Recovery of zeta-chain expression and changes in spontaneous IL-10 production after PSA-based vaccines in patients with prostate cancer.

Circulating T lymphocytes of patients with prostate cancer have been reported to have functional deficits, including low or absent zeta-chain expression. To determine whether these functional impairments could be reversed by prostate specific antigen-based vaccination therapy, 10 patients treated with recombinant human prostate specific antigen plus GM-CSF and eight others receiving prostate specific antigen plus oil emulsion in two pilot clinical trials were evaluated prior to and after vaccination for several immunologic end points, including zeta-chain expression and cytokine production by circulating T cells as well as the frequency of T cells able to respond to prostate specific antigen in ELISPOT assays. The flow cytometry assay for zeta-chain expression was standardized to allow for a reliable comparison of pre- vs post-vaccination samples. Prior to therapy, the patients were found to have significantly lower zeta-chain expression in circulating CD3(+) cells and a higher percentage of zeta-chain negative CD3(+) and CD4(+) cells than normal donors. The patients\u27 peripheral blood mononuclear cells spontaneously produced more IL-10 ex vivo than those of normal controls. After vaccination, recovery of zeta-chain expression was observed in 50% of patients in both clinical trials. Also, spontaneous IL-10 secretion by peripheral blood mononuclear cells decreased following immunotherapy in patients treated with prostate specific antigen and GM-CSF. The frequency of prostate specific antigen-reactive T cells was detectable in 7 out of 18 patients vs 4 out of 18 patients prior to vaccination. Only one of 18 patients was a clinical responder. The vaccine had stimulatory effects on the patients\u27 immune system, but post-vaccine immune recovery could not be correlated to progression-free survival in this small cohort of patients with prostate cancer

Dietary Restriction Promotes Vessel Maturation in a Mouse Astrocytoma

Mature vasculature contains an endothelial cell lining with a surrounding sheath of pericytes/vascular smooth muscle cells (VSMCs). Tumor vessels are immature and lack a pericyte sheath. Colocalization of vascular endothelial growth factor receptor 2 (VEGFR-2) and platelet-derived growth factor receptor beta (PDGF-Rβ) reduces pericyte ensheathment of tumor vessels. We found that a 30% dietary restriction (DR) enhanced vessel maturation in the mouse CT-2A astrocytoma. DR reduced microvessel density and VEGF expression in the astrocytoma, while increasing recruitment of pericytes, positive for alpha-smooth muscle actin (α-SMA). Moreover, DR reduced colocalization of VEGF-R2 and PDGF-Rβ, but did not reduce total PDGF-Rβ expression. These findings suggest that DR promoted vessel normalization by preventing VEGF-induced inhibition of the PDGF signaling axis in pericytes. DR appears to shift the tumor vasculature from a leaky immature state to a more mature state. We suggest that vessel normalization could improve delivery of therapeutic drugs to brain tumors

Recovery of ζ-chain expression and changes in spontaneous IL-10 production after PSA-based vaccines in patients with prostate cancer

Circulating T lymphocytes of patients with prostate cancer have been reported to have functional deficits, including low or absent ζ-chain expression. To determine whether these functional impairments could be reversed by prostate specific antigen-based vaccination therapy, 10 patients treated with recombinant human prostate specific antigen plus GM-CSF and eight others receiving prostate specific antigen plus oil emulsion in two pilot clinical trials were evaluated prior to and after vaccination for several immunologic end points, including ζ-chain expression and cytokine production by circulating T cells as well as the frequency of T cells able to respond to prostate specific antigen in ELISPOT assays. The flow cytometry assay for ζ-chain expression was standardized to allow for a reliable comparison of pre- vs post-vaccination samples. Prior to therapy, the patients were found to have significantly lower ζ-chain expression in circulating CD3+ cells and a higher percentage of ζ-chain negative CD3+ and CD4+ cells than normal donors. The patients' peripheral blood mononuclear cells spontaneously produced more IL-10 ex vivo than those of normal controls. After vaccination, recovery of ζ-chain expression was observed in 50% of patients in both clinical trials. Also, spontaneous IL-10 secretion by peripheral blood mononuclear cells decreased following immunotherapy in patients treated with prostate specific antigen and GM-CSF. The frequency of prostate specific antigen-reactive T cells was detectable in 7 out of 18 patients vs 4 out of 18 patients prior to vaccination. Only one of 18 patients was a clinical responder. The vaccine had stimulatory effects on the patients' immune system, but post-vaccine immune recovery could not be correlated to progression-free survival in this small cohort of patients with prostate cancer

Neoadjuvant concurrent chemoradiotherapy with and without hyperthermia in retroperitoneal sarcomas: feasibility, efficacy, toxicity, and long-term outcome

