[Prognostic value of erythrocyte polyamines levels in renal cell carcinoma. Prospective study in 418 cases]. / Valeur pronostique des polyamines érythrocytaires dans le cancer du rein. Etude chez 418 patients.

OBJECTIVES: Polyamines: Spermine (Spm) and Spermidine (Spmd), are essential for cell proliferation and differentiation. A measurement of erythocytes polyamines (EPA) was developed in our institution. Our objective was to evaluate this marker as a new prognostic factor in renal cell carcinoma. PATIENTS AND METHODS: A blood sample was prospectively taken before surgery, among 418 patients who had an enlarged nephrectomy (n=318) or a partial nephrectomy (n=100) to quantify EPA rates by using the HPLC technique. The qualitative and quantitative variables have been compared using chi(2) and Student statistical analyses. The survivals have been normalized by the Kaplan Meier and Cox methods. RESULTS: The average age of our population was 64 years (21-88). The average decline was 41 months (1-214). The median size of tumors was 6.5cm (1-24). The median rate of Spm and Spmd were respectively 4.7 (1-83) and 9 (2-86)nmol/8.10(9) erythrocytes. Spm and Spmd were linked to the T stage (p=0.001), and the ECOG (p=0.001 and 0,008). Spm was not linked at N and M stages but at the Fuhrman grade (p=0.001). Spmd was linked to the N, M stages (p=0.04). With univariate analysis, the tumor diameter, the TNM stage, the Fuhrman grade as well as Spm and Spmd (p<0.0001) were predictors of specific survival. With multivariate analysis, some prognostic factors remained independent: the TNM stage, the ECOG and Spmd, a continuous variable (p=0.0001), pushing the rank of Fuhrman out of the model. When Spm and Spmd were dichotomized in quantitative variables, they were both independent factors. CONCLUSION: The EPA is a new prognostic tool, before surgery, which will be tested for its integration into prognostic normograms.

Effects of CD40 binding on ovarian carcinoma cell growth and cytokine production in vitro.

The poor prognosis associated with ovarian carcinoma (OVCA) is linked to the high incidence of local recurrence. There is a pressing need to identify factors that can play a role in OVCA growth and spread. Here, we focused on CD40, a member of the tumour necrosis factor (TNF) receptor superfamily with important functions in immune response. The expression of CD40 has been reported on various types of carcinoma cells, but its biological role is still poorly understood. The aim of the present study was to investigate the expression and function of the CD40 in OVCA cell lines. Detectable CD40 levels ranging from low to very high were found on the cell surface of several OVCA cell lines by flow cytometry analysis. Co-culture with a murine cell line transfected with CD40 ligand (CD40L) inhibited cell growth and up-regulated the secretion of proinflammatory cytokines interleukin (IL)-6, IL-8 and TNF-alpha in high-level CD40-expressing OVCA cell lines. Similarly, an increase of IL-6 and IL-8 release could be obtained by adding a soluble form of CD40L to the OVCA cultures. These results suggest that CD40-CD40L interaction is an important pathway affecting growth regulation and cytokine production in OVCA.

Interleukin-6 and vascular endothelial growth factor release by renal cell carcinoma cells impedes lymphocyte-dendritic cell cross-talk.

Anti-tumour T cell response requires antigen presentation via efficient immunological synapse between antigen presenting cells, e.g. dendritic cells (DC), and specific T cells in an adapted Th1 cytokine context. Nine renal cell carcinoma (RCC) primary culture cells were used as sources of tumour antigens which were loaded on DC (DC-Tu) for autologous T cell activation assays. Cytotoxic activity of lymphocytes stimulated with DC-Tu was evaluated against autologous tumour cells. Assays were performed with 75 grays irradiated tumour cells (Tu irr) and with hydrogen peroxide +/- heat shock (Tu H(2)O(2) +/- HS) treated cells. DC-Tu irr failed to enhance cytotoxic activity of autologous lymphocytes in seven of 13 assays. In all these defective assays, irradiated tumour cells displayed high interleukin (IL)-6 and vascular endothelial growth factor (VEGF) release. Conversely, when tumour cells released low IL-6 levels (n = 4), DC-Tu irr efficiently enhanced CTL activity. When assays were performed with the same RCC cells treated with H(2)O(2) + HS, DC-Tu stimulation resulted in improved CTL activity. H(2)O(2) + HS treatment induced post-apoptotic cell necrosis of tumour cells, totally abrogated their cytokine release [IL-6, VEGF, transforming growth factor (TGF)-beta1] and induced HSP70 expression. Taken together, data show that reduction in IL-6 and VEGF release in the environment of the tumour concomitantly to tumour cell HSP expression favours induction of a stronger anti-tumour CTL response.

