Combined metallothioneins and p53 proteins expression as a prognostic marker in patients with Dukes stage B and C colorectal cancer.

Our study aimed to evaluate metallothionein and p53 expression in colorectal cancer and to correlate their combined expression with selected clinical and pathologic variables of the disease, to define their prognostic significance. Colorectal cancer specimens from 99 patients were retrospectively analyzed by immunohistochemistry for metallothionein and p53 expression. Survival curves were generated according to the Kaplan-Meier method, and univariate survival distributions were compared with the use of the log-rank test. Multivariate models were computed using Cox proportional hazards regression. This research was approved by the institutional review boards of all centers. Tumors showing concomitant high metallothionein expression and negative p53 (metallothionein(H)/p53(-)) were significantly inversely related to depth of invasion, frequency of nodal metastasis, and Dukes stage (P < .01). In univariate analysis, patients with metallothionein(H)/p53(-) phenotype showed a better overall survival (hazard ratio [HR], 2.83; P < .05) and disease-free survival (HR, 2.03; P < .05). In multivariate analysis, considering staging, metallothionein, and metallothionein + p53 variables, in 83 patients with Dukes stages B and C, metallothionein(H)/p53(-) combination was the sole factor showing an independent prognostic value for overall survival (HR, 3.88; P < .1) and disease-free survival (HR, 2.56; P < .1). In conclusion, the combined analysis of metallothionein and p53 may enhance the prognostic power of each individual marker by predicting the progression of the disease and contributing to a better identification of patients at low risk for mortality, especially for those with Dukes stage B and C colorectal cancer.

IL-15 and IL-2 increase Cetuximab-mediated cellular cytotoxicity against triple negative breast cancer cell lines expressing EGFR.

Triple negative breast cancer (TNBC) patients are not likely to benefit from anti-estrogen or anti-HER2 therapy and this phenotype is associated with a more aggressive clinical course and worse clinical outcome. Taking into account the limited treatment possibilities in TNBC, the aim of the present work was to study a potential therapy based on Cetuximab-mediated immune activity by natural killer (NK) cells. We performed in vitro studies on human breast cancer (BC) cell lines, IIB-BR-G, and the in vivo metastatic variant IIB-BR-G MT. The immunohistochemical analysis showed a TNBC phenotype with high but different levels of EGFR expression on each cell line, measured by flow cytometry. DNA sequencing showed that both cell lines have a mutated K-RAS status, 38 G > A at codon 13. Consequently, Cetuximab did not inhibit cellular proliferation or induce apoptosis. We investigated if Cetuximab could trigger immune mechanisms, and we determined that both cell lines treated with 1 µg/ml Cetuximab were susceptible to antibody dependent cellular cytotoxicity (ADCC), mediated by peripheral blood mononuclear cells (PBMC). At 50:1 effector:target ratio, lytic activity was 34 ± 2% against IIB-BR-G and 27 ± 6% against IIB-BR-G MT cells. PBMC pretreatment with IL-2 allowed reaching 65 ± 3% of Cetuximab-mediated ADCC against IIB-BR-G and 63 ± 6.5% against IIB-BR-G MT. Furthermore, IL-15 pretreatment increased the ADCC up to 71 ± 3% in IIB-BR-G and 79 ± 3.5% in IIB-BR-G MT. We suggest that NK cells are the effectors present in PBMC since they were able to induce ADCC at lower effector:target ratios. Besides, IL-2- and mainly IL-15-induced upregulation of NK activating receptors CD16 and NKG2D and enhanced IFN-γ production. EGFR-expressing TNBC could be killed by Cetuximab-mediated ADCC at clinically achievable concentrations. IL-15 could advantageously replace IL-2 in most of its immunologic activities, stimulating the ability to produce IFN-γ, and paralleling the up-regulation of activating receptors.

