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Supplemental Figure 1. The SFRP1 mRNA (A) and protein (B) expression of CNE-2 and SUNE-1 cells transfected with plasmid pcDNA3.1-SFRP1 compared to the original cell lines and the pcDNA3.1-vector.

Supplemental Figure 2. (A)The SFRP1 mRNA and protein expression of NP69 cells transfected with SFRP1 siRNA compared with the original cell lines and the control siRNA-NC; Effects of SFRP1 siRNA on NP69 cell proliferation(B), migration (C and D).

Supplemental Methods and Materials

Distant metastasis remains the predominant mode of treatment failure in nasopharyngeal carcinoma (NPC). Unfortunately, the molecular events underlying NPC metastasis remain poorly understood. Secreted frizzled-related protein 1 (SFRP1) plays an important role in tumorigenesis and progression. However, little is known about the function and mechanism of SFRP1 in NPC. Immunohistochemistry was used to determine SFRP1 expression levels in patients with NPC. SFRP1 function was evaluated using MTT, colony formation, wound-healing, Transwell assays, and in vivo models. The methylation level of SFRP1 in NPC cells was examined using bisulfate pyrosequencing; the Wnt/β-catenin signaling pathway genes were studied using Western blotting. Compared with patients with high SFRP1 expression, patients with low SFRP1 expression had worse overall survival [HR, 2.32; 95% confidence interval (CI), 1.36–3.94; P = 0.002], disease-free survival (HR, 1.98; 95% CI, 1.23–3.18; P = 0.005), and distant metastasis-free survival (HR, 2.07; 95% CI, 1.19–3.59; P = 0.009). Multivariate Cox regression analysis indicated that SFRP1 was an independent prognostic factor. Furthermore, SFRP1 was significantly downregulated in NPC cell lines. SFRP1 overexpression suppressed NPC cell proliferation, migration, and invasion in vitro and lung colonization in vivo. SFRP1 expression was restored after treatment with a demethylation agent, and the SFRP1 promoter region was hypermethylated in NPC cells. β-Catenin, c-Myc, and cyclin D1 were downregulated after SFRP1 restoration, which suggested that SFRP1 suppressed growth and metastasis by inhibiting the Wnt/β-catenin signaling pathway in NPC. SFRP1 provides further insight into NPC progression and may provide novel therapeutic targets for NPC treatment. Cancer Prev Res; 8(10); 968–77. ©2015 AACR.

Relationship between CNA level and TMB in (A) MSK pan-cancer non-ICI treated cohort and (B) other three ICI-treated cohorts (MSK NSCLC, SKCM, and Dana-Farber ICI cohort). For each cancer type and each cohort, a plot is shown containing the CNA level versus tumor mutation burden. The spearman correlation coefficient and P value are shown.

Demographic and clinicopathologic characteristics of three cohorts treated with immune checkpoint inhibitors.

Altered DNA methylation is a key feature of cancer, and aberrant methylation is important in nasopharyngeal carcinoma (NPC) development. However, the methylation mechanisms underlying metastasis of NPC remain unclear. Analyzing data from public databases and conducting our own experiments, we report here that promoter hypermethylation of SHISA3 is common and contributes to the downregulation of this gene in many types of tumors, including NPC. SHISA3 suppressed NPC cell invasion and metastasis in vitro and in vivo by impeding the E3 ubiquitin ligase tripartite motif containing 21 (TRIM21)–mediated ubiquitination and degradation small G protein signaling modulator 1 (SGSM1) and by inhibiting the MAPK pathway activation. Silencing SGSM1 abrogated the inhibitory effect of SHISA3 on NPC cell migration and invasion. This newly identified SHISA3–TRIM21–SGSM1 axis could be a novel therapeutic target in NPC.Significance:These findings highlight the mechanism by which a newly identified tumor suppressor SHISA3 suppresses invasion and metastasis of nasopharyngeal carcinoma.

Supplementary Figures S1-S8: -Figure S1. The SHISA3 promoter is hypermethylated in NPC. Figure S2. The SHISA3 promoter is hypermethylated in NPC cell lines. Figure S3. SHISA3 is hypermethylated in 18 primary solid tumors. Figure S4. Correlations between SHISA3 promoter methylation and mRNA expression in primary solid tumors. Figure S5. SHISA3 has little effect on NPC cell proliferation in vitro. Figure S6. SHISA3 stabilizes SGSM1 by impeding TRIM21-mediated deubiquitination. Figure S7. The effects of SHISA3 overexpression on nasopharyngeal carcinoma aggressiveness in vivo. Figure S8. SHISA3 has little effect on the canonical Wnt signaling pathway.

Supplementary figure legends

Purpose:Although tumor mutation burden (TMB) has been well known to predict the response to immune checkpoint inhibitors (ICI), lack of randomized clinical trial data has restricted its clinical application. This study aimed to explore the significance and feasibility of biomarker combination based on TMB and copy-number alteration (CNA) for the prognosis of each tumor and prediction for ICI therapy in metastatic pan-cancer milieu.Experimental Design:Non-ICI–treated MSK pan-cancer cohort was used for prognosis analysis. Three independent immunotherapy cohorts, including non–small cell lung cancer (n = 240), skin cutaneous melanoma (n = 174), and mixed cancer (Dana-Farber, n = 98) patients from previous studies, were analyzed for efficacy of ICI therapy.Results:TMB and CNA showed optimized combination for the prognosis of most metastatic cancer types, and patients with TMBlowCNAlow showed better survival. In the predictive analysis, both TMB and CNA were independent predictive factors for ICI therapy. Remarkably, when TMB and CNA were jointly analyzed, those with TMBhighCNAlow showed favorable responses to ICI therapy. Meanwhile, TMBhighCNAlow as a new biomarker showed better prediction for ICI efficacy compared with either TMB-high or CNA-low alone. Furthermore, analysis of the non-ICI–treated MSK pan-cancer cohort supported that the joint stratification of TMB and CNA can be used to categorize tumors into distinct sensitivity to ICI therapy across pan-tumors.Conclusions:The combination of TMB and CNA can jointly stratify multiple metastatic tumors into groups with different prognosis and heterogeneous clinical responses to ICI treatment. Patients with TMBhighCNAlow cancer can be an optimal subgroup for ICI therapy.