Is magnetic resonance imaging texture analysis a useful tool for cell therapy in vivo monitoring?

Assessment of anti-tumor treatment efficiency is usually done by measuring tumor size. Treatment may however induce changes in the tumor other than tumor size. Magnetic Resonance Imaging Texture Analysis (MRI-TA) is presently used to follow activated lymphocyte cell therapy. We used a 7T microimager to acquire high-resolution MR images of an experimental liver metastasis from colon carcinoma in rats treated (n = 4) or not (n = 3) with a cell therapy product. MRI-TA was then performed with Linear Discriminant Analysis and showed: i) a significant variation of tumor texture with tumor growth and ii) a significant modification in the texture of tumors treated with activated lymphocytes compared with untreated tumors. T2-weighted images or volume calculation did not evidence any difference. MRI-TA appears as a promising method for early detection and follow-up of response to cell therapy.

Interleukin-2 expanded lymphocytes from lymph node and tumor biopsies of human renal cell carcinoma, breast and ovarian cancer.

Adoptive immunotherapy with immune effector cells has proved to be potent for treatment of tumors, however neither the attendant criteria for potential clinical efficacy of the injected cells, nor the method to prepare these cells are presently well established. Our procedure of collecting lymphocytes from biological samples, was based on the use of low IL-2 concentrations (90 to 150 IU/ml) and on the stringent separation of lymphocytes from tumor cells at the very early stages of their outgrowth in culture. When lymphocytes were derived from tumor biopsies (TIL), we observed differences depending on the histological type of tumor. In renal cell carcinoma, natural killer cells were expanded in 4/11 biopsies contrary to what was observed in breast cancer (92 +/- 5% of T lymphocytes from 9 biopsies). The outgrowth of lymphocytes from breast tumors was slower and lower than from renal carcinomas. The autologous tumor cell line was more difficult to obtain from breast carcinoma (23%) than from renal cell carcinoma (61%) biopsies. For ovarian cancer, short-term culture of tumor cells could be obtained for half of the tumor-invaded biological samples. Eight of the 23 tumor-derived cultures contained more than 40% CD8 T. TIL were consistently cytolytic each time they could be evaluated. For ascitic and pleural fluids, data were of similar range. In ascitic-derived cultures, tumor cells and antigen-presenting cells are present and can be supposed to rechallenge T cells with tumor antigens. Lymphocytes derived from lymph nodes could be expanded to a larger number than TIL. However, only 1/18 of these cultures contained more than 40% CD8 T. The presence of few tumor cells in this culture was in favor of significant specific and non-specific cytotoxicity in RCC lymph node cultures and higher percentages of CD8 T in breast cancer lymph nodes. Correlations could not be established between CD8 T percentages and specific in vitro cytotoxicity in our polyclonal populations. Our conclusion is that phenotypic and functional quality of lymphocytes is of interest when the T cells are derived 1) from tumors (RCC, breast or ovarian cancer) and isolated very early to avoid inhibitor factors secreted from tumor cells or 2) from lymph nodes and ascitic and pleural fluids when very few tumor cells are co-cultivated with lymphocytes at initial steps of culture. Final expansion to a number of lymphocytes suitable for therapy (> 109) could be attained in a second step of the procedure by the use of 1,000 IU/ml IL-2 each time it was assayed with 50.106 lymphocytes. In view of these data it appears that phenotypic and functional changes occur during culture depending on the presence of a particular ratio of tumor antigens. This could be artificially reproduced.

Polyamines in human breast cancer and its relations to classical prognostic features: clinical implications.

Experimental evidence suggest an important role of polyamines in breast cancer development. Polyamines have been determined in tissue and erythrocyte samples from 100 patients with primary invasive breast cancer and 30 patients with fibroadenomas. Statistical analysis was performed in order to determine the prognostic value of the polyamine patterns of tumor tissues and erythrocytes in comparison with clinical and histological prognostic factors. In malignant tissues, polyamine levels were significantly higher than in benign tissues. They correlated with markers of tumor aggressivity (axillary node involvement and especially with markers of high mitotic rate as Ki-67 staining, histological grade). No correlation was found between estrogen and progesterone status, tumor size and polyamine concentrations. Erythrocyte polyamines levels were identical between cancer patients and controls. The knowledge of the polyamine pattern in breast cancer could become useful in clinical practice particularly if polyamine metabolism is targeted as a therapeutic approach.

