No abstract

Purpose In order to investigate Poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NP) as potential vehicles for efficient tumor antigen (TA) delivery to dendritic cells (DC), this study aimed to optimize encapsulation/release kinetics before determining immunogenicity of antigen-containing NP. Methods Various techniques were used to liberate TA from cell lines. Single (gp100) and multiple (B16-tumor lysate containing gp100) antigens were encapsulated within differing molecular weight PLGA co-polymers. Differences in morphology, encapsulation/release and biologic potency were studied. Findings were adopted to encapsulate fresh tumor lysate from patients with advanced tumors and compare stimulation of tumor infiltrating lymphocytes (TIL) against that achieved by soluble lysate. Results Four cycles of freeze-thaw + 15 s sonication resulted in antigen-rich lysates without the need for toxic detergents or protease inhibitors. The 80KDa polymer resulted in maximal release of payload and favorable production of immunostimulatory IL-2 and IFN-γ. NP-mediated antigen delivery led to increased IFN-γ and decreased immunoinhibitory IL-10 synthesis when compared to soluble lysate. Conclusions Four cycles of freeze-thaw followed by 15 s sonication is the ideal technique to obtain complex TA for encapsulation. The 80KDa polymer has the most promising combination of release kinetics and biologic potency. Encapsulated antigens are immunogenic and evoke favorable TIL-mediated anti-tumor responses.

Objective.  To determine whether nanoparticles (NP) made from FDA‐approved biopolymers could be incorporated into a novel clinically translatable dendritic cell (DC)‐based therapy for HNSCC. Methods.  Head and neck squamous cell carcinoma lines were used to make lysates employing multiple techniques, and with varying amounts of protein so as to improve encapsulation within differing molecular weights of Poly (lactic‐co‐glycolic acid) (PLGA) polymers. NP were fabricated by a double emulsion solvent‐evaporation technique with flash freeze‐lyophilization. Encapsulation and release profile were studied to determine the ideal formulation. These findings were adopted for encapsulating tumour material from five patients with advanced HNSCC. Pattern of protein release was detected using Silver stained SDS‐PAGE gels and immunoblotting for p53. GM‐CSF/IL‐4 DC made from blood monocyte precursors were loaded with either the soluble or encapsulated versions of tumour lysate, and used for stimulation of magnetic bead‐isolated autologous CD8+ T‐cells. Differences in Th1/Th2 responses were estimated by Cytometric bead array (CBA). NP morphology and DC loading were evaluated by scanning electron microscopy. Results.  Optimal release was recorded from 80 K PLGA NP containing a lysate made using four cycles of freeze‐thaw plus 15‐s sonication followed by brief lyophilization. NP were small enough to be phagocytosed by DC. Encapsulation and release was directly proportional to the lysate concentration with no major antigen degradation. In four patients, CBA showed significantly increased IFN‐γ or decreased IL‐10 (P = 0.012) production associated with NP encapsulation. Conclusions.  Biopolymer NP can efficiently deliver the panoply of unknown tumour antigens. This strategy has been optimized for use in a clinical trial. References.  1 Sandor M., Enscore D., Weston P et al. (2001) Effect of protein molecular weight on release from micron‐sized PLGA microspheres. J. Control. Release 76, 297–311 2 Elamanchili P., Lutsiak C., Hamdy S et al. (2007) ‘Pathogen‐mimicking’ nanoparticles for vaccine delivery to dendritic cells. J. Immunother.30, 378–395

Introduction and Aim: Cancer related inflammation is recognized as a critical multifaceted player in tumour initiation and progression. The neutrophil to lymphocyte ratio (NLR) and platelet to lymphocyte ratio (PLR) are important markers of systemic inflammatory burden in malignancy. The present retrospective study aimed to evaluate the prognostic value of pre-treatment baseline (NLR) and (PLR) for survival in HNC patients.   Materials and Methods: Analysis of data of 257 patients with head and neck cancer treated with definitive therapy over 3.5 yrs. Neutrophil, lymphocyte and platelet counts before treatment of all patients were collected and NLR and PLR were calculated.   Results: Median value of NLR and PLR were 3.7 and 170.63 respectively. Receiver operator characteristics (ROC) curve analysis showed the predictive cut-off value of NLR and PLR as 3.9 and 158.3. Univariate analysis using Cox-regression model showed NLR (p < 0.001) and PLR (p = 0.001) significantly influenced the locoregional recurrence free survival (LRRFS) and overall survival (OS). The multivariate analysis showed NLR is the independent prognostic factors influencing LRRFS (p = 0.007) and OS (p = 0.002). Kaplan-Meier survival curve (Log rank test; Chi-square (?2) value) showed OS is significantly influenced by NLR group (LRRFS; ?2 = 23.9 and OS; ?2 = 33.7) and PLR group (LRRFS; ?2 = 11.2 and OS; ?2 = 19.3) in contrast to LRRFS.   Conclusion: NLR can be strongly used as biomarker for prognostication for outcome and survival in head neck cancer. However, a well-designed, larger studies with longer follow-up is warranted.

The interaction of HPV16 E6 and PTP-BL is a potentially important mechanism of HPV-related cancer subject to targeting by small molecule therapy in the future. SUPPORT: NIH K08 award Veterans Administration Merit Award.

Objective.  To establish proof of principle of transimmunization (T.I.) in an animal model prior to use in a dendritic cell (DC)‐based immunotherapy clinical trial for HNSCC. Methods.  Pooled blood from syngeneic Fischer 344 rats was photoactivated in the presence of 8‐methoxypsoralen and Ultraviolet‐A, followed by overnight incubation to facilitate conversion of monocyte precursors to DC. Cells were matured using pathogen associated molecular patterns (PAMPs) – LPS, CpG DNA, flagellin and Poly I: C. Cells were either labeled with Indium‐111 and injected peritumorally followed by in vivo tracking using micro‐Single Photon Emission CT (micro‐SPECT), or used unlabeled in randomized double‐blinded vaccination studies in tumor‐bearing animals. Treatment groups included: T.I. cells ± PAMPs, T.I. cells + lysate ± PAMPs and T.I. cells + tumor lysate (TL) containing‐NP ± PAMPs. Outcome measures included: mean tumor size, survival and ELISA to study differences in production of IFN‐γ and IL‐10. Results.  There were statistically significant differences in mean tumor size at 2 weeks post‐injections (P = 0.033) and overall survival (P = 0.009) in the group treated with T.I. cells alone. Addition of PAMPs made no significant impact on outcome measures. Micro‐SPECT images indicated that T.I. cells remain at the site of injection between 24 and 96 h. Conclusions.  (1) Immature T.I. cells on their own appear to be significantly more effective than those loaded with antigen, or mature ones. (2) T.I. cells remain at the site of injection for at least 24 h prior to migration to regional lymph nodal area for classical antigen cross‐presentation.

No abstract

No abstract