High-Dimensional Characterization of the Systemic Immune Landscape Informs on Synergism Between Radiation Therapy and Immune Checkpoint Blockade.

PURPOSE: Improved antitumor responses have been observed in patients after combination radiation therapy (RT) and immune checkpoint blockade (ICB). Whether these clinical responses are linked to the host systemic immune system has not been elucidated. METHODS AND MATERIALS: In this single-institution prospective observational study, peripheral blood was longitudinally collected from 10 patients with metastatic disease who had responded to anti-PD-1/anti-PD-L1 ICB and received RT (8-50 Gy in 1-5 fractions) upon disease progression at the following timepoints: baseline (pre-RT), 1 to 2 weeks post-RT, and post-ICB (cycle 1) on reintroduction post-RT. To thoroughly characterize the interaction between combined RT-ICB and the host immune system, we performed high-dimensional, mass cytometry-based immunophenotyping of circulating lymphocytes using a 40-marker panel addressing lineage, differentiation, activation, trafficking, cytotoxicity, and costimulatory and inhibitory functions. Phenotypic expression of circulating lymphocytes was compared across patients and time points and correlated with post-RT tumor responses. RESULTS: Foremost, we demonstrated excellent posttreatment clinical responses, including 4 local responses with >50% reduction in radiated tumor size, 1 out-of-field response, and 4 patients who resumed ICB for >1 year. Baseline and post-RT immune states were highly heterogeneous among patients. Despite this interindividual heterogeneity in baseline immune states, we observed a systemic immune reaction to RT-ICB common across patients, histology, and radiation sites; a subset of pre-existing Ki-67+ CD8+ T cells were increased post-RT and further expanded upon reintroduction of ICB post-RT (2.3-fold increase, P = .02). Importantly, RT did not alter the phenotypic profile of these Ki-67+ CD8+ T cells, which was characterized by a distinct activated and differentiated effector phenotype. CONCLUSIONS: Collectively, these findings point toward a sustained reinvigoration of host antitumor immunity after RT-ICB and suggest an expansion in activated Ki-67+ CD8+ T cells as a possible demonstration of this synergy, thereby providing new insights that may support the development of optimal sequencing strategies.

Germline Polymorphisms and Length of Survival of Nasopharyngeal Carcinoma: An Exome-Wide Association Study in Multiple Cohorts.

Germline polymorphisms are linked with differential survival outcomes in cancers but are not well studied in nasopharyngeal carcinoma (NPC). Here, a two-phase association study is conducted to discover germline polymorphisms that are associated with the prognosis of NPC. The discovery phase includes two consecutive hospital cohorts of patients with NPC from Southern China. Exome-wide genotypes at 246 173 single nucleotide polymorphisms (SNPs) are determined, followed by survival analysis for each SNP under Cox proportional hazard regression model. Candidate SNP is replicated in another two independent cohorts from Southern China and Singapore. Meta-analysis of all samples (n = 5553) confirms that the presence of rs1131636-T, located in the 3'-UTR of RPA1, confers an inferior overall survival (HR = 1.33, 95% CI = 1.20-1.47, P = 6.31 × 10-8). Bioinformatics and biological assays show that rs1131636 has regulatory effects on upstream RPA1. Functional studies further demonstrate that RPA1 promotes the growth, invasion, migration, and radioresistance of NPC cells. Additionally, miR-1253 is identified as a suppressor for RPA1 expression, likely through regulation of its binding affinity to rs1131636 locus. Collectively, these findings provide a promising biomarker aiding in stratifying patients with poor survival, as well as a potential drug target for NPC.

Upconversion superballs for programmable photoactivation of therapeutics.