Mutant p53 protein in serum could be used as a molecular marker in human breast cancer.

p53 wild-type is a tumor suppressor gene involved in DNA gene transcription or DNA repair mechanisms. When damage to DNA is unrepairable, p53 induces programmed cell death (apoptosis). The mutant p53 gene is the most frequent molecular alteration in human cancer, including breast cancer. Here, we analyzed the genetic alterations in p53 oncogene expression in 55 patients with breast cancer at different stages and in 8 normal women. We measured by ELISA assay the serum levels of p53 mutant protein and p53 antibodies. Immunohistochemistry and RT-PCR using specific p53 primers as well as mutation detection by DNA sequencing were also evaluated in breast tumor tissue. Serological p53 antibody analysis detected 0/8 (0%), 0/4 (0%) and 9/55 (16.36%) positive cases in normal women, in patients with benign breast disease and in breast carcinoma, respectively. We found positive p53 mutant in the sera of 0/8 (0.0%) normal women, 0/4 (0%) with benign breast disease and 29/55 (52.72%) with breast carcinoma. Immunohistochemistry evaluation was positive in 29/55 (52.73%) with mammary carcinoma and 0/4 (0%) with benign breast disease. A very good correlation between p53 mutant protein detected in serum and p53 accumulation by immunohistochemistry (83.3% positive in both assays) was found in this study. These data suggest that detection of mutated p53 could be a useful serological marker for diagnostic purposes.

Androgen receptors in human melanoma cell lines IIB-MEL-LES and IIB-MEL-IAN and in human melanoma metastases.

The presence and characteristics of androgen receptors (ARs) have been described by our group in one human melanoma cell line. We have now investigated their presence in two other human melanoma cell lines, IIB-MEL-LES and IIB-MEL-IAN, as well as in biopsies from human metastatic melanoma. Scatchard analysis revealed a single binding component for both cell lines, the apparent dissociation constant obtained being 15 nM, with a binding capacity of 280 fmol/mg total cell protein, for IIB-MEL-LES cells and 14 nM, with a binding capacity of 206 fmol/mg total cell protein for IIB-MEL-IAN cells. When specificity was assessed, not only androgen and anti-androgen but also non-androgenic compounds were able to compete for [3H]R1881 binding, as seen before. When immunocytochemistry of IIB-MEL-LES and IIB-MEL-IAN cells was performed for ARs, both cell lines were deeply stained in the nucleus, whereas no staining was found for oestrogen or progesterone receptors. Every specimen of melanoma metastases tested for the presence of ARs was deeply stained, and in the majority the intensity of the staining was high. Several hormones and anti-hormones were tested for their ability to affect cell proliferation. In both cell lines, testosterone, dihydrotesterone, oestradiol and progesterone significantly stimulated cell proliferation, and this was reversed by hydroxyflutamide, bicalutamide or tamoxifen.

Inflammatory changes after cryosurgery-induced necrosis in human melanoma xenografted in nude mice.

There is growing evidence that necrosis, instead of apoptosis, could act as a natural adjuvant, which could activate an immune response. In this work we have investigated if induction of tumor necrosis could trigger the affluence of inflammatory cells at the tumor site, and thus induce an immune response. For this purpose, a liquid N2 spray was applied on human melanoma (IIB-MEL-J cell line) xenografted in nude mice and 24 h later some mice received intratumorally a single 500 U dose of recombinant murine granulocyte macrophage-colony-stimulating factor. 77-100% of the tumor mass underwent necrosis. Congestion, edema, and endothelial cell activation were the first noticeable events. A quick infiltrative response of polymorphonuclear leukocytes around the tumor was detected 24 h after liquid N2 application, peaking at day 3. Massive macrophage recruitment was observed since day 3. An early intratumoral infiltration with inflammatory cells was only detected in the group that received recombinant murine granulocyte macrophage- colony-stimulating factor after necrosis induction by liquid N2. Coexisting DEC 205- and F4/80-positive cells increased in number, and their localization was predominantly peritumoral after necrosis. Antibody response was only detected in the groups with tumor-induced necrosis. Our results suggest that cryosurgery-induced necrosis could be a useful model to analyze the interaction among necrosis, inflammation, and the generation of an immune response.

Differential lytic and agglutinating activity of the anti-Lewis(x) monoclonal antibody FC-2.15 on human polymorphonuclear neutrophils and MCF-7 breast tumor cells. In vitro and ex vivo studies.

