The use of gamma-irradiation and ultraviolet-irradiation in the preparation of human melanoma cells for use in autologous whole-cell vaccines

<p>Abstract</p> <p>Background</p> <p>Human cancer vaccines incorporating autologous tumor cells carry a risk of implantation and subsequent metastasis of viable tumor cells into the patient who is being treated. Despite the fact that the melanoma cell preparations used in a recent vaccine trial (Mel37) were gamma-irradiated (200 Gy), approximately 25% of the preparations failed quality control release criteria which required that the irradiated cells incorporate ³H-thymidine at no more than 5% the level seen in the non-irradiated cells. We have, therefore, investigated ultraviolet (UV)-irradiation as a possible adjunct to, or replacement for gamma-irradiation.</p> <p>Methods</p> <p>Melanoma cells were gamma- and/or UV-irradiated. ³H-thymidine uptake was used to assess proliferation of the treated and untreated cells. Caspase-3 activity and DNA fragmentation were measured as indicators of apoptosis. Immunohistochemistry and Western blot analysis was used to assess antigen expression.</p> <p>Results</p> <p>UV-irradiation, either alone or in combination with gamma-irradiation, proved to be extremely effective in controlling the proliferation of melanoma cells. In contrast to gamma-irradiation, UV-irradiation was also capable of inducing significant levels of apoptosis. UV-irradiation, but not gamma-irradiation, was associated with the loss of tyrosinase expression. Neither form of radiation affected the expression of gp100, MART-1/MelanA, or S100.</p> <p>Conclusion</p> <p>These results indicate that UV-irradiation may increase the safety of autologous melanoma vaccines, although it may do so at the expense of altering the antigenic profile of the irradiated tumor cells.</p

Long-term disease-free survival in advanced melanomas treated with nitrosoureas: mechanisms and new perspectives

BACKGROUND: Median survival of metastatic malignant melanoma is 6.0 to 7.5 months, with a 5-year survival of ~6.0%. Although long-term complete remissions are rare, few reports describe cases after chemotherapy. Fifty-three patients with metastatic melanoma were treated with Cystemustine, a chloroethyl nitrosourea (CENU) (60 or 90 mg/m(2)). CASE PRESENTATION: We describe 5 cases, presenting with complete response with long-term disease-free survival of long-term remission of 14, 12, 9, 7 and 6 years after Cystemustine therapy alone. CONCLUSION: Long-term survival has already been described in literature, but in all cases they have been obtained after chemotherapy associated with or followed by surgery. But despite these noteworthy and encouraging but also rare results, it appears essential to increase cystemustine efficiency

Tumor-derived interleukin-10 as a prognostic factor in stage III patients undergoing adjuvant treatment with an autologous melanoma cell vaccine.

OBJECTIVES: Interleukin-10 (IL-10) downregulates T-cell-mediated immune responses. We studied the association between IL-10 production by freshly isolated melanoma cell suspensions in vitro and overall survival in patients undergoing adjuvant treatment with a vaccine prepared from the same autologous melanoma cells modified with a hapten, dinitrophenyl (DNP). METHODS: Forty-four patients with cutaneous melanoma (29 stage III and 15 stage IV) were prospectively evaluated. Tumor cells were extracted from metastatic deposits for production of DNP-modified autologous melanoma cell vaccine. Small aliquots of the melanoma cell suspensions were separated prior to vaccine processing and cultured overnight for IL-10 production. Based on a blind assessment of the distribution of IL-10 levels in the culture supernatants, a cutoff of 200 pg/ml was used to define high versus low IL-10 producers. Cox regression model was used for multivariate analysis. Overall survival was calculated using the Kaplan-Meier method, and survival curves were compared with the log-rank test. RESULTS: Out of 44 patients, 29 were low and 15 were high IL-10 producers. The median OS was significantly worse for high compared with low IL-10 producers (10.5 months vs. 42 months; P = 0.022). In stage III patients, the multivariate hazard ratio for high versus low IL-10 producers was 2.92 (95% CI, 1.04-8.20; P = 0.041). The corresponding hazard ratio in stage IV patients was 0.92 (95% CI, 1.04-8.20; P = 0.888). CONCLUSIONS: High IL-10 production in the tumor microenvironment could be a determinant of clinical outcomes in stage III melanoma patients receiving autologous melanoma cell vaccine

Phase III multicenter clinical trial of the sialyl-TN (STn)-keyhole limpet hemocyanin (KLH) vaccine for metastatic breast cancer

PURPOSE. This double-blind, randomized, phase III clinical trial evaluated time to progression (TTP) and overall survival in women with metastatic breast cancer (MBC) who received sialyl-TN (STn) keyhole limpet hemocyanin (KLH) vaccine. Secondary endpoints included vaccine safety and immune response. EXPERIMENTAL DESIGN. The study population consisted of 1,028 women with MBC across 126 centers who had previously received chemotherapy and had had either a complete or a partial response or no disease progression. All women received one-time i.v. cyclophosphamide (300 mg/m(2)) 3 days before s.c. injection of 100 μg STn-KLH plus adjuvant (treatment group) or 100 μg KLH plus adjuvant (control group) at weeks 0, 2, 5, and 9. Subsequently, STn-KLH without adjuvant or KLH without adjuvant was then administered monthly for 4 months, and then quarterly until disease progression, without cyclophosphamide. RESULTS. STn-KLH vaccine was well tolerated; patients had mild to moderate injection-site reactions and reversible flu-like symptoms. Week-12 antibody testing revealed high specific IgG titers and a high rate of IgM-to-IgG seroconversion; the median IgG titers in STn-KLH recipients were 320 (anti-ovine submaxillary mucin) and 20,480 (anti-STn), with no detectable antimucin antibodies in the control group. The TTP was 3.4 months in the treatment group and 3.0 months in the control group. The median survival times were 23.1 months and 22.3 months, respectively. CONCLUSIONS. Although STn-KLH was well tolerated in this largest to date metastatic breast cancer vaccine trial, no overall benefit in TTP or survival was observed. Lessons were learned for future vaccine study designs