DNA methylation is an important epigenetic change in carcinogenesis. However, the function and mechanism of DNA methylation dysregulation in nasopharyngeal carcinoma (NPC) is still largely unclear. Our previous genome-wide microarray data showed that NFAT1 is one of the most hypermethylated transcription factor genes in NPC tissues. Here, we found that NFAT1 hypermethylation contributes to its down-regulation in NPC. NFAT1 overexpression inhibited cell migration, invasion, and epithelial-mesenchymal transition in vitro and tumor metastasis in vivo. We further established that the tumor suppressor effect of NFAT1 is mediated by its inactivation of ITGA6 transcription. Our findings suggest the significance of activating NFAT1/ITGA6 signaling in aggressive NPC, defining a novel critical signaling mechanism that drives NPC invasion and metastasis and providing a novel target for future personalized therapy.

Current anatomic TNM stage classification fails to capture the immune heterogeneity of oral squamous cell carcinoma (OSCC). Increasing evidence indicates the strong association between epithelial-mesenchymal transition (EMT) and tumor immune response. In this study, we employed an EMT signature to classify OSCC patients into epithelial- (E-) and mesenchymal- (M-) phenotypes using TCGA and GSE41613 transcriptome data. The ESTIMATE and CIRBERSORT analyses implied that the EMT signature genes originated from the stroma of the bulk tissue. The M-subtype tumors were characterized as “immune-hot” with more immune cell infiltration than the E-subtype ones. The low infiltration of active immune cells, the high infiltration of inactive immune cells, and the high expressions of immune checkpoints demonstrated an immunosuppressive characteristic of the M-subtype tumors. Moreover, we developed and validated a novel prognostic classifier based on the EMT score, the expressions of seven immune checkpoints, and the TNM stages, which could improve the prediction efficiency of the current clinical parameter. Together, our findings provide a better understanding of the tumor immune heterogeneity and may aid guiding immunotherapy in OSCC.

Altered DNA methylation is a key feature of cancer, and aberrant methylation is important in nasopharyngeal carcinoma (NPC) development. However, the methylation mechanisms underlying metastasis of NPC remain unclear. Analyzing data from public databases and conducting our own experiments, we report here that promoter hypermethylation of SHISA3 is common and contributes to the downregulation of this gene in many types of tumors, including NPC. SHISA3 suppressed NPC cell invasion and metastasis in vitro and in vivo by impeding the E3 ubiquitin ligase tripartite motif containing 21 (TRIM21)–mediated ubiquitination and degradation small G protein signaling modulator 1 (SGSM1) and by inhibiting the MAPK pathway activation. Silencing SGSM1 abrogated the inhibitory effect of SHISA3 on NPC cell migration and invasion. This newly identified SHISA3–TRIM21–SGSM1 axis could be a novel therapeutic target in NPC. Significance: These findings highlight the mechanism by which a newly identified tumor suppressor SHISA3 suppresses invasion and metastasis of nasopharyngeal carcinoma.

Objectives: Clinical benefits of immune-checkpoint blockade (ICB) versus standard chemotherapy have been established in unselected non-small cell lung cancer (NSCLC). However, the response to ICB therapy among patients is heterogeneous in clinical practice. Materials and Methods: We retrospectively assessed the predicitive effect of the primary and metastatic lesion spectrum (baseline sum of the longest diameters [SLD], number of metastatic sites and specific organ metastases) on the efficacy of atezolizumab over docetaxel in OAK and POPLAR trial cohorts. A decision model, termed DSO (Diameter-Site-Organ), based on the spectrum was developed and validated for guiding ICB. Results: Higher SLD (>38 mm) and more metastatic sites (≥2) were characterized with pronounced overall survival (OS) benefits from atezolizumab versus docetaxel. Specifically, adrenal gland and brain metastases were identified as favorable predictors of atezolizumab treatment. The DSO model was developed in the discovery cohort to integrate the directive effect of the primary and metastatic lesion spectrum. Remarkably, a general pattern of enhanced efficacy of atezolizumab versus docetaxel was observed along with the increase of the DSO score. For patients with DSO score > 0, atezolizumab yielded a significantly prolonged OS than docetaxel, whereas OS was generally similar between two treatments in patients with DSO score ≤ 0. Equivalent findings were also seen in the internal and external validation cohorts. Conclusions: The response to anti-PD-L1 therapy among patients varied with the primary and metastatic lesion spectrum. The DSO-based system might provide promising medication guidance for ICB treatment in NSCLC patients.

