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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.

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.

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.

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.

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.

Background: Genetic variations in nonsquamous NSCLC displayed significant impact on immune microenvironment and response to programmed cell death protein 1 (PD-1) blockade immunotherapy. We undertook an unbiased analysis to develop a gene mutation-based signature (GMS) and predict the efficacy of anti-PD-1 treatment. Methods: Two independent cohorts (MSK-IMPACT and CheckMate 012) consist of 265 nonsquamous NSCLC patients treated with anti-PD-1 were analyzed for gene mutation via next-generation sequencing. A GMS was built in a randomly selected 123 samples from MSK-IMPACT training cohort, using multivariate cox analysis of high-frequency mutation genes (≥10%) associated with progression-free survival (PFS) after anti-PD-1 treatment. We then validated our findings in the remaining 83 samples (internal validation cohort) and in CheckMate-012 external validation cohort (n=59). Results: A GMS that consisted of 6 genes (KRAS, EGFR, TP53, STK11, PTPTD and KMT2C), 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 PFS (p Conclusion: Our study highlights the potential predictive value of GMS for immunotherapeutic benefit in nonsquamous NSCLC. The combination of GMS and PD-L1 may be a feasible and promising biomarker in guiding treatment decisions for anti-PD-1 therapy. Table 1Univariate and multivariable Cox regression analysis of predictive factorsUnivariate analysisMultivariate analysisVariableHR95%CIP-valueHR95%CIP-valueMSK-IMPACT cohortGMS (High vs. low)0.360.19-0.680.0020.380.20-0.740.004PD-L1 (High vs. low)0.520.31-0.860.0110.570.33-0.960.034TMB (High vs. low)0.590.36-0.990.046Gender (Male vs. female)0.810.49-1.350.423Age (≥ 65 vs. Citation Format: Zhong-Yi Dong, Xin-Ran Tang, Li Liu, De-Hua Wu. Development and validation of a gene mutation-based signature to predict response to PD-1 inhibitors in nonsquamous NSCLC [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 4066.