The Lewis(x) (Le(x)) trisaccharide (CD15) linked to proteins and glycolipids is highly expressed on the surface of normal human polymorphonuclear neutrophils (PMN) and several human neoplasias, such as breast and gastrointestinal carcinomas and chronic myeloid leukemias. FC-2.15 is an IgM murine mAb that specifically recognizes Le(x) and has been previously shown to mediate the in vitro lysis of Le(x)(+) cells by human complement. In a phase I clinical trial of FC-2.15, a temporary neutropenia was the main toxicity, and antitumor responses were observed. In order to characterize FC-2.15 further and determine the physiological relevance of Le(x) binding, the reactivity of FC-2.15 on PMN was investigated under several conditions. Flow cytometry revealed a strong reactivity of FC-2.15 with almost 100% of PMN, and Scatchard analysis demonstrated an affinity constant of 5.14 x 10(9) M(-1) and 1.11 x 10(6) antigen sites/cell. In vitro, the binding of Le(x) epitopes by FC-2.15 induced PMN homotypic aggregation, only 28.4 +/- 4.1% remaining as single cells. When PMN and the Le(x)(+) MCF-7 breast cancer cells were co-incubated, FC-2.15 induced heterotypic aggregation. In 51Cr-release assays employing human complement, FC-2.15 lysed 93.4 +/- 7.9% of PMN and 87.8 +/- 10.7% of MCF-7 cells. However, when the effect of FC-2.15 was tested in ex vivo circulating blood, no lytic activity against PMN was detected, whereas MCF-7 cells were still lysed. Blood smears demonstrated that FC-2.15 induced PMN agglutination and heterotypic aggregates when MCF-7 cells were present. A pretreatment of PMN with colchicine impaired PMN agglutination both in vitro (single PMN = 81.15 +/- 4.35%) and in ex vivo circulating blood. In the latter condition, FC-2.15-lytic activity was restored, suggesting that PMN homotypic aggregation by FC-2.15, but not lysis, is dependent on microtubule integrity and that PMN agglutination hinders their lysis. Moreover, when 51Cr-release assays were performed following agglutination, FC-2.15 cytotoxicity was restricted to isolated PMN. It is suggested that crosslinking of Le(x) epitopes by FC-2.15 induces PMN to form homotypic aggregates. It is suggested that the neutropenia observed in FC-2.15-treated patients would be due to PMN agglutination and margination, rather than lysis. In addition, FC-2.15 appears to be able to lyse Le(x)(+) tumor cells in circulation.

Different efficacy of in vivo herpes simplex virus thymidine kinase gene transduction and ganciclovir treatment on the inhibition of tumor growth of murine and human melanoma cells and rat glioblastoma cells.

Initial studies have demonstrated the therapeutic efficacy for cancer treatment of in vivo transfer of the herpes simplex virus thymidine kinase gene followed by ganciclovir (GCV) treatment. However, recent studies have questioned the validity of this approach. Using retroviral vector-producing cells (VPC) as a source for in vivo gene transfer, we evaluated the efficacy of in vivo transduction of malignant cells using three different tumor cell models: B16 murine and IIB-MEL-LES human melanomas and a C6 rat glioblastoma. In vitro studies showed a bystander effect only in C6 cells. In vivo studies showed an inhibition of tumor growth in the two melanoma models when tumor cells were coinjected with VPC-producing retroviral vectors carrying the herpes simplex virus thymidine kinase gene, followed by GCV treatment; however, 100% of mice developed tumors in both models. Under similar experimental conditions, 70% (7 of 10) of syngeneic rats completely rejected stereotactically transferred C6 tumor cells; most of them (5 of 10) showed a prolonged survival. Treating established C6 tumors with VPC-producing retroviral vectors carrying the herpes simplex virus thymidine kinase gene and GCV led to the cure of 33% (4 of 12) of the animals. Rats that rejected tumor growth developed an antitumor immune memory, leading to a rejection of a stereotactic contralateral challenge with parental cells. The immune infiltrate, which showed the presence of T lymphocytes, macrophages, and polymorphonuclear cells at the site of the first injection and mainly T lymphocytes and macrophages at the site of tumor challenge, strengthened the importance of the immune system in achieving complete tumor rejection.

A new epitope on human melanoma-associated antigen CD63/ME491 expressed by both primary and metastatic melanoma.