Phase I-II study of everolimus and low-dose oral cyclophosphamide in patients with metastatic renal cell cancer

<p>Abstract</p> <p>Background</p> <p>For patients with metastatic renal cell cancer (mRCC) who progressed on vascular endothelial growth factor (VEGF) receptor tyrosine kinase inhibitor therapy, the orally administered mammalian target of rapamycin (mTOR) inhibitor everolimus has been shown to prolong progression free survival. Intriguingly, inhibition of mTOR also promotes expansion of immunosuppressive regulatory T cells (Tregs) that can inhibit anti-tumor immune responses in a clinically relevant way in various tumor types including RCC. This study intends to investigate whether the antitumor efficacy of everolimus can be increased by preventing the detrimental everolimus induced expansion of Tregs using a metronomic schedule of cyclophosphamide.</p> <p>Methods/design</p> <p>This phase I-II trial is a national multi-center study of different doses and schedules of low-dose oral cyclophosphamide in combination with a fixed dose of everolimus in patients with mRCC not amenable to or progressive after a VEGF-receptor tyrosine kinase inhibitor containing treatment regimen. In the phase I part of the study the optimal Treg-depleting dose and schedule of metronomic oral cyclophosphamide when given in combination with everolimus will be determined. In the phase II part of the study we will evaluate whether the percentage of patients progression free at 4 months of everolimus treatment can be increased from 50% to 70% by adding metronomic cyclophosphamide (in the dose and schedule determined in the phase I part). In addition to efficacy, we will perform extensive immune monitoring with a focus on the number, phenotype and function of Tregs, evaluate the safety and feasibility of the combination of everolimus and cyclophosphamide, perform monitoring of selected angiogenesis parameters and analyze everolimus and cyclophosphamide drug levels.</p> <p>Discussion</p> <p>This phase I-II study is designed to determine whether metronomic cyclophosphamide can be used to counter the mTOR inhibitor everolimus induced Treg expansion in patients with metastatic renal cell carcinoma and increase the antitumor efficacy of everolimus.</p> <p>Trial Registration</p> <p>ClinicalTrials.gov Identifier <a href="http://www.clinicaltrials.gov/ct2/show/NCT01462214">NCT01462214</a>, EudraCT number 2010-024515-13, Netherlands Trial Register number <a href="http://www.trialregister.nl/trialreg/admin/rctview.asp?TC=2040">NTR3085</a>.</p

Kroppenstedtia pulmonis sp. nov. and Kroppenstedtia sanguinis sp. nov., isolated from human patients

Three human clinical strains (W9323T, X0209T and X0394) isolated from lung biopsy, blood and cerebral spinal fluid, respectively, were characterized using a polyphasic taxonomic approach. Comparative analysis of the 16S rRNA gene sequences showed the three strains belonged to two novel branches within the genus Kroppenstedtia: 16S rRNA gene sequence analysis of W9323T showed closest sequence similarity to Kroppenstedtia eburnea JFMB-ATE T (95.3 %), Kroppenstedtia guangzhouensis GD02T (94.7 %) and strain X0209T (94.6 %); sequence analysis of strain X0209T showed closest sequence similarity to K. eburnea JFMB-ATE T (96.4 %) and K. guangzhouensis GD02T (96.0 %). Strains X0209T and X0394 were 99.9 % similar to each other by 16S rRNA gene sequence analysis. The DNA-DNA relatedness was 94.6 %, confirming that X0209T and X0394 belong to the same species. Chemotaxonomic data for strains W9323T and X0209T were consistent with those described for the genus Kroppenstedtia: whole-cell peptidoglycan contained LLdiaminopimelic acid; the major cellular fatty acids were iso-C15 and anteiso-C15; and the major menaquinone was MK-7. Different endospore morphology and carbon utilization profiles of strains W9323T and X0209T supported by phylogenetic analysis enabled us to conclude that the strains represent two new species within the genus Kroppenstedtia, for which the names Kroppenstedtia pulmonis sp. nov. (type strain W9323T =DSM 45752 T) and Kroppenstedtia sanguinis sp. nov. (type strain X0209T =DSM 45749T=CCUG 38657 T) are proposed

Whole-cell cancer vaccination: from autologous to allogeneic tumor- and dendritic cell-based vaccines

The field of tumor vaccination is currently undergoing a shift in focus, from individualized tailor-made vaccines to more generally applicable vaccine formulations. Although primarily predicated by financial and logistic considerations, stemming from a growing awareness that clinical development for wide-scale application can only be achieved through backing from major pharmaceutical companies, these new approaches are also supported by a growing knowledge of the intricacies and minutiae of antigen presentation and effector T-cell activation. Here, the development of whole-cell tumor and dendritic cell (DC)-based vaccines from an individualized autologous set-up to a more widely applicable allogeneic approach will be discussed as reflected by translational studies carried out over the past two decades at our laboratories and clinics in the vrije universiteit medical center (VUmc) in Amsterdam, The Netherlands