BackgroundGenetic variations of some driver genes in non-small cell lung cancer (NSCLC) had shown potential impact on immune microenvironment and associated with response or resistance to programmed cell death protein 1 (PD-1) blockade immunotherapy. We therefore undertook an exploratory analysis to develop a genomic mutation signature (GMS) and predict the response to anti-PD-(L)1 therapy.MethodsIn this multicohort analysis, 316 patients with non-squamous NSCLC treated with anti-PD-(L)1 from three independent cohorts were included in our study. Tumor samples from the patients were molecularly profiled by MSK-IMPACT or whole exome sequencing. We developed a risk model named GMS based on the MSK training cohort (n=123). The predictive model was first validated in the separate internal MSK cohort (n=82) and then validated in an external cohort containing 111 patients from previously published clinical trials.ResultsA GMS risk model consisting of eight genes (TP53, KRAS, STK11, EGFR, PTPRD, KMT2C, SMAD4, and HGF) was generated to classify patients into high and low GMS groups in the training cohort. Patients with high GMS in the training cohort had longer progression-free survival (hazard ratio (HR) 0.41, 0.28–0.61, p<0.0001) and overall survival (HR 0.53, 0.32–0.89, p=0.0275) compared with low GMS. We noted equivalent findings in the internal validation cohort and in the external validation cohort. The GMS was demonstrated as an independent predictive factor for anti-PD-(L)1 therapy comparing with tumor mutational burden. Meanwhile, GMS showed undifferentiated predictive value in patients with different clinicopathological features. Notably, both GMS and PD-L1 were independent predictors and demonstrated poorly correlated; inclusion of PD-L1 with GMS further improved the predictive capacity for PD-1 blockade immunotherapy.ConclusionsOur study highlights the potential predictive value of GMS for immunotherapeutic benefit in non-squamous NSCLC. Besides, the combination of GMS and PD-L1 may serve as an optimal partner in guiding treatment decisions for anti-PD-(L)1 based therapy.

Background: Clinical benefits of immune-checkpoint blockade (ICB) versus standard chemotherapy have been established in unselected non-small cell lung cancer (NSCLC). However, the response to ICB therapy among patients is still heterogeneous in clinical practice. Methods: We retrospectively assessed the impact of baseline sum of the longest diameters (SLD), number of metastatic sites and specific organ metastases on the efficacy of atezolizumab versus docetaxel in the pooled population from OAK and POPLAR studies. An assistant decision model based on the machine-learning method, incorporating these indicators, termed DSO (Diameter-Site-Organ), was developed and validated in OAK and POPLAR cohorts. Findings: Higher SLD (> 38mm) and more metastatic sites (≥ 2) were characterized with pronounced OS benefits from atezolizumab versus docetaxel. Specifically, brain and adrenal gland metastases were identified as favorable predictors of atezolizumab treatment. The DSO model for guiding second-line treatment was developed based on SLD and metastatic sites and organs in the discovery cohort. Remarkably, a general pattern of enhanced efficacy of atezolizumab versus docetaxel was observed along with the increase of the DSO score. For patients with DSO score > 0, atezolizumab yielded a prolonged OS than docetaxel (hazard ratio [HR] 0.67, 95% CI 0.54-0.84; P = 0.0003), whereas OS was generally similar between two treatments in patients with DSO score ≤ 0 (HR 1.46, 95% CI 0.89-2.39; P = 0.1346). Equivalent findings were seen in the internal and external validation cohorts. Interpretation: Our study for the first time revealed that patients with higher tumor burden were more suitable for ICB compared with standard chemotherapy. More importantly, the integrative DSO decision model might provide promising medication guidance for second-line ICB treatment in unselected NSCLC patients. Funding: This study was supported by the National Natural Science Foundation for YoungScientists of China (Grant No. 81802863 and 81902353), the Natural Science Foundation of Guangdong Province (Grant No. 2018030310285), and the Outstanding Youths Development Scheme of Nanfang Hospital, Southern Medical University (Grant No. 2017J003). Conflict of Interest: The authors have no actual or potential conflicts of interest to declare. Ethical Approval: The study was approved by theInstitutional Ethical Review Boards of Nanfang Hospital. All patients enrolled in OAK and POPLAR provided signed informed consent in accordance with the protocols of the corresponding studies.

Purpose: Epigenetic alterations play important roles in metastasis and drug resistance through gene regulation. However, the functional features and molecular mechanisms of epigenetic changes remain largely unclear in nasopharyngeal carcinoma (NPC) metastasis. Experimental Design: Gene regulatory network analysis was used to identify metastatic-specific dysregulated genes between normal and NPC tissues and the expression was validated in published Gene-Expression Omnibus data set. The regulatory and functional role of RAB37 downregulation was examined in NPC and was validated in vitro and in vivo, and downstream target of RAB37 was explored. The clinical value of RAB37 methylation was evaluated in NPC metastasis and chemosensitivity. Results: We identified RAB37 as a specific hypermethylated gene that is most commonly downregulated in NPC. Moreover, RAB37 downregulation was attributed to hypermethylation of its promoter and was significantly associated with metastasis- and docetaxel chemoresistance-related features in NPC. Ectopic RAB37 overexpression suppressed NPC cell metastasis and enhanced chemosensitivity to docetaxel. Mechanistically, RAB37 colocalized with TIMP2, regulated TIMP2 secretion, inhibited downstream MMP2 activity, and consequently altered NPC cell metastasis. Furthermore, RAB37 hypermethylation was correlated with poor clinical outcomes in patients with NPC. We developed a prognostic model based on RAB37 methylation and N stage that effectively predicted an increased risk of distant metastasis and a favorable response to docetaxel-containing induction chemotherapy (IC) in NPC patients. Conclusions: This study shows that RAB37 hypermethylation is involved in NPC metastasis and chemoresistance, and that our prognostic model can identify patients who are at a high risk of distant metastasis and might benefit from for docetaxel IC.