Upconversion nanoparticles (UCNPs) are the preferred choice for deep-tissue photoactivation, owing to their unique capability of converting deep tissue-penetrating near-infrared light to UV/visible light for photoactivation. Programmed photoactivation of multiple molecules is critical for controlling many biological processes. However, syntheses of such UCNPs require epitaxial growth of multiple shells on the core nanocrystals and are highly complex/time-consuming. To overcome this bottleneck, we have modularly assembled two distinct UCNPs which can individually be excited by 980/808 nm light, but not both. These orthogonal photoactivable UCNPs superballs are used for programmed photoactivation of multiple therapeutic processes for enhanced efficacy. These include sequential activation of endosomal escape through photochemical-internalization for enhanced cellular uptake, followed by photocontrolled gene knockdown of superoxide dismutase-1 to increase sensitivity to reactive oxygen species and finally, photodynamic therapy under these favorable conditions. Such programmed activation translated to significantly higher therapeutic efficacy in vitro and in vivo in comparison to conventional, non-programmed activation.

Vandetanib sensitizes head and neck squamous cell carcinoma to photodynamic therapy through modulation of EGFR-dependent DNA repair and the tumour microenvironment.

BACKGROUND: Epidermal growth factor receptor (EGFR) overexpression is characteristic in head and neck cancers and is associated with tumour regrowth following photodynamic therapy (PDT). PURPOSE: We investigated vandetanib, which selectively blocks EGFR and vascular endothelial growth factor receptor-2 (VEGFR-2), to enhance the efficacy of PDT. METHODS: We assessed the in vitro therapeutic efficacy of: 1) vandetanib; 2) PDT with the photosensitizer Chlorin e6 (Fotolon®); and 3) combined PDT + vadetanib treatment in CAL-27 oral squamous cell carcinoma (OSCC) cell line by cell viability, γH2AX foci immunostaining, cell cycle arrest and western blot. We also performed in vivo tumour regression study and immunohistochemical staining of formalin-fixed paraffin-embedded (FFPE) regressed and regrown tumour tissues. RESULTS: First, we observed significantly higher cytotoxicity and residual DNA damage in vandetanib + PDT-treated CAL-27 OSCC cells than tumour cells treated with PDT alone. This is due to impaired DNA DSB repair caused by downregulation of EGFR-mediated DNA-dependent protein kinase catalytic subunit (DNA-PKcs) activation. Next, combined vandetanib and PDT resulted in significant tumour growth delay in vivo that is linked to reduction of PDT-induced EGFR phosphorylation and cellular proliferation, along with loss of tumour vasculature. In particular, we observed significant revascularisation of the microenvironment that is associated with upregulated ERK1/2 phosphorylation in regrown tumours post-vandetanib + PDT, thereby corroborating the importance of microenvironmental modification for the observed drug-PDT synergistic interaction. CONCLUSION: Taken together, our data suggests that vandetanib enhances the efficacy of PDT through both direct and indirect effects on the cellular DNA repair machinery and tumour microenvironment, respectively.

Combinatorial strategies of radiotherapy and immunotherapy in nasopharyngeal carcinoma.

Immunotherapy and radiation therapy (RT) have each demonstrated clinical success in the treatment of nasopharyngeal carcinoma (NPC) when utilized independently. Several characteristics of NPC make it particularly well suited for immunotherapeutic strategies, such as the association with viral infections like EBV and human papilloma virus (HPV), upregulation of PD-L1 expression, and the high number of tumor infiltrating lymphocytes. Immune checkpoint blockade is one such immunotherapeutic strategy that is gaining popularity rapidly. However, clinical benefit of immunotherapy using immune checkpoint inhibitors has been limited to only a small subset of patients with existing T cell responses. Additionally, they are frequently associated with dose-limiting immune-related toxicities. On the other hand, RT is a conventional strategy for NPC treatment, which has demonstrated high efficacy in local tumor control and has also been reported to exhibit immune modulatory effects. However, the abscopal effect of RT alone, i.e., the regression of distant metastases outside of the irradiation field, remains a rare phenomenon. Furthermore, RT treatment efficacy is also limited by radioresistance and radiation-related toxicities. Hence, the combination of RT and immunotherapy has the potential to improve treatment efficacy over either individual therapies alone. Here, we reviewed the clinical problem in locally advanced and recurrent/metastatic NPC, and discussed how combinatorial RT and immunotherapeutic strategies can be relevant to NPC treatment in each clinical scenario by examining the underlying mechanisms involved in the different strategies.