Purpose Retroperitoneal (RPS) sarcomas are associated with poor local and abdominal tumor control. However, the benefit of preoperative radio- or chemotherapy alone for these entities is currently unclear. Moreover, as intermediate- and high-grade sarcomas have a tendency toward early metastasis, exploration of neoadjuvant strategies is of high importance. This analysis reports the results of our 20-year single-institution experience with preoperative neoadjuvant concurrent chemoradiation. Methods From 2000–2019, 27 patients with intermediate- or high-grade RPS (12 dedifferentiated liposarcoma, 10 leiomyosarcoma, 5 others) were treated with radiotherapy (median dose: 50.4 Gy; range 45–75 Gy) and two cycles of chemotherapy (doxorubicin 50 mg/m2 BSA/d3 q28 and ifosfamide 1.5 g/m2 BSA/d1‑5 q28) in neoadjuvant intent. Chemotherapy consisted of doxorubicin alone in two cases and ifosfamide alone in one case. Fifteen patients (56%) additionally received deep regional hyperthermia. Results The median follow-up time was 53 months (±56.7 months). 92% of patients received two cycles of chemotherapy as planned and 92% underwent surgery. At 5 and 10 years, abdominal-recurrence-free survival was 74.6% (±10.1%) and 66.3% (±11.9%), distant metastasis-free survival was 67.2% (±9.7%) and 59.7% (±11.1%), and overall survival was 60.3% (±10.5%) and 60.3% (±10.5%), respectively. CTC grade III and IV toxicities were leukocytopenia (85%), thrombocytopenia (33%), and anemia (11%). There were no treatment-related deaths. Conclusion Neoadjuvant chemoradiotherapy with and without hyperthermia for retroperitoneal sarcomas is feasible and provided high local control of intermediate- and high-grade sarcoma

Harmonization guidelines for HLA-peptide multimer assays derived from results of a large scale international proficiency panel of the Cancer Vaccine Consortium

PURPOSE: The Cancer Vaccine Consortium of the Cancer Research Institute (CVC-CRI) conducted a multicenter HLA-peptide multimer proficiency panel (MPP) with a group of 27 laboratories to assess the performance of the assay. EXPERIMENTAL DESIGN: Participants used commercially available HLA-peptide multimers and a well characterized common source of peripheral blood mononuclear cells (PBMC). The frequency of CD8+ T cells specific for two HLA-A2-restricted model antigens was measured by flow cytometry. The panel design allowed for participants to use their preferred staining reagents and locally established protocols for both cell labeling, data acquisition and analysis. RESULTS: We observed significant differences in both the performance characteristics of the assay and the reported frequencies of specific T cells across laboratories. These results emphasize the need to identify the critical variables important for the observed variability to allow for harmonization of the technique across institutions. CONCLUSIONS: Three key recommendations emerged that would likely reduce assay variability and thus move toward harmonizing of this assay. (1) Use of more than two colors for the staining (2) collect at least 100,000 CD8 T cells, and (3) use of a background control sample to appropriately set the analytical gates. We also provide more insight into the limitations of the assay and identified additional protocol steps that potentially impact the quality of data generated and therefore should serve as primary targets for systematic analysis in future panels. Finally, we propose initial guidelines for harmonizing assay performance which include the introduction of standard operating protocols to allow for adequate training of technical staff and auditing of test analysis procedures

Redirecting T Cells to Ewing's Sarcoma Family of Tumors by a Chimeric NKG2D Receptor Expressed by Lentiviral Transduction or mRNA Transfection

We explored the possibility to target Ewing's sarcoma family of tumors (ESFT) by redirecting T cells. To this aim, we considered NKG2D-ligands (NKG2D-Ls) as possible target antigens. Detailed analysis of the expression of MICA, MICB, ULBP-1, -2, and -3 in fourteen ESFT cell lines revealed consistent expression of at least one NKG2D-L. Thus, for redirecting T cells, we fused a CD3ζ/CD28-derived signaling domain to the ectodomain of NKG2D, however, opposite transmembrane orientation of this signaling domain and NKG2D required inverse orientation fusion of either of them. We hypothesized that the particularly located C-terminus of the NKG2D ectodomain should allow reengineering of the membrane anchoring from a native N-terminal to an artificial C-terminal linkage. Indeed, the resulting chimeric NKG2D receptor (chNKG2D) was functional and efficiently mediated ESFT cell death triggered by activated T cells. Notably, ESFT cells with even low NKG2D-L expression were killed by CD8pos and also CD4pos cells. Both, mRNA transfection and lentiviral transduction resulted in high level surface expression of chNKG2D. However, upon target-cell recognition receptor surface levels were maintained by tranfected RNA only during the first couple of hours after transfection. Later, target-cell contact resulted in strong and irreversible receptor down-modulation, whereas lentivirally mediated expression of chNKG2D remained constant under these conditions. Together, our study defines NKG2D-Ls as targets for a CAR-mediated T cell based immunotherapy of ESFT. A comparison of two different methods of gene transfer reveals strong differences in the susceptibility to ligand-induced receptor down-modulation with possible implications for the applicability of RNA transfection