Gene expression patterns can be used as prognostic biomarkers in various types of cancers. We aimed to identify a gene expression pattern for individual distant metastatic risk assessment in patients with locoregionally advanced nasopharyngeal carcinoma.In this multicentre, retrospective, cohort analysis, we included 937 patients with locoregionally advanced nasopharyngeal carcinoma from three Chinese hospitals: the Sun Yat-sen University Cancer Center (Guangzhou, China), the Affiliated Hospital of Guilin Medical University (Guilin, China), and the First People's Hospital of Foshan (Foshan, China). Using microarray analysis, we profiled mRNA gene expression between 24 paired locoregionally advanced nasopharyngeal carcinoma tumours from patients at Sun Yat-sen University Cancer Center with or without distant metastasis after radical treatment. Differentially expressed genes were examined using digital expression profiling in a training cohort (Guangzhou training cohort; n=410) to build a gene classifier using a penalised regression model. We validated the prognostic accuracy of this gene classifier in an internal validation cohort (Guangzhou internal validation cohort, n=204) and two external independent cohorts (Guilin cohort, n=165; Foshan cohort, n=158). The primary endpoint was distant metastasis-free survival. Secondary endpoints were disease-free survival and overall survival.We identified 137 differentially expressed genes between metastatic and non-metastatic locoregionally advanced nasopharyngeal carcinoma tissues. A distant metastasis gene signature for locoregionally advanced nasopharyngeal carcinoma (DMGN) that consisted of 13 genes was generated to classify patients into high-risk and low-risk groups in the training cohort. Patients with high-risk scores in the training cohort had shorter distant metastasis-free survival (hazard ratio [HR] 4·93, 95% CI 2·99-8·16; p0·0001), disease-free survival (HR 3·51, 2·43-5·07; p0·0001), and overall survival (HR 3·22, 2·18-4·76; p0·0001) than patients with low-risk scores. The prognostic accuracy of DMGN was validated in the internal and external cohorts. Furthermore, among patients with low-risk scores in the combined training and internal cohorts, concurrent chemotherapy improved distant metastasis-free survival compared with those patients who did not receive concurrent chemotherapy (HR 0·40, 95% CI 0·19-0·83; p=0·011), whereas patients with high-risk scores did not benefit from concurrent chemotherapy (HR 1·03, 0·71-1·50; p=0·876). This was also validated in the two external cohorts combined. We developed a nomogram based on the DMGN and other variables that predicted an individual's risk of distant metastasis, which was strengthened by adding Epstein-Barr virus DNA status.The DMGN is a reliable prognostic tool for distant metastasis in patients with locoregionally advanced nasopharyngeal carcinoma and might be able to predict which patients benefit from concurrent chemotherapy. It has the potential to guide treatment decisions for patients at different risk of distant metastasis.The National Natural Science Foundation of China, the National ScienceTechnology Pillar Program during the Twelfth Five-year Plan Period, the Natural Science Foundation of Guang Dong Province, the National Key Research and Development Program of China, the Innovation Team Development Plan of the Ministry of Education, the HealthMedical Collaborative Innovation Project of Guangzhou City, China, and the Program of Introducing Talents of Discipline to Universities.

The American Joint Committee on Cancer (AJCC) staging system is inadequate for an accurate prognosis in nasopharyngeal carcinoma (NPC). Thus, new biomarkers are under intense investigation. Here, we investigated whether the density of TILs could predict prognosis in NPC. First, we used 1490 cases of nasopharyngeal carcinoma samples from two independent cohorts to evaluate the density and distribution of tumor-infiltrating lymphocytes (TILs). Second, in one cohort, we assessed associations between TILs and clinical outcomes in 593 randomly selected samples (defined as the training set) and validated findings in the remaining 593 samples (defined as the validation set). Furthermore, we confirmed the prognostic value of TILs in a second independent cohort of 304 cases (defined as the independent set). Based on multivariable Cox regression analysis, we also established an effective prognostic nomogram including TILs to improve accuracy in predicting disease-free survival (DFS) for patients with nondisseminated NPC. We found that high TILs in the training set were significantly associated with favorable DFS [hazard ratio (HR) 0.41, 95% confidence interval (CI) 0.28-0.58, p < 0.001], overall survival (OS, HR 0.42, 95% CI 0.27-0.64, p < 0.001), distant metastasis-free survival (DMFS, HR 0.37, 95% CI 0.23-0.58, p < 0.001) and local-regional recurrent free survival (LRRFS, HR 0.43, 95% CI 0.25-0.73, p = 0.002). Multivariate analysis showed that TILs are an independent prognostic indicator for DFS in all cohorts. In summary, this study indicated that TILs may reflect the immunological heterogeneity of NPC and could represent a new prognostic biomarker.

