Treatment patterns, outcomes, and resource utilization of patients with metastatic melanoma in the U.K.: the MELODY study.

BACKGROUND: Advanced melanoma is an aggressive disease with a poor prognosis. Approved therapy is limited in the U.K. and, until recently, no treatment had improved survival over best supportive care. A deeper understanding of current clinical practice will help new agents find a place in future treatment pathways. OBJECTIVES: To document U.K. clinical practice for the treatment of patients with unresectable stage III/IV (advanced) melanoma. METHODS: MELODY (melanoma treatment patterns and outcomes among patients with unresectable stage III/IV disease: a retrospective longitudinal survey) compiled registries of consecutive patients with malignant melanoma (any stage) between 1 July 2005 and 30 June 2006 from France, Italy and the U.K. Patients with advanced melanoma and ≥ 2 months of follow-up were eligible for analysis. RESULTS: There were 220 eligible patients identified in the U.K., of whom 117 (53.2%) received systemic therapy outside of clinical trials. Over half of these patients received dacarbazine as first- or second-line therapy. Healthcare-resource utilization was extensive and patients had short survival times: 1- and 2-year survival rates after first-line systemic treatment were 45.5% [95% confidence interval (CI) 37.1-53.6] and 24.7% (95% CI 17.7-32.3), respectively. CONCLUSIONS: Systemic and palliative treatments used to manage advanced melanoma in the U.K. are associated with considerable healthcare resource utilization and poor short-term survival.

Lenalidomide enhances the anti-prostate cancer activity of docetaxel in vitro and in vivo.

BACKGROUND: In this study, we investigated the effects of combining lenalidomide and docetaxel on in vitro and in vivo models of prostate cancer as a potential strategy for treatment of castrate resistant prostate cancer (CRPC). METHODS: The effects of combining lenalidomide and docetaxel on proliferation, apoptosis, invasive potential, anchorage independent growth, and p53 activation in the PC3 and DU145 prostate cell lines were investigated. The effects of the lenalidomide and docetaxel combination on LNCaP prostate cancer cell growth and invasiveness in vitro was also studied. The combination of these two agents was finally tested on a xenograft model of PC3 tumor growth in nude mice. RESULTS: Lenalidomide decreased the IC(50) of docetaxel by up to 50% (P < 0.05) and also decreased invasion in PC3, LNCaP, and DU145 cells and anchorage independent growth in PC3 cells (P < 0.01). Apoptosis in lenalidomide/docetaxel-treated cells was increased by 2.2-fold over single agent docetaxel and a corresponding increase in p53, p38, and BAD activation was observed in Western blots (P < 0.001). When PC3 challenged mice were treated with lenalidomide and docetaxel, median survival increased from 48 to 59 days and the rate of tumor growth was significantly reduced (P < 0.05). CONCLUSIONS: Lenalidomide may be a promising candidate for combination with docetaxel in the treatment of CRPC.

Supernatants derived from chemotherapy-treated cancer cell lines can modify angiogenesis.

BACKGROUND: There is evidence that tumours produce substances such as cytokines and microvesicular bodies bearing bioactive molecules, which support the carcinogenic process. Furthermore, chemotherapy has also been shown to modify these exudates and in doing so, neutralise their tumourigenic influence. METHODS: In the current study, we have investigated the effect of chemotherapy agents on modifying the cytokine profile and microvesicular cargo of supernatants derived from cancer cell lines. In addition, we have explored the effect of these tumour-derived supernatants on angiogenesis, and how chemotherapy can alter the supernatants rendering them less pro-angiogenic. RESULTS: Herein, we show that supernatants contain a rich cocktail of cytokines, a number of which are potent modulators of angiogenesis. They also contain microvesicular bodies containing RNA transcripts that code for proteins involved in transcription, immune modulation and angiogenesis. These supernatants altered intracellular signalling molecules in endothelial cells and significantly enhanced their tubulogenic character; however, this was severely compromised when supernatants from tumours treated with chemotherapy was used instead. CONCLUSION: This study suggests tumour exudates and bioactive material from tumours can influence cellular functions, and that treatment with some chemotherapy can serve to negate these pro-tumourigenic processes.

Supernatants from lymphocytes stimulated with Bacillus Calmette-Guerin can modify the antigenicity of tumours and stimulate allogeneic T-cell responses.

