The circulating auto-antibodies to p53 protein in the follow-up of lymphoma patients.

Mutant p53 proteins may be targets of the host immune system - consequently a certain proportion of cancer patients (the percentage varies according to the type of cancer) with tumors that carry p53 missense mutations develop circulating p53 antibodies. The present study was aimed at defining the occurrence of circulating antibodies to p53 protein in patients with various types of non-Hodgkin's lymphomas (NHL). Altogether, the sera of 108 cases with various histological types of NHL and of 20 healthy controls were assessed for the presence of antibodies to p53 protein with an ELISA method. In 73 cases of NHL, also the immunohistochemical staining for p53 antigen was performed to make a rough estimation of the frequency of mutational events. The development of autoantibodies to p53 protein was observed in approximately 7% of NHL patients (predominantly in the more aggressive variants of the disease, but also in one case of small lymphocytic lymphoma). This proportion represents roughly one third of the number of patients assessed (immunohistochemically) to carry a missense p53 mutation in their tumors. The autoantibodies to p53 protein can be used as a tumor marker (early appearance, high specificity) in the follow-up of a subset of NHL patients, but, unfortunately, this subset comprises only approximately 7% of NHL patients.

Effectiveness of a simply designed tumor vaccine in prevention of malignant melanoma development.

We investigated the efficacy of a simple syngeneic tumor vaccine to induce specific antitumor immunity in female C57Bl/6 mice. Tumor vaccine was prepared by mixing irradiated B-16 melanoma tumor cells with the pleiotropic biological response modifier-maleic anhydride divinyl ether (MVE-2). Experimental animals were pretreated with the vaccine in order to prevent the development of intraperitoneal (i.p.) B-16 melanoma tumors after inoculation of viable tumor cells. More than 40% of prevaccinated animals challenged i.p. with 5 x 10(5) viable tumor cells were completely protected from tumor development and remained tumor-free 100 days after tumor cell inoculation. The percentage of tumor-free animals (survivors) rose to as much as 90% when the application of tumor vaccine was repeated two weeks after the first vaccination (i.e. one week after the inoculation of viable tumor cells). The induced antitumor response depended predominantly upon macrophage function, since vaccinated animals which were depleted of peritoneal macrophages died within the same time range as animals in the control group. Also, tumor-type specificity of the vaccine was confirmed by the fact that the animals vaccinated with B-16 melanoma vaccine were not protected from the development of another type of tumor. In conclusion, comparison of the experimental data with the data from the literature suggests that our simple tumor vaccine may be as effective as genetically engineered tumor vaccines. At the same time, this kind of vaccine is easier to control and thus safer to apply in humans when compared to genetically engineered vaccines.

An effective tumor vaccine against malignant melanoma: irradiated autologous tumor cells admixed with MVE-2.

The aim of this study was to develop as effective as possible autologous tumor vaccine which would be at the same time easy to produce, highly controllable, and without undesired side effects on normal tissue. Therefore, irradiated autologous - syngeneic B-16 tumor cells admixed with a non-specific immunomodulator MVE-2 (a polymer fraction of 1,2-co-polymer of divinyl ether and maleic anhydride) were used for subcutaneous (s.c.). or intraperitoneal (i.p.) prevaccination of experimental mice. Compared to the control mice, a statistically significant delay in tumor development of s.c. tumors was achieved in prevaccinated mice (p<0.05). An even better effect was observed in mice challenged i.p. with viable tumor cells. Using a single prevaccination complete protection was obtained in between 40-85% of the experimental mice. When the survivors from the groups injected once with the tumor vaccine were rechallenged with viable tumor cells (101 day after the first tumor challenge, no additional prevaccination), 15.7% remained free of tumor, while the survivors from the groups injected with the tumor vaccine twice and 101 day later rechallenged with viable tumor cells remained free of tumor in 60% of the cases. Based on these results we can postulate that our vaccine is effective for prevention of tumor development. The achieved protection can be augmented with serial vaccinations and can be maintained for a longer period of time.

Transfection of mammalian cells by the methods of receptor mediated gene transfer and particle bombardment.

