kDa.
The elevated levels of CIC are present in the sera of tumor
patients. The level of CIC partly depends on the histological
grade of malignancy. Thus, determination of CIC levels can be
helpful in prognosis, but cancer-derived atypical proteins present
in CIC generated during the course of the cancer process
still await analysis.
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Source of support: This work was supported in part by a research grant
from The Ludwik Rydygier Collegium Medicum in Bydgoszcz Nicolaus Copernicus
University in Torun, Poland (BW 127/97, BW 44/2007).
Acknowledgment. To Kazimierz Madaliński, Professor, for English revision of