International Urology and Nephrology 22 (6), pp. 501--506 (1990)

Serum and Urine Levels of Tumour Necrosis Factor in Patients with Genitourinary Cancer and their Relevance to Disease Status A. AKDAS,L. T~RKERI,T. AKO~LU* Department of Urology, *Department of Internal Medicine, Marmara University School of Medicine, Istanbul, Turkey (Received November 9, 1989) Serum and urine levels of tumour necrosis factor (TNF) which is a lymphokine was measured in patients with genitourinary cancer (either prostatic, bladder or testicular carcinoma) in order to find out whether there were elevated levels of TNF in those patients and if it was related to disease status. Although the preliminary results are not conclusive, further research on TNF may prove this immunoprotein to be a potential diagnostic and therapeutic agent.

Introduction

It was demonstrated by O'Malley et al. in 1962 that serum from animals injected with endotoxin when administered to other animals could cause haemorrhagic necrosis of tumours [1 ]. The same phenomenon had also been reported by Coley as early as a century ago in a patient with sarcoma who contracted an intercurrent streptococcal infection [2]. This activity of serum was later confirmed [3] and a unique protein was purified and subsequently named cachectin [4] because of its ability to suppress lipoprotein lipase leading to striking lipaemia and profound wasting diathesis in rabbits with tryponosomiasis [2]. It was subsequently demonstrated that this factor may have both direct cytotoxic and cytostatic effects on the tumour with indirect antitumour effects mediated by the immune system and is derived f r o m myeloid cell line [2, 5], and renamed as tumour necrosis factor (TNF). Recently, endogenous receptors on cell surface to cytokines including T N F have been isolated [6, 7, 8]. Since then, studies on detecting serum levels of T N F both in normal subjects and in cancer patients, with inconclusive results in the latter case, have been reported [9, 10]. In this clinical study we have determined the serum and urine levels of endogenous T N F in patients with genitourinary cancer showing disease activity within the range of complete remission to high-stage active malignancy. 1"

VSP, Utrecht Akad6miai Kiadd, Budapest

502

Akdas et al.: Turnout necrosis factor

Materials and methods Serum and urine samples have been obtained from a total of 50 patients with genitourinary cancer (Table 1). Their age ranged between 19 and 85 years (mean 48.5); four of them (8 ~o) were females. At the time of sampling, 19 (38 ~ ) patients were in complete remission without any evidence of disease. Three (9.7 ~ ) of the remaining 31 patients with different disease activities were under treatment and 16 (51.6 ~ ) were in posttreatment period at the time of the study. The remaining 12 (38.7 ~ ) did not receive any treatment yet (Table 2).

Table 1 Distribution of tumours Testis

Total number of patients Pretreatment group Treatment group Posttreatment group

11 0 3 8*

Bladder

24 4 1 19"

Prostate

13 7 0 6

* None of the patients showed evidence of disease

A s s a y procedure

In this study the C E L L F R E E | T N F * test kit was used. it is a sandwich enzyme immunoassay for the determination of released T N F levels. An anti-TNF monoclonal coating antibody is first adsorbed on to polystyrene microtiter wells. Soluble T N F present in the sample or standard binds to antibody on the coated well; unreacted sample components are removed by washing. An enzyme conjugated anti-TNF monoclonal antibody directed against a second epitope on the T N F molecule is then added and it binds to the T N F captured by the first antibody, completing the sandwich. U n b o u n d enzyme conjugated a n t i - T N F is removed during a wash step and substrate solution is added to the wells. A coloured product is formed in proportion to the amount of T N F present in the sample. The reaction is terminated by addition of stop solution and absorbance at 490 nm is measured. A standard curve is prepared f r o m five T N F standards. Unknown values are determined from the standard curve.

* T Cell Sciences Inc., 840 Memorial Drive, Cambridge, MA 02139, USA. International Urology and Nephrolo#y 22, 1990

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Table 2 Stages of turnours Stage

No. of patients

Testicular tumours according to Peckham I IIA IIB IIC III IV

2

1

NED

8

Bladder tumours according to Marshall 0 A B~

1 2 5

B2

C D1 D~ NED

1 3 1 11

Prostatic tumours according to Whitmore 0 A

3

B

1

C D NED

6 3

NED: No evidence of disease

Results

Two patients only in the whole study group had elevated serum levels of T N F : one from the group with no disease activity (5.3 %), the other from the patient group (3.4 %) (40 and 70 pg/ml, respectively). One of these patients had a bladder turnout with superficial muscle wall invasion, who did not receive any treatment yet at the time of sampling, and did not have concomitant T N F rise in the urine. The other one has been free of his testicular tumour for over 3 years and accepted to be cured. Again there was no elevation in urine levels of TNF. The only patient (5.3 %) who had a high level o f T N F in the urine (255 pg/ml) was a young male in the group with no disease International Urology and Nephrology 22, 1990