A monoclonal antibody (MAb) designated FC-5.01 (IgG2a) was generated that binds to several human carcinomas and malignant melanoma. It has revealed no or very low reactivity with most human normal tissues, except for the fact that FC-5.01 binds to some cells from the neuroendocrine system, macrophages, and some renal proximal convolute tubules with an intracellular pattern. Biochemical studies indicate that FC-5.01 recognizes a heterogeneous glycoprotein (broadband between 30-60 kDa) in melanoma tumors. The epitopes reside in the protein core and are presumably conformational, with disulphide bonds implicated in MAb recognition. The current study presents evidence that MAb FC-5.01 reacts with CD63 antigen (Ag), which has been initially described as a melanoma associated Ag, and is a member of the tetraspan family. Reactivity of MAb FC-5.01 with CD63 was demonstrated by Western blot, immunodepletion assay, and FACS analysis of the CD63-negative melanoma cells (KM3) after transfection with the genomic copy of CD63. The epitope recognized by MAb FC-5.01 was shown to be different from the epitope recognized by another anti-CD63 MAb, ME491, by an inhibition radioimmunoassay. Opposite to what has been stated for MAb ME491, no significant differences were found in CD63 expression between primary and metastatic melanoma using MAb FC-5.01.

[Antitumor gene therapy using suicide genes]. / Terapia génica antitumoral mediante el uso de genes suicidas.

Tumor cells transduced with retrovirus carrying the herpes simplex-1 virus thymidine kinase (HSV-tk) are capable of transforming the antiviral drug ganciclovir (GVC) into a metabolic form only toxic to dividing cells. The efficiency of this suicide gene therapy is increased by a "bystander" effect resulting not only in the death of the recipient cell, but also in the death of non modified surrounding cells. Even though the mechanism of this "bystander" effect remains to be elucidated, strong evidence suggest that the immune system plays a main role to achieve complete tumor eradication. In the present study we evaluate the efficiency of this suicide system on three different tumor models: one human melanoma, one murine melanoma, and a rat glioblastoma. Tumors were established by injection of tumor cells s.c. in nude and C57Bl/6 mice, respectively, and stereotactically into the brain of Sprague Dawley rats. Animals in the treated group were co-injected with packaging cells producing recombinant retrovirus carrying the HSV-tk gene, and followed by i.p. administration of GVC. In short term studies, we observed inhibition of tumor growth for all the tumor models evaluated (p < 0.01). In long term studies, using the C6 rat glioma line, 50% of the animals survived longer than 75 days (p < 0.0001), and were able to reject a contralateral challenges with C6 parental cells. Histological and immunohistochemical analysis showed the presence at an inflammatory infiltrate composed by T lymphocytes, macrophages and polymorphonuclear cells. These data demonstrate that suicide genes might represent an attractive form of cancer gene therapy in the treatment of brain tumors and their intracerebral dissemination.

The human breast cancer cell line IIB-BR-G has amplified c-myc and c-fos oncogenes in vitro and is spontaneously metastatic in vivo.

IIB-BR-G is an undifferentiated, highly heterogeneous, hormone receptor negative human breast cancer cell line previously established in our laboratory from a patient's primary tumor. An in vitro growing cell line (IIB-BR-G) and a xenotransplanted tumor growing in nude mice (IIB-BR-G(NUDE)) were derived. To further characterize these systems, immunocytochemical analysis was performed for differentiation antigens (PEM 200 kDa, CEA, NCA 90 kDa), blood-group related antigens (Le(x), sTn), oncogenes and tumor suppressor gene products (Her-2/neu protein, p53), metastasis-related cathepsin D and CD63/5.01 Ag, and the chemokine monocyte chemotactic protein 1 (MCP-1). Expression of markers was heterogeneous in these different systems. Previously reported karyotypic analysis has shown extensive chromosomal alterations including double min. Searching for oncogene amplification, we detected augmented copy number of c-myc and c-fos, the last one with two rearranged fragments. No amplification was found for c-erbB-2 in the cell line or in IIB-BR-G(NUDE), although this oncogene was amplified in the patient's primary tumor DNA. The differences observed between the patient's tumor, the cell line and the IIB-BR-G(NUDE) tumors are probably due to clonal expansion of cell variants not present in the original tumor. Electron microscopy of IIB-BR-G growing cells revealed epithelial characteristics with abundant dense granules, presumably secretory, distributed all over the cytoplasm and great nuclear pleomorphism. In vitro, IIB-BR-G cells showed a significant number of invading cells by Matrigel assay. After nearly 40 sequential subcutaneous passages of the original xenograft through nude mice, 80% of recipients developed spontaneous metastases, primarily to the lung and lymph nodes. Since this experimental model allowed to analyze changes produced in cancer cells from the primary tumor during adaptation to in vitro and in vivo growth, our results provide novel insights on the behaviour of hormone independent metastatic breast cancer.

