PTU - Polskie Towarzystwo Urologiczne

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 [16]. Mavligit and Stuckey have found the presence of CEA in CIC isolated from the serum of colorectal patients [17]. 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].

CIC isolations

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 [11]. The CIC suspension in PBS was then diluted in the borate buffer and used for polyacrylamide gel electrophoresis.

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 [28]. 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 [29].

Data analysis

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 [8]. 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 [6]. 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 [24]. In 1991 Wiederkehr and Bueler [20] 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 [18] 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 [18] 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 [18]. 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 [29]. 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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adres autorów

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
the manuscript.