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Additional file 6: Fig. S1. Functional enrichment and network analysis in the TCGA-HNSC dataset. A Heatmap of the top 20 functional enrichment terms by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis based on differentially expressed therapeutic response-related genes (TRRGs). B Protein-protein interaction subnetworks of 20 functional enrichment terms.

Additional file 7: Fig. S2 Expression information of AREG, L1CAM and GPRC5D. A-C Kaplan Meier analysis showed that AREG, L1CAM and GPRC5D had a good prognostic efficacy in the Human Protein Atlas (HPA) database. D-F mRNA expression of AREG, L1CAM and GPRC5D was not significantly different between cancer and normal tissues in the TCGA database.

Additional file 8: Fig. S3. Relationships between the risk score and immune checkpoint blockade (ICB)-associated genes and mutation rate. A ICB-associated genes and nonsilent mutation rate in the Risk-H and Risk-L groups. B Tumor purity, ESTIMATE score, immune score and stromal score in the Risk-H and Risk-L groups. ns, not significant; *, p < 0.05; **, p < 0.01; *** p < 0.001; ****, p< 0.0001.

Additional file 3: Supplementary Table S1. Comparison of RNF38 expression between nasopharyngeal carcinoma and normal nasopharyngeal epithelia tissues.

Additional file 5: Supplementary Table S3. Characteristics of patients with nasopharyngeal carcinoma grouped by RNF38 expression.

Huocong He*,1 Shuo Qie,*,2,3 Qiaojuan Guo,4 Shuyang Chen5,6 Changyan Zou,1 Tianzhu Lu,4 Ying Su,1 Jingfeng Zong,4 Hanchuan Xu,4 Dan He,5 Yun Xu,4 Bijuan Chen,4 Jianji Pan,4 Nianli Sang2,5 Shaojun Lin41Department of Radiation Biology, Fujian Cancer Hospital & Fujian Medical University, Fujian Provincial Key Laboratory of Translational Cancer Medicine, Fuzhou, Fujian, People’s Republic of China; 2Department of Pathology and Laboratory Medicine, Drexel University College of Medicine, Philadelphia, PA 19104, USA; 3Department of Biochemistry & Molecular Biology, Medical University of South Carolina, Charleston, SC 29425, USA; 4Department of Radiation Oncology, Fujian Cancer Hospital & Fujian Medical University, Fujian Provincial Key Laboratory of Translational Cancer Medicine, Fuzhou, Fujian, People’s Republic of China; 5Department of Biology, Drexel University College of Arts & Sciences, Philadelphia, PA 19104, USA; 6Department of Pharmacology & Experimental Therapeutics, Boston University School of Medicine, Boston, MA 02118, USA*These authors contributed equally to this workBackground: Stanniocalcin 2 (STC2) expression is upregulated under multiple stress conditions including hypoxia, nutrient starvation and radiation. Overexpression of STC2 correlates with tumor progression and poor prognosis.Purpose: We previously demonstrated that overexpression of STC2 in nasopharyngeal carcinomas (NPC) positively correlates with radiation resistance and tumor metastasis, two major clinical obstacles to the improvement of NPC management. However, it remains elusive whether STC2 expression is a critical contributing factor for post-radiation survival and metastasis of NPC cells.Materials and methods: Using the radiation resistant CNE2 cell line as a model, we examined the importance of STC2 expression for post-radiation survival, migration and invasion. Here, we report the establishment of STC2 knockout lines (CNE2-STC2-KO) using the CRISPR/Cas9-based genome editing technique.Results: Compared with the parental line, STC2-KO cells showed similar proliferation and morphology in normal culture conditions, and loss of STC2 did not compromise the cell tumorigenicity in nude mice model. However, STC2-KO lines demonstrated increased sensitivity to X-radiation under either normoxic or hypoxic conditions. Particularly, upon X-radiation, parental CNE2 cells only slightly whereas STC2-KO cells remarkably decreased the migration and invasion ability. Cell cycle analysis revealed that loss of STC2 accumulated cells in G1 and G2/M phases but decreased S-population.Conclusion: These data indicate that the expression of STC2, which can be stimulated by metabolic or therapeutic stresses, is one important factor to promote survival and metastasis of post-radiation NPC cells. Therefore, targeting STC2 or relative downstream pathways may provide novel strategies to overcome radiation resistance and metastasis of NPC.Keywords: metabolic stress, metastasis, migration, nasopharyngeal carcinoma, radiation resistance, stanniocalcin 2