The gastrointestinal polyamine source depletion enhances DFMO induced polyamine depletion in MCF-7 human breast cancer cells in vivo.

Due to the polyamine requirement for cell growth, blockade of polyamine biosynthesis is considered a potential anticancer target. The lack of efficacy of DFMO in vivo, has been attributed to other sources of polyamines, mainly from the gastrointestinal tract (alimentary and bacterial). An experiment was designed to test the role of intestinal polyamine deletion in addition to DFMO (a specific inhibitor of ODC) in established MCF-7 tumors in nude mice. Using DFMO and polyamine-free diet, the tumor putrescine concentrations were more profoundly decreased in comparison to DFMO alone and cellular spermine was also depleted, as has never been observed with DFMO alone. The blockade of the gastrointestinal sources of polyamines enhances the intracellular polyamine depletion induced by DFMO on MCF-7 tumor in nude mice.

Neomycin inhibition of intestinal putrescine uptake.

Enhanced intestinal polyamine uptake was confirmed in tumor-bearing rats. Intestinal brush border membrane from rats bearing a Mat Lylu prostatic adenocarcinoma exhibited 46% increase in 14C putrescine uptake. After oral ingestion of 14C putrescine, radioactivity was recovered in the tumor (about 2% of total amount ingested) and was enhanced in liver and in blood. Radioactivity in tumor-bearing rat red blood cells (RBC) was two-fold higher than in healthy control rats indicating that polyamines originating in food can contribute to the enhancement of RBC polyamine levels observed after tumor development. Neomycin-induced inhibition of intestinal putrescine absorption was observed both in vitro and in vivo. In intestinal brush border membrane 500 microM neomycin inhibited putrescine uptake by 30-35%. In vivo, a single dose of neomycin given per os modified final 14C radioactivity distribution: a) The final accumulation of radioactivity in the tumor was reduced by 50%. b) Neomycin treatment lowered the high levels of radioactivity in the blood of cancerous animals. This reduction was correlated with a significant reduction of RBC polyamine levels. c) Radioactivity accumulated in the colon after neomycin treatment in both healthy and cancerous rats. The role of neomycin as an intestinal antibiotic and the presently described effect of this drug on intestinal polyamine uptake emphasize the importance of neomycin in polyamine deprivation treatments.

Tumour growth modifies intravascular polyamine transport by plasma lipoproteins in the mouse.

Polyamines are polycationic compounds which are implicated in cell division and tumor growth. We have evaluated the potential role of plasma lipoproteins in the transport of major polyamines, spermine, spermidine and putrescine, and the effect of tumor growth on such transport. Plasmas of healthy male BL6/DBA2 mice and of mice bearing Lewis lung carcinoma (3LL) were fractionated by isopycnic density gradient ultracentrifugation, and polyamine content determined in lipoprotein fractions. Spermidine was the most abundant polyamine in the lipoproteins of both control and tumor-bearing mice and was principally associated with HDL (d: 1.046-1.136 g/ml); approx. 40% of total plasma polyamines was lipoprotein-associated in control mice and 60% in cancerous mice. Only minor amounts were transported by LDL (< 10% of total lipoprotein-associated polyamines), while VLDL were devoid of these substances. Marked elevations of circulating levels of LDL were found in 3LL grafted mice: in these particles however, the contents of spermidine and spermine were significantly reduced. A preferential uptake of polyamines by red blood cells could in part explain this marked reduction of LDL polyamine content, but the consequence of this reduction on the net electrical charge and biochemical function of LDL remains unknown. Elevations of plasma LDL and HDL levels in 3LL-grafted mice underlie the finding that only minor modification was detected in the putrescine content of these particles. However, it is evident that elevated total amounts of putrescine were present in the plasma of such animals. Finally, the density profile of polyamines was modified in cancerous mice in which a shift to transport in lighter apo.AI-containing HDL particles was observed for spermidine; an even more marked shift was found for spermine. In conclusion, our data demonstrate that HDL particles constitute the major plasma vehicle for polyamine transport in both control and in tumor-bearing mice.

Synergistic activation of macrophage activity by polyamine deprivation and cyclophosphamide.

A synergistic reduction in tumor growth rate and of metastasis dissemination together with an increased life span, were observed when polyamine deprivation was combined with a low dose of cyclophosphamide. When rats were treated with this combination treatment, TNF and NO release was enhanced and phagocytic activity was increased. In addition, PGE2 release by macrophages was enhanced, whereas PGE2 plasma levels were restored to normal values. The decrease in PGE2 plasma levels presumably indicates the synergistic effect of the combination treatment on tumor cell metabolism. The enhancement of macrophage tumoricidal activities indicates immunostimulating synergistic effects, which may contribute to the anti-tumoral effect of the treatment.