Objectives To investigate the feasibility of contralateral lower neck sparing intensity modulation radiated therapy (IMRT) for nasopharyngeal carcinoma patients (NPC) with unilateral cervical lymph node metastasis. Materials and methods Retrospective review of 546 patients with unilateral cervical lymph node metastasis treated between November 2009 and February 2012 at one institution. All patients were staged using magnetic resonance imaging and received radical IMRT. Patients were classified into two groups: the inferior border of the negative neck irradiation field only covered Levels III to Va in Group 1; the inferior border covered entire neck down to Levels IV to Vb in Group 2. Results Median follow-up was 49.9 months (range, 1.3–69.2 months). Four-year overall survival (OS:89.3% vs. 88.9%, P = 0.91), disease-free survival (DFS:81.7% vs. 81.0%, P = 0.91), distant metastasis-free survival (DMFS:88.2% vs. 87.9%, P = 0.95), local relapse-free survival (LRFS:96.7% vs. 94.7%, P = 0.70) and nodal relapse-free survival (NRFS: 96.1% vs. 95.9%, P = 0.94) were not significantly different between Group 1 and Group 2. Twenty-two patients developed cervical lymph node relapse; of whom 20/22 (91.0%) developed unilateral relapse within pretreatment positive neck. Only one patient developed out-of-field relapse, though this patient also relapsed within the neck irradiation field (Level II). No clinicopathological feature tested had significant prognostic value for NRFS in multivariate analysis. Conclusions In the IMRT and MRI era, contralateral lower neck sparing IMRT seems to be feasible for NPC patients with unilateral cervical lymph node metastasis.

Nasopharyngeal carcinoma (NPC) is characterized by a high rate of local invasion and early distant metastasis. Increasing evidence indicates that epigenetic abnormalities play important roles in NPC development. However, the epigenetic mechanisms underlying NPC metastasis remain unclear. Here we investigate aberrantly methylated transcription factors in NPC tissues, and we identify the HOP homeobox HOPX as the most significantly hypermethylated gene. Consistently, we find that HOXP expression is downregulated in NPC tissues and NPC cell lines. Restoring HOPX expression suppresses metastasis and enhances chemosensitivity of NPC cells. These effects are mediated by HOPX-mediated epigenetic silencing of SNAIL transcription through the enhancement of histone H3K9 deacetylation in the SNAIL promoter. Moreover, we find that patients with high methylation levels of HOPX exhibit poor clinical outcomes in both the training and validation cohorts. In summary, HOPX acts as a tumour suppressor via the epigenetic regulation of SNAIL transcription, which provides a novel prognostic biomarker for NPC metastasis and therapeutic target for NPC treatment., HOPX is a transcription factor epigenetically silenced in several cancers. Here the authors, by analysing methylation profiles, identify HOPX as a suppressor of metastasis in nasopharyngeal carcinoma: mechanistically HOPX inhibits SNAIL transcription through deacetylation-mediated silencing.

Objective To compare hospital costs and clinical outcomes between transradial intervention (TRI) and transfemoral intervention (TFI) in elderly patients aged over 65 years. Methods We identified 1229 patients aged over 65 years who underwent percutaneous coronary intervention (PCI) in Fuwai Hospital, Beijing, China, between January 1 and December 31, 2010. Total hospital costs and in-hospital outcomes were compared between TRI and TFI. An inverse probability weighting (IPW) model was introduced to control potential biases. Results Patients who underwent TRI were younger, less often female, more likely to receive PCI for single-vessel lesions, and less likely to undergo the procedure for ostial lesions. TRI was associated with a cost saving of CNY7495 (95%CI: CNY4419-10 420). Such differences were mainly driven by lower PCI-related costs. TRI patients had shorter length of stay (1.9 days, 95%CI: 1.1-2.7 days), shorter post-procedural stay (0.7 days, 95%CI: 0.3-1.1 days), and fewer major adverse cardiac events (adjusted odds ratio = 0.47, 95%CI: 0.31-0.73). There was no statistical significance in the incidence of post-PCI bleeding between TRI and TFI (P>0.05). Such differences remained consistent in clinically relevant subgroups of acute myocardial infarction, acute coronary syndrome, and stable angina. Conclusion The use of TRI in patients aged over 65 years was associated with significantly reduced hospital costs and more favorable clinical outcomes.

Purpose: Although tumor mutation burden (TMB) has been well known to predict the response to immune checkpoint inhibitors (ICI), lack of randomized clinical trial data has restricted its clinical application. This study aimed to explore the significance and feasibility of biomarker combination based on TMB and copy-number alteration (CNA) for the prognosis of each tumor and prediction for ICI therapy in metastatic pan-cancer milieu. Experimental Design: Non-ICI–treated MSK pan-cancer cohort was used for prognosis analysis. Three independent immunotherapy cohorts, including non–small cell lung cancer (n = 240), skin cutaneous melanoma (n = 174), and mixed cancer (Dana-Farber, n = 98) patients from previous studies, were analyzed for efficacy of ICI therapy. Results: TMB and CNA showed optimized combination for the prognosis of most metastatic cancer types, and patients with TMBlowCNAlow showed better survival. In the predictive analysis, both TMB and CNA were independent predictive factors for ICI therapy. Remarkably, when TMB and CNA were jointly analyzed, those with TMBhighCNAlow showed favorable responses to ICI therapy. Meanwhile, TMBhighCNAlow as a new biomarker showed better prediction for ICI efficacy compared with either TMB-high or CNA-low alone. Furthermore, analysis of the non-ICI–treated MSK pan-cancer cohort supported that the joint stratification of TMB and CNA can be used to categorize tumors into distinct sensitivity to ICI therapy across pan-tumors. Conclusions: The combination of TMB and CNA can jointly stratify multiple metastatic tumors into groups with different prognosis and heterogeneous clinical responses to ICI treatment. Patients with TMBhighCNAlow cancer can be an optimal subgroup for ICI therapy.