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Akdas et al.: Turnout necrosis factor

activity, who had received combination chemotherapy because of high volume metastatic testicular tumour and was free of any evidence of disease at the time of sampling. There was no detectable elevation of the serum level of T N F in this patient. Serum levels of T N F in the remaining 45 cases were between 0 and 12 pg/ml with a median value of 1.1 pg/ml. There was no detectable T N F in the urine of the study group, except in one case with an elevated value.

Discussion The tumour necrotic effects of bacterial endotoxins have been known for a long time [11]. Subsequently, macrophages and macrophage cell lines were shown to produce a monokine in response to lipopolysaccharide (LPS) and the oncolytic effect was demonstrated to be due to this factor which was termed tumour necrosis factor and was then isolated and purified [12]. The major inducer of T N F is LPS, while others are reported to be muramyl dipeptide, other bacterial and protozoan products, mitogens and viruses [12, 13]. T N F is a protein with a molecular weight of 17 kilo Dalton and is composed of 157 amino acids. The genes that are coding T N F are located on the sixth chromosome [1 ]. The released T N F acts as a hormone, binding to specific high-affinity receptors and eliciting biological responses [141. T N F appears to modulate the transcription and release of other cytokines, including interleukin-1 tu macrophages and endothelial cells [15]. There is strong evidence for a wide variety of effects of T N F on body structures including the immune system, which are responsible for the signs and symptoms of pathological conditions, especially infections. Under normal circumstances, little biologically active T N F is present within the macrophages, because the gene or genes controlling its expression are subject to strong repression. Plasma concentrations of T N F are usually less than 35 pg/ml in normal subjects [16]. In a recent study using a sensitive enzyme-linked immunosorbent assay for TNF, which can detect 40 pg/ml, measurable T N F levels have been found in 7.9 % of sera from normal human subjects, and in a similar percentage of sera from patients having a variety of neoplastic diseases and representing a range of disease activity from clinical remission to progressive cancer [17]. In the same study there were detectable levels of T N F in 66 ~o of patients with visceral leishmaniasis and m 70~o of patients with malaria. Once produced T N F is cleared from the plasma with a half-life of 6.5 to 17 minutes [4, 16, 18]. In our clinical work we tried to estimate the levels of T N F both in serum and urine of patients with a specific system malignancy. In our patient group the presence of one of the genitourinary tumours was proposed to be the possible potential cause of detectable levels of TNF. But our results are inconclusive since elevated levels of T N F were demonstrated in only 2 patients; one of them was not bearing a tumour at all at that time. The only high level of urine T N F was seen in a International Urology and Nephrology 22, 1990

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patient with high v o l u m e m e t a s t a t i c testicular t u m o u r initially, b u t he was in c o m plete remission when the urine sample was o b t a i n e d . The role o f T N F as a p a r t o f the i m m u n e system, which is t h o u g h t to have a central effect in the p a t h o g e n e s i s o f m a l i g n a n c y , is still n o t clear. Yet there is a m a j o r interest focussed on the utilization o f T N F as an a n t i n e o p l a s t i c agent. T h e necrotizing action o f T N F in vivo is t h o u g h t to be related mainly to capillary injury [19]. O n the basis o f in vivo a n t i t u m o u r tests it is speculated t h a t T N F also induced a h o s t - m e d i a t e d factor which c o n t r i b u t e d to a n t i t u m o u r activity [19]. T h e cytotoxic effect was e x a m i n e d b y using a m i c r o s p e c t r o p h o t o m e t r i c assay for the l y s o s o m o t r o p h i c p r o b e - acridine o r a n g e - a n d the results suggested t h a t T N F exerts its effect also b y e n h a n c i n g e n d o g e n o u s t u m o u r l y s o s o m a l activity [19]. W h e t h e r e n d o g e n o u s T N F p r o d u c t i o n is r e m a r k a b l e a n d has a n y effect on the o u t c o m e o f the m a l i g n a n c y is still obscure a n d needs further evaluation.