Polyamine deprivation prevents the development of tumour-induced immune suppression.

Mice grafted with the 3LL (Lewis lung) carcinoma exhibit immune suppression: spleen cells showed decreased spontaneous interleukin 2 (IL-2) production and T-CD4+ and T-CD8+ lymphocyte populations; in addition the polyamine content in the spleen was increased. By treating the mice with a polyamine-deficient diet containing neomycin, metronidazole and inhibitors of ornithine decarboxylase and polyamine oxydase, tumour growth was reduced and the immune abnormalities were reversed. The spleen cells overproduced IL-2 by reducing exogenous sources of polyamines, but total blockade of all major polyamine sources was necessary to obtain an optimal effect both on IL-2 production and on spleen polyamine content. Irrespective of whether polyamine deprivation was started at an early or at an advanced stage of tumour growth, T-lymphocyte populations were restored to normal values, demonstrating that polyamine deprivation not only prevents tumour-induced immune suppression, but reverses established immunological disorders. In contrast to what was observed regarding IL-2 production by spleen cells and natural killer (NK) cell activity, the polyamine oxidase (PAO) inhibitor did not enhance the number of T lymphocytes. These findings are consistent with a direct effect of the polyamines on immune effector cell metabolism. They suggest an important role of the gastrointestinal polyamines and of PAO activity in the regulation of IL-2 production.

Erythrocyte spermine levels: a prognostic parameter in childhood common acute lymphoblastic leukemia.

Polyamines have been implicated to play a role in cell proliferation and in cancer development. Ninety percent of the circulating spermidine (Spd) and spermine (Spm) are transported by red blood cells (RBC). RBC Spd and Spm levels were prospectively determined in 63 unselected children with common acute lymphoblastic leukemia. The Spm and Spd levels were not correlated with white blood cell (WBC) count. On the basis of the polyamine levels it was possible to discriminate four groups with P< 10(-3). In C1, C2, C3 and C4 group the Spm level was respectively 90 (39-597), 3.75 (1-7.45), 9.95 (2.9-12.6) and 17(6.3-33.8). The probability of relapse-free survival (RFS) of the 58 children who entered complete remission was 55% +/- 9. For the groups C1 (n = 6), C2 (n = 16), C3 (n = 21) and C4 (n= 15) groups, the RFS was 25% +/- 20, 73% +/- 12, 73% +/- 13 and 32% +/- 13 respectively. For children with Spm levels <13/> or = 13nmol/8 x 10(9) RBC, event-free survival (EFS) was 54% +/- 11/33% +/- 10 and RFS was 64% +/- 12/38% +/- 11 respectively (P < 0.03, P < 0.005). Our clinical study shows clearly that an RBC spermine level could be used as parameter of prognosis at the time of diagnosis, particularly for patients with intermediary WBC count.

Inhibition of tumor growth and polyamine uptake by tetracyclic amidines bearing a putrescine moiety.

Tetracyclic amidines (tetrahydroquino[4.3-b][l]benzazepine: compound 11 and tetrahydrobenzo[k]naphthyridine: compound 12) bearing a putrescine moiety possess significant DNA-binding affinity. We report here that these compounds and their a and b isomers inhibit tumor cell growth and putrescine uptake in 3LL carcinoma cells in vitro. Moreover, compound 11 reduced by 50% the accumulation of putrescine in intestinal brush border membrane vesicles. In CHO-MG, a cell line deficient for the specific polyamine uptake system, the cytotoxicity of these compounds was significantly reduced compared to the CHO wild cell line. The IC50 for CHO-MG was significantly higher than for CHO, demonstrating that the polyamine transport system increased the efficacy of these compounds. The efficacy of compounds 11 and 12 might therefore be related to their ability to interact with DNA as well as their structural analogy with polyamines. Moreover, we clearly show that DFMO enhances the efficacy of these tetracyclic amidines in vivo. Potential mechanisms include: a) lower intracellular polyamine levels reduces polyamine DNA-stabilizing functions, increasing accessibility for DNA-binding drugs; b) DFMO enhances the polyamine uptake system in tumor cells, increasing the entry of tetracyclic amidines bearing a putrescine moiety as well as their accessibility to final DNA-binding sites. The fact that natural polyamine uptake is reduced by the same compounds constitutes an additive mechanism for antitumoral efficiency.