Background: Genetic variations in non-small cell lung cancer (NSCLC) have been shown potential impact on immune microenvironment, and associated with response or resistance to programmed cell death protein 1 (PD-1) blockade immunotherapy. We therefore undertook an unbiased analysis to develop a gene mutation-based signature (GMS) and predict the response to anti-PD-(L1) therapy. Methods: In this retrospective multicohort analysis, 316 patients with nonsquamous NSCLC treated with anti-PD-(L1) based therapy from three independent cohorts were included in our study. We developed a risk model named GMS based on the MSK training cohort (n=123). The predictive model was first validated in the separate internal MSK cohort (n=82), and then validated in Checkmate-012 (n=59) and Keynote/SU2C (n=52) external clinical trial cohorts. Tumor samples from the patients were molecularly profiled by MSK or whole exome sequencing. Findings: A GMS that consisted of 8 genes (TP53, KRAS, STK11, EGFR, PTPRD, KMT2C, SMAD4 and HGF) was generated to classify patients into high and low GMS groups in the training cohort. Patients with high GMS in the training cohort had longer progression-free survival (HR 0·44, 0·30-0·65, p=0·0002) and overall survival (HR 0·56, 0·33-0·94, p=0·0412) compared with low GMS. We noted equivalent findings in the internal validation cohort and in the external validation cohort. The GMS was demonstrated as an independent predictive factor for anti-PD-1(L1) therapy, without affecting the prognosis of non-squamous NSCLC. Meanwhile, GMS showed undifferentiated predictive value in patients with different clinicopathological features. Notably, both GMS and PD-L1 were independent predictors and demonstrated poorly correlated, inclusion of PD-L1 with GMS furtherly improved the predictive capacity for PD-1 blockade immunotherapy. Interpretation: Our study highlights the potential predictive value of GMS for immunotherapeutic benefit in non-squamous NSCLC. Besides, the combination of GMS and PD-L1 may serve as optimal partner in guiding treatment decisions for anti-PD-(L1) based therapy. Funding Statement: The National Natural Science Foundation for Young Scientists of China, the Natural Science Foundation of Guangdong Province, and the Outstanding Youths Development Scheme of Nanfang Hospital, Southern Medical University. Declaration of Interests: The authors have no actual or potential conflicts of interest to declare. Ethics Approval Statement: This study was approved by the Institutional Ethical Review Boards of Nanfang Hospital. Patients provided signed informed consent in accordance with their respective trial protocols or MSK-IMPACT sequencing project.

Dysregulation of miRNAs has been shown to contribute to the carcinogenesis and progression of nasopharyngeal carcinoma (NPC). Our previous microarray data showed that miR-16 expression is significantly decreased in archived NPC tissues. Here, we confirmed that miR-16 was reduced in NPC cell lines and freshly-frozen samples. Ectopic expression of miR-16 suppressed NPC cell proliferation, migration, and invasion in vitro and inhibited tumor growth and metastatic colonization in the lung in vivo. Furthermore, fibroblast growth factor 2 (FGF2) was identified as a direct target of miR-16, and both phosphoinositide-3- kinase/AKT (PI3K/AKT) and mitogen-activated protein kinase (MAPK) signaling pathways were repressed after miR-16 overexpression. In addition, the restoration of FGF2 reversed the suppressive effects of miR-16. Together, these results indicated that miR-16 suppresses NPC carcinogenesis and progression by targeting FGF2, thereby representing a potential target for miRNA-based therapy for NPC in the future.

Distant metastasis remains the predominant mode of treatment failure in nasopharyngeal carcinoma (NPC). Unfortunately, the molecular events underlying NPC metastasis remain poorly understood. Secreted frizzled-related protein 1 (SFRP1) plays an important role in tumorigenesis and progression. However, little is known about the function and mechanism of SFRP1 in NPC. Immunohistochemistry was used to determine SFRP1 expression levels in patients with NPC. SFRP1 function was evaluated using MTT, colony formation, wound-healing, Transwell assays, and in vivo models. The methylation level of SFRP1 in NPC cells was examined using bisulfate pyrosequencing; the Wnt/β-catenin signaling pathway genes were studied using Western blotting. Compared with patients with high SFRP1 expression, patients with low SFRP1 expression had worse overall survival [HR, 2.32; 95% confidence interval (CI), 1.36–3.94; P = 0.002], disease-free survival (HR, 1.98; 95% CI, 1.23–3.18; P = 0.005), and distant metastasis-free survival (HR, 2.07; 95% CI, 1.19–3.59; P = 0.009). Multivariate Cox regression analysis indicated that SFRP1 was an independent prognostic factor. Furthermore, SFRP1 was significantly downregulated in NPC cell lines. SFRP1 overexpression suppressed NPC cell proliferation, migration, and invasion in vitro and lung colonization in vivo. SFRP1 expression was restored after treatment with a demethylation agent, and the SFRP1 promoter region was hypermethylated in NPC cells. β-Catenin, c-Myc, and cyclin D1 were downregulated after SFRP1 restoration, which suggested that SFRP1 suppressed growth and metastasis by inhibiting the Wnt/β-catenin signaling pathway in NPC. SFRP1 provides further insight into NPC progression and may provide novel therapeutic targets for NPC treatment. Cancer Prev Res; 8(10); 968–77. ©2015 AACR.