Reactivity of monoclonal antibody FC-2.15 against drug resistant breast cancer cells. Additive cytotoxicity of adriamycin and taxol with FC-2.15.

Monoclonal antibody (MAb) FC-2.15 recognizes Lewis x antigen (Le(x)-Ag) expressed on the cell surface of most human breast cancer cells. FC-2.15 displays important human complement (C')-mediated cytotoxicity (CMC) against its target cells. In this study the reactivity of FC-2.15 against drug resistant-breast cancer cells was investigated, as well as the possibility to combine the antitumor activities of this MAb with adriamycin (Adr) or taxol. Since resistant clones with altered expression of tumor-associated antigens usually emerge after chemotherapy, the expression of Le(x)-Ag was analyzed in Adr(R) MCF-7 breast cancer cells (Adr resistant subline) and in tumor samples from nine patients with locally advanced breast carcinoma who were treated with FEC chemotherapy. A flow cytometry assay showed that most of Adr(R) MCF-7 cells, as well as wild type (WT) cells, expressed Le(x)-Ag; however, the Le(x) epitope is probably bound to different backbones in these cells. When the cytotoxic ability of FC-2.15 against WT and Adr(R) MCF-7 cells was compared, it was found that a 90 min treatment with FC-2.15 plus C' induced similar CMC against both cell lines. An important cytolysis was obtained at 5 microg/ml FC-2.15, reaching a plateau at 25 microg/ml, at which cell population was diminished to 21.1% for WT and 27.9 for Adr(R) MCF-7 cells. Regarding human tumors, Le(x)-Ag expression was evaluated in samples obtained before and in most cases after chemotherapy, and it could be observed that: 1) before treatment, tumor samples from all patients analyzed (responders and non-responders to chemotherapy) were FC-2.15-positive; 2) the presence of Le(x)-Ag was not modified after treatment. The combined action of Adr or taxol with FC-2.15 was then evaluated. WT and Adr(R) MCF-7 cells were cultured with Adr or taxol followed by an incubation with different FC-2.15 concentrations plus C'. When the effect of Adr alone was determined, ID50 were 1 x 10(-7) M for WT and 4.2 x 10(-5) M for Adr(R) MCF-7 cells. The cytotoxic ability of taxol alone was also tested, and ID50 were 6.4 x 10(-9) M for WT and 3.1 x 10(-6) M for Adr(R) MCF-7 cells. When FC-2.15 was added to Adr or taxol, the cytotoxicity of the drug-FC-2.15 combined treatment was always higher than the isolated effects, showing additive cytotoxicity at the different concentrations tested and with both cell lines. Our results suggest that FC-2.15 may be a useful agent against breast tumor cells which survive chemotherapy with Adr or taxol.

FC-2.15, a monoclonal antibody active against human breast cancer, specifically recognizes Lewis(x) hapten.

FC-2.15 is a murine IgM monoclonal antibody that recognizes breast and colon human carcinomas, chronic myeloid leukemias, Sternberg cells of Hodgkin's lymphoma and some normal cells, such as peripheral polymorphonuclear granulocytes. It has been previously demonstrated that FC-2.15 recognizes the carbohydrate moiety of different glycoproteins. FC-2.15 is able to mediate the in vitro lysis of Ag-2.15+ cells by human complement. In a phase I clinical trial, FC-2.15 induced antitumor responses and reversible neutropenia was its main toxicity. In this work, analysis of epitope specificity has demonstrated that FC-2.15 specifically recognizes terminally exposed Lewis(x) trisaccharide but not sialyl-Lewis(x), Lewis(a), trifucosylated Lewis(y), blood-group antigens A and B, globo H and gangliosides. In polymorphonuclear granulocytes (PMN), myeloid leukemic cells and colon carcinoma T84 cells, Lewis(x) was found to be almost exclusively N-linked to the protein core, whereas in breast carcinoma MCF-7 cells, Lewis(x) appeared to be mostly O-linked. Treatment with neuraminidase increased detection by FC-2.15 in normal PMN, myeloid leukemia cells and T84 cells but not in MCF-7 cells.

