Germline Variants Associated with Nasopharyngeal Carcinoma Predisposition Identified through Whole-Exome Sequencing.

The current understanding of genetic susceptibility factors for nasopharyngeal carcinoma (NPC) is still incomplete. To identify novel germline variants associated with NPC predisposition, we analysed whole-exome sequencing data from 119 NPC patients from Singapore with a family history of NPC and/or with early-onset NPC, together with 1337 Singaporean participants without NPC. Variants were prioritised and filtered by selecting variants with minor allele frequencies of <1% in both local control (n = 1337) and gnomAD non-cancer (EAS) (n = 9626) cohorts and a high pathogenicity prediction (CADD score > 20). Using single-variant testing, we identified 17 rare pathogenic variants in 17 genes that were associated with NPC. Consistent evidence of enrichment in NPC patients was observed for five of these variants (in JAK2, PRDM16, LRP1B, NIN, and NKX2-1) from an independent case-control comparison of 156 NPC patients and 9770 unaffected individuals. In a family with five siblings, a FANCE variant (p. P445S) was detected in two affected members, but not in three unaffected members. Gene-based burden testing recapitulated variants in NKX2-1 and FANCE as being associated with NPC risk. Using pathway analysis, endocytosis and immune-modulating pathways were found to be enriched for mutation burden. This study has identified NPC-predisposing variants and genes which could shed new insights into the genetic predisposition of NPC.

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.

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.

Correction: Early assessment of tumor response to photodynamic therapy using combined diffuse optical and diffuse correlation spectroscopy to predict treatment outcome.

[This corrects the article DOI: 10.18632/oncotarget.15720.].

Early assessment of tumor response to photodynamic therapy using combined diffuse optical and diffuse correlation spectroscopy to predict treatment outcome.

Photodynamic therapy (PDT) of cancer involves the use of a photosensitizer that can be light-activated to eradicate tumors via direct cytotoxicity, damage to tumor vasculature and stimulating the body's immune system. Treatment outcome may vary between individuals even under the same regime; therefore a non-invasive tumor response monitoring system will be useful for personalization of the treatment protocol. We present the combined use of diffuse optical spectroscopy (DOS) and diffuse correlation spectroscopy (DCS) to provide early assessment of tumor response. The relative tissue oxygen saturation (rStO2) and relative blood flow (rBF) in tumors were measured using DOS and DCS respectively before and after PDT with reference to baseline values in a mouse model. In complete responders, PDT-induced decreases in both rStO2 and rBF levels were observed at 3 h post-PDT and the rBF remained low until 48 h post-PDT. Recovery of these parameters to baseline values was observed around 2 weeks after PDT. In partial responders, the rStO2 and rBF levels also decreased at 3 h post PDT, however the rBF values returned toward baseline values earlier at 24 h post-PDT. In contrast, the rStO2 and rBF readings in control tumors showed fluctuations above the baseline values within the first 48 h. Therefore tumor response can be predicted at 3 to 48 h post-PDT. Recovery or sustained decreases in the rBF at 48 h post-PDT corresponded to long-term tumor control. Diffuse optical measurements can thus facilitate early assessment of tumor response. This approach can enable physicians to personalize PDT treatment regimens for best outcomes.

Novel delivery of Chlorin e6 using anti-EGFR antibody tagged virosomes for fluorescence diagnosis of oral cancer in a hamster cheek pouch model.

PURPOSE: Overexpression of epidermal growth factor receptor (EGFR) is observed in oral squamous cell carcinoma (OSCC) and is associated with increased proliferation, metastasis and therapeutic resistance. We aim to develop a novel drug delivery system comprised of a photosensitizer Chlorin e6 (Ce6) that is encapsulated in a viral envelope and tagged with anti-EGFR antibody to target OSCC. METHODS: Ce6 was encapsulated in both virosomes (Ce6-Vir) and virosomes tagged with anti-EGFR antibody (Ce6-Vir-EGFR'). In vitro studies were conducted to assess the cellular uptake and bioavailability of the photosensitizer in OSCC cells. Ce6 alone or in constructs was then administered in a hamster cheek pouch model and fluorescence imaging and spectroscopy was performed. RESULTS: In vitro results showed that the uptake of Ce6-Vir-EGFR' was lower than that for Ce6-Vir and Ce6 possibly due to its large size. Nevertheless, in vivo results showed significant tumor specificity of Ce6-Vir-EGFR' compared to Ce6. The tumor to normal mucosa ratio showed that Ce6-Vir-EGFR' can successfully target OSCC lesions and therefore shows potential for use in fluorescence diagnosis of OSCC. CONCLUSIONS: Both the virosome-Ce6 constructs were internalized by OSCC cells and successfully used for fluorescence imaging. Tagging with anti-EGFR antibody further improved the targeting ability toward OSCC.