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Supplementary Figure Legends and Supplementary Tables from A Four-Gene Promoter Methylation Marker Panel Consisting of GREM1, NEURL, LAD1, and NEFH Predicts Survival of Clear Cell Renal Cell Cancer Patients

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posted on 2023-03-31, 20:12 authored by Iris J.H. van Vlodrop, Sophie C. Joosten, Tim De Meyer, Kim M. Smits, Leander Van Neste, Veerle Melotte, Marcella M.L.L. Baldewijns, Leo J. Schouten, Piet A. van den Brandt, Jana Jeschke, Joo Mi Yi, Kornel E. Schuebel, Nita Ahuja, James G. Herman, Maureen J. Aarts, Fred T. Bosman, Wim Van Criekinge, Manon van Engeland

File containing Supplementary Tables S1-S7 and Legends to Supplementary Figures S1-S4. Supplementary Table S1. Primers for promoter methylation analysis by MSP of the 43 candidate genes. Supplementary Table S2. Primers for mRNA expression analysis by qRT-PCR for 22 genes. Supplementary Table S3. Candidate gene selection. Supplementary Table S4. Validation of expression microarrays and MBD-NGS by MSP. Supplementary Table S5. Candidate gene promoter methylation associated with patient and tumor characteristics - hospital-based series. Supplementary Table S6. Hazard ratio's with 95% confidence intervals and p-values, including model fit according to Harrell's C and AIC, for the single markers, all possible two- and three-marker panels, and the four-marker panel for both the age- and sex-adjusted models and the multivariable models in the hospital-based series. Supplementary Table S7. Hazard ratio's with 95% confidence intervals and p-values, including model fit according to Harrell's C and AIC, for the single markers, all possible two- and three-marker panels, and the four-marker panel for both the age- and sex-adjusted models and the multivariable models in the population-based series. Supplementary Figure S1. Methylation analysis of GREM1, LAD1, NEFH and NEURL by methylation-specific PCR (MSP) in the hospital- and population-based series and in the TCGA series. Supplementary Figure S2. Validation of up-regulation of gene expression after demethylation with DAC by qRT-PCR for 22 genes. Supplementary Figure S3. Cause-specific overall survival curves for candidate genes in hospital-based ccRCC series (related to Supplementary Table S5). Supplementary Figure S4. Cause-specific overall survival curves for GREM1, LAD1, NEFH, and NEURL in hospital- and population-based ccRCC series (related to Table 1 and Table 2).

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ARTICLE ABSTRACT

Purpose: The currently used prognostic models for patients with nonmetastatic clear cell renal cell carcinoma (ccRCC) are based on clinicopathologic features and might be improved by adding molecular markers. Epigenetic alterations occur frequently in ccRCC and are promising biomarkers. The aim of this study is to identify prognostic promoter methylation markers for ccRCC.Experimental Design: We integrated data generated by massive parallel sequencing of methyl-binding domain enriched DNA and microarray-based RNA expression profiling of 5-aza-2′-deoxycytidine–treated ccRCC cell lines to comprehensively characterize the ccRCC methylome. A selection of the identified methylation markers was evaluated in two independent series of primary ccRCC (n = 150 and n = 185) by methylation-specific PCR. Kaplan–Meier curves and log-rank tests were used to estimate cause-specific survival. HRs and corresponding 95% confidence intervals (CI) were assessed using Cox proportional hazard models. To assess the predictive capacity and fit of models combining several methylation markers, HarrellC statistic and the Akaike Information Criterion were used.Results: We identified four methylation markers, that is, GREM1, NEURL, LAD1, and NEFH, that individually predicted prognosis of patients with ccRCC. The four markers combined were associated with poorer survival in two independent patient series (HR, 3.64; 95% CI, 1.02–13.00 and HR, 7.54; 95% CI, 2.68–21.19). These findings were confirmed in a third series of ccRCC cases from The Cancer Genome Atlas (HR, 3.60; 95% CI, 2.02–6.40).Conclusions: A four-gene promoter methylation marker panel consisting of GREM1, NEURL, LAD1, and NEFH predicts outcome of patients with ccRCC and might be used to improve current prognostic models. Clin Cancer Res; 23(8); 2006–18. ©2016 AACR.

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