BACKGROUND: Reduced expression of class 1 human leucocyte antigens (HLA1) is often a mechanism by which tumours evade surveillance by the host immune system. This is often associated with an immune function that is unable to mount appropriate responses against disease, which can result in a state that favours carcinogenesis. METHODS: In the current study, we have explored the effects of Bacillus Calmette-Guerin (BCG) on the cytokine output of leucocytes, which is a key determinant in generating antitumour action, and have also assessed the effect of these cytokine cocktails on HLA1 expression in solid tumour cell lines. RESULTS: BCG potently activated a broad range of leucocytes, and also enhanced the production of cytokines that were Th(1)-predominant. Supernatants from BCG-treated leucocytes significantly increased the expression of HLA1 on the surface of cancer cell lines, which correlated with increased cytolytic T-cell activity. We also showed that the increased HLA1 expression was associated with activation of intracellular signalling pathways, which was triggered by the increases in the Th(1)-cytokines interferon-γ and tumour necrosis factor-α, as counteracting their effects negated the enhancement. CONCLUSION: These studies reaffirm the role of BCG as a putative immunotherapy through their cytokine-modifying effects on leucocytes and their capacity to enhance tumour visibility.

Anti-tumour activity of afatinib, an irreversible ErbB family blocker, in human pancreatic tumour cells.

BACKGROUND: The combination of the reversible epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) erlotinib with gemcitabine obtained FDA approval for treating patients with pancreatic cancer. However, duration of response is often limited and there is currently no reliable predictive marker. METHODS: We determined the sensitivity of a panel of human pancreatic tumour cell lines to treatment with afatinib, erlotinib, monoclonal antibody (mAb) ICR62, and gemcitabine, using the Sulforhodamine B colorimetric assay. The effect of these agents on cell signalling and cell-cycle distribution was determined by western blot and flow cytometry, respectively. RESULTS: At 200 nM, ICR62 had no effect on growth of these tumour cells with the exception of BxPC-3 cells. BxPC-3 cells were also sensitive to treatment with afatinib and erlotinib with respective IC(50) values of 11 and 1200 nM. Compared with erlotinib, afatinib was also more effective in inhibiting the growth of the other human pancreatic tumour cell lines and in blocking the EGF-induced phosphorylation of tyrosine, EGFR, MAPK, and AKT. When tested in BxPC-3 xenografts, afatinib induced significant delay in tumour growth. CONCLUSION: The superiority of afatinib in this study encourages further investigation on the therapeutic potential of afatinib as a single agent or in combination with gemcitabine in pancreatic cancer.

Effect of Gemcitabine based chemotherapy on the immunogenicity of pancreatic tumour cells and T-cells.

PURPOSE: Chemotherapy for advanced pancreatic cancer has limited efficacy due to the difficultly of treating established tumours and the evolution of tumour resistance. Chemotherapies for pancreatic cancer are typically studied for their cytotoxic properties rather than for their ability to increase the immunogenicity of pancreatic tumour cells. In this study Gemcitabine in combination with immune modulatory chemotherapies Oxaliplatin, zoledronic acid and pomalidomide was studied to determine how combination therapy alters the immunogenicity of pancreatic tumour cell lines and subsequent T-cell responses. METHODS: Pancreatic tumour cell lines were stimulated with the chemotherapeutic agents and markers of immune recognition were assessed. The effect of chemotherapeutic agents on DC function was measured using uptake of CFSE-stained PANC-1 cells, changes in markers of maturation and their ability to activate CD8+ T-cells. The effect of chemotherapeutic agents on T-cell priming prior to activation using anti-CD3 and anti-CD28 antibodies was determined by measuring IFN-γ expression and Annexin V staining using flow cytometry. RESULTS: These agents demonstrate both additive and inhibitory properties on a range of markers of immunogenicity. Gemcitabine was notable for its ability to induce the upregulation of human leukocyte antigen and checkpoints on pancreatic tumour cell lines whilst inhibiting T-cell activation. Pomalidomide demonstrated immune modulatory properties on dendritic cells and T-cells, even in the presence of gemcitabine. DISCUSSION: These data highlight the complex interactions of different agents in the modulation of tumour immunogenicity and immune cell activation and emphasise the complexity in rationally designing chemo immunogenic combinations for use with immunotherapy.

Pre-treatment with chemotherapy can enhance the antigenicity and immunogenicity of tumours by promoting adaptive immune responses.