Monoclonal antibody FC-5.01, directed against CD63 antigen, is internalized into cytoplasmic vesicles in the IIB-BR-G human breast cancer cell line.

Monoclonal antibody (MAb) FC-5.01, raised against the undifferentiated breast cancer cell line IIB-BR-G, has been recently shown to react with CD63. The antigen (Ag) recognized by MAb FC-5.01 is expressed in plasma membranes of IIB-BR-G and other neoplastic cells, as well as in activated platelets and endothelial cells, as detected by indirect immunofluorescence performed at 4 degrees C on live cells. In permeabilized cells, MAb FC-5.01 colocalizes with acridine orange in acidic vesicles (lysosomal/endosomal compartment). Scatchard plot analysis performed on IB-BR-G cells demonstrated a 1.4+/-0.4 x 10(7) M(-1) affinity constant and 2.1 x 10(6) antigenic sites per cell. MAb FC-5.01 is not able to mediate C fixation or ADCC toward CD63+ cells, but the FC-5.01-CD63 complex is efficiently internalized into cytoplasmic vesicles, as shown by an acid wash immunofluorescence assay. Cellular catabolism of the antibody bound by IIB-BR-G cells was studied using [125I]-FC-5.01. At 18 h, >70% of the radioactivity was present in the supernatant as degraded fragments (TCA-soluble). After internalization, rapid Ag re-expression could be demonstrated in IIB-BR-G cells. MAb FC-5.01 diminished migration of CD63+ cells in a Boyden chamber assay. Some of the above-mentioned properties would enable the use of MAb FC-5.01 as a vehicle to target different compounds inside CD63+ cells.

Suppression of SPARC expression by antisense RNA abrogates the tumorigenicity of human melanoma cells.

Acquisition of invasive/metastatic potential is a key event in tumor progression. Cell surface glycoproteins and their respective matrix ligands have been implicated in this process. Recent evidence reveals that the secreted glycoprotein SPARC (secreted protein, acidic and rich in cysteine) is highly expressed in different malignant tissues. The present study reports that the suppression of SPARC expression by human melanoma cells using a SPARC antisense expression vector results in a significant decrease in the in vitro adhesive and invasive capacities of tumor cells, completely abolishing their in vivo tumorigenicity. This is the first evidence that SPARC plays a key role in human melanoma invasive-metastatic phenotype development.

The expression of the secreted protein acidic and rich in cysteine (SPARC) is associated with the neoplastic progression of human melanoma.

SPARC (secreted protein acidic and rich in cysteine) is an extracellular protein associated with tissues exhibiting high rates of cell proliferation and matrix remodeling. The current work shows that the human melanoma cell lines IIB-MEL-LES, IIB-MEL-IAN, and IIB-MEL-J and different human metastatic melanomas expressed high levels of SPARC mRNA and protein. By western blot analysis we detected a single secreted 42-kDa band in human diploid fibroblasts-conditioned medium and a 45- to 40-kDa doublet in the three melanoma cell lines and all the metastatic melanomas tested. Part of the melanoma samples and cell lines showed an additional doublet of 36-34 kDa. SPARC mRNA was expressed by the three established cell lines, 14 metastatic melanoma samples, and tumors raised in nude mice, and no spliced variants were found. The heterogeneous pattern of SPARC secreted by human melanoma cells is the result of post-translational glycosylation and a specific extracellular leupeptin-inhibitable cleavage. Unlike human fibroblasts, melanoma cells did not overexpress SPARC on addition of TGF-beta. Immunohistochemical analysis showed that SPARC was strongly expressed in 100% of primary melanomas (7 of 7) and metastatic melanomas (29 of 29), moderately expressed in most of the positive dysplastic nevi (13 of 14), and only weakly expressed in nevocellular nevi (4 of 25). Normal melanocytes did not express SPARC. The data suggest that the expression of SPARC is associated with the neoplastic progression of human melanoma.

Allogeneic cells vaccine increases disease-free survival in stage III melanoma patients. A non randomized phase II study.