Objectives: To investigate the prognostic value of residual tumor based on Magnetic resonance imaging(MRI) and establish an effective prognostic nomogram model referring to clinical,pathological and other related factors for predicting prognosis in nasopharyngeal carcinoma. Methods: Overall, 538 patients with non-metastatic, histologically-confirmed nasopharyngeal carcinoma were retrospectively examined. Data from 397 patients were used for the construction and validation of a nomogram based on the presence of residual tumor. A concordance index (C-index) was employed to assess the predictive accuracy and discriminative ability of the nomogram. Results: The 3-year survival rates in the non-residual and residual tumor cohorts were as follows: progression-free survival, 73.4% vs. 61.0%, P = 0.009; locoregional recurrence-free survival, 81.9% vs. 72.0%, P = 0.02; and distant metastasis-free survival, 80.7% vs. 73.5%, P = 0.11. Nine significant factors were included in the nomogram model. The calibration curve for the probability of progression-free survival showed that the nomogram-based predictive values had good concordance with the actual observations. Conclusion: The results showed that the patients in the residual tumor cohorts had a worse prognosis.The proposed nomogram may predict the prognosis and guide clinical decision-making concerning local residual tumors in nasopharyngeal carcinoma patients. Patients with a high risk of progression require more timely and aggressive treatment.

Human nasopharyngeal carcinoma (NPC) has the highest metastatic rate in head and neck. However, the mechanisms underlying NPC metastasis remain unclear. Here using propensity-score-matched miRNA microarray analysis, miR-142-3p is identified to be the most correlated with distant-metastasis-free survival and downregulated in paraffin-embedded NPC with distant metastasis, which is validated in both internal cohort and external GEO dataset from Canada. miR-142 locus hypermethylation was observed and found to be associated with miR-142-3p downregulation in metastatic NPC. Furthermore, miR-142-3p was epigenetically silenced by EZH2-recruited DNMT1 and suppressed NPC cell metastasis and EMT. Intersecting PCR array gene profiling with bioinformatic prediction, we identify ZEB2 as a direct and functional target of miR-142-3p in NPC. Reversal of miR-142-3p silencing efficiently suppresses NPC cell invasion and metastasis. Moreover, epigenetic miR-142 hypermethylation is correlated with unfavorable prognosis in both training and validation cohorts. This study identifies miR-142-3p as a key suppressive regulator in NPC metastasis and reveals a DNMT1-mediated epigenetic mechanism for miR-142-3p silencing, providing a potential prognostic marker and therapeutic target to combat NPC metastasis.

Altered DNA methylation is a key feature of cancer, and aberrant methylation is important in nasopharyngeal carcinoma (NPC) development. However, the methylation mechanisms underlying metastasis of NPC remain unclear. Analyzing data from public databases and conducting our own experiments, we report here that promoter hypermethylation of

Background Alternative polyadenylation (APA) is a widespread phenomenon in the posttranscriptional regulation of gene expression that generates mRNAs with alternative 3′-untranslated regions (3’UTRs). APA contributes to the pathogenesis of various diseases, including cancer. However, the potential role of APA in the development of nasopharyngeal carcinoma (NPC) remains largely unknown. Methods A strategy of sequencing APA sites (SAPAS) based on second-generation sequencing technology was carried out to explore the global patterns of APA sites and identify genes with tandem 3’UTRs in samples from 6 NPC and 6 normal nasopharyngeal epithelial tissue (NNET). Sequencing results were then validated using quantitative RT-PCR in a larger cohort of 16 NPC and 16 NNET samples. Results The sequencing data showed that the use of tandem APA sites was prevalent in NPC, and numerous genes with APA-switching events were discovered. In total, we identified 195 genes with significant differences in the tandem 3’UTR length between NPC and NNET; including 119 genes switching to distal poly (A) sites and 76 genes switching to proximal poly (A) sites. Several gene ontology (GO) terms were enriched in the list of genes with switched APA sites, including regulation of cell migration, macromolecule catabolic process, protein catabolic process, proteolysis, small conjugating protein ligase activity, and ubiquitin-protein ligase activity. Conclusions APA site-switching events are prevalent in NPC. APA-mediated regulation of gene expression may play an important role in the development of NPC, and more detailed studies targeting genes with APA-switching events may contribute to the development of novel future therapeutic strategies for NPC. Electronic supplementary material The online version of this article (10.1186/s12929-018-0477-6) contains supplementary material, which is available to authorized users.

Background Epigenetic abnormalities play important roles in nasopharyngeal cancer (NPC), however, the epigenetic changes associated with abnormal cell proliferation remain unclear. Methods We detected epigenetic change of ZNF671 in NPC tissues and cell lines by bisulfite pyrosequencing. We evaluated zinc finger protein 671 (ZNF671) expression in NPC cell lines and clinical tissues using real-time PCR and western blotting. Then, we established NPC cell lines that stably overexpressed ZNF671 and knocked down ZNF671 expression to explore its function in NPC in vitro and in vivo. Additionally, we investigated the potential mechanism of ZNF671 by identifying the mitotic spindle and G2/M checkpoint pathways pathway downstream genes using gene set enrichment analysis, flow cytometry and western blotting. Results ZNF671 was hypermethylated in NPC tissues and cell lines. The mRNA and protein expression of ZNF671 was down-regulated in NPC tissues and cell lines and the mRNA expression could be upregulated after the demethylation agent 5-aza-2′-deoxycytidine treatment. Overexpression of ZNF671 suppressed NPC cell proliferation and colony formation in vitro; silencing ZNF671 using a siRNA had the opposite effects. Additionally, overexpression of ZNF671 reduced the tumorigenicity of NPC cells in xenograft model in vivo. The mechanism study determined that overexpressing ZNF671 induced S phase arrest in NPC cells by upregulating p21 and downregulating cyclin D1 and c-myc. Conclusions Epigenetic mediated zinc finger protein 671 downregulation promotes cell proliferation and enhances tumorigenicity by inhibiting cell cycle arrest in NPC, which may represent a novel potential therapeutic target.

The American Joint Committee on Cancer (AJCC) staging system is inadequate for an accurate prognosis in nasopharyngeal carcinoma (NPC). Thus, new biomarkers are under intense investigation. Here, we investigated whether the density of TILs could predict prognosis in NPC. First, we used 1490 cases of nasopharyngeal carcinoma samples from two independent cohorts to evaluate the density and distribution of tumor-infiltrating lymphocytes (TILs). Second, in one cohort, we assessed associations between TILs and clinical outcomes in 593 randomly selected samples (defined as the training set) and validated findings in the remaining 593 samples (defined as the validation set). Furthermore, we confirmed the prognostic value of TILs in a second independent cohort of 304 cases (defined as the independent set). Based on multivariable Cox regression analysis, we also established an effective prognostic nomogram including TILs to improve accuracy in predicting disease-free survival (DFS) for patients with nondisseminated NPC. We found that high TILs in the training set were significantly associated with favorable DFS [hazard ratio (HR) 0.41, 95% confidence interval (CI) 0.28-0.58, p 0.001], overall survival (OS, HR 0.42, 95% CI 0.27-0.64, p 0.001), distant metastasis-free survival (DMFS, HR 0.37, 95% CI 0.23-0.58, p 0.001) and local-regional recurrent free survival (LRRFS, HR 0.43, 95% CI 0.25-0.73, p = 0.002). Multivariate analysis showed that TILs are an independent prognostic indicator for DFS in all cohorts. In summary, this study indicated that TILs may reflect the immunological heterogeneity of NPC and could represent a new prognostic biomarker.

Objective To compare hospital costs and clinical outcomes between transradial intervention (TRI) and transfemoral intervention (TFI) in elderly patients aged over 65 years. Methods We identified 1229 patients aged over 65 years who underwent percutaneous coronary intervention (PCI) in Fuwai Hospital, Beijing, China, between January 1 and December 31, 2010. Total hospital costs and in-hospital outcomes were compared between TRI and TFI. An inverse probability weighting (IPW) model was introduced to control potential biases. Results Patients who underwent TRI were younger, less often female, more likely to receive PCI for single-vessel lesions, and less likely to undergo the procedure for ostial lesions. TRI was associated with a cost saving of CNY7495 (95%CI: CNY4419-10 420). Such differences were mainly driven by lower PCI-related costs. TRI patients had shorter length of stay (1.9 days, 95%CI: 1.1-2.7 days), shorter post-procedural stay (0.7 days, 95%CI: 0.3-1.1 days), and fewer major adverse cardiac events (adjusted odds ratio = 0.47, 95%CI: 0.31-0.73). There was no statistical significance in the incidence of post-PCI bleeding between TRI and TFI (P0.05). Such differences remained consistent in clinically relevant subgroups of acute myocardial infarction, acute coronary syndrome, and stable angina. Conclusion The use of TRI in patients aged over 65 years was associated with significantly reduced hospital costs and more favorable clinical outcomes.

Background: Cancer immunotherapy has provided durable responses and improved survival in a subset of patients with advanced disease. Tumor mutation burden (TMB) and copy number alterations (CNA) have been shown to be associated with response to immune checkpoint inhibitors (ICI). However, the prognostic and predictive role of TMB and CNA across different types of advanced cancer has not yet been clarified. Methods: We analyzed the prognostic value of TMB and CNA respectively and in combination across different types of advanced cancers from MSK-IMPACT sequencing cohort. For predictive analysis, non-small cell lung cancer (NSCLC, n=240) and skin cutaneous melanoma (SKCM, n=174) patients from previous studies were analyzed for efficacy to ICIs by TMB and CNA respectively and in combination. Kaplan-Meier curve and log-rank tests were used to estimate correlations of these molecular classifiers with progression-free survival (PFS), and overall survival (OS). Findings: Patients with low TMB level had longer OS in most cancer types including NSCLC, head and neck squamous cell carcinoma (HNSCC), colon and rectal adenocarcinoma (COAD) and prostate adenocarcinoma (PRAD) compared with patients with high TMB level. Similarly, patients with low CNA level had longer OS in most cancer types including NSCLC, bladder urothelial carcinoma (BLCA), breast cancer (BRCA), PRAD and glioblastoma (GBM) compared with patients with high CNA level as well. Furthermore, the combination of TMB and CNA level could serve as a better prognosis biomarker in almost all of the cancer types above. In predictive cohort, high TMB (p=0.009) level and low SCNA (p=0.052) level was associated with longer PFS in NSCLC patients receiving anti-PD1/L1 treatment respectively. For SKCM cohort, patients with low CNA level (p Interpretation: This study shows that TMB and CNA represent both a prognostic and predictive factor of outcomes. Furthermore, the combination of TMB and CNA can jointly stratify metastatic cancers into groups with different prognosis and clinical responses to ICI treatment. It might guide immunotherapeutic decisions for patients with metastatic cancer. Citation Format: Xin-Ran Tang, Zhong Yi Dong, Dehua Wu. Prognostic and predictive role of tumor mutation burden and copy number alterations across metastatic cancer: Immunotherapeutic implications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4065.