BACKGROUND: Some cancer patients are immuno-compromised, and it has been long felt that immune-intervention is not compatible with standard chemotherapies. However, increasing evidence suggests that standard chemotherapy drugs may stimulate beneficial changes in both the immune system and tumour. METHODS: We have assessed the expression of human leucocyte antigen class 1 (HLA1) on tumour cells before and after chemotherapy agents (cyclophosphamide, oxaliplatin or gemcitabine). In addition, we show that chemotherapy-stressed tumour cells may release cytokines that enhance the interactions between dendritic cells (DCs) and T cells into growth media. RESULTS: Here we report that some chemotherapy agents can increase HLA1 expression in tumour cells, even when expression is low. Increases were associated with killing by cytotoxic T cells, which were negated by HLA1-blockade. Furthermore, T-cell function, as indicated by increased proliferation, was enhanced as supernatants derived from tumours treated with chemotherapy augmented DC-maturation and function. CONCLUSION: There is evidence that a facet of immune surveillance can be restored by appropriate chemotherapy agents. Also, tumours exposed to some chemotherapy may secrete cytokines that can mature DCs, which ultimately enhances T-cell responses.

Investigator-initiated trials of targeted oncology agents: why independent research is at risk?

BACKGROUND: Drug development traditionally has relied upon the complementary contributions of clinicians and scientists at academic institutions and at pharmaceutical companies. Greater regulatory burdens, increased bureaucratic requirements, restricted reimbursement, and spiralling research and development costs are exerting pressure on the drug development pipeline. The result is a de-emphasis of exploratory research, particularly independent academic research, despite its proven value in identifying new drug targets and developing innovative cancer therapies. DESIGN: An expert panel assembled by the Biotherapy Development Association-a nonprofit international forum for academic and industry researchers, patients, and government regulatory and postregulatory agencies-examined the growing schism between academia and industry and identified several causes of declining academic research. RESULTS: The authors propose solutions to sustain investigator-initiated research and provide a new model whereby expert organisations provide a forum for academia and industry to plan studies within a regulatory framework to support licensure/authorisation and reimbursement for new molecularly targeted agents and biomarkers. CONCLUSIONS: Investigator-initiated trials have led to the discovery and development of innovative, safe, and effective cancer treatments. To ensure that such research continues, action will be required on the parts of legislative and regulatory bodies, industry, universities, patient advocacy organisations, and preclinical and clinical academic scientists.

The effect of colorectal cancer upon host peripheral immune cell function.

OBJECTIVE: Colorectal cancer is immunogenic. However, it is also associated with suppression of host immunity. Identifying the mechanisms involved in immune suppression is necessary to develop future immunotherapeutic strategies. The aim of this study was to assess immune cell function in colorectal cancer patients. METHOD: A total of 80 colorectal cancer patients (41 male) prior to treatment and 38 matched controls (21 male) were recruited. Venous blood samples were taken. White blood cell composition was determined using monoclonal antibodies. Levels of cytokines IFN-gamma, TNF-alpha, IL-2, IL-10, IL-4 and IL-6 were measured from the supernatants of activated peripheral blood mononuclear cells (PBMC) following thawing and re-suspension. Peripheral blood mononuclear proliferation was measured using 3H-Thymidine. RESULTS: Stage I-III cancer patients had elevated percentages of CD8 T cell (P = 0.004) whilst stage IV patients had low total lymphocyte percentages (P = 0.016). Monocyte and NKT cell percentage decreased with advanced tumour stages (P = 0.013 and P = 0.038). Patients had lower PBMC proliferation and production of the TH1 cytokines (IFN-gamma and TNF-alpha) (P < 0.001) than that of the controls. IL-6 and IL-4 production were not significantly different. IFN-gamma and TNF-alpha concentrations reduced with tumour vascular invasion (P = 0.011 and P = 0.019). CONCLUSION: Colorectal cancer induces an immunological response, shifting the cytokine balance. The most profound changes are seen once disease has spread systemically.

Inhibition of metastatic potential in colorectal carcinoma in vivo and in vitro using immunomodulatory drugs (IMiDs).