The efficacy of currently developed methods for gene transfer into mammalian cells depends primarily on the transfection technique, and also on the type of targeted cells. Considering the importance of gene transfer in the creation of gene therapies, our study was aimed at the assessment of transfection capacity of receptor mediated gene transfer method (RMGT), and method of particle bombardment (helios gene gun system--HGG) in different normal and malignant mammalian cells ex vivo. In addition, the HGG was also assessed for its ability to transfect tumor cells of subcutaneous (s.c.) tumors in C57Bl/6 mice in vivo. Using RMGT an average ex vivo transfection rate of 35.7%, and 20.4% was achieved in malignant melanoma B-16, and human breast adenocarcinoma MCF7, respectively. However, in normal fibroblast L929 cells the transfection by RMGT succeeded only in 2.1% of the cells. On the other hand, the transfection efficacy of HGG was comparable in both malignant cell lines resulting in an average gene transfer to 9.6% of B-16 and 10.5% of MCF7 cells, while only 3.9% of normal fibroblasts were successfully transfected. Application of HGG for an in vivo gene transfer into s.c. B-16 melanoma tumors in C57Bl/6 mice resulted in a successful but limited transfection of the epithelium as well as of the superficially sited tumor cells. Taking into consideration both methods, RMGT is more appropriate for ex vivo transfection of cells, under the condition that target cells express a specific receptor for the molecule attached to the carrier. On the other hand, HGG is not complicated to use, no requirements for specific structures on target cells are necessary (potentially usable in different cells), and it has applicability in direct in vivo transfection processes.

New TNF-alpha analogues: a powerful but less toxic biological tool against tumours.

Our approach to the modification of recombinant human tumour necrosis factor alpha (rhTNF-alpha) comprised changes in flexible loop regions on the surface of the TNF molecule. Using this approach, two different rhTNF-alpha analogues LK 801 and LK 805 were synthesized and tested for their ability to affect the growth of Sa-1 tumour cells. Results obtained in vitro indicate that neither rhTNF-alpha nor its analogues have a direct cytotoxic effect. In vivo experiments were performed on subcutaneous Sa-1 tumours in A/J mice, where the antitumour effect and the toxic side effects of the cytokines were followed. There was no significant difference between growth delay of tumours in animals treated with native rhTNF-alpha and in animals treated with one of the analogues. On the contrary, the LD50 for rhTNF-alpha was 29.1 microg, for LK 801 59.3 microg, and for LK 805 even 66.1 microg, indicating that LK 801 and especially LK 805 were significantly better tolerated. The results confirm that the rhTNF-alpha molecule has been successfully modified resulting in two new analogues with a potent antitumour activity and much lower systemic toxicity. A particularly low systemic toxicity and a strong antitumour effect were observed after treatment with LK 805 suggesting that this analogue merits further investigation in pre-clinical and clinical trials.

Clinical evaluation of potential usefulness of CEA, CA 15-3, and MCA in follow-up of breast cancer patients.

The potential usefulness of MCA, CA 15-3 and CEA in monitoring of breast cancer patients was evaluated in 135 female patients with histologically confirmed breast cancer. The patients were classified into two groups as follows: group of patients with no evidence of disease, NED; and group of patients with progressive disease, PD. In total, 2106 measurements of CEA, CA 15-3, and MCA were performed using an enzyme immunoassay. Serum levels of all three markers in the NED group differed significantly from those of patients with PD. The observed differences in the sensitivity and specificity of CEA, CA 15-3, and MCA tests were not significant. The serum concentrations of a particular marker correlated well with the concentrations of the other two markers, except when CEA was correlated with MCA or CA 15-3 in NED group patients. The elevation of tumor markers preceded by some 7 months the clinical evidence of dissemination, and marker levels reflected at a high percentage the response to therapy in PD patients. Therefore, this clinical study confirmed that MCA, CA 15-3 and also CEA are suited to discriminate between disease and disease-free periods, and also validated the usefulness of markers for treatment response monitoring.

Interactions of interferon and vinblastine on experimental tumor model melanoma B-16 in vitro.

In this study, we tried to define in vitro interactions of two antitumor agents that have different sites and different mechanisms of action. Vinblastine (VLB) in combination with human recombinant interferon-alpha A/D (rHuIFN-alpha A/D) and in combination with murine recombinant interferon-gamma (rMuIFN-gamma) was studied. The effect of the combination was determined with cell growth kinetics assay on B-16 melanoma and the interaction defined by means of Spector's formula. Both the combination of rHuIFN-alpha A/D with VLB and the combination of rMuIFN-gamma with VLB synergistically inhibited cell growth in vitro. There was a positive biochemical modulation between the two drugs, but it is still unknown whether it occurred at the level of uptake into the cell, metabolism within the cell or egress from the cell.