Background: The aberrant expression of surface receptors on immunocytes may represent potential markers of tumor escape for nasopharyngeal carcinoma (NPC). The aim of this study was to investigate the expression of representative receptors on natural killer (NK) cells and NK group 2, member D (NKG2D) on immunocytes in the peripheral blood of patients with NPC. Methods: Patients (n = 64) with NPC prior to initiation of treatment were defined as the study group. Healthy volunteers (n = 31) served as the control group. The expression of NK cells and NKT cells; the triggering receptors NKp30, NKp44, and NKp46 on NK cells; the activating receptor NKG2D on NK cells, CD4+ T cells, and CD8+ T cells; and the inhibitory receptors CD158b and CD159a on NK cells were analyzed by flow cytometry in the two groups. Results: Here, our study showed that no differences were observed in terms of the numbers of NK cells or NKT cells, or the expression of CD158b and CD159a on the surface of NK cells between the two groups. Nevertheless, the expression levels of NKp30 and NKp46 on NK cells in the NPC patients were significantly lower than in the healthy individuals (P < 0.05). No differences existed in the expression of NKG2D on NK cells, but NKG2D on CD8+ T cells showed a markedly lower expression in the study group (P < 0.001). Conclusions: Our findings may reflect a possible mechanism of immune evasion for NPC. The enhancement of immunotherapy concerning NKp30, NKp46, and NKG2D may be an innovative treatment strategy for patients with NPC.

Tianzhu Lu,1,* Yiping Chen,2,* Jieyu Li,3 Qiaojuan Guo,2 Wansong Lin,3 Yuhong Zheng,4 Ying Su,5 Jingfeng Zong,2 Shaojun Lin,1,2 Yunbin Ye,3,6 Jianji Pan1,2 1The School of Clinical Medicine, Fujian Medical University, Fuzhou, People’s Republic of China; 2Department of Radiation Oncology, Fujian Cancer Hospital, Fujian Medical University, Fuzhou, People’s Republic of China; 3Laboratory of Immuno-Oncology, Fujian Cancer Hospital, Fuzhou, People’s Republic of China; 4Department of Clinical Laboratory, Fujian Cancer Hospital, Fujian Medical University, Fuzhou, People’s Republic of China; 5Department of Radiation Biology, Fujian Cancer Hospital, Fujian Medical University, Fuzhou, People’s Republic of China; 6The School of Basic Medical Sciences, Fujian Medical University, Fuzhou, People’s Republic of China*These authors contributed equally to this workCorrespondence: Jianji PanThe School of Clinical Medicine, Fujian Medical University, Fuzhou, People’s Republic of ChinaTel +86 591-83638732Fax +86 591-83928767Email panjianji1956@fjmu.edu.cnYunbin YeThe School of Basic Medical Sciences, Fujian Medical University, Fuzhou 350014, People’s Republic of ChinaTel +86 591-83638732Fax +86 591-83928767Email zjyunbin@sina.comPurpose: Immune checkpoint proteins in the tumor microenvironment can enter the blood circulation and are potential markers for liquid biopsy. The aims of this study were to explore differences in immune checkpoint protein expression between patients with nasopharyngeal carcinoma (NPC) and healthy controls and to investigate the prognostic value of the soluble form of programmed death-ligand 1 (sPD-L1) in NPC.Methods: In total, 242 patients were included in the disease group. Plasma samples from 23 NPC patients and 15 healthy control were used for immune checkpoint protein panel assays. Samples from 219 patients with NPC including 30 paired pre-treatment and post-radiotherapy samples were evaluated by enzyme-linked immunosorbent assay to determine sPD-L1 levels.Results: A total of 14 immune checkpoint proteins, including sPD-L1were upregulated in 23 patients with NPC (all p< 0.001) compared with 15 healthy controls. Among 219 patients, the median follow-up time was 50 months (7– 82 months). Based on the optimal cutoff value of 93.7 pg/mL, patients with high expression of sPD-L1 had worse distant metastasis-free survival (87.5% vs 74.0%, p=0.006) than those of patients with low expression. Multivariate analysis showed that sPD-L1 (HR=1.99, p=0.048) and EBV-DNA (HR=2.51, p=0.030) were poor prognostic factors for DMFS. In the group with high EBV-DNA expression, DMFS was worse for patients with high sPD-L1 expression than those with low sPD-L1 expression (56.4% vs 82.6%, p=0.002).Conclusion: Plasma immune checkpoint protein expression differed significantly between patients with NPC and healthy donors. Plasma sPD-L1 levels are a candidate prognostic biomarker, especially when combined with EBV-DNA.Keywords: nasopharyngeal carcinoma, programmed death-ligand 1, Epstein-Barr virus, immune checkpoint protein