BACKGROUND: Thalidomide and lenalidomide are FDA approved for the treatment of multiple myeloma and, along with pomalidomide, are being investigated in various other cancers. Although these agents display immunomodulatory, anti-angiogenic and anti-apoptotic effects, little is known about their primary mode of therapeutic action in patients with cancer. METHODS: As part of a continuing research effort, we have investigated the effects of these agents on the metastatic capacity of murine colorectal cancer cell lines both in vivo and in vitro. Allied to these, we have studied their effects on the molecular pathways associated with metastasis. RESULTS: Results indicate that thalidomide, lenalidomide and pomalidomide significantly inhibit the metastatic capability of colorectal carcinoma cells. Anchorage-independent growth, used as a coarse indicator of transformation, was significantly reduced, as were migratory capacity and invasive competence. In addition, an in vivo experimental metastasis model also showed that treatment with the drugs resulted in a significantly lower number of metastatic pulmonary nodules relative to control mice. Allied to these cellular and phenotypic changes were alterations in molecular markers of metastasis and in intracellular signalling competency. CONCLUSIONS: These results provide evidence that in addition to their immunomodulatory effects, thalidomide, lenalidomide and pomalidomide can impair the metastatic capacity of tumours, and that this mechanism may involve alterations to cell signalling functionality.

Epitopes of the CD4 antigen and HIV infection.

The CD4 (or T4) surface antigen of human T lymphocytes is an important part of the receptor for the human immunodeficiency virus (HIV). After binding to the receptor, the HIV may enter the T cell and induce the formation of syncytia. In an attempt to identify the receptor site more closely, monoclonal antibodies (Mab's) to CD4 were tested for their ability to block HIV infection in a syncytium formation assay, and the CD4 epitopes so identified were mapped by antibody cross-blocking. The antibodies that showed strong inhibition of HIV fell into two main families while a third group of Mab's blocked syncytia formation weakly or not at all. Several different isolates of HIV as well as the laboratory strain CBL1 grown in CEM cells were used to induce the syncytia. The data indicate that only some epitopes of CD4 are important for virus binding and imply that the virus-binding site for CD4 is conserved in different isolates of HIV with substantially divergent env gene sequences. Preliminary studies of patients suggest that polymorphism of these epitopes does not play a role in determining susceptibility to infection.

How immunotherapy can enhance the response to other modalities and improve outcome and quality of life.

Early studies suggested that the induction of an effective immune response could lead to elimination of residual tumour. Over a hundred years ago Coley invented his eponymous named "toxins" that appeared to induce a strong inflammatory response, leading to tumour reduction. Subsequent attempts to enhance the immune response have essentially been on a vaccine basis, trying to induce a specific response against the tumour. Numerous vaccine approaches have claimed to give significant clinical benefit in clinical response but very few of these have survived a randomised trial. A major reason for this is the heterogeneity of many tumours, as well as the various forms of defence against an immune response that they employ. It was thought that chemotherapy and radiotherapy were mutually exclusive for immunotherapy using the vaccine approach. More recently, however, it has become appreciated that vaccine approaches may enhance subsequent responses to radiotherapy and that certain chemotherapies actually enhance responses to vaccines. It has been suggested that one of the mechanisms of action of chemotherapy is to reduce the cells that suppress T-cells. These cells primarily defend the tumour from an immunological attack, but more recently it has been suggested that the benefit may encompass other aspects, such as enhancing antiangiogenic responses. One reason why immunostimulatory approaches may be so useful in cancer is that many cancers evolve out of a chronic inflammatory environment that actively suppresses cell mediated immune responses and enhances tumour angiogenesis. An ideal cancer drug would therefore be expected to have these properties. One such drug is lenalidomide, which features include marked immune stimulatory properties as well being able to inhibit regulatory T-cells. They have also been shown to enhance anticancer activity with vaccines in both preclinical models and more recently in clinical observations, where the responses to vaccines in patients with myeloma is much higher when they are on lenalidomide than other treatments. A number of regularly used chemotherapy regimens have marked activity in modulating the immune response. These maybe of benefit and the regimens will be reviewed, which include gemcitabine, cyclophosphamide and the IMiDs.

Topical imiquimod and intralesional interleukin-2 increase activated lymphocytes and restore the Th1/Th2 balance in patients with metastatic melanoma.

BACKGROUND: Patients with metastatic skin disease in malignant melanoma are difficult to treat, with unresectable lesions proving the biggest challenge. We have recently published data showing a significant clinical response in patients with multiple in-transit melanoma metastases treated with a combination of topical imiquimod and intralesional interleukin (IL)-2. Here we report the results of immunological analysis with the aim of highlighting correlations with our clinical findings. OBJECTIVES: To investigate the systemic effects of our localized combination treatment in patients with accessible metastases of melanoma, and to correlate this with their clinical responses. METHODS: The peripheral blood mononuclear cells of patients were collected at various time points throughout the treatment. Using antibodies to T-cell subsets we measured the changes in cell populations, and along with polyclonal stimulation, changes in cytokine production from these cells over a treatment course. RESULTS: We report an increase in the mean CD4/CD8 ratio from 2.78 to 3.54 with treatment (P < 0.01), and a rise in the percentage of CD25+ cells in the CD4+ population from 14.52% to 38.56%. Furthermore, staining with activation and T-regulatory markers showed that the majority of this population is activated T cells. Cytokine analysis on polyclonally stimulated peripheral blood mononuclear cells showed an increase in the ability of cells to produce interferon (IFN)-gamma over the treatment course, with an initial rise in the IFN-gamma/IL-5 ratio in five of six patients. CONCLUSIONS: The results of this study provide evidence that, in the majority of patients with in-transit metastases of melanoma, therapy with a combination of topical imiquimod and intralesional IL-2 induces a systemic immunological effect by reversing some of changes noted in patients with malignant disease.

Gemcitabine alters the proteasome composition and immunopeptidome of tumour cells.

The antigenic makeup of tumour cells can have a profound effect on the progression of cancer and success of immunotherapies. Therefore, one strategy to improve the efficacy of cancer treatments is to augment the antigens displayed by tumours. The present study explores how the recognition of tumour cells may be altered by non-cytotoxic concentrations of gemcitabine (GEM). Testing a panel of chemotherapeutics in human cancer cell lines in vitro, it was found that GEM increased surface expression of HLA-A,B,C and that underlying this were specific increases in ß-2-microglobulin and immunoproteasome subunit proteins. Furthermore, the peptide antigen repertoire displayed on HLA class I was altered, revealing a number of novel antigens, many of which that were derived from proteins involved in the DNA-damage response. Changes in the nature of the peptide antigens eluted from HLA-A,B,C after GEM treatment consisted of amino acid anchor-residue modifications and changes in peptide length which rendered peptides likely to favour alternative HLA-alleles and increased their predicted immunogenicity. Signalling through the MAPK/ERK and NFκB/RelB pathways was associated with these changes. These data may explain observations made in previous in vivo studies, advise as to which antigens should be used in future vaccination protocols and reinforce the idea that chemotherapy and immunotherapy could be used in combination.

Priming of human CD4+ antigen-specific T cells to undergo apoptosis by HIV-infected monocytes. A two-step mechanism involving the gp120 molecule.

The study of the pathology of HIV-1 infection in chimpanzees supports the idea of the crucial role of HIV-infected monocytes in the pathogenesis of AIDS, although viral mechanisms that lead to T cell dysfunction and deletion during HIV infection are still unclear. We show here that HIV-1-infected antigen-presenting monocytes (APCs) are able to prime in vitro non-HIV-infected antigen-specific CD4+ T cell lines or peripheral blood CD4+ T cells to undergo apoptosis after antigen-specific restimulation. The priming of T cells for apoptosis occurs in the absence of HIV replication in the T cells. Priming for apoptosis required two concomitant signals present on the same APC, an antigenic stimulus and a second signal provided by the HIV gp120 protein as demonstrated by the use as APCs of EBV-LCLs infected with different recombinant deleted proviruses or transfected with different HIV proteins. These results provide a mechanism for the priming for apoptosis of T cells in HIV-infected patients, implicating a role for HIV-infected APCs in the induction of T cell dysfunction and depletion in AIDS.

Immunobiological aspects of HIV treatment.

Recent information on the efficacy of anti-retroviral therapy and vaccination strategies has been disappointing as well as confusing. The recently announced Concorde study suggested that there is no advantage to early treatment of asymptomatic HIV infection with azidothymidine alone, even though the levels of CD4+ cells in the treated group were consistently higher than in the untreated group. This will lead to increasing attention being paid to the mechanisms whereby HIV causes AIDS, which have sadly been sidelined in the rush to produce classically based therapies and vaccines. Over the last year many different theories on how HIV kills CD4+ cells and leads to AIDS have been discussed and tentatively explored.

HIV types and testing.

The past year has seen the discovery of new isolates which do not readily fit into the established categories HIV-1, HIV-2 and SIV, as well as the availability of new assays including the use of the polymerase chain reaction. New combination enzyme-linked immunosorbent assays can detect viruses from two or more groups where specific enzyme-linked immunosorbent assays have been developed using peptides and monoclonal antibodies.

Toxicity profile of the immunomodulatory thalidomide analogue, lenalidomide: phase I clinical trial of three dosing schedules in patients with solid malignancies.

Thalidomide is an anti-angiogenic agent currently used to treat patients with malignant cachexia or multiple myeloma. Lenalidomide (CC-5013) is an immunomodulatory thalidomide analogue licensed in the United States of America (USA) for the treatment of a subtype of myelodysplastic syndrome. This two-centre, open-label phase I study evaluated dose-limiting toxicities in 55 patients with malignant solid tumours refractory to standard chemotherapies. Lenalidomide capsules were consumed once daily for 12 weeks according to one of the following three schedules: (I) 25 mg daily for the first 7 d, the daily dose increased by 25 mg each week up to a maximum daily dose of 150 mg; (II) 25mg daily for 21 d followed by a 7-d rest period, the 4-week cycle repeated for 3 cycles; (III) 10 mg daily continuously. Twenty-six patients completed the study period. Two patients experienced a grade 3 hypersensitivity rash. Four patients in cohort I and 4 patients in cohort II suffered grade 3 or 4 neutropaenia. In 2 patients with predisposing medical factors, grade 3 cardiac dysrhythmia was recorded. Grade 1 neurotoxicity was detected in 6 patients. One complete and two partial radiological responses were measured by computed tomography scanning; 8 patients had stable disease after 12 weeks of treatment. Fifteen patients remained on treatment as named patients; 1 with metastatic melanoma remains in clinical remission 3.5 years from trial entry. This study indicates the tolerability and potential clinical efficacy of lenalidomide in patients with advanced solid tumours who have previously received multi-modality treatment. Depending on the extent of myelosuppressive pre-treatment, dose schedules (II) or (III) are advocated for large-scale trials of long-term administration.

Review article: immune suppression and colorectal cancer.

BACKGROUND: Advances in immunology and molecular biology have shown that colorectal cancer is potentially immunogenic and that host immune responses influence survival. However, immune surveillance and activation is frequently ineffective in preventing and/or controlling tumour growth. AIM: To discuss potential ways in which colorectal cancer induces immune suppression, its effect upon prognosis and avenues for therapeutic development. METHOD: A literature review was undertaken for evidence of colorectal cancer-induced immune suppression using PubMed and Medline searches. Further studies were identified from the reference lists of identified papers. RESULTS: Immune suppression occurs at a molecular and cellular level and can result in a shift from cellular to humoral immunity. Several mechanisms for immune suppression have been described affecting innate and adaptive immunity with suppression linked to poorer clinical outcome. CONCLUSIONS: Colorectal cancer causes direct inhibition of the host's immune response with a detrimental effect upon prognosis. Immunotherapy offers a therapeutic strategy to counteract these effects with promising results seen particularly in precancerous conditions and early tumours. This review strongly suggests that immunotherapy should be incorporated into adjuvant therapeutic trials for stage 2 tumours and be considered as adjuvant treatment in conjunction with standard chemotherapy regimes for advanced disease.

Human tumour and dendritic cell hybrids generated by electrofusion: potential for cancer vaccines.

Hybrid cells created by fusion of antigen presenting and tumour cells have been shown to induce potent protective and curative anti-tumour immunity in rodent cancer models. The application of hybrid cell vaccines for human tumour therapy and the timely intervention in disease control are limited by the requirement to derive sufficient autologous cells to preserve homologous tumour antigen presentation. In this study, the efficiency of various methods of electrofusion in generating hybrid human cells have been investigated with a variety of human haemopoietic, breast and prostate cell lines. Cell fusion using an electrical pulse is enhanced by a variety of stimuli to align cells electrically or bring cells into contact. Centrifugation of cells after an exponential pulse from a Gene Pulser electroporation apparatus provided the highest yield of mixed cell hybrids by FACS analysis. An extensive fusogenic condition generated in human cells after an electrical pulse contradicts the presumption that prior cell contact is necessary for cell fusion. Alignment of cells in a concurrent direct current charge and osmotic expansion of cells in polyethylene glycol also generated high levels of cell fusion. Waxing of one electrode of the electroporation cuvette served to polarize the fusion chamber and increase cell fusion 5-fold. Optimisation of a direct current charge in combination with a fusogenic pulse in which fusion of a range of human cells approached or exceeded 30% of the total pulsed cells. The yield of hybrid prostate and breast cancer cells with dendritic cells was similar to the homologous cell fusion efficiencies indicating that dendritic cells were highly amenable to fusion with human tumour cells under similar electrical parameters. Elimination of unfused cells by density gradient and culture is possible to further increase the quantity of hybrid cells. The generation and purification of quantities of hybrid cells sufficient for human vaccination raises the possibility of rapid, autologous tumour antigen presenting vaccines for trial with common human tumours.