The incidence of melanoma is increasing rapidly, and in many cases the primary tumor is excised after metastatic spreading. In 80% of the cases, the first metastatic site is in regional lymph nodes (AJCC Stage III). After excision of these nodes, the patient is clinically disease-free, but the chances of recurrency vary between 40-80%. Thirty patients with stage III melanoma were treated in a non-randomized Phase II adjuvant trial with a vaccine consisting of a mixture of three allogeneic cell lines: IIB-MEL-J, IIB-MEL-LES and IIB-MEL-IAN (5 x 10(6) cells each). The cells were irradiated (5,000 cGy) and BCG was used as nonspecific stimulant. Before each vaccination (72 hr) the patients received cyclophosphamide (300 mg/sqm). The untreated control group was composed of 24 Stage III melanoma patients. Vaccination started within 60 days after surgery, and patients received 4 vaccinations, one every 21 days and then 1 every two months during the 1st year; 1 every three months during the 2nd year, and 1 every 6 months during the 3rd, 4th and 5th years. The treated group was composed by 19 men (63.3%) and 11 women (36.7%); average age: 47.6 +/- 14.1 years (range: 16-70 yr). The control group was composed by 18 men (75%) and 6 women (25%); average age 49.8 +/- 14.2 yr (range: 26-73 yr). The median disease free survival (DFS) calculated according to Kaplan-Meier was 7.0 months in the control group vs 20.0 months in the treated group (p < 0.001). The results of this clinical trial suggest that treatment with allogeneic cell vaccines increases DFS in stage III melanoma patients.

Tumor cells engineered to express interleukin-6 exhibit a reduced tumorigenicity depending on the tumor cell model.

Cytokine gene transfer to tumor cells has been demonstrated to induce tumor rejection in different murine models. However, controversial results were presented for different cytokines. In order to study the antitumorigenic activity that has been proposed for IL-6, the poorly immunogenic melanoma B16 and the colon adenocarcinoma CT26-murine cell lines, were transduced with recombinant retrovirus expressing rat IL-6. In vivo studies showed that IL-6-producing-B 16 cells inoculated s.c. in syngeneic mice, exhibited reduced tumorigenicity compared to vector-transduced B 16 cells. The histology of growing IL-6-producing tumors showed a "pseudo-nodular" pattern which correlated with a strong inhibition of the in vitro invasive capacity of these cells. IL-6-producing-B 16 cells did not develop tumors in athymic nude mice suggesting that the antitumor effect is not mediated by a normal host-T- and B-cell response. In contrast, IL-6-producing CT26 cells grew as tumors in syngeneic mice with a faster growth rate than parental and vector-transduced cells, in accordance with an increased in vitro growth kinetics. These results indicate that IL-6 expression by tumor cells demonstrate different effects depending on the tumor cell model.

Identification and characterization of a 14 kDa immunosuppressive protein derived from IIB-MEL-J, a human melanoma cell line.

The causes of decreased immune competence in melanoma patients as well as in other cancer patients are incompletely understood. The identification of the factor(s) responsible for this behaviour remains elusive. The present report demonstrates that an immunosuppressive activity (ISA), manifested in vitro as an inhibition of proliferation of human peripheral blood lymphocytes (PBL) induced both by phytohemagglutinin (PHA) or the cytokine IL-2, was exhibited by serum-free conditioned medium (SFCM) from the human melanoma cell line IIB-MEL-J, as well as by two other melanoma cell lines, IIB-MEL-LES and IIB-MEL-IAN, established in our laboratory. The ISA was found to be exerted by a protein, which co-eluted with serum albumin in anionic exchange Mono-Q, gel filtration chromatography and Blue Sepharose columns. It showed a molecular weight (Mw) of 14 kDa when separated from albumin traces by means of a Sephacryl S 200 column. It is not recognized by a pan-specific anti-transforming growth factor-beta (TGF-beta) antibody as determined by Western blots assays performed on the SFCM. The immunosuppressive factor (ISF) is secreted by IIB-MEL-J cell line in soluble from and in very scarce amounts, non-detectable by polyacrylamide gel electrophoresis (PAGE). This characteristic difficults its obtention in adequate quantities to sequentiation. Since this inhibitory factor may have a role in protecting melanoma tumors from attack by the host immune system, preparative isolation will be attempted. But we consider these results only as preliminary one's.