References 1. Aggarwal, B B., Aiyer, R. A., Pennica, D., Gray, P. W., Goeddel, D. V.: Human tumor necrosis factors: Structure and receptor interactions. In: Tumor Necrosis Factor and Related Cytotoxins. Ciba Foundation Symposium 131. Wiley and Sons Ltd., Chichester 1987. 2. Beutter, B., Cerami, A.: Cachectin: More than a tumor necrosis factor. N. Enyl. J. Med., 316, 379 (1987). 3. Carswell, E., Old., L. J., Kassel, R. L., Green, S., Fiore, N., Wilfiamson, B.: An endotoxin induced serum factor that causes necrosis of tumors. Proe. Natl. Acad. Sci., 72, 3666 (1975). 4. Beutler, B., Mahoney, J., LeTrang, N., Pekala, P., Cerami, A. : Purification of cachectin, a lipoprotein lipase suppressing hormone secreted by endotoxin induced RAW 264.7 cells. J. Exp. Med., 161, 984 (1985). 5. Palladino, M. A., Patton, J. S., Figari, I. S., Shalaby, M. R.: Possible relationships between in vivo antitumor activity and toxicity of tumor necrosis factor-alpha. In: Tumor Necrosis Factor and Related Cytotoxins. Ciba Foundation Symposium 131. Wiley and Sons Ltd., Chichester 1987. 6. Hass, P. E., Hotchkiss, A., 1 Mohler, M., Aggarwal, B. B.: Characterization of specific high affinity receptors for human tumor necrosis factor on mouse fibroblasts, ar. Biol. Chem., 260, 12214 (1985). 7. Aggarwal, B. B., Eessalu, T. E., Hass, P. E.: Characterization of receptors for human tumor necrosis factor and their regulation by gamma-interferon. Nature, 318, 665 (1985). 8. Shalaby, M. R., Palladino, M. R., Hirabayashi, S. E.: Receptor binding and activation of polymorphonuclear neutrophits by tumor necrosis factor alpha. J. Leukocyte Biol., 41, 196 (1987). 9. Petersen, C. M., Moller, B. K. : Immunological reactivity of tumor necrosis factor. Lancet, 1, 935 (1988). 10. Sherman, M. L., Spriggs, D. R., Arthur, K. A., Imamura, K., Frei, E., Kufe, D. W.: Recombinant human tumor necrosis factor administered as a five day continuous infusion in cancer patient: Phase I toxicity and effects on lipid metabolism. J. Clin. Oncol., 6, 344 (1988). 11. Ruff, M. R., Gifford, G. E.: Tumor necrosis factor. In: Pick, E. (ed.): Lymphokines. Academic Press, New York 1981 ; Vol. 2, 1988, p. 235. International Urolooy and Nephrolopy 22, 1990

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12. Gifford, G. E., Flick, D. A.: Natural production and release of tumor necrosis factor. In: Tumor Necrosis Factor and Related Cytotoxins. Ciba Foundation Symposium 131. Wiley and Sons Ltd., Chichester 1987. 13. Ziegler, E. J.: Tumor necrosis factor in humans. N. Engl. J. Med., 318, 1533 (1988). 14. Tracey, K. J., Lowry, S. F., Cerami, A. : Physiological response to cachectin. In: Tumor Necrosis Factor and Related Cytotoxins. Ciba Foundation Symposium 131. Wiley and Sons Ltd., Chichester 1987. 15. Nawroth, P. P., Bank, I., Handley, D., Cassimeris, J., Ckess, L., Stern, D." Tumor necrosis factor/cachectin interacts with endothelial cell receptors to induce release of interleukin 1. J. Exp. Med., 163, 1363 (1986). 16. Michie, H. R., Manogue, K. R., Spriggs, D. R. : Detection of circulating tumor necrosis factor after endotoxin administration. N. EngL J. Med., 318, 1481 (1988). 17. Scuderi, P., Lain, K. S., Ryan, K. J.: Raised serum levels of tumor necrosis factor in parasitic infections. Lancet, 2, 1364 (1986). 18. Flick, D. A., Gifford, G. E.: Pharmacokinetics of murine tumor necrosis factor. J. l m munopharmacol., 8, 89 (1986). 19. Haranaka, K., Satomi, N., Sakurai, A., Haranaka, R. : Antitumor effects of tumor necrosis factor: Cytotoxic or necrotizing activity and its mechanism. In: Tumor Necrosis Factor and Related Cytotoxins. Ciba Foundation Symposium 131. Wiley and Sons Ltd., Chichester 1987.

International Urology and Nephrology 22, 1990

Serum and urine levels of tumour necrosis factor in patients with genitourinary cancer and their relevance to disease status.

Serum and urine levels of tumour necrosis factor (TNF) which is a lymphokine was measured in patients with genitourinary cancer (either prostatic, bla...
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