Advanced Stage IV and IIIc melanoma has a dismal survival, with or without, standard chemotherapy. New therapies are required to improve survival and reduce morbidity. Repeated vaccine dosing does not appear to have been explored, so Vaccinia Melanoma Cell Lysate (VMCL) vaccine repetitive therapy was tested, either alone, or combined with chemotherapy. 37 patients (31 Stage IV [M1a(6), b(7), c(18)] and 6 Stage IIIc) were studied using intra-dermal VMCL vaccine therapy. If disease progressed, vaccine was continued with standard chemotherapy (DTIC and/or Fotemustine). Overall survival was assessed and clinical responses were also recorded. From vaccine commencement, median overall follow-up was 10 months. Survivals ranged from 4 to 73 months. Median (mean) overall survival was 10 (23.5) months; overall survival at 1, 2 and 3 years was 40.5%, 21.6% and 10.8% respectively. CR and PR occurred in 18.9% (7) and 18.9% (7) of patients; these were durable for up to 6.1 years in 4 patients. Stable disease was noted in a further 17 patients (45.9%). In 6 patients (16.2%) no response to therapy was apparent. Repeated vaccinations with or without chemotherapy produced strong, durable clinical responses with overall survival > 23 months occurring in nearly 25% of advanced melanoma patients. The overall disease control rate (CR, PR and SD) was 83.7%, including CR in very advanced cases. These results, in a largely unselected population of advanced metastatic melanoma patients, compare very favourably with other regimens, and notably were associated with minimal, if any, toxicity. Further analysis of this approach appears warranted

BackgroundRepetitive long-term Vaccinia Melanoma Cell Lysate (VMCL) vaccination schedules have proved clinically effective in producing Complete Responses and strong durable survivals for up to 6.1 years in a previous study of patients with advanced Stage IV and Stage IIIc melanoma. These studies were expanded to include 54 patients for further evaluation of these findings.Methods54 patients comprising 48 Stage IV (6 M1a, 14 M1b, 28 M1c) and 6 advanced Stage III (5 IIIc; 1 IIIb) were studied using repeated intra-dermal VMCL vaccine therapy. If disease progressed, vaccine was continued together with standard chemotherapy (DTIC and/or Fotemustine). Overall survival was the primary end-point assessed, with clinical responses and toxicity recorded.ResultsFrom vaccine commencement, median overall survival was 14 months, ranging from 4 to 121 months. Kaplan-Meier survival analysis demonstrated overall 1, 2 and 3-year survival estimates of 57%, 26% and 18.5% respectively, and overall 5-year survival of 15.4%. No appreciable toxicity was observed. Complete Responses (CR) occurred in 16.7% (9) and partial responses (PR) in 14.8% (8) of patients. Stable disease was noted in a further 25 patients (46.3%). No response to therapy was apparent in 12 patients (22.2%). The overall response rate was 31.5% (CR + PR), with clinically significant responses (CR + PR + SD) in 77.8% of patients. Strong, durable clinical responses with overall survivals ≥ 23 months occurred in 29.6% of patients treated with repeated VMCL vaccine for advanced melanoma, (+/- concurrent chemotherapy).ConclusionsProlonged, repetitive VMCL vaccination immunotherapy appears to be a clinically effective means of generating relatively high CR rates, useful clinical responses and long-term survivals, with little toxicity, but remains notably under-explored. Successive immunomodulation might explain the results. Closer analysis of repetitive dosing is required to improve clinical response rates and survival, perhaps by optimising the timing of immunotherapy delivery.Trial registrationAustralian and New Zealand Clinical Trials Registry ANZCTRN12605000425695.Electronic supplementary materialThe online version of this article (doi:10.1186/2051-1426-2-9) contains supplementary material, which is available to authorized users.

In several animal species a sublethal dose of x-rays, from 100 to 200 R on, causes maximum depression of the primary antibody response if antigen is injected 1-2 days after total-body irradiation. The depressive effect of sublethal doses of x-rays is transient, however; recovery starts at 1 week and may be complete 2 months after radiation doses as large as 500-700 R (1).Recovery of the immune system after a supralethal dose of x-rays can be achieved by transplantation of hemopoietic cells. Animals so treated are denominated chimeras because they possess, often for their life span, cells of graft origin (2). Antigenic stimulation of radiation chimeras may result in antibody production by cells of donor type (3). Recovery of the antibody response of bone marrow chimeras is faster in syngeneic than in allogeneic combinations. In some experiments, the antibody response of syngeneic chimeras could attain normal levels when antigen was injected 30-60 days (4) or 60-90 days (5) after bone marrow transplantation. In other experiments, however, the antibody response of syngeneic chimeras immunized 16060 days after transplantation was still subnormal although higher than the response of allogeneic chimeras-of the same age (6). The lower antibody titer observed in allogeneic as compared to syngeneic chimeras was attributed to incomplete repopulation of the immune system, although it could not be ruled out that immunologically competent cells were less efficient in antibody production when stimulated in allogeneic hosts (5). That immunologic mechanisms are abnormally operating in allogeneic bone marrow chimeras was suggested by the finding that these animals, 90-120 days after transplantation, had a decreased ability to synthesize 7S antibodies to certain antigens (7).In all these studies on the antibody response in sublethally irradiated mice and in bone marrow radiation chimeras, antibody affinity was not examined. Affinity, that is, the binding Irradiation. Total-body x-irradiation of the animals was performed as described (9). Mice were either sublethally irradiated (450 R) or lethally irradiated (900 R).Chimeras. Within 2 hr after radiation exposure, lethally irradiated mice were injected intravenously with I ml of a cell suspension containing 1 X 107 nucleated bone marrow cells from normal donors. Suspensions of bone marrow cells were prepared in Eagle's medium as described (9). Allogeneic chimeras were obtained using DBA donors and C3H recipients, and designated DBA/C3H. Syngeneic chimeras were of the following types: C3H/C3H and DBA/DBA. In each strain combination, donors and hosts were of the same sex. In a previous study (5), erythropoietic and antibody-forming cells of all allogeneic chimeras prepared as the present ones and tested 60-150 days after transplantation were of donor type only.Immunization. The antigen used for primary and secondary immunization was dinitrophenyl-keyhole-limpet-hemocyanin (Dnp-KLH), 62 moles of Dnp per mole of KLH, from Calbiochem.Normal C3H and DBA mice, sublethally irradiated C3H m...

A simple, quick, easy and cheap tandem mass spectrometry (MS/MS) method for the determination of adenosine monophosphate (AMP) and cyclic adenosine monophosphate (cAMP) has been newly developed. This novel MS/MS method was applied for the evaluation of the inhibitory effect of a novel 2-oxo-1,2-dihydropyridine-3-carbonitrile derivative, also named DF492, on PDE3 enzyme activity in comparison to its parent drug milrinone. Molecule DF492, with an IC 50 of 409.5 nM, showed an inhibition of PDE3 greater than milrinone (IC 50 = 703.1 nM). To explain the inhibitory potential of DF492, molecular docking studies toward the human PDE3A were carried out with the aim of predicting the binding mode of DF492. The presence of different bulkier decorating fragments in DF492 was pursued to shift affinity of this novel molecule toward PDE3A compared to milrinone in accordance with both the theoretical and experimental results. The described mass spectrometric approach could have a wider potential use in kinetic and biomedical studies and could be applied for the determination of other phosphodiesterase